INSECTS AND OTHER TERRESTRIAL ARTHROPODS:

BIOLOGY, CHEMISTRY AND BEHAVIOR

LEPIDOPTERA
CLASSIFICATION,
BEHAVIOR AND ECOLOGY

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 INSECTS AND OTHER TERRESTRIAL
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INSECTS AND OTHER TERRESTRIAL ARTHROPODS:
BIOLOGY, CHEMISTRY AND BEHAVIOR

LEPIDOPTERA
CLASSIFICATION,
BEHAVIOR AND ECOLOGY

ELIA GUERRITORE
AND
JOHANNES DESARE
EDITORS

New York
Copyright © 2013 by Nova Science Publishers, Inc.

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Lepidoptera : classification, behavior and ecology / editors: Elia Guerritore and Johannes DeSare.
[Link].
Includes index.
ISBN:  (eBook)
1. Lepidoptera. I. Guerritore, Elia. II. DeSare, Johannes.
QL542.L43 2013
595.78--dc23
2012040817
ISBN: 978-1-62417-248-9

Published by Nova Science Publishers, Inc. † New York

 CONTENTS

Preface vii
Chapter 1 The Molecular Components of Diapause in the Moth Sesamia
Nonagrioides (Lepidoptera: Noctuidae) 1
Anna Kourti, Theodoros Gkouvitsas
and Dimitrios Kontogiannatos
Chapter 2 Current Issues in Integrated Pest Management of Lepidoptera
Pest Threats in Industrial Crop Models 45
Petros T. Damos
Chapter 3 Odor Plumes and Odor Source Interactions: Behavioral
and Physiological Implications on Phytophagous Insects 87
Muhammad Binyameen
Chapter 4 Immune Response in Lepidoptera 119
Eileen Uribe-Querol, Nancy Mora
and Carlos Rosales
Chapter 5 Grape Vine Moth, Lobesia Botrana: Classification,
Behavior and Ecology 149
Vanesa Ortega-López, Mariano Amo-Salas
and Belén Alonso
Chapter 6 South American Lonomia Obliqua Caterpillars: Morphological
Aspects and Venom Biochemistry 169
Ana Marisa Chudzinski-Tavassi
and Miryam Paola Alvarez-Flores
Chapter 7 Ecology of the Blue-Striped Grub Moth Parasa Lepida
(Lepidoptera: Limacodidae) in Japan 187
Kazuo Yamazaki, Kosuke Nakanishi and Hiroichi Sawada
Chapter 8 Biotransformation of Secondary Plant Metabolites by Lepidoptera 203
Clécio Souza Ramos
Index 217
 PREFACE

Lepidoptera is a large order of insects that includes moths and butterflies which inhabit
all terrestrial habitats ranging from desert to rainforest, from lowland grasslands to mountain
plateaus. In this book, the authors discuss the classification, behavior and ecology of
lepidoptera. Topics include the molecular components of diapause in the moth sesamia
nonagrioides; integrated pest management of lepidoptera in industrial crop models; odor
plume behavioral and physiological implications in phytophagous insects; immune response
in lepidoptera; grape vine moth behavior and ecology; South American caterpillars; and
ecology of the blue-striped grub moth parasa lepida.
Chapter 1 - Insects enter in diapause in response to diverse environmental cues (nutrients,
day length or temperature). During diapause, insects arrest their development and many genes
are down-regulated while a small number of genes uniquely expressed at this time. The
knowledge of the mechanism of diapause is essential for understanding the seasonal biology
of insect species. This review presents available data regarding the rythmisity of diapause in
the moth Sesamia nonagrioides (Lepidoptera: Noctuidae). Studying the transcriptional
regulation of several genes (five heat shock proteins, two storage proteins and one juvenile
hormone esterase) the authors showed that these genes may play various roles in the diapause
programming.
The heat shock protein genes respond differently to diapause conditions in S.
nonagrioides. SnoHsp19.5 gene was consistently expressed, while SnoHsp20.8 was down-
regulated in deep diapause and was up-regulated at the termination of diapause, suggesting
that these two genes play distinctive roles in the regulation of diapause. SnoHsp70 is down
regulated during diapause, while SnoHsc70 is induced as the larvae enter in deep diapause.
High temperature stress during diapause has no further effect on transcript levels of
SnoHsc70. The authors’ results show that SnoHsc70 may play important roles in assisting
protein conformation during specific stages of diapause. SnoHsp83 displays a similar pattern
to SnoHsc70 under diapause conditions, when extra larval moults occur, indicating that could
be involved in the developmental process that occurs between two moults.
To date, two hexamerin encoding genes, SnoSP1 and SnoSP2 have been characterized in
corn stalk borer. Expression of both hexamerin genes was observed throughout diapause, for
as long as 130 days. The results confirm the importance of SnoSP1 and SnoSP2 biosynthesis
and finally lead us to the conclusion that larval diapause of S. nonagrioides is associated with
continuous synthesis and accumulation of storage proteins. However, enhanced levels of
SnoSP2, but not SnoSP1 transcripts were detected at the initiation of diapause. In contrast,
viii Elia Guerritore and Johannes DeSare

levels of SnoSP1 were detected in deep diapause phase and greatly increased during post-
diapause phase. The SnoSP1 and SnoSP2 genes have the same transcriptional pattern in non-
diapausing conditions and almost the opposite pattern during diapausing conditions. The
authors’ data suggest that individual members of the methionine rich storage proteins family
may have distinct roles in diapause of corn stalk borer. SnoSP2 associated with the onset of
diapause, while the SnoSP1 with the termination of diapause.
The SnoJHER transcriptlevels were higher in long day non-diapausing larvae than in
short day diapausing ones. During the fifth instar of the non-diapausing and the ninth instar of
the diapausing larvae, SnoJHER mRNAs reached higher expression levels on the days close
to each larval molt. In the last (sixth) nondiapausing larval instar, SnoJHER mRNA levels
peaked during the intermolt period but were lower than during the fifth instar.
Our results suggest that these genes could play major roles in the developmental process,
regulating important physiological mechanisms, as diapause.
Chapter 2 - The sweeping intensification of agriculture using industrial models that grows
annual crops in monoculture is characterized by strong dependency of influxes. Pest
management in particular relies heavily upon the use of synthetic chemical compounds and
related energy inputs. Common side effects of pesticides include not only resistance
development but also environmental impacts on the diversity, composition and functioning of
natural ecosystems and rural areas throughout the world. This chapter explores current
Integrated Pest Management (IPM) strategies to control Lepidoptera pests-threats that are
damaging industrial crops models including sugar beet (Beta vulgaris), potato (Solanum
tuberosum), cotton (Gossypium hirsutum) and corn (Zea mays). The information is in
particular designed to encourage the development alternative control options for rational
management of moths with high economic significance for crop production, including:
Agrotis sp. (Lepidoptera: Noctuidae), Phthorimaea operculella (Lepidoptera: Gelechiidae),
Helicoverpa armigera (Lepidoptera: Noctuidae) and Sesamia nonagrioides (Lepidoptera:
Noctuidae). Representative examples of decision making tools, utile in IPM, are discussed
such as monitoring and scouting procedures, the use of phenological models and the
development of economic injury levels and thresholds. Moreover, efforts are made to
integrate most of the available-alternative pest control options on a combined manner. The
significance of cultivation measures and biological control option, as well as the use of
semiochemicals and pheromones are emphasized. Additionally, since pesticides rotation is
essential for IPM, to avoid resistance development, the major pesticides groups are classified
according to their mode of action and chemical structure. The IPM compatibility of
biorational products is outlined, while the sustainable use of pesticides is discussed according
to the latest directives of the European Parliament upon their future use in Agriculture.
Although the authors addressed the subject primarily from a perspective to manage
Lepidoptera pests, most IPM principles of the current chapter are presented on a holistic
manner in the view to be wider adopted in several industrial crop models to control several
pests.
Chapter 3 - In nature, insects live in an olfactory landscape of diverse semiochemicals.
When odor molecules are released from “attractive” and “inhibitory” sources, they can mix
together, and travel in the form of complex plumes similar to those seen in the form of smoke
plumes. To locate and select hosts or conspecifics and to avoid non-hosts or danger, insects
must discriminate between these “positive” and “negative” signals present in the odor plumes.
Various factors affect this discrimination of “positive” and “negative” signals, of which odor
 Preface ix

plume structure and odor source interactions are of importance. Odor dispersion takes place
by molecular diffusion and wind turbulence. With molecular diffusion, random movement of
molecules gradually causes them to move away from each other with time, and creates clines
of concentration on very small scales (mm- cm). However, odor dispersal strongly depends
on wind and turbulence, which are the most frequent odor dispersal features that an insect has
to deal with in order to find its odor source over distance. In an odor plume, odors travel in
package or filament laden air interspersed by clean air. These filaments maintain their highest
concentrations near the odor source and, as they travel downwind, become elongated and torn
apart by eddies and wind turbulence. Consequently, the odor concentration within plumes
decreases as they move downwind, but as odor filaments are interspersed with pockets of
clean air and due to their filamentous nature, compound ratios stay very similar. Insects
infrequently or never utilize time-averaged concentrations and instead use near-instantaneous
concentrations. With distance from the odor source, the plume width and height increase,
while filament intensity and intermittency decrease and distance between filaments increase.
This implies that the interval between filaments and the concentration within filaments may
both provide important information for insects trying to locate odor sources. Behavioral
studies suggest that odor-induced and wind-guided (anemotaxis) behaviors are the key
components for insects to locate the odor sources. The insect olfactory system identifies odors
of interest in complex plumes for successful selection of mate or host. Many electro-
physiological (extracellular as well as intracellular) studies also suggest that precise temporal
or spatio-temporal patterns of olfactory neurons underlie the spatio-temporal neural codes for
odors. Among these, several studies suggest that co-localization of disparate olfactory
receptor neurons in the same sensillum, specificity of olfactory receptor proteins, and
neuronal interactions in the primary olfactory centre (antennal lobe) and higher brain centre
(mushroom body and calyx) all play roles in the coding of complex mixture of odors in a
plume. Thus, odor plume structure and interaction between plumes may play a key role in
host or mate selection in phytophagous insects. Plume structure can be analyzed with
advanced technologies and analytical tools such as photo ionization detectors and
electroantennography as well as by those that are simple and less expensive, like soap bubbles
generators. However, there is very little, if any, understanding of interactions of odor plumes
and their implications on insect’s behaviors and sensory physiologies; these areas should be a
focus of future research.
Chapter 4 - Insects like many other organisms are exposed to a wide range of infectious
agents. Defense against these agents is provided by innate immune systems, which include
physical barriers, humoral responses, and cellular responses. Much of what we know about
insect immune systems comes from studies on Lepidopteran insects. Caterpillars are also
among the most important agricultural pests, and understanding their immune systems has
enormous implications. Upon infection, Lepidopteran larvae activate cellular and humoral
defense responses. The cellular responses include nodulation, encapsulation, melanization,
and phagocytosis, while the humoral responses lead to the production of antimicrobial
peptides. An early response includes hemocyte adhesion, leading to phagocytosis, nodule
formation, or encapsulation. Microorganisms are recognized by binding of hemocytes or
hemolymph proteins to microbial surface components. This “pattern recognition” initiates
phagocytosis, activates prophenoloxidase and melanization, and also triggers Serine
proteinase cascade pathways that lead to activation of the cytokine Spätzle, which in turn
initiates the Toll pathway leading to expression of antimicrobial peptides. This chapter
x Elia Guerritore and Johannes DeSare

describes Lepidoptera innate immune functions with emphasis on recent advances on the
authors understanding of humoral and cellular responses and the receptors involved in the
recognition of microorganisms.
Chapter 5 - The grape vine moth, Lobesia botrana, a microlepidoptera member of the
family Tortricidae, is one of the most important pests that attacks vineyards in Southern
Europe and Northern Africa. It is a polyvoltine species with three or four generations per year
depending on the latitude. The number of generations is mainly modulated by the temperature
and photoperiod but other factors including relative humidity also have an important
influence on its development. Temperature and photoperiod operate on the growth rate and
the induction of diapause, respectively. L. botrana passes through eight development stages in
its cycle: egg, five larval stages, pupa and adult.
The efficiency of the control methods (mating disruption with pheromones and chemical
treatments) depends on the treatment of pest populations at their most susceptible stages, the
prediction of the moth’s development cycle would therefore greatly help in determining an
optimal treatment schedule. Pheromone traps provide useful information on male moth
activity in vineyards, but by themselves, do not provide a reliable basis for timing control
methods. However, together with the method of temperature summation, trap samples are
important components of phenological models which could be helpful in identifying the peak
flight of [Link], achieving better timing of treatments. Considering the increasing interest
for biorational insecticides where precise timing of treatments is important, daily temperature
models could be a useful tool in improving their efficiency.
Chapter 6 - Lepidoptera species are widely distributed around the world and the majority
of medically important envenoming occurs after the contact of human skin with the hairs or
bristles of the larva form (caterpillars). In most occasions, the adverse effects are self-limited
and the treatment is based on the use of antipruritics and oral antihistamines. However,
envenomation caused by the South American Lonomia obliqua caterpillar (family:
Saturniidae), can lead to a hemorrhagic syndrome characterized by coagulation disorders. A
specific anti-serum had to be produced for an effective treatment against the caterpillar
venom and for re-establishing the coagulation parameters in poisoned patients. The biological
cycle of L. obliqua is composed by 4 phases with distinct durations (egg, larva with 6 instars,
pupa, and adult). Human envenoming usually occurs with larvae between instars 4 to 6. After
the contact with the human skin, caterpillar bristles are broken and the toxic principle quickly
reaches the bloodstream, affecting mainly the hemostatic system. Recent studies have
demonstrated that the mechanisms triggered by L. obliqua toxins are complex and some
multifunctional toxins have been related to the pathophysiology of envenoming but also
related to the development process of the animal, such as regulation of the cell cycle. The
main toxins isolated from this venom (a prothrombin activator and a factor X activator) act at
different levels on the coagulation cascade. This chapter reviews the information currently
available about distribution, morphological aspects, adverse effects in humans and venomous
components related to the Lonomia obliqua caterpillar that have been described so far.
Chapter 7 - The blue-striped grub moth Parasa lepida (Cramer) is an invasive alien
species in Japan; serious infestations occur in various street and fruit trees. To better
understand its present ecological impacts and to devise efficient control methods for such
alien pests, studies that compare their ecology among districts are useful. In this chapter, the
authors summarize the bionomics of P. lepida in Japan for comparison with their native
ranges. This species was first found in southern Japan in 1921; its distribution spread
 Preface xi

northeast into the eastern mainland, where it is generally restricted to urban and suburban
areas. It emerges twice a year, but a portion of the population has a univoltine life cycle and
infests dozens of broad-leaved tree species, but not palms. The larvae have venomous spines
and modified setae that cause dermatitis in humans. Various natural enemies such as
pathogenic viruses, fungi, invertebrate predators, and birds attack immature stages of this
moth. Parasitoids play only a minor role in the regulation of these moth populations. The
cocoons are so durable that we can use them to examine population dynamics. Intensive
studies using life table analyses suggested that nuclear polyhedrosis virus (NPV) might kill
considerable numbers of second-generation larvae, reducing the number of adults emerging
from overwintering cocoons. Populations of this moth are controlled by spraying insecticides
and removing overwintering cocoons and aggregations of early-instar larvae. Future
perspectives for ecological studies of and control methods for this moth are discussed.
Chapter 8 - It is presumed that the first insects in the fossil record from the middle of the
Carboniferous period were not herbivores, but rather saprophages, and that the habit of
feeding on green plants arose during the co-evolution of insects and plants. In response to the
change in the saprophagous habit of insects, plants developed chemical defenses against them
by accumulating toxic compounds known as secondary metabolites. In response, insects
developed strategies to overcome the chemical defense arsenal of plants. There is still much
discussion over exactly how this occurred, but there is evidence that insects developed the
ability to use the biotransformation mechanism to detoxify these plant defense substances.
The successful exploitation of plants as food by the second largest order of insects,
Lepidoptera, with over 160,000 species, can be associated with the capacity of herbivorous
species to minimize the harmful effects of toxic plant substances through biotransformation as
the main detoxification mechanism. Since Lepidoptera species choose the host plant during
the adult phase, their larvae must adapt to the diet imposed on them by overcoming the
possible chemical and physical barriers. Despite a dearth of reports, studies of the metabolism
of plant compounds by Lepidoptera show that biotransformation of compounds to their less
toxic and more polar forms is a strategy employed by these insects. For example, larvae of the
butterfly Heraclides brasiliensis biotransform the compound 4-nerolidylcatechol, a natural
antioxidant and insecticide, the main constituent of Piper umbellata leaves, to its respective
acid, which is less toxic and more polar than 4-nerolidylcatechol The study of the metabolism
of plant compounds by Lepidoptera, besides generating information that can help understand
the feeding habits of insects and how they overcome plants’ chemical defense mechanisms,
can also help in the search for new secondary metabolites with potential as drugs or
agricultural chemicals.
Of the extraordinary diversity found in the world’s biota, 60% corresponds to the Insecta,
of which 46% are phytophagous. Lepidoptera is the second largest order of insects, with more
than 160,000 identified species and its caterpillars are strongly attracted to plants. Initially, it
was presumed that the first insects, including Lepidoptera, in the fossil record, dating from
the middle of the Carboniferous period, were not herbivores but rather saprophages, and that
the habit of feeding on green plants arose during the co-evolution of insects and plants. In
response to the change in the saprophagous habit of insects, plants developed chemical
defenses against them by accumulating toxic compounds known as secondary metabolites,
causing insects to develop strategies to overcome the chemical defense arsenal of plants.
There is still much discussion over exactly how this occurred, but there is evidence that
xii Elia Guerritore and Johannes DeSare

insects developed the ability to use the biotransformation mechanism to detoxify these plant
defense substances
The chemical interaction between Lepidoptera and plants is a well-studied subject in
chemical ecology, with several papers available. It is well known from early studies that
several deterrent chemicals from plants, such as cardenolides, pyrrolizidine alkaloids, iridoid
glycosides and aristolochic acids, are sequestrated by Lepidoptera, which use these
substances for their own purposes. In danaid butterflies, cardenolides have often been
implicated in chemical defense, and pyrrolizidine alkaloids in both chemical defense and
pheromone production. However, the metabolic pathway of toxic compounds from plants in
Lepidoptera and other insect orders is underexplored, mainly involving the natural diet of
herbivorous insects, and is almost nonexistent when involving the volatile chemical
constituents of the host plants, although this study is a crucial step to understanding how they
overcome plant chemical defenses and adapt to host plants.
The successful exploitation of plants as food by the second largest order of insects can be
associated with the capacity of these herbivorous to minimize the harmful effects of toxic
plant substances through biotransformation as the main detoxification mechanism. Since
Lepidoptera species choose the host plant during the adult phase, their larvae must adapt to
the diet imposed on them by overcoming the possible chemical and physical barriers. Despite
the dearth of reports, studies of the metabolism of plant compounds by Lepidoptera show that
biotransformation of compounds to their less toxic and more polar forms is a strategy
employed by these insects. This chapter describes the metabolism of several plant secondary
metabolites and their possible biotransformation during the digestive process in Lepidoptera
larvae.
In: Lepidoptera ISBN: 978-1-62417-248-9
Editors: Elia Guerritore and Johannes DeSare © 2013 Nova Science Publishers, Inc.

Chapter 1

THE MOLECULAR COMPONENTS

OF DIAPAUSE IN THE MOTH SESAMIA
NONAGRIOIDES (LEPIDOPTERA: NOCTUIDAE)

Anna Kourti, Theodoros Gkouvitsas

and Dimitrios Kontogiannatos
Department of Agricultural Biotechnology,
Agricultural University of Athens, Athens, Greece

ABSTRACT
Insects enter in diapause in response to diverse environmental cues (nutrients, day
length or temperature). During diapause, insects arrest their development and many genes
are down-regulated while a small number of genes uniquely expressed at this time. The
knowledge of the mechanism of diapause is essential for understanding the seasonal
biology of insect species. This review presents available data regarding the rythmisity of
diapause in the moth Sesamia nonagrioides (Lepidoptera: Noctuidae). Studying the
transcriptional regulation of several genes (five heat shock proteins, two storage proteins
and one juvenile hormone esterase) we showed that these genes may play various roles in
the diapause programming.
The heat shock protein genes respond differently to diapause conditions in S.
nonagrioides. SnoHsp19.5 gene was consistently expressed, while SnoHsp20.8 was
down-regulated in deep diapause and was up-regulated at the termination of diapause,
suggesting that these two genes play distinctive roles in the regulation of diapause.
SnoHsp70 is down regulated during diapause, while SnoHsc70 is induced as the larvae
enter in deep diapause. High temperature stress during diapause has no further effect on
transcript levels of SnoHsc70. Our results show that SnoHsc70 may play important roles
in assisting protein conformation during specific stages of diapause. SnoHsp83 displays a
similar pattern to SnoHsc70 under diapause conditions, when extra larval moults occur,
indicating that could be involved in the developmental process that occurs between two
moults.


Iera Odos 75, 11855, Athens, Greece, Tel. +30 210-5294615. E-mail: akourti@[Link].
2 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

To date, two hexamerin encoding genes, SnoSP1 and SnoSP2 have been
characterized in corn stalk borer. Expression of both hexamerin genes was observed
throughout diapause, for as long as 130 days. The results confirm the importance of
SnoSP1 and SnoSP2 biosynthesis and finally lead us to the conclusion that larval
diapause of S. nonagrioides is associated with continuous synthesis and accumulation of
storage proteins. However, enhanced levels of SnoSP2, but not SnoSP1 transcripts were
detected at the initiation of diapause. In contrast, levels of SnoSP1 were detected in deep
diapause phase and greatly increased during post-diapause phase. The SnoSP1 and
SnoSP2 genes have the same transcriptional pattern in non-diapausing conditions and
almost the opposite pattern during diapausing conditions. Our data suggest that individual
members of the methionine rich storage proteins family may have distinct roles in
diapause of corn stalk borer. SnoSP2 associated with the onset of diapause, while the
SnoSP1 with the termination of diapause.
The SnoJHER transcriptlevels were higher in long day non-diapausing larvae than in
short day diapausing ones. During the fifth instar of the non-diapausing and the ninth
instar of the diapausing larvae, SnoJHER mRNAs reached higher expression levels on the
days close to each larval molt. In the last (sixth) nondiapausing larval instar, SnoJHER
mRNA levels peaked during the intermolt period but were lower than during the fifth
instar.
Our results suggest that these genes could play major roles in the developmental
process, regulating important physiological mechanisms, as diapause.

Keywords: Sesamia nonagrioides, diapause, heat shock proteins, storage proteins, juvenile
hormone esterase

INTRODUCTION
Diapause, a developmental program that has existed in metazoans for millions of years, is
characterized by processes opposite to those of reproductive growth, such as the arrest or
slowing of cell division in response to an immediate or anticipated stress, reduction of
metabolism and enhancement of stress tolerance, (Adedokun & Denlinger, 1985; Baker
&Russell, 2000). Diapause is a more profound, endogenously and centrally mediated
interruption that routes the developmental programme away from direct morphogenesis into
an alternative diapause programme of succession of physiological events; the start of diapause
usually precedes the advent of adverse conditions and the end of diapause need not coincide
with the end of adversity (Danks, 1987; Kostal, 2006). Among different insect groups
diapause may occurs at any stage of the development, although in every species the
diapausing stage is specific (Denlinger, 1985; Nijhout, 1994).
Diapause is divided into overlapping stages encompassing induction, preparation,
initiation, maintenance and termination, with the latter followed by post-diapause quiescence
if environmental conditions are not conducive to growth (Almbro & Kullberg, 2008). The
entry into and progression through diapause are mediated by molecular mechanisms similar to
those that guide cellular reproductive growth; these include differential gene expression, post-
transcriptional events, post-translational protein modifications and protein localization to
specific regions within cells and organisms. The ability to execute these events in a
coordinated manner implies the function of molecular switches that exert regulatory roles,
directing development along the appropriate pathways. Identifying the switches and the extent
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 3

to which they are shared between organisms are central issues in the study of diapause
(Bharucha, 2009).
A typical indicator of diapause is the low rate of protein synthesis, while shortly before
and after diapause this rate is significantly higher (Chippendale, 1988). In many insect
species, the onset of diapause is also correlated with the synthesis of selected proteins. In
nondiapausing individuals those proteins are either not detectable, or found in much lower
concentrations (Salama & Miller, 1992; De Kort, 1996). Such facts led researchers to ask
whether diapause can be associated with a specific pattern of gene expression rather than a
shutdown of genetic activity. Several of the diapause up-regulated genes that have been
identified so far, encode proteins that may be involved in stress responses or may contribute
to the protection of the insect during its long period of dormancy. It is known that genes
characteristic of reproductive growth are down-regulated during diapause, while others genes,
often termed diapause, are up-regulated, and the expression of still others is either not altered
or changes intermittently (Lee et al., 2002).
A glimpse of the molecular mechanisms underlying diapause has begun to emerge in a
number of insect species, providing us with tantalizing directions for future research. From a
genetic perspective, it appears that genes involved in clock function, insulin signaling, stress
resistance, and development have been co-opted into insect diapause pathways. Finally, there
are similarities between insect diapauses and other dormancies such as dauer formation in
nematodes, hibernation in mammals, and mammalian embryonic diapause. This review
presents recent data regarding the rythmisity of diapause in the moth Sesamia nonagrioides
(Lepidoptera: Noctuidae) and furthermore reviews the molecular events dictating the
expression of diapause. Studying the transcriptional regulation of several genes involved in
stress resistance and development (five heat shock proteins, two storage proteins and one
juvenile hormone esterase) we showed that these genes may play various roles in the diapause
programming.
The Mediterranean Corn Borer Sesamia nonagrioides (Lefèbvre) is a lepidopteran of the
Noctuidae family that feeds mainly on maize, sugar cane and sorghum. It is found in almost
all Mediterranean countries, from the northern border at the 45th parallel to the southern
border north of Africa. S. nonagrioides is a multivoltine species. In Greece, three to four
generations are completed per year (Fantinou et al., 1995). Stavrakis (Stavrakis, 1967)
reported that the pupation of the overwintering population in Greece takes place in April-
May. The importance of this fourth generation depends on the percentage of second-
generation larvae that enter diapause, a hormonally regulated state with altered or reduced
metabolic activity. This state is genetically determined and is a response to a series of stimuli
indicating forthcoming adverse conditions for the insect. It has been reported that a short-day
photoperiod induces larval diapause during the first and second instars and that the effect of
the photoperiod is being modulated by the temperature and the quality of the nutrients
(Eizaguirre et al., 1994; Fantinou et al., 1995).
Diapausing larvae continue to develop and overwinter in the stubbles of maize. While
non-diapausing larvae mostly molt to pupae after the sixth instar, diapausing larvae feed,
move and molt with an indeterminate number of supernumerary molts (Fantinou et al., 1995;
Gadenne et al., 1997). Diapause in S. nonagrioides is related to increased levels of juvenile
hormone (JH) in the hemolymph [Eizaguirre et al., 1995; Eizaguirre et al., 2005; Pérez-Hedo
et al., 2011 a, b). During diapause, the larvae maintain JH at a titer that allows retention of
larval characters during the stationary molts that occur throughout diapause (Chippendale,
4 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

1997). Hemolymph is an open system that allows the dispersion of the hormones
throughought the insect body, and has always attracted the attention of insect physiologists
(Wyatt, 1961). The last (sixth) instar was chosen to carry out our study because it is the one in
which the larva continues its normal development leading to pupation (non-diapausing) or
remains as a larva, molting to another larval stage (diapausing).
The insects in this study were obtained from an established laboratory colony of S.
nonagrioides, derived from larvae collected in Kopais (latitude 38 14΄, Central Greece) in
2007, maintained at 25±1°C and reared on an artificial diet (Tsitsipis et. al., 1984). The colony
of non-diapausing insects was reared under long day (LD) conditions (16:8, light: dark) at
25°C, while diapausing larvae were reared under short day (SD) conditions (10:14, light:
dark) at 25°C. In the following text, the ontogeny that includes diapause will be divided into
three main phases: (1) Pre-diapause: larvae grow through the 6th instar. (2) Deep-diapause:
during the deep-diapause phase, larvae of S. nonagrioides undergo supernumerary moults and
increased their body weights until the 9th instar. (3) Post-diapause: after diapause
development, larvae remain under a post-diapause phase. Body weight decreased, larvae
exhibit supernumerary moults and proceed to pupation. Larvae of S. nonagrioides under SD
conditions exhibit ~13 instars (Gkouvitsas et al., 2008). Two different temperature treatments
were performed: (i) Larvae under LD or SD were placed in a polystyrene tube and submerged
in a water bath at 40 C for 15–60 min. (ii) Larvae under LD were exposed to -5°C, 0°C, 5°C,
10°C, or 17°C, for 1 h. The cold/heat shocked larvae of S. nonagrioides were maintained after
treatment at 25 C at LD. The age of the analysed larvae within each instar was measured in
days after the preceding ecdysis and in respect to physiological markers such as body mass
and head capsule width(Fantinou et al.,1996). The nomenclature describing larval stages
follows the pattern of designation of the instar followed by day of the stadium (e,g. L5d2
denotes larvae of the 5th instar, 2 days after ecdysis). To obtain synchronously growing
animals, newly molted larvae were removed from the colony every day during the 6th-8th hour
of the photophase. The selected larvae had mean weight and mean head capsule width as
follows: for non-diapause conditions (LD) 101.3 mg and 1.74 mm (L5d0); 160.4 mg and 2.32
mm (L6d0). For diapause conditions (SD) 180.1 mg and 2.32 mm (L6d0); 268.4 mg and 2.53
mm (L7d0); 324.1 mg and 2.63 mm (L8d0); 343.7 mg and 2.74 mm (L9d0); 321.7 mg and
2.74 mm (L11d0) and 308.9 mg and 2.68 mm (L13d0) (Kontogiannatos et al., 2011).

A. S. NONAGRIOIDES DIAPAUSE IN RESPONSE

TO STRESS AXES

The level of stress tolerance achieved during diapause varies, along with the degrees of
morphogenesis and behavioural modifications contributing to survival under adverse
conditions. We have investigated the stress resistance of S. nonagrioides during diapause.
Heat shock proteins (Hsps) comprise a diverse group of different classes of proteins present in
almost all forms of life and are expressed in high levels in response to a sudden increase in
temperature (Lindquist, 1986; Nover & Scharf, 1997). Some Hspsare constitutively expressed
in unstressed cells, while others are induced in cells exposed to stressful stimuli such as heat,
cold, desiccation or oxygen deprivation (Feder & Hofmann, 1999; Kregel, 2002). The Hsps
are molecular chaperones acting to prevent misfolding and aggregation of unfolded or
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 5

partially folded proteins (Parsell & Lindquist, 1993; Hart & Hayar-Hartl, 2002). In insects
there are four major heatshock gene families: the small Hsp family with molecular masses
ranging from 20-30 kDa, the Hsp60 family with molecular masses of approximately 60 kDa,
the Hsp70 family with molecular masses of approximately 70 kDa and the Hsp90 family with
higher masses (Denlinger et al., 2001).

1. Expression Pattern of SnoHsp19.5 and SnoHsp20.8 Genes

throughout Diapause

Small heat shock proteins (sHsps) range in size from 12 to 43 kDa and play important
roles in the cellular defence of prokaryotic and eukaryotic organisms against a variety of
internal and external stressors (Narberhaus, 2002). Multimerization of sHsps is assumed to be
crucial for their function as molecular chaperones ensuring correct folding, assembly and
transport of newly synthesized polypeptides as well as removing abnormal cellular proteins.
Therefore, increased expression of sHsps can improve an organism's tolerance to a variety of
environmental challenges such as heat, cold, salt, desiccation and oxidants (Feder and
Hofmann, 1999; Hayward et al., 2004). sHsps appear to be involved in various biological
processes including cytoskeletal dynamics, cell differentiation, aging and apoptosis Dierick et
al., 2005; Arrigo, 2007). In insects, are assumed to play a role in the regulation of diapause
(Yocum et al., 1998; Rinehart & Denlinger, 2000). During diapause, many genes are down-
regulated and a small number of genes are uniquely expressed at this time (Denlinger, 2002).
It has been demonstrated that genes encoding certain stress proteins (Hsp23 and 70) are
highly upregulated during diapause, while others are either unaffected (Hsc70) or are
downregulated (Hsp90) (Denlinger et al., 2001). Precisely what role Hsps may play in the
long-term developmental arrest associated with diapause is unclear. Yocum et al. (1998)
suggested that Hsps may persist for long periods during diapause and this is very interesting
because extended expression of Hsps can lead to deleterious effects, including retardation and
cessation of development (Feder et al., 1992). sHsps has been studied in insects, including
Drosophila triauraria, and D. melanogaster (Goto et al.,1998; Goto and Kimura, 2004), the
flesh fly, Sarcophaga crassipalpis (Yocum et al., 1998; Rinehart et al., 2000), the
endoparasitic wasp, Venturia canescens (Reineke, 2005), the leaf beetle, the intertidal
copepod Tigriopus japonicus (Seo et al., 2006) Gastrophysa atrocyanea (Atungulu et al.,
2006), the Indianmeal moth, Plodia interpunctella (Shirk et al., 1998) and the silkworm,
Bombyx mori (Sakano et al., 2006). The up-regulation of sHsps appears to be common to
diapause in species representing diverse insect orders, including Diptera, Lepidoptera,
Coleoptera, and Hymenoptera that occurs in different developmental stages (embryo, larva,
pupa, adult) (Rinehart et al., 2007).However, recent studies propose that this is not the case in
all insect species studied so far (Goto & Kimura, 2004; Tachibana et al., 2005; Sonoda et al.,
2006).
In order to unravel the potential contribution of sHsps transcripts to diapause of S.
nonagrioides, we isolated two full-lengths cDNA sequences of S. nonagrioides ,(SnoHsp19.5
and SnoHsp20.8) (Gkouvitsas et al., 2008). The lengths of cDNA for Hsp19.5 and Hsp20.8
were 787 and 768 nucleotides, respectively. Hsp19.5 cDNA containing an open-reading
frame of 522 bp, produced a putative protein of 174 amino acids with 5.97 pI and 19.5 kDa.
There is a 5'-untranslated region of 144 bp preceding the initiation codon (ATG). The
6 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

polyadenylation signal (AATAAA) is present at nucleotide 740. This gene was registered in
GenBank with accession number EU668902. The cDNA of Hsp20.8 containing an open-
reading frame of 555 bp, produced a putative protein of 185 amino acids with 6.24 pI and
20.8 kDa. There is a 5'-untranslated region of 93 bp preceding the initiation codon (ATG).
The polyadenylation signal (AATAAA) is present at nucleotide 741. This gene was registered
under DQ336356 in GenBank.
The expression patterns showed that these genes are rapidly and highly induced after heat
stress and down-regulated after cold stress (Figure 1, 2).

40oC

Figure 1. Expression of Sno19.5 and Sno20.8 in response to heat shock in non-diapausing larvae (5th
instar-25 days since hatching). Larvae exposed to 40oC for 15, 30, and 45 min. UC: untreated control. S.
nonagrioides β-tubulin was used as an internal control for normalization.

In order to investigate the expression pattern of SnoHsp19.5 and SnoHsp20.8 genes

during diapause, RNA was isolated from 5th instar non-diapausing or diapausing larvae and
analyzed by semi-quantitative RT-PCR. At 25°C, a non-stress temperature, the presence of
mRNAs encoding sHsps is linked to the entire diapause period.

Figure 2. Semiquantitative RT-PCR expression analysis of Sno19.5 and Sno20.8 genes in non-
diapausing larvae (5th instar-25 days since hatching) upon recovery 1 hour to LD 16:8 at 25oC, after 1
hour cold shock at -5, 0, 5, 10 or 17oC. UC: untreated control. S. nonagrioides β-tubulin was used as an
internal control for normalization.
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 7

Figure 3. Expression of Sno19.5 and Sno20.8 in non-diapausing (5o instar-25 days since hatching) and
diapausing larvae, reared under LD 16:8 at 25iC and LD 10:14 at 25oC, respectively. S. nonagrioides β-
tubulin was used as an internal control for normalization. HS: non-diapausing larvae exposed to 40oC
for 15min.

As shown in Figure 3, expression of SnoHsp19.5 begins with the onset of larval diapause,
and the mRNAs are present until post-diapause (120 days since hatching). During the post-
diapuse period, the larvae of S. nonagrioides will not initiate development. SnoHsp20.8
transcript levels were up-regulated in pre-diapause (15 days since hatching), down regulated
during deep larval diapause and up-regulated in post-diapause stage. These results suggested
that SnoHsp19.5 and SnoHsp20.8 genes had different regulation regime throughout the course
of diapause.
In order to determine the responsiveness of the two genes to heat stress S. nonagrioides
larvae, during diapause or non-diapause, were exposed to 40o C for 1h (Figure 4). However,
under non-diapause conditions, SnoHsp20.8 transcripts remained for longer period than
SnoHsp19.5 mRNAs. This phenomenon was reversed when larvae were in diapause. The
SnoHsp19.5 transcripts remained for longer period than SnoHsp20.8 mRNAs, indicating that
the former should play a crucial role in [Link] additionally tested the effects of cold
shock (-5°C for 15, 30 or 60 min) on the expression pattern of SnoHsp19.5 and SnoHsp20.8
genes in diapausing and non-diapausing larvae (Figure 5).
There was an obvious induction of mRNA accumulation within 60 min after recovery at
°
25 C when diapausing or non diapausing insects were cold-stressed for 15 or 30 min.
However, when larvae were stressed for longer periods (60 min) at 5°C, there was no obvious
induction of mRNA accumulation 6h after recovery, except for the SnoHsp20.8 gene in
diapausing larvae (Figure 5). Our results clearly show that the expression levels of
SnoHsp19.5 and SnoHsp20.8 are regulated in a different way during diapause. In diapausing
larvae of S. nonagrioides, SnoHsp19.5 transcripts showed no difference in levels throughout
the course of diapause, whereas SnoHsp20.8 transcripts undergo regulated changes.
SnoHsp20.8 transcripts were relatively high during the initiation and termination phases of
diapause. This pattern of accumulation clearly defines the early stages of diapause as well as
the culmination of the diapause process. Our understanding of how diapause ends is still
incomplete.
8 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

Hours past heat shock (40oC  1hr)

Figure 4. Semiquantitative RT-PCR expression analysis of Sno19.5 and Sno20.8 genes in non-
diapausing (5th instar) and diapausing larvae (50 days after hatching) upon recovery to LD 16:8 at 25oC,
after a heat shock at 40oC for 1 hour. Numbers indicate the hours after returning. S. nonagrioides β-
tubulin was used as an internal control for normalization.

Figure 5. Semiquantitative RT-PCR expression analysis of Sno19.5 and Sno20.8 genes after recovery
from cold shock in diapausing larvae (50 days after hatching) and nondiapausing (25 days since
hatching) upon recovery after cold shock at -5oC for: a =15 min, b = for 30 min and c= 60 min.
Numbers indicate the hours after returning to LD 16:8 at 25 C. HS = non-diapausing larvae exposed to
40oC for 15 min. S. nonagrioides β-tubulin was used as an internal control for normalization.

It is recognized that diapause maintenance may culminate in spontaneous termination and

resumption of direct development in many insects and mites which are under constant
laboratory conditions, such us in the diapause of S. nonagrioides (Fantinou et al., 1998;
Kostal, 2006). We propose that the expression level increase of SnoHsp20.8 could serve as a
molecular marker of diapause termination in S. nonagrioides. These results indicatethat
Sno19.5 and Sno20.8 genes may have distinctive roles in the initiation, continuation and
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 9

termination of the dynamic process of diapause. SnoHsp19.5 transcripts remained at high

levels after heat treatment during diapause. In contrast, SnoHsp20.8 transcripts remained at
low levels after heat treatment during diapause but were elevated after heat treatment in non-
diapausing [Link] clear distinction in mRNA accumulation of the two genes in diapause
and non-diapause larvae could indicate distinctive roles of the two genes during the process of
diapause and development. Members of the small Hsps families are up-regulated during
dormancies in several noninsect organisms (Bonato et al., 1987; Pla et al., 1998; Cherkasova
et al., 2000). A possible role for small heat shock proteins in diapause is the involvement in
the regulation of the cell cycle arrest (Tammariello & Denlinger, 1998).
Denlinger et al. (2001) suggested that small Hsps are involved in protecting organisms
against low temperature stress and in the regulation of cell cycle arrest. The fact that sHsps
are up-regulated during diapause in a variety of insect species, at different diapause stages,
suggests that sHsps are key players in the overwintering response of many insects. In S.
crassipalpis, a sHsps (Hsp23) is highly up-regulated during fly diapause and this occurs
simply because the pupa enters diapause and is not a function of temperature stress (Rinehart
et al., 2007). Rinehart et al. (2007) proposed that up-regulation of sHsps during diapause is a
major factor contributing to coldhardiness of overwintering insects. They consider the up-
regulation of sHsps to provide a ‘‘back-up’’ mechanism guaranteeing that development will
remain arrested during diapause. By contrast, sHsp genes were not up-regulated in response
to diapause in D. triauraria (Goto et al., 1998; Goto and Kimura, 2004), Lymantria dispar
(Yocum et al., 1991; Denlinger et al., 1992), Lucilia sericata (Tachibana et al., 2005) and
Chilo suppressalis (Sonoda et al., 2006). Some sHsps are induced while others are
suppressed. For the question why are there so many sHsps, in S. nonagrioides it seems that
SnoHsp19.5 and SnoHsp20.8 have specific functions. Most likely, the different sHsps are
interacting differently with specific groups of proteins (Sun & MacRae, 2005). Our results
suggest that these two sHsps contribute to diapause and to cold response in slightly different
ways. The SnoHsp19.5 gene was consistently expressed in response to diapause, while
SnoHsp20.8 gene was down-regulated in deep diapause and up-regulated at the termination of
diapause. Therefore, an analysis of sHsps can provide significant information for
understanding diapause in insects.

2. Expression Pattern of SnoHsc/Hsp70 during Diapause

The Hsp70 family is one of the most highly conserved gene families and its proteins are
the most widely studied (Gupta & Golding, 1993; Boorstein et al., 1994). The Hsp70 gene
family contains both stress inducible and constitutively (Hsc70) expressed genes that share
many common structural features. The relationship between diapause and Hsp70 has been
studied in several insects. Hsp70 genes were found to be up-regulated during diapause in a
number of species, including adults diapause of the flesh fly, Sarcophaga crassipalpis
(Rinehart et al., 2000) and the Colorado potato beetle, Leptinotarsa decemlineata (Yocum,
2001); pupal diapauses of the solitary bee, Megachile rotundata (Yocum et al., 2005) and the
onion maggot Delia antique (Chen et al., 2006); larval diapause of the rice stem borer Chilo
suppressalis (Sonoda et al., 2006), and embryonic diapause of the silkmoth Bombyx mori
(Hwang et al., 2005). However, limited studies have reported on the expression of Hsc70
during insect diapause.
10 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

We determined the full Hsc70 and Hsp70 cDNA sequences and their deduced amino acid
sequences of S. nonagrioides (Gkouvitsas et al., 2009). RT-PCR was performed to clone the
DNA fragment corresponding to Hsc/Hsp70. The amplified fragments were isolated and the
nucleotide sequences were determined. One full-length cDNA sequence was 2166 bp long
and contained an open reading frame (ORF) spanning nucleotides 104-2065 and producing a
putative protein of 653 amino acids with a deduced molecular weight of 71.5 kDa and
putative pI 5.38. There was a 5'-untranslated region of 103 bp preceding the initiation codon
(ATG). It has a polyadenylation signal (AATAAA) at nucleotides 2134-2139, 12 nucleotides
upstream from the poly(A) tail. Amino acid sequence comparisons with both the NCBI
GenBank and PROSITE database showed that this protein belongs to cytosolic Heat shock
cognate 70 kDa protein family. Therefore, this S. nonagrioides gene will be referred as
SnoHsc70. The cDNA sequence was deposited in GenBank with accession number
DQ004584. The second full-length cDNA sequence was 2318 bp long and contained an open
reading frame (ORF) spanning nucleotides 132-2090, producing a putative protein of 633
amino acids with a deduced molecular weight of 70.2 kDa and putative pI 5.76. There was a
5'-untranslated region of 131 bp preceding the initiation codon (ATG). It has a
polyadenylation signal (AATAAA) at nucleotides 2272-2277, 23 nucleotides upstream from
the poly(A) tail. Amino acid sequence comparisons with both the NCBI GenBank and
PROSITE database showed that this protein is an Hsp70 protein. Therefore, this S.
nonagrioides gene will be referred as SnoHsp70. The cDNA was deposited in GenBank with
accession number EU430480 (Gkouvitsas et al., 2009).
To investigate the expression patterns of SnoHsc/Hsp70 genes and to correlate expression
with diapause, RNA was isolated from 5th instar non-diapausing or diapausing larvae and
analyzed by semi-quantitative RT-PCR and Real-Time PCR assays. At 25°C, SnoHsp70
transcripts were consistently low and there was almost no difference in the levels as a
function of the developmental stage of non-diapause larvae or during diapause (Figure 6. A,
C). In contrast, the SnoHsc70 gene was upregulated during deep larval diapause (45-75 days
since hatching). SnoHsc70 showed constitutive expression in pre-diapause (15 days and 25
days since hatching) and post-diapause (120 days since hatching). At these stages, expression
levels were similar to the levels observed in non-diapausing 5th instar larvae (Figure 6A, B).
Transcript accumulation increased in deep-diapause, showed a gradient increase with a peak
in 60 day-old larvae and thereafter a gradual decrease. There was an apparent 2-fold increase
in Hsc70 expression levels when compared to SnoHsc70 mRNAs present in 5th instar
[Link] results indicated that the SnoHsc70 transcript levels differed throughout the
course of diapause. In contrast, the pattern of SnoHsp70 transcript accumulation remained
similar in all stages of diapause.
To determine whether the SnoHsc/Hsp70 genes respond to heat treatments during
diapause, diapausing larvae (50 days since hatching) were transferred to 40°C for 1 h (Figure
7). We further tested the effects upon recovery to ambient temperature. The levels of
SnoHsp70 transcripts were different between diapausing and non- diapausing larvae. Higher
levels of mRNA accumulation were evident in non-diapausing larvae. In non-diapausing
larvae, high levels of SnoHsp70 transcripts were apparent immediately after heat shock.
However, the decline in mRNA accumulation thereafter was rapid (1h). Furthermore, the
transcripts gradually decreased over a period of 4h, reaching levels detected in non-
diapausing larvae.
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 11

Figure 6. Expression of SnoHsc70/Hsp70 in nondiapausing (5th instar-25 days since hatching) and
diapausing larvae, reared under LD 16:8 at 25 oC and LD 10:14 at 25°C, respectively. HS: non-
diapausing larvae exposed to 40oC for 15 min. A. Semiquantitative RT-PCR expression analysis of
SnoHsc70/Hsp70 genes. Snoβ-tubulin was used as the control gene. B. Real-Time PCR analysis of
SnoHsc70/Hsp70 genes. The relative mRNA copy number of the SnoHsc70/Hsp70 genes transcripts are
shown in relation to the number of Snoβ-tubulin transcripts for each cDNA sample. The error bars refer
to the standard error of the mean.
12 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

In diapausing larvae, up-regulation of the gene was minimal; transcripts remained high
levels 2 h after recovery but were undetected at 4h (Figure 7).
In diapausing larvae, levels of SnoHsc70 transcripts were similar after recovery (0, 1, 2,
4h). In non-diapausing larvae the SnoHsc70 transcripts were high at 0 and 1h after heat
treatment, but rapidly declined thereafter (Figure 7). These results show that the SnoHsc70
gene responds to heat shock differently in the two stages tested. While it responds to heat
shock in non-diapausing larvae, it does not respond in deep diapause, indicating that
expression of the gene is also regulated by the diapause/non-diapause conditions.
This study showed that these two members of 70 kDa heat shock protein family are
differentially expressed during diapause in S. nonagrioides. SnoHsp70 transcripts were
consistently low, almost undetectable, at all stages tested. Accumulated data from different
species have resulted in contradictory conclusions. Hsp70 is not upregulated as a function of
diapause in Drosophila triauraria and Lucilia sericata (Goto et al., 1998; Tachibana et al.,
2005) while it is slightly upregulated in the diapausing adults of Colorado potato beetle
Leptinotarsa decemlineata (Yocum, 2001) and is only expressed in the diapausing pharate
first instar larva of the gypsy moth Lymantria dispar after exposure to low temperature
(Yocum et al., 1991). In contrast, in a number of species including S. crassipalpis (Rinehart et
al., 2000), L. decemlineata (Yocum, 2001), M. rotundata (Yocum et al., 2005), D. antiqae
(Chen et al., 2005), C. suppressalis (Sonoda et al., 2006) and B. mori (Hwang et al., 2005),
the transcripts of Hsp70 are upregulated during diapause, even without thermal stress, and
their expression persists throughout diapause. Rinehart et al. (2007) reported five additional
species in which Hsp70 was up-regulated during diapause (two dipterans and three
lepidopteran species with diverse diapause programs: the obligate embryonic diapause of
Lymantria dispar, the facultative larval diapause of Ostrinia nubilalis, and the facultative
pupal diapause of Manduca sexta). Hsp70 up-regulation appears to be more common in
preadult diapause than in adult (reproductive) diapause.

Figure 7. Semiquantitative RT-PCR expression analysis of SnoHsc70/Hsp70 genes in nondiapausing

(5th instar-25 days since hatching) and diapausing larvae (50 days since hatching) after heat shock at
40oC for 1 hour. Numbers indicate the hours upon recovery to LD 16:8 at 25oC. Nondiapausing and
diapausing larvae were reared under LD 16:8 at 25oC and LD 10:14 at 25oC, respectively. Snoβ-tubulin
was used as the control gene.

Our results show that SnoHsc70 is induced during deep diapause. Only a few studies in
insect diapause have reported on Hsc70. In S. crassipalpis the transcripts of Hsc70 showed no
difference in levels throughout the course of diapause and remained the same in diapausing
and nondiapausing individuals (Rinehart et al., 2000), and in M. rotundata, Hsc70 is a normal
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 13

part of both the diapause and post-diapause developmental programs (Yocum et al., 2005).
Therefore different members of the Hsp70 gene family respond differently during
development or specific stages of insect diapause. Since most insects have a number of Hsp70
members, the contradictory results observed in different species may be the consequence of
different members being examined in the respective studies.
The response to heat stress was different for SnoHsp70 when larvae were in diapause or
non-diapause conditions. Under diapause conditions induction of SnoHsp70 was moderate,
indicating that the physiological status of the larvae is a prerequisite even for the bona fide
Hsp genes. Under non-diapause conditions SnoHsc70 is induced while under diapause
conditions the gene is not further induced in response to temperature stress. This result shows
that the regulation of the SnoHsc70 is under strict developmental regulation. The latter,
combined with results obtained under normal deep diapausing conditions, indicates that
SnoHsc70 may play a crucial role during the diapause of S. nonagrioides, most likely
assisting the low but vital protein proper conformation during this specific developmental
period. Possibly SnoHsc70 transcripts during deep-diapause are hormonally controlled.
Larval diapause is characterized by certain endocrine events, such as the presence of
important juvenile hormone (JH) activity (Yin & Chippendale, 1979). According to
Eizaguirre et al. (1998), JH is concerned in the maintenance of diapause in S. nonagrioides. In
the presence of JH insect tissues maintain their developmental status quo when exposed to
ecdysteroids (the molting hormones), whereas in the absence of JH, tissues change their
commitment to that of the next metamorphic stage upon exposure to ecdysteroids (Riddiford,
1996; Nijhout, 1994). In M. sexta, the brain neuropeptide prothoracicotropic hormone
(PTTH) stimulates a rapid increase in ecdysteroid hormone synthesis that is accompanied by
general and specific increases in protein synthesis, including that of a 70 kDa cognate Heat
shock protein (Hsc70) (Rybczynski & Gilbert, 2000). In this species, protein and mRNA data
suggest that Hsc70 could be involved in a negative feedback loop regulating assembly of the
ecdysone receptor complex. SnoHsc70 could possibly be involved in a loop regulating the
ecdysone receptor complex, as in M. sexta. The resolution of the function of Hsc70 in
ecdysteroidogenesis or other cellular processes will require development of model systems in
which Hsc70 levels can be readily manipulated under a variety of conditions.
The results presented here for the corn stalk borer and other examples from the literature
clearly demonstrate that the regulation of the heat shock response is not a simple on/off
reaction but is finely tuned to developmental and environmental conditions. The differential
expression of SnoHsc70 and SnoHsp70 during diapause and in response to high and low
temperatures indicates that individual members of the 70 kDa heat shock protein family may
well have varying roles in diapause of S. nonagrioides.

3. Expression Pattern of SnoHsp83 during Diapause

Hsp90 proteins are essential molecular chaperones involved in signal transduction, cell
cycle control, stress management, and folding, degradation, and transports of proteins. Hsp90
proteins have been found in a variety of organisms suggesting that they are ancient and
conserved (Chen et al., 2006). A unique feature of Hsp90 proteins is that they are
constitutively expressed at substantial level during non-stress conditions (Arbona, 1993). In
unstressed cells, Hsp90 can account for 1–2% of the total cytosolic protein (Picard, 2002).
14 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

The upregulation of Hsp90 in response to heat shock is not as robust as for other heat shock
proteins. In most cases, a 10-15 fold increase in protein levels can be expected when
comparing heat-stressed organisms to unstressed controls (Arbona, 1993). Hsp90 appears to
have a specific function during recovery from stress, being especially important in the
reactivation of stress-inactivated protein (Nathan et al., 1997). The conserved Hsp90
chaperone family includes the Hsp90 of the eykaryotic cytosol Hsp90α and β in humans,
Hsp86 and Hsp82 in mice, Hsp83 in Drosophila and Hsc82 and Hsp82 in yeast (Young et al.,
2001). In D. melanogaster and most other insects, Hsp90 proteins are approximately 83 kDa
in size (Blackman & Meselson, 1986). Unlike Hsp90 in vertebrates, Hsp90 genes for all
insects studied so far exist as a single copy, except of Anopheles albimanus, which contains
two (Benedict et al., 1996). Hsp90 genes are also implicated in developmental regulation,
such as morphological evolution and arrest of reproduction in Drosophila (Rutheford &
Lindquist, 1998; Marcus, 2001), during the dauer stage of Chaenorhabditis elegans (Dalley &
Golomb, 1992), and during the diapause of Chillo suppressalis and Dellia antiquα (Sonoda et
al., 2006; Chen et al., 2005). Studies in D. triauraria and the blowfly, Lucillia sericata, have
not found evidence of Hsp90 involvement in diapause (Goto & Kimura, 2004; Tachibana et
al., 2005), whereas in studies with Sarcophaga crassipalpis Hsp90 transcripts have been
found to be down-regulated during diapause (Rinehart & Denlinger, 2000). This discrepancy
needs to be elucidated by using additional species and different types of diapauses. For most
insects, short day length evokes a stage-specific developmental arrest, known as diapause
(Tauber et al., 1986; Danks, 1987; Denlinger et al., 2005).
We determined the full lenght of Hsp83 cDNA of S. nonagrioides, assempled from the
tag sequence and both 5' and 3' ends, is 2481 bp long and contains a unique open reading
frame (ORF), which produced a putative protein of 717 amino acids with a predicted
molecular mass of 82.6 kDa. The start codon was located at position 173. There was a 5'
untranslated region (5' UTR) of 173 bp and a complete 3' untranslated region (3' UTR) which
contains a canonical poly(A) addition site (AATAAA) located 18 nucleotides upstream from
the start of the poly(A) tract. The termination codon (TAA) occurred at nucleotide 2325. The
cDNA and its deduced protein sequence were deposited in GenBank/EMBL/DDBJ with
accession numbers DQ198859 and ABA54273 respectively. All five highly conserved
segments defining the Hsp90 family signature of known eukaryotes (Gupta, 1995), were well
conserved in the S. nonagrioides sequence. The terminal amino acid sequence MEEVD,
constituting the core of the Hsp90 interaction surface for the tetratricopeptide repeats of
Hsp90 co-chaperones (Ramsey et al., 2000; Scheufler et al., 2000) was strictly conserved and
shared with the Hsp70 gene family (Gkouvitsas et al., 2009).
It had been shown that SnoHsp70 was down regulated during diapause, while SnoHsc70
is induced as the larvae enter deep diapause (Gkouvitsas et al., 2009). This provoked us to
investigate whether other families of Hsps, such as Hsp90, are also up-regulated during
diapause of S. nonagrioides. To answer this we studied the accumulation of SnoHsp83
mRNA under heat or cold stress in diapausing and non-diapausing larvae. SnoHsp83 showed
constitutive expression in pre-diapause phase (15 days and 25 days since hatching) and post-
diapause phase (90 days and 120 days since hatching). At these stages, the levels were similar
to the ones observed at the 5th instar larvae of non-diapausing development (Figure 8A, B).
The transcript accumulation increased in deep-diapause period, showed a gradient increase
with a peak at 60 day-old larvae and thereafter a gradual decrease. There was an apparent 2-
fold increase in Hsp83 expression levels during deep diapause when compared to SnoHsp83
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 15

mRNAs present in 5th instar. These results showed that the SnoHsp83 transcript levels
differed throughout the course of diapause. Hsp90 during diapause could be regulated by
ecdysteroids. In D. melanogaster the presence of ecdysteroids leads to upregulation of Hsp90
(Thomas & Lengyel, 1986) and the Hsp90 chaperone is required for ecdysone receptors
activity (Arbeitman & Hogness, 2000). In the C. elegans, dauer larvae are enriched in Hsp90
gene transcripts and the corresponding protein could interact with steroid hormone receptors
(Dalley & Golomb, 1992; Cherkasova et al., 2000).
The ecdysteroid titer rose before each extra larval molt in S. nonagrioides and a burst at
the passing 8th to 9th instar (Eizaguirre et al., 2007) that is at day 60 of our collection time
(Fantinou at al., 1996). Since S. nonagrioides larvae continue supernumerary molts during
diapause, their physiological state cyclically changes. The periodic patterns of the SnoHsp83
expression could be correlated to the supernumerary molts during diapause or to the cyclic
changes of ecdysteroid titre. The up-regulation of SnoHsp83, as well as that of SnoHsc70
studied previously (Gkouvitsas et al., 2009) could represent important molecular cues for corn
borer diapause. It is very interesting that the two genes display a similar pattern when larvae
are under diapause conditions. SnoHsc70 could possibly be involved in a loop regulating the
ecdysone receptor complex (Gkouvitsas et al., 2009) and SnoHsp83 and SnoHsc70 could be
required for ecdysone activity. Consistent with this possibility is the up-regulation of the two
genes in deep-diapause phase of S. nonagrioides, when extra larval molts go through. These
results might indicate that Hsp90 transcripts were regulated developmentally in diapausing
larvae of S. nonagrioides, i.e., they were up-regulated before entering to 9th instar, suggesting
that it is involved in a developmental process that occurs between two molts. A more
extensive survey is necessary to elucidate how the expression of SnoHsp83 changes between
two molts in order to shed light to diapause at the molecular level. The regulation and
function of the various Hsp families during diapause of corn stalk borer could provide
insights into the molecular mechanisms involved in the diapause of this species.

B. S. NONAGRIOIDES DIAPAUSE IN RESPONSE

TO THE DEVELOPMENT

1. Expression of Two Storage Proteins during Diapause

Many holometabolous insects have been shown to accumulate large quantities of

hexameric proteins in their hemolymph during larval development, and sequestered in fat
body where they serve as sources of nitrogen and amino acids for utilization by pupae and
adults during metamorphosis and reproduction. These hemolymph proteins are generally
referred to as “storage proteins”. Most of these storage proteins are hemocyanin-related
macromolecules and generally referred as hexamerins, because of the composition of six
identical or similar subunits in the 72.000- to 90.000-dalton size range (Telfer & Kunkel,
1991; Burmester et al., 1998).
Insect hexamerins belong to a phylogenetically old arthropod family of proteins that
includes hemocyanins, prophenoloxidases and arylphorin-receptor proteins (Burmester,
2002). They have been characterized mainly in dipteran and lepidopteran species. These
proteins are of fundamental importance for insect development and, therefore, studies
16 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

concerning their structure, biosynthesis, regulation and evolution have been conducted
(Haunerland, 1996; Burmester, 1999; Burmester, 2001). In insects, before the initiation of
metamorphosis, a characteristic family of proteins has been found in storage tissues such as
the hemolymph and fat body. Most of these storage proteins are hemocyanin-related
macromolecules and generally referred as hexamerins, because of the composition of six
identical or similar subunits in the 72,000- to 90,000-dalton size range (Telfer & Kunkel,
1991; Burmester et al., 1998). Hexamerins have long been proposed to serve as a source of
amino acids for tissue reconstruction during pupal development and have been shown to be a
component of the sclerotizing system of the cuticle (Peter & Scheller, 1991). They serve as
ecdysteroid carrier in the haemolymph (Enderle et al., 1983),function in nutrient uptake and
storage, and some are capable of binding the insect morphogenetic hormone juvenile
hormone (JH) (Brawn and Wyatt, 1996; Gilbert et al., 2000; Tawnfik et al., 2006).

Figure 8. Expression of SnoHsp83 in non-diapausing (5th instar-25 days since hatching) (ND) and
diapausing larvae, reared under LD 16:8 at 25oC and LD 10:14 at 25oC, respectively. HS: non-
diapausing larvae exposed to 40oC for 15 min. A. Semiquantitative RT-PCR expression analysis. S.
nonagrioides β-tubulin was used as the control gene. B. Real-Time PCR analysis. The relative
quantities indicate the levels of Snohsp83 transcripts normalized to the internal standard S.
nonagrioides β-tubulin. Each column is a mean±S.E.M. of six repeats. The differences in mRNA levels
were assessed by ANOVA followed by Duncan’s multiple comparison test (P<0.05) (columns flanked
by different letters differ significantly).
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 17

The hexamerins of insects belong to a growing protein superfamily that also comprises
the arthropod hemocyanins and prophenoloxidases, and dipteran hexamerin receptors
(Burmester, 1999 Phylogenetic analysis revealed that they share a common ancestry with
arthropod phenoloxidases such as tyrosinases and the hemocyanins of the Crustacea, which
are copper-containing oxygen carriers occurring in the hemolymph of arthropods (Burmester
et al., 1998; Burmester, 2001). These proteins are generally classified into three groups: (i)
the arylphorins, polypeptides rich in the aryl groups tyrosine and phenylalanine, (ii)
methionine-rich proteins and (iii) proteins which are neither rich in aromatic amino acids nor
in methionine (Scheller et al., 1990; Haunerland and Bowers, 1986; Pan & Telfer, 1996).
Induction of diapause is correlated with many physiological, biochemical and behavioral
traits. Among different insects groups diapause may occur at any stage of development,
although in every species the diapausing stage is specific (Derlinger, 1985; Nijhout, 1994). A
typical indicator of diapause is the low rate of protein synthesis, while shortly before and after
diapause this rate is significantly higher (Chippendale, 1988). In many insect species, the
onset of diapause is also correlated with the synthesis of selected proteins. In nondiapausing
individuals those proteins are either not detectable, or found in much lower concentrations
(Salama & Miller, 1992; De Kort, 1996). Such facts led researchers to hypothesize that
diapause can be associated with a specific pattern of gene expression rather than a shutdown
of genetic activity. A few previous studies have shown that diapause associated proteins may
be hexameric storage proteins (Sula et al., 1995; Kludkiewicz et al., 1996).
Here we report the isolation of two complete hexamerin-cDNA clones (SnoSP1and
SnoSP2) and their deduced amino acid sequence in S. nonagrioides. We examined the
suppression of their mRNA by treatment with methroprene (JH analog) and the
developmental profiles of SnoSP1 and SnoSP2 expression in fat body from diapausing and
non-diapausing larvae. The complete sequence of SnoSP1 cDNA contains 2403 nucleotides
(Spyliotopoulos et al., 2008). Within the cDNA sequence is an open reading frame of 2255
nucleotides. The methionine corresponding to translation initiation codon ATG lies at
positions 24–26, while the translation stop codon TAA at positions 2277–2279. The amino
acid sequence of the protein deduced from SnoSP1 cDNAs has 751 amino acids. The
predicted molecular mass for SnoSP1 is 88.3 kDa and the calculated isoelectric point from the
deduced amino acid sequence is 8.72. SnoSP1 protein was found to be most closely related in
amino acid sequence (71% identity) with Trichoplusia ni basic juvenile hormone-suppressible
protein 2 precursor (TniSP2) (Jones et al., 1993) and with Spodoptera litura moderately
methionine rich storage protein (SliSP2 beta gene and SliSP1alpha gene) (71% and 68%
identity respectively) (Zheng et al., 2000). The physiological and chemical characteristics of
SnoSP1 is similar to the basic juvenile hormone-suppressible proteins (TniSP1 and TniSP2)
of T. ni. Hence, the SnoSP1 belongs to the group of “juvenile hormone suppressible
hexamerins” (Spyliotopoulos et al., 2008). The complete sequence of SnoSP2 cDNA
containes 2332 nucleotides (Gkouvitsas & Kourti, 2009). Within the cDNA sequence is an
open reading frame of 2244 nucleotides. The methionine corresponding to translation
initiation codon ATG lies at positions 25–27, while the translation stop codon TAA at
positions 2269–2271. The amino acid sequence of the protein deduced from SnoSP2 cDNAs
has 748 amino acids. The predicted molecular mass for SnoSP2 is 87.89 kDa and the
calculated isoelectric point from the deduced amino acid sequence is 9.41. The SnoSP2 seems
to fall into the class of hexameric proteins based on its subunit structure and its sequence
similarity to other known hexamerins (Gkouvitsas & Kourti., 2009).
18 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

1.1. Effects of JHA on SnoSP1 and SnoSP2 Expression

We treated unsexed fifth instar larvae with JHA, methoprene, to determine whether the
SnoSP1 and SnoSP2 genes respond to juvenile hormone of. The high degree of similarity
between SnoSP1 and TniSP2 suggested that the SnoSP1 gene may be regulated by JH, as has
been proposed for other Met-rich storage proteins (Haunerland, 1996). In allatectomized
Manduca sexta larvae, female specific storage protein (FSP) mRNA appears precociously,
whereas its appearance is prevented by application of the JH mimic, methoprene, indicating
that JH down-regulates the appearance of FSP mRNA (Webb & Riddiford, 1988). Jones et al.
(1993), demonstrated that maintenance of a high JH titer from the penultimate stadium into
the final stadium of T. ni suppressed transcripts for the basic proteins to less than 1% of their
normal level on day 2. Also in the fall webworm, Hyphantria cunea, topical application of the
methoprene, suppressed transcription of the SP-2 gene (a juvenile hormone-suppressible
hexamerin) to less than 30% of its normal level (Hwang et al., 2001). Our results (Figure 9)
are consistent with hypothesis that the SnoSP1 gene exhibit negative regulation by JH. Figure
9 shows that within fat body, SnoSP2 transcription levels did not change significantly in
comparison with the acetone-treated controls.

1.2. SnoSP1 mRNA Expression Levels

To investigate the expression of the SnoSP1 and SnoSP2 transcripts during different
developmental stages and photoperiods (LD16:8 and LD10:14), we determined the levels of
the corresponding transcripts usingand semi-quantitative and real-time PCR on RNA isolated
from fat body. SnoSP1 mRNA is present in corn borer from late larval to pupal stages (Fig
10A). In larval stages, SnoSP1 transcript levels were abundant (28 days since hatching), but
progressively were reduced during pupal development, reaching the lowest level at the end of
pupal stage and beginning of adult life.
In diapausing conditions (Figure 10A) semiquantitative RT-PCR shows that SnoSP1
mRNA is present from deep diapause (55 days since hatching) until the end of post diapause
development (130 days since hatching). The presence of SnoSP1-mRNA in different
developmental stages was also examined by a series of real-time PCR analyses.
Developmental expression levels of SnoSP1 mRNAs were determined in fat body from larvae
reared under photoperiod LD10:14 and LD 16:8. In non-diapausing larvae (LD16:8) the
results showed that SnoSP1 mRNA expression levels are very low in fifth instar larvae, but
they increased rapidly, reaching a peak in sixth instar larvae (prepupae). In the pupal stage the
abundance of early pupa is greatly decreased and maintained throughout late pupa (Figure
10B).
Expression of SnoSP1 has been confirmed in both diapausing and non-diapausing
periods. In non-diapausing insects, the abundance of SnoSP1 in fat body was found in high
levels during the last larval stage and decreased gradually during the pupal stage. The
persistence of SnoSP1 transcript through the early pupal stage suggests that mRNA might be
stable or expressed during the pupal stage (Figure10A, B). During diapause induced by short
days (10L:14D), initiation of expression of SnoSP1 gene was a little different. SnoSP1
transcripts did not occur before day 38 since hatching (prediapause development). The
expression of SnoSP1 detected from the beginning of diapause and increases until at least day
130 since hatching (Figure 10A, B). The SnoSP1 mRNA expression levels were higher in the
non-diapausing than the diapausing larvae and declined when the insect initiated
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 19

metamorphosis (Figure 11). SnoSP1 serves as amino acid reservoirs at the termination of
diapause and during adult development is a distinct possibility.
Larval diapause is characterized by certain endocrine events, such as the presence of
important juvenile hormone (JH) activity (Yin & Chippendale, 1979). Many researchers
suggest that high levels of JH are necessary only at the initiation phase of diapause rather than
in the long lasting “deep” diapause of some lepidoptera species (Chippendale & Yin, 1979;
Bean and Beck, 1980). Thus we suggest that the absence of SnoSP1 expression into the first
days of diapause period (prediapause) is caused by a relatively high level of juvenilizing
factors (JH or its metabolites). After its concentration decreases, SnoSP1 transcription begins.

Figure 9. Semiquantitative RT-PCR expression analysis of SnoSP1 (1) and SnoSP2 (2) genes in fifth
instar larvae treated with methoprene. C. Normal (untreated) larvae. Tubulin is shown as an internal
control. 1. mRNAs from normal (C) and JHA-treated (0.1 and 1.0 μg) fifth instar larvae. 72 h after fifth
instar larvae (28 days since hatching) were ligated, methoprene (a juvenile hormone analog) was
topically administered. Fat body was collected for RNA extraction 12 h after hormone treatment. 2. A.
Animals treated with 0.5 mg methoprene for different times. B. Animals treated for 12 h with different
concentrations of methoprene.

According to Eizaguirre et al. (1998) diapausing larvae of S. nonagrioides have a higher

JH titer in the hemolymph than non-diapausing larvae, which shows that JH plays a basic
rolein the maintenance of diapause in this species. In S. nonagrioides, diapause programming
includes an increase of juvenile hormone (JH) titer in the hemolymph from about 20 to 50 nM
in the 4th and 5th instar larvae (18-25 days since hatching). The JH titer drops to zero before
pupation but remains around to 20 nM during diapause. The increase in the levels of the
SnoSP1mRNAs seems to be correlated with the commitment peak of ecdysteroids in the 5th
instar (25 days since hatching) (Eizaguire et al., 2005).
When the juvenile hormone (JH) titer decreases (days 35 since hatching), it allows the
expression of SnoSP1 mRNAs (35 days since hatching). It is likely that when the last instar
larvae commit to become pupae, the juvenile hormone (JH) titer decreases, which allows the
20 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

expression of SnoSP1 mRNAs. This kind of JH suppression of larval specific proteins has
been reported for related proteins, TniSP1 and TniSP2 (Jones et al., 1993; Hwang et al.,
2001). Since JH is generally considered necessary for maintaining the insect in the larval
state, it is probable that the absence of JH allows the expression of SnoSP1 mRNA.
Expression of SnoSP1 gene is observed throughout diapause, for as long as 130 days since
hatching. The results confirm the importance of LSP biosynthesis and finally lead us to the
conclusion that the larval diapause of S. nonagrioides is associated with continuous synthesis
and accumulation of storage proteins. However, in numerous species investigated so far,
storage proteins were synthesized only previous to diapause and continue in the hemolymph
of diapausing individuals (Sula et al., 1995; Palli et al, 1998).

1.3. SnoSP2 Gene Expression during Larval Development

We examined the expression of SnoSP2 mRNA in the fat bodies during different
developmental stages and photoperiods (LD, 16:8 and LD, 10:14). In non-diapausing
conditions (LD, 16:8), semi-quantitative RT-PCR shows (Figure 12A) that SnoSP2 mRNA
transcripts started to be detected 24 days since hatching (DSH). However SnoSP2 expression
increased in 26 DSH, reaching the maximum in 28 DSH and progressively decreased during
pupal development, reaching the lowest level at the end of pupal stage (and beginning of adult
life). In diapausing conditions (LD, 10:14) semi-quantitative RT-PCR shows (Figure 12A)
that SnoSP2 mRNAtranscripts were present at low levels in 24 DSH and reached a peak in 28
DSH (pre-diapause phase). In deep-diapause phase SnoSP2 mRNA was constitutively
expressed at high levels and started decreasing in the post- diapause phase, at the end of
which (130 DSH), only a trace amount could detected. The expression pattern of the SnoSP2-
gene was also examined by real-time PCR in different developmental stageson RNA isolated
from larvae fat body reared under photoperiod LD10:14 and LD 16:8.
In non-diapausing larvae (L[Link]) the PCR analysis shows that SnoSP2 mRNA
expression levels are low in early fifth instar larvae, exhibited a 8-fold increase during late
fifth instar and reac a peak (28-fold increase) in sixth instar larvae (28 DSH). In the pupal
stage SnoSP2 expression steadily decreased from early to late pupae (Figure 12B).
We also monitored the changes in SnoSP2 transcripts abundance in the fat bodies during
pre-diapause, deep-diapause and post-diapause phases. In diapausing larvae, SnoSP2
transcripts appear in day 24 since hatching and increased 2-fold in day 28. After pre-diapause
phase, mRNA level started togradually decrease from the deep diapause phase until the end of
post-diapause phase (Figure 13B).
The pattern of SnoSP2 mRNA levels changed considerably duringthe different diapause
phases . The levels detected in 24 day since hatching and greatly increased in day 28 (pre-
diapause phase). The level of SnoSP2 mRNA remained high until day 55 (deep diapause) and
started decreased following the post diapause phase, at the end of which disappeared. The up-
regulation of SnoSP2 gene expression during this period indicates that is associated with the
onset of diapause. The pattern of SnoSP2 mRNA is quite similar to the pattern of Lhp76 gene
of Galleria mellonella, in relation to diapause (Godlewski et al., 2001) and CfDAP2 gene of
Choristoneura fumiferana (Pali et al., 1998).
Using real time quantitative PCR (qPCR), we monitored theexpression levels ofthe
SnoSP2 and SnoSP1 genes (Spyliotopoulos et al., 2007) (Figure 14). The expression profile
of the two genes is almost the same in non-diapausing period, but the levels of the transcripts
were different. There was an apparent 2-fold increase in SnoSP1 expression levels when
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 21

compared to SnoSP2 mRNAs. In diapausing period, the transcript levels of the two genes are
almost the same, but the patterns are different. In the pre-diapause phase the induction of
SnoSP2 was different and high transcript levels were apparent, while the SnoSP1 mRNA
levels were very low. On the contrary, in the post-diapause phase, were apparent very high
SnoSP1 mRNA levels, while the levels of SnoSP2 decline and were not detactable at the end
of the phase. The pattern of SnoSP2 mRNA levels changed considerably during the different
diapause phases. The levels detected in 24 day since hatching and greatly increased in day 28
(pre-diapause phase). The level of SnoSP2 mRNA remained high until day 55 (deep diapause)
and started decreased following the post diapause phase, at the end of which disappeared. The
up-regulation of SnoSP2 gene expression during this period indicates that is associated with
the onset of diapause. The pattern of SnoSP2 mRNA is quite similar to the pattern of Lhp76
gene of Galleria mellonella, in relation to diapause (Godlewski et al., 2001) and CfDAP2
gene of Choristoneura fumiferana (Pali et al., 1998).

Figure 10. mRNA accumulation of SnoSP1. Analysis performed in fat body of diapause larvae (LD
10:14). A. Semiquantitative RT-PCR expression analysis of SnoSP1 gene during different
developmental stages as indicated. To ensure equal amounts of template, S. nonagrioides β-tubulin was
used as a reference gene (lower panel). The products of the amplification reaction were analyzed on a
1.5% agarose gel. First stand cDNAs were synthesized from fat body RNA extracted from larvae: 24
Days since Hatching (DSH) (1), 38 DSH (2), 70 DSH (3), 130 DSH (4). B. Real-Time PCR analysis of
expression was performed in fat body in different developmental stages. The relative mRNA copy
number of the SnoSP1 gene transcripts is shown in relation to the number of S. nonagrioides β-tubulin
transcripts for each cDNA sample. The error bars refer to the standard error of the mean.
22 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

Figure 11. Changes in mRNA accumulation on SnoSP1 during non diapause and diapause development
by Real time PCR analysis. Non diapause include larval and pupal stages. Fold regulation of the
SnoSP1 gene is expressed compared to 24days- sample.

During diapause, initiation of expression of SnoSP2 and SnoSP1 genes was different.
Although expression of SnoSP2 lasts from the beginning of diapause until the last day 130,
SnoSP1 transcripts were very low in larvae preparing to enter diapause and increased drama-
tically in those that were in diapause as well as in those that terminate diapause
It has been established that SnoSP1 gene is juvenile hormone-suppressible
(Spyliotopoulos et al., 2007). Our findings do not confirm the above statement for the SnoSP2
gene. The same concentration of JHA is high enough to silence SnoSP1 gene, but SnoSP2
transcripts are still present. There are a few probable explanations for this discrepancy.
Distinct modes of regulation of the two genes to JHA, different stability of the transcripts, or
even insensitivity of SnoSP2 to the hormone are possibilities. Further studies will be required
to further investigate the basis for this unexpected discrepancy . Juvenile hormone (JH), along
with ecdysone, has a profound effect on insect development and behavior. The presence of JH
maintains insects in the larval stage, and its absence allows metamorphosis to proceed. In the
adult forms of insects JH stimulates reproductive maturation (Gilbert et al., 2000). Hexamerin
genes provide excellent systems for understanding hormonally regulated expression on
transcriptional and posttranscriptional levels. Transcriptional and posttranscriptional
regulation of hexamerin production by JH has been studied in different species of insects. In
some experiments, exogenous JH did not produce any notable effects (Riddiford & Hice,
1985; Kumaran et al., 1987). However, there are several cases where hexamerin biosynthesis
was demonstrated to be regulated by JH. In general, it has been shownthat the hormone
represses the biosynthesis of this class of proteins (Riddiford and Hice, 1985; Jamroz et al.,
1996; Jones et al., 1990; Memmel et al., 1994; Hwang et al., 2001). As a general rule, a
decrease in the titers of both, hormones and ecdysteroids, is the signal for the metamorphic
molt of holometabolous insects. Larval diapause is characterized by certain endocrine events,
such as the presence of important juvenile hormone (JH) activity (Yin & Chippendale, 1979).
Many researchers suggest that high levels of JH are necessary only at the initiation phase of
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 23

diapause rather than in the long lasting “deep” diapause of some lepidoptera species
(Chippendale & Yin, 1979; Bean & Beck, 1980).
SnoSP2 mRNA levels did not change significantly in response to JHA application. Thus
we suggest that the SnoSP2 expression into the first days of diapause period (pre-diapause) is
induced by a relatively high level of juvenilizing factors (JH or its metabolites). After its
concentration decreases, SnoSP2 transcription declines. When the juvenile hormone (JH) titer
decreases (days 35 since hatching), the expression of SnoSP2 mRNAs (35 days since
hatching) decreases also. It is likely that when the last instar larvae commit to become pupae,
the juvenile hormone (JH) titer decreases, which also happen in the expression of SnoSP2
mRNAs. Since JH is generally considered necessary for maintaining the insect in the larval
state, it is probable that the presence of JH induce the expression of SnoSP2.

Figure 12. Profiling of SnoSP2 gene transcripts. Analysis performed in fat body of non- diapause larvae
and pupae (LD 16:8). A. Semiquantitative RT-PCR analysis of SnoSP2 expression during S.
nonagrioides different developmental stages. A S. nonagrioides β-tubulin was used as a housekeeping
gene. Fat body RNA extracted from larvae: 24 Days Since Hatching (DSH) (1), 26 DSH (2), 28 DSH
(3), 34 DSH -early pupa (4) and 38 DSH -late pupa (5). B. Real-Time PCR analysis of expression was
performed in fat body in different developmental stages. The relative mRNA copy number of the
SnoSP2 gene transcripts is shown in relation to the number of S. nonagrioides β-tubulin transcripts for
each cDNA sample. The error bars refer to the standard error of the mean.

To date, two hexamerin encoding genes, SnoSP1 and SnoSP2 were characterized in corn
stalk borer. Their structures as well as expression patterns in non-diapause and diapause
conditions were demonstrated to be very distinct. Consistent with a function in
metamorphosis, both mRNAs were abundant during last larval stages, and were gradually
depleted during pupal stage. Expression of both hexamerin genes in S. nonagrioides is
24 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

observed throughout diapause, for as long as 130 days. The results confirm the importance of
SnoSP1 and SnoSP2 biosynthesis and finally lead us to the conclusion that larval diapause of
S. nonagrioides is associated with continuous synthesis and accumulation of storage proteins.
However, enhanced levels of SnoSP2, but not SnoSP1 were detected at the initiation of
diapause. In contrast, levels of SnoSP1 were detected in deep-diapause phase and greatly
increased during post-diapause phase. Our present data support different roles of SnoSP2 and
SnoSP2 gene in diapause of S. nonagrioides.

2. The Role of a Carboxylesterase Gene (Juvenile Hormone Esterase Related

Gene) in the Diapause of S. nonagrioides

Esterases are hydrolytic enzymes that cleave ester bonds in a diversity of biomolecules
(Oakeshott et al., 2005). Many insect esterases have well-defined biological functions, such as
those involved in xenobiotic, lipid, acetylcholine, and JH metabolism. Carboxylesterases
(COEs) are a multifunctional superfamily and ubiquitous in all living organisms, including
insects and other animals, plants and microbes (Ranson et al., 2002). COEs have been the
subject of intense research, in terms of their catalytic mechanism, molecular evolution and
developmental regulation (Gibney et al., 1990). Based on sequence similarity and substrate
specificity, insect COE genes can be subdivided into eight subfamilies: α-esterases, β-
esterases, juvenile hormone esterases, gliotactins, acetylcholinesterases, neurotactins,
neuroligins, and glutactin class (Ranson et al., 2002). Juvenile hormone esterases, α-esterases,
β-esterases, and acetylcholinesterases account for the majority of the catalytically active
COEs (Ranson et al., 2002). However, the physiological role of the vast majority of insect
COEs is unknown (Teese et al., 2010). Insect COEs can be divided into three major classes
(intracellular catalytic, secreted catalytic, and neurodevelopmental classes) based on the
topology of a phylogenetic tree of known COEs (Yu et al., 2009). Juvenile hormone esterase
(JHE) integument esterase, β-esterase, and glutactin belong to the secreted catalytic class.
Juvenile hormone esterase (JHE), is a carboxylesterase that has attracted great interest
regarding its critical role in regulating larval to adult transition in insects and other
arthropods. JHE hydrolyzes the key developmental and reproductive hormone, juvenile
hormone (JH), and partially regulates its titer (Hammock, 1985; Roe & Venkatesh, 1990;
Goodman & Granger, 2005). Juvenile hormone (JH) plays a major role in the control of
growth, development, metamorphosis, diapause and reproduction in insects (Denlinger, 1985;
Riddiford et al., 2003). The onset of metamorphosis is preceded by a decrease in the
biosynthesis of JH and an increase in JH esterase activity (de Kort & Granger, 1996). This
then sets the stage for the elevation of ecdysteroids titers (Mizoguchi, 2001). JH is normally
present at the time of increase in ecdysteroid titres for larval molts and ensures that larvae
molt to the next larval stage (instar). However, at the time of final larval molt, JH disappears,
allowing ecdysone to induce metamorphosis (Riddiford, 1996). JHE is crucial for JH
hemolymph titre reduction and thereby for the initiation of metamorphosis in diverse insects,
but strong inhibition of JHE activity in S. nonagrioides larvae has no effect on the onset of
metamorphosis (Schafellner et al. 2008).
Lepidopteran JHEs are composed of a single polypeptide of approximately 63–67 kDa
and show a high affinity for the juvenile hormone (20–200 nM Km), combined with a
moderate turnover (Goodman & Granger, 2005). JHEs contain characteristic motifs such as
RF, DQ, E and GxxHxxD/E (Feng et al., 1999; Munyiri and Ishikawa, 2007). Furthermore,
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 25

the GxSxG consensus sequence around the active site serine, that is a critical element of
COEs, is present as GQSAG. These various motifs can therefore be used to distinguish JHEs
from other general COEs. Cloning of JHE cDNAs has been performed for several
lepidopteran species: Heliothis virescens (Harshman et al., 1994), Trichoplusia ni (Jones et
al., 1994), Choristoneura fumiferana (Feng et al., 1999), Manduca sexta (Hinton and
Hammock, 2001) and Bombyx mori (Hirai et al., 2002).

Figure 13. Profiling of SnoSP2 gene transcripts. Analysis performed in fat body of diapause larvae (LD
10:14). A. Semiquantitative RT-PCR analysis of SnoSP2 expression during S. nonagrioides different
developmental stages. S. nonagrioides β-tubulin was used as a housekeeping gene. Fat body RNA
extracted from larvae: 28 Days Since Hatching (DSH) (1), 38 DSH (2), 45 DSH (3), 55 DSH (4), 70
DSH (5), 100 DSH (6), 130 DSH (7). B. Real-Time PCR analysis of expression was performed in fat
body in deferent developmental stages. The relative mRNA copy number of the SnoSP2 gene
transcripts is shown in relation to the number of S. nonagrioides β-tubulin transcripts for each cDNA
sample. The error bars refer to the standard error of the mean.

In this review we report the isolation of a full-length cDNA clone of a JHER

carboxylesterase in S. nonagrioides, which is structurally distinguishable from other JHEs
based on certain residues at the active site. Furthermore, we nvestigate the transcriptional
responses of the S. nonagrioides JHER mRNAs to changes in larval development status under
non-diapause and diapause conditions.
A member of the carboxylesterase superfamily has been identified in the corn stalk borer
S. nonagrioides, SnJHER (Kontogiannatos et al., 2011). The cloned SnJHER cDNA is 1838
nucleotides long. The deduced amino acid sequence of this cDNA has high identity (65%)
with T. ni JHE-Related sequence (TnJHER) (Jones et al., 1994), 49-46% with other
lepidopteran juvenile hormone esterases (JHEs) and more than 28% similarity with other
insect COEs. SnJHER has all the typical motifs of JHEs (RF, DQ, E, GxxHxxD/E; Ward et
al., 1992). The primary structure of the deduced amino acid sequence of the cDNA showed
that the catalytic site of SnJHER has a cysteine residue next to the catalytic serine (GQSCG),
26 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

while most other described juvenile hormone esterases have alanine at this position
(GQSAG). Another JHE sequence with the same particularity at its catalytic site is the
TnJHER (Jones et al., 1994). Kadono-Okuda et al (2000) examined the enzyme, TnJHER,
and found that it is related to, but structurally, biochemically, and kinetically distinct from,
the classical TnJHE.

Figure 14. Changes in mRNA accumulation on SnoSP1 and SnoSP2 during non diapause (A) and
diapause (B) development by Real time PCR analysis. Non diapause includes larval and pupal stages.
The relative mRNA copy number of the SnoSP2 gene transcripts is shown in relation to the number of
S. nonagrioides β-tubulin transcripts for each cDNA sample.

Yu et al. (2009) constructed a neighbor-joining tree among known COEs in lepidopteran

insects in which the catalytic triad was predicted by BLAST searching and a GXSXG
consensus sequence around the active site serine was presented. Based on this phylogenetic
tree, insect carboxylesterases were divided into three major classes: intracellular catalytic,
secreted catalytic and neurodevelopmental class. According to this tree (Yu et al., 2009),
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 27

SnJHER (ABW24129) grouped with other lepidopteran JHEs together in a wider

phylogenetic category, the secreted catalytic COEs. SnJHER was the only carboxylesterase
with a GQSCG motif in that tree. In the phylogenetic tree of this study (Figure 3), SnJHER
grouped with other lepidopteran JHEs, and this clade was distinct from other species JHEs, as
reported previously (Claudianos et al., 2006). On the other hand, SnJHER were located
together with TnJHER in a distinct subclade of the lepidopteran JHEs and both contained the
GQSCG motif instead of the characteristic GQSAG motif of the other lepidopteran JHEs
(Figure 15). The appearance of the GQSCG in SnJHER, instead of the GQSAG motif, is the
first major finding of our study.
To determine the potential influence of different insect hormone analogs on SnJHER
gene expression, 6th instar larvae (L6d2) were topically treated with several amounts of
methoprene, RH-5992 and with a combination of methoprene/ RH-5992.
Additionally L6d2 larvae were injected with bisphenol A. The S. nonagrioides larvae
were exposed to these hormones for 3, 6, 12 or 24 h (Figure 16). We first tested the potential
effects on SnJHER gene expression after the topical application of several concentrations of
the JH analog methopene (1, 10, 25 μg respectively) sampling larvae 3, 6, 12 and 24 hours
post treatment. None of these concentrations induced SnJHER gene expression (data not
shown). 25 μg of the juvenile hormone analogue methoprene did not stimulate SnJHE mRNA
synthesis 12 or 24 hours post treatment (Figure 16A). In contrast, when a mixture of 25 and
2.5 μg of methoprene and RH-5992 was applied topically on S. nonagrioides larvae, SnJHER
mRNA levels up-regulated slightly after 12 hours post treatment (Figure 16A). Additionally,
topical application of 10 and 100 μg of the ecdysteroid analog RH-5992, resulted in high
mRNA levels 3 and 6 hours post treatment (Figure 16B). This induction seems to be time-
independent; there was little difference in mRNA levels at 3 and 6 hours post treatment
(Figure 16B). Additionally, the endocrine disruptor with ecdysteroidal activity bisphenol- A
was seen to be a strong inducer of SnJHER (Figure 16C). Only the higher dose of 120 μg
induced SnJHER mRNA synthesis in a time-independent manner with little difference in
expression apparent at 3 and 6 hours post treatment (Figure 16C).
The JH analog methoprene did not affect SnJHER gene expression (Figure 16A), whereas
ecdysteroids induced it (Figure 16B, C). Both effects were experimentally demonstrated by
topical application to larvae of the JH analog methoprene, and, RH-5992 and BPA
(ecdysteroid agonist and endocrine disruptor with ecdysteroidal activity, respectively). The
transcripts of the JHE encoding genes, that have already been described in insects, are
strongly induced by JH, e.g. T. ni (Venkataraman et al., 1994), H. virescens (Wroblewski et
al., 1990), Leptinotarsa decemlineata (Vermunt et al., 1997), C. fumiferana (Feng et al.,
1999), B. mori (Hirai et al., 2002), D. melanogaster (Kethidi et al., 2005) and Nilaparvata
lugens (Liu et al., 2008). In contrast to these studies with these insects, in S. nonagrioides, the
JH analog methoprene did not affect SnJHER gene expression (Figure 16A). The lack of a
JH-inductive effect observed here on SnJHER transcription suggests that this gene is not
sensitive to JH or JH analogs. Previous studies in T. ni (Jones et al., 1994) showed that
exogenous treatment with the powerful JH analog, fenoxycarb, does not affect the levels of
JHER mRNAs, consistent with our results.
Here we found that RH-5992 (ecdysteroid agonist) induced SnJHER (Figure 16 B). In
studies of C. fumiferana, JH-I increased and 20-E decreased CfJHE mRNA in CF-203 culture
cells (Feng et al., 1999). The SnJHER gene, in contrast to the JHE genes of other insects,
seems to be ecdysteroid sensitive. According to Schafellner et al (2008), the JHE activity in S.
28 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

nonagrioides is suppressed after the treatment with the powerful ecdysteroid agonist RH-
2485.

Figure 15. Neighbor-joining tree using as query the SnJHER amino acid sequence (ABW24129) and
known COEs in other insects. COEs sequences were found with BLAST (NCBI). The GXSXG
consensus sequence around the active site serine is presented. The SnJHER/TnJHER cluster is
[Link] full names of species, the abbreviations and theaccessions are: SnJHER (Juvenile hormone
esterase-related of S. nonagrioides accession number ABW24129); TnJHER (Juvenile hormone
esterase-related of T. ni, S55233), MsJHE (Juvenile hormone esterase of M. sexta, AF327882), BmJHE
(Juvenile hormone esterase of B. mori, AF287267), HvJHE (Juvenile hormone esterase of H. virescens,
AAC38822); CfJHE (Juvenile hormone esterase of C. fumiferana, AF153367); BmCOE-6
(carboxylesterase-6 of B. mori, ABX46627); MysJHE (juvenile hormone esterase of Mythimna
separata, ACO81854); NvCOECA (carboxylesterase clade A, member 9 of Nasonia vitripennis,
NP_001165951); OfJHE (juvenile hormone esterase of Omphisa fuscidentalis, ACB12192), ArCOE
(carboxylesterase of Athalia rosae, BAD91555); Bma-est48 (alpha-esterase 48 isoform 1of B. mori,
NP_001165227); Bma COE4 (carboxylesterase 4 variant 1 of Bombyx mandarina, ACF98320);
HaCCE014a (carboxyl/cholinesterase CCE014a of Helicoverpa armigera, ADF43475); HaCCE016a
(carboxyl/cholinesterase CCE016a of H. armigera, ADF43478); DmJHE (juvenile hormone esterase of
D. melanogaster, AF304352); CqJHE (juvenile hormone esterase of Culex quinquefasciatus,
XP_001863697); AaJHE (juvenile hormone esterase of Aedes aegypti, XP_001650480); AmJHEL
(juvenile hormone esterase like of Apis mellifera, NP_001011563); LcE3 (E3 esterase of Lucilia
cuprina, AAB67728); AcCOE (carboxylesterase of Anisopteromalus calandrae, AAC36245); GmCOE
(carboxylesterase of Glossina morsitansmorsitans, ADD19755); ChCOEE3 (carboxylesterase E3of
Cochliomyia hominivorax, ACR56068); CsEst (esterase of Chilo suppressalis, ABD62772).

This discrepancy suggests that SnJHER gene is not a conventional JHE. In addition to
RH-5992, BPA (endocrine disruptor with ecdysteroidal activity) increased SnJHER mRNA
(Figure 16C). Bisphenol A (BPA) is a xenobiotic commonly employed in the manufacture of
polycarbonate plastic and epoxy resin and acts as an endocrine disruptor (ED) (Crain et al.,
2007). BPA proved to be ED due to its ecdysteroidal activity (Lemos et al., 2010). Endocrine,
immune and neuronal systems, as well as reproductive organs, are frequently identified as
targets of EDs in both vertebrates and invertebrates (von Saal & Welshons, 2006).
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 29

2.2. Developmental Regulation of SnJHER Gene under LD and SD Photoperiod

In order to assess the developmental expression of SnJHER during insect development
under diapause or non-diapause conditions, mRNA levels were examined by semi-
quantitative RT-PCR (Figure 17, 18). In non-diapausing conditions (LD photoperiod), larvae
were examined during the 5th and 6th instar (Figure 17). In the L5d1, the SnJHER mRNAs
were low and increased gradually until L5d3, just before the larval ecdysis. In the 6th (last)
larval instar, SnJHER mRNAs were lower than those of the 5th and increased gradually from
L6d4 to L6d5, when they peaked; on the next days, the transcripts declined and disappeared
on L6d8. These results showed that SnJHER mRNAs reached peak expression on the day
before larval molt for the 5th instar, while for the last (6th) larval instar, SnJHER mRNA levels
were lower throughout the instar and peaked during the intermolt period. In diapausing
conditions (SD photoperiod), SnJHER mRNAs (Figure 18A) showed a peak on L8d10. The
levels of SnJHER mRNAs at any diapausing stage tested were lower than those of the non-
diapausing controls (L5d3) (Figure 18A). By increasing the RT-PCR cycles (from 32 to 37)
we were able to measure the SnJHER gene expression during the 9th instar of the diapausing
period (Figure 19B). In the 9th instar, the SnJHER mRNA levels were lowest on L9d1 and
increased gradually, reaching peak expression on L9d12, a day before the molt into the next
(10th) instar (Figure 18B).
Expression of SnJHER mRNA throughout the 5th to 6th (last) instar was examined in non-
diapausing conditions (LD photoperiod) (Figure 18). In the 5th instar, the SnJHER gene was
expressed at constitutive levels during the first two days and then dramatically increased to a
maximum level on the instar's final day (Figure 18). In the 6th instar, SnJHER mRNAs were
low at the first days and increased to a peak level on day 5, then declined and were
undetectable on the final days (Figure 18). The SnJHER transcripts throughout the 6th (last;
pre-pupal) instar were lower than during the 5th instar. SnJHER mRNA levels throughout the
5th to 6th instar did not coincide with JHE activity that Schafellner et al (2008) had reported
for non-diapausing larval S. nonagrioides. According to Schafellner et al (2008) JHE activity
was low in the 4th, higher in the 5th and maximal in the 6th instar. Positive correlation between
JHE mRNA levels and JHE activities has been observed in a number of insects, e.g., B. mori
(Hirai et al., 2002), Psacothea hilaris (Munyiri and Ishikawa, 2007), Gryllus assimilis (Crone
et al., 2007) and Nilaparvata lugens (Liu et al., 2008).
The developmental profile of SnJHER transcript in the 6th (last) instar (Figure 18), does
not show peak expression during the second half of the final larval instar, as has been shown
for the JHE genes of other lepidopteran species, e.g. T. ni (Jones & Hammock, 1983), C.
fumiferana (Feng et al., 1999) and B. mori (Hirai et al, 2002). The expression profile of
SnJHER in the last instar of non-diapausing larvae (Figure 18), is similar to that of JHER in
the final larval stage of T. ni (Jones et al., 1994). The transcript of JHER in the cabbage
looper is present on the first days of the final larval stage and is undetectable on the final
days. TnJHER, in contrast to the JH specific esterase of T. ni (TnJHE), did not show a
transcriptional peak during the prepupal stage (Jones et al., 1994). This second esterase gene
in T. ni, TnJHER, which was more closely related on the basis of similarity of structure to the
“classical” JH specific esterase than to any other esterase, raised the question of whether two
related, but functionally distinct esterases exist and are expressed during insect
metamorphosis.
30 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

Figure 16. Effects of methoprene (JHanalog), RH-5992 (ecdysteroid agonist) and bisphenol A (BPA, 2,
2-Bis (4-hydroxyphenyl) propane, 4, 40-isopropylidenediphenol) (ecdysteroidal activity) on SnJHER
gene expression. (A) 6th instar larvae (last instar larvae, L6d2) topically treated with 25 μg of
methoprene and 2.5 μg RH5992/25 μg methoprene. Controls were topically treated with DMSO. (B) 6 th
instar larvae (last instar larvae, L6d2) topically treated with 10 μg and 100 μg RH-5992. Controls were
topically treated with DMSO. (C) 6th instar larvae (last instar larvae, L6d2) injected with 12 μg and 120
μg BPA. Controls were injected with 1 μL of 100% (v/v)e thanol. B-tubulin is shown as an internal
control. Abbreviations: BPA: bisphenol A, C: control, Met: Methoprene, RH:RH5992.

Figure 17. Semiquantitative RT-PCR expression analysis of SnJHER gene in non-diapausing larvae
(LD 16:8) from the fifth to the sixth (last) instar. S. nonagrioides β-tubulin was used as the control
gene.

TnJHER and TnJHE, closely related “classical” JH specific esterases, may have a distinct
role in the catabolism of JH-like structures for proper insect development (Okuda et al.,
2000). After the biochemical characterization of the TnJHER enzyme, Okuda et al. (2000)
proposed that TnJHER is expressed just prior to metamorphosis and may be implicated in a
catabolic cascade of a JH-like substrate with a hydrophobic backbone, a proximal ester, and a
terminal epoxide or related substitution, a chemical structure similar to that of JH. Similarly,
it is possible that SnJHER is the hydrolyzing enzyme of a JH related natural compound which
has not yet been characterized.
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 31

Figure 18. Semiquantitative RT-PCR expression analysis of SnJHER gene in diapausing larvae (LD
10:14), “32 cycles” (A) vs. “37 cycles” (B). A. Semiquantitative RT-PCR expression analysis of
SnJHER gene in diapausing larvae from the 6th sixth up to 13th (last) diapausing instar. A fifth instar
non-diapausing larva (L5d3) it has been used as control (C). B. Semiquantitative RT-PCR expression
analysis of the SnJHER gene in 9th instar diapausing larvae. S. nonagrioides β-tubulin was used as the
control gene.

During diapause (SD photoperiod), the SnJHER mRNA levels for diapausing larvae
(Figure 18A) were lower than for non-diapausing larvae (Figure 17). According to
Schafellner et al (2008) in S. nonagrioides, the diapausing and non-diapausing larvae exhibit
similar levels of JHE activity. These levels of JHE activity did not coincide with SnJHER
mRNA levels observed during diapause. Despite the low levels of SnJHER during the
diapausing period, it seems that SnJHER presents the higher levels during the L8d10 of
diapause. This peak of expression of the SnJHER in the 8th instar of diapause (Figure 18A)
seems to be coincided with the ecdysteroid peaks in diapausing larvae of S. nonagrioides
measured by Eizaguirre et al (2007). According to Eizaguirre et al. (2007), in S. nonagrioides
larvae, the ecdysteroid titer rose before each extra larval moult and peaks during the molting
of the 8th to the 9th diapausing instar.
Additionally, increasing the cycle number of the semiquantitative RT-PCR, to amplify
low levels of SnJHER expressed in diapausing 9th instars, showed that expression was highest
on the last day of the instar (Figure 18B), prior to molting to a 10th larval instar in diapause.
This gene expression pattern was similar to that of non-diapausing 5th instars (Figure 18).
Thus, the SnJHER mRNA appeared to peak on the final day of both non-diapausing (Figure
18) and diapausing (Figure 18B) larval-stage instars.
Considered together, the data presented here show that SnJHER is not a “classical” JH
specific esterase gene. SnJHER, a carboxylesterase gene, with the GQSCG catalytic motif,
could be ecdysteroid sensitive and juvenile hormone insusceptible, in contrast to other JHEs
genes with the GQSAG motif, which are juvenile hormone inducible and ecdysteroid
suppressible. Furthermore, SnJHER could be the hydrolyzing enzyme of a JH related natural
compound which has not been characterized as yet. A characterization of the potential JH like
natural substrate of SnJHER, in combination with a biochemical characterization of this
enzyme, will clarify the role of this new carboxylesterase gene in the developmental
physiology of S. nonagrioides.
32 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

REFERENCES
Adedokun T. A., Denlinger D. L. 1985. Metabolic reserves associated with pupal diapause in
the flesh fly, Sarcophaga crassipalpis.. J. Insect Physiol 31:229–33.
Arbeitman, M. N. and Hogness, D.S. 2000. Molecular chaperones activate the Drosophila
ecdysone receptor, an RXR heterodimer. Cell 101:67-77.
Arbona, M., de Frutos, R. and Tanguay, R. M. 1993. Transcriptional and translational study
of the Drosophila subobscura hsp83 gene in normal and heat-shock conditions. Genome
36: 694–700.
Arrigo, A. P. 1998. Small stress proteins: chaperones that act as regulators of intracellular
redox state and programmed cell death. Biological Chemistry 379: 19–26.
Arrigo, A. P. and Landry, J. 1994. Expression and function of the low-molecular-weight heat
shock proteins. In:. Morimoto R. I, Tissieres A. and Georgopoulos C., Editors. The
Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, pp. 335–373.
Atungulu, E., Tanaka, H., Fujita, K., Yamamoto, K., Sakata, M., Sato, E., Hara, M.,
Yamashita,, T., Suzuki, K. 2006. A Double Chaperone Function of the sHsp Genes
against Heat-Based Environmental Adversity in the Soil-Dwelling Leaf Beetles. Journal
of Insect Biotechnology and Sericology 75: 15-22.
Baker D., Russell S. 2009. Gene expression during Drosophila melanogaster egg
development before and after reproductive diapause. BMC Genomics 10:242.
Bean, D. W., Beck, S. D. 1980. The role of juvenile hormone in the larval diapause of the
European corn borer, Ostrinia nubilalis. Journal of Insect Physiology 26: 579–584.
Beintena, J. J., Stam, W. T., Hazes, B., Smidt, M. D. 1994. Evolution of arthropod
hemocyanins and insect storage proteins (hexamerins). Molecular Biology and Evolution.
11: 493-503.
Benedict, M. Q., Levine, B. J., Ke, Z. X., Cockburn, A. F. and Seawright, J. A. 1996. Precise
limitation of concerted evolution to ORFs in mosquito Hsp82 genes. Insect Mol Biol 5:
73–79.
Bharucha K. N. 2009. The epicurean fly: using Drosophila melanogaster to study
metabolism. Pediatr. Res. 65:132–37.
Blackman, R. K. and Meselson, M. 1986. Interspecific nucleotide sequence comparisons used
to identify regulatory and structural features of the Drosophila hsp 82 gene. J Mol Biol
188: 499–515.
Buchner, J. 1999. Hsp90 – a holding for folding. Trends Biochem Sci 24: 136–141.
Bonato, M. C., Silva, A. M., Gomes, S. L., Maia, J. C., Juliani, M. H. 1987. Differential
expression of heat-shock proteins and spontaneous synthesis of HSP70 during the life
cycle of Blastocladiella emersonii. European Journal Biochemistry 163: 211–220.
Boorstein, W. R., Ziegelhoffer, T. And Craig, E. A. 1994. Molecular evolution of the Hsp70
multigene family. J. Mol. Evol. 38: 1–17.
Brawn, R. P., Wyatt, G.W. 1996. Sequence of the hexamerin juvenile hormone-binding
protein from the hemolymph of Locusta migratoria. Journal of Biological Chemistry 6:
31756-31762.
Bradshaw W. E., Holzapfel C. M. 2007. Evolution of animal photoperiodism. Annu. Rev.
Ecol. Syst. 38:1–2.
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 33

Burmester, T., Massey, H. C., Zakharkin, J. S. O., H. Benes, H. 1998. The evolution of
hexamerins and the phylogeny of insects. Journal of Molecular Evolution 47: 93–108.
Burmester, T., Scheller, K. 1999. Ligands and receptors: common theme in insect storage
protein transport. Naturwissenschaften 84: 468-474.
Burmester, Τ., 1999. Evolution and function of the insect hexamerins. European Journal of
Entomology 96: 213–225.
Burmester, T. 2001. Molecular evolution of the arthropod hemocyanin superfamily.
Molecular Biology and Evolution 18: 184–195.
Burmester, T. 2002. Origin and evolution of arthropod hemocyanins and related proteins,
Journal of Comparative Physiology B 172: 95–107.
Caplan, A. J. 1999. HSP90’s secrets unfold: new insights from structural and functional
studies. Trends Cell Biol 9: 262-268.
Cavener, D. R. 1987. Comparison of the consensus sequence flanking translational start sites
in Drosophila and vertebrates. Nucleic Acids Res 15: 1353–1361.
Chen, B., Kayukawa, T., Monteiro, A., Ishikawa, Y. 2005. The expression of the HSP90 gene
in response to winter and summer diapauses and thermal-stress in the onion maggot,
Delia antique. Insect Molecular Biology 14: 697-702.
Chen, B., Kayukawa, T., Monteiro, A. and Ishikawa, Y. 2006. Cloning and characterization
of the Hsp70 gene, and its expression in response to diapauses and thermal stress in the
onion maggot, Delia antiqua. J. Bioch. Mol. Biol. 39: 749-758.
Cheon, H. M., Hwang, S. J., Kim, H. J., Jin B. R., Chae, K. S., Yun, C. Y., Seo, S. J. 2002.
Two juvenile hormone suppressible storage proteins may play different roles in
Hyphantria cunea Drury. Archives of Insect Biochemistry and Physiology 50: 157-172.
Cherkasova, V., Ayyadevara, S., Egilmez, N., Shmookler, Reis, R.2000. Diverse
Caenorhabditis elegans genes that are upregulated in dauer larvae also show elevated
transcript levels in long-lived, aged, or starved adults. Journal of Molecular Biology 300:
433–448.
Chippendale, G. M., 1977. Hormonal-regulation of larval diapause. An Rev Entomol 22: 121–
138.
Chippendale, G. M., Yin, C.-M. 1979. Larval diapause of the European corn borer, Ostrinia
nubilalis: Further experiments examining its hormonal control. Journal of Insect
Physiology 25: 53–58.
Chippendale, G. M. 1988. Role of proteins in insect diapause. In: Sehnal, F., Zabza, A. and
Denlinger, D. L., Editors. Endocrinological Frontiers in Physiological Insect Ecology,
Technical University Press, Wroclaw, pp. 331–346.
Cordero, A., Malvar, R. A., Button, A., Revilla, P., Velasco, P., Ordas, A. 1998. Population
dynamics and life-cycle of corn borers in South Atlanic European coast. Maydica 43: 5-
12.
Corpuz, L. M., Choi, H., Muthukrishnan, S. and Kramer, K.J. 1993. Sequences of two cDNAs
and expression of the genes encoding methionine-rich storage proteins of Manduca sexta.
Insect Biochemistry 21: 265–576.
Craig, E. A., Inglolia, T. D. and Manseau, L. J. 1983. Expression of Drosophila heat-shock
cognate genes during heat shock and development. Dev. Biol. 99: 418-426.
Craig, E. A., Gambill, B. D. and Nelson R. J. 1993. Heat shock proteins: molecular
chaperones of protein biogenesis. Microbiol Mol Biol Rev 57: 402-414.
34 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

Crain, A. D., Eriksen, M., Iguchi, T., Jobling, S., Laufer, H., LeBlanc, G. A., Louis, J.,
Guillette, J. 2007. An ecological assessment of bisphenol-A: Evidence from comparative
biology. Reproductive Toxicology 24: 225–239.
Cycler, M., Schrag, J. D., Sussman, J. L., Harel, M., Silman, I., Gentry, M. G., Doctor, B. P.
1993. Relationship between sequence conservation and three-dimensional structure in a
large family of esterases, lipases, and related proteins. Prot. Sci. 2: 366–382.
Dalley, B. K. and Golomb, M. 1992. Gene expression in the Caenorhabditis elegans dauer
larva: developmental regulation of hsp90 and other genes. Dev Biol 151: 80 90.
Danks H. V. 1987. Insect Dormancy: An ecological Perspective (Biological Survey, Ottava,
Canada).
Denlinger, D. L. 1985. Hormonal control of diapause. In Comprehensive insect physiology,
biochemistry and pharmacology, eds. Kerkut G. A. and Gilbert L. I., Vol.8, pp 353–412.
Denlinger, D. L., Lee, R. E., Yocum G. D., Kukal, O. 1992. Role of chilling in the accuisition
of cold tolerance and the capacitation to express stress proteins in diapausing pharate
larvae of the gypsy moth Lymantria dispar. Archives of Insect Biochemistry and
Physiology 21: 271-280.
Denlinger, D. L., Rinehart J. P, Yocum G. D. 2001. Stress proteins: a role in insect diapause?
In: Insect Timing: Circadian Rhythmicity to Seasonality. Ed. by Denlinger, D. L.,
Giebultowicz, J., Saunders, D. S., Elsevier, Amsterdam, 155–171.
Denlinger, D. L., 2002. Regulation of diapause. Annual Review of Entomology 47: 93-122.
Denlinger, D. L., Yocum, G. D. and Rinehar, J. P. 2005. Hormonal control of diapause. In:
Comprehensive Molecular Insect Science, vol. 3. Gilbert, L. I., Iatrou, K.,and Gill, S.
(Eds). Elsevier, Amsterdam, pp. 615-650.
Ehrnsperger, M., Graber, S., Gaestel, M., Buchner, J. 1997. Binding of non-native protein to
Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation,
EMBO Journal 16: 221–229.
Eizaguirre, M., Lopez, C., Asin, L., Albajes, R. 1994. Thermoperiodism, pfotoperiodism and
sensitive stage in the diapause induction of Sesamia nonagrioides (Lepidoptera:
Noctuidae). J. Insect Physiol 40: 113-119.
Eizaguirre, M., Prats, J., Abellana, M., Lopez, C., Llovera, M., Canela, R. 1998. Juvenile
hormone and diapause in the Mediterranean corn borer Sesamia nonagrioides. J. Insect
Physiol 44: 419-425.
Eizaguirre, M., Schafellner, C., López, C., Sehnal, F. 2005. Relationship between an increase
of juvenile hormone titer in early instars and the induction of diapause in fully grown
larvae of Sesamia nonagrioides. J. Insect Physiol. 51: 1127–1134.
Eizaguirre, M., López, C., Schafellner, C., Sehnal, F. 2007. Effects of ecdysteroid agonist
RH-2485 reveal interactions between ecdysteroids and juvenile hormones in the
development of Sesamia nonagrioides. Arch. Insect Biochem. Physiol. 65: 74–84.
Fantinou, A. A., Karandinos, M. G., Tsitsipis, J. A. 1995. Diapause induction in the Sesamia
nonagrioides (Lepidoptera: Noctuidae) effect of photoperiod and temperature.
Environmental Entomology 24: 1458-1466.
Fantinou, A. A., Tsitsipis, J. A. and Karandinos, M. G. 1996. Effects of short- and long-day
photoperiods on growth and development of Sesamia nonagrioides (Lepidoptera:
Noctuidae). Environm Entomoly 25: 1337-1343.
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 35

Fantinou, Tsitsipis, J. A., A. A., Karandinos, M. G., 1998. Diapause termination in Sesamia
nonagrioides (Lepidoptera: Noctuidae) under laboratory and field conditions.
Environmental Entomology 27: 53-58.
Feder, M. E. and Hofmann, G. E. 1999. Heat-shock proteins, molecular chaperones, and the
stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61: 243–
282.
Felsenstein, J., 2001. PHYLIP (Phylogeny Inference Package) version 3.6alpha. Distributed
by the author. Department of Genetics, University of Washington, Seattle, WA.
Feng, O. L., Ladd, T. R., Tomkins, B. L, Sundaram, M., Sohi, S. S., Retnakaran, A., Davey,
K. G., Palli, S. R. 1999. Spruce budworm (Choristoneura fumiferana) juvenile hormone
esterase: hormonal regulation, developmental expression and cDNA cloning. Mol. Cell
Endocrin. 148: 95–108.
Flanagan, R. D., Tammariello, S. P., Joplin, K. H., Cirka-Ireland, R. A., Yocum, G. D.,
Denlinger, G. D. 1998. Diapause specific expression in pupae of the flesh fly Sarcophaga
crassipalpis. Proc Natl Acad Sci USA 95: 5616-5620.
Fujii, T., Sautai, H., Izumi, S., Tomino, S. 1989. Structure of the gene for the arylphorin-type
protein SP2 of Bombyx mori. Journal of Biological Chemistry 264: 11020-11025.
Gadenne C., Dufour M. C., Rossignol F., Bécard J. M., Couillaud F.1997. Occurrence of non-
stationary larval moult during diapauses in the corn-stalk borer, Sesamia nonagrioides
(Lepidoptera: Noctuidae). J Insect Physiol 43:425-431.
Galichet, P. F. 1982. Hibernation d’une population de Sesamia nonagrioides Lef. (Lep.
Noctuidae) en France Meridionale. Agronomie (Paris). 2: 561-566.
Garnier, J., Gibrat, J. F., Robson, B. 1996. GOR secondary structure prediction method
version IV. In Methods in Enzymology. Ed. R. F. Doolittle, vol 266: 540-533.
Gilbert, L. I., Granger, N. A., Roe, R. M. 2000. The juvenile hormones: historical facts and
speculations on future research directions. Insect Biochemistry and Molecular Biology
30: 617-644.
Gibney, G., Camp, S., Dione, M., Mac-Phee-Quigley, K., Taylor, P. 1993. Mutagenesis of
essential functional residues in acetylcholinesterase. Proc. Natl. Acad. Sci. USA 87: 7546-
7550.
Gkouvitsas, T., Kontogiannatos, D. and Kourti, A. 2008. Differential expression of two small
Hsps during diapause in the corn stalk borer Sesamia nonagrioides (Lef.). J Insect
Physiol 54: 1503-1510.
Gkouvitsas, T., Kontogiannatos D. and Kourti, A. 2009. Cognate Hsp70 gene is induced
during deep diapause in the moth Sesamia nonagrioides. Insect Mol Biol 14: 697-702.
Gkouvitsas, T., and Kourti, A. 2009. Juvenile hormone induces the expression of the SnoSP2
gene encoding a methionine-rich hexamerin in Sesamia nonagrioides (Lepidoptera).
Comp Biochem Physiol B Biochem Mol Biol 153: 32-41.
Gkouvitsas T., Kontogiannatos D., Kourti A. 2009. Expression of the Hsp83 gene in response
to diapause and thermal stress in the moth Sesamia nonagrioides. Insect Mol. Biol. 18,
759-768.
Godlewski, J., Kludkiewicz, B., Grzelak, k., Cymborowski, B. 2001. Expression of larval
hemolymph proteins (Lhp) genes and protein synthesis in the fat body of greater wax
moth (Galleria mellonella) larvae during diapause. Journal of Insect Physiology 47: 759-
766.
36 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

Goto, S. G., Yoshida, K. M. and Kimura, M. T. 1998. Accumulation of Hsp70 mRNA under
environmental stresses in diapausing and nondiapausing adults of Drosophila triauraria.
J. Insect Physiol. 44: 1009–1015.
Goto, S. G., Kimura, M. T. 2004. Heat-shock-responsive genes are not involved in the adult
diapause of Drosophila triauraria. Gene 326: 117–122.
Goodman, W. G., Granger, N. A. 2005. The juvenile hormones. In: Gilbert, L. I., Iatrou, K.,
Gill, S. S., (Eds.), Comprehensive Molecular Insect Science. Elsevier, Amsterdam, vol. 3,
pp. 319–406.
Gupta, R. S. and Golding, G. B. 1993. Evolution of Hsp70 gene and its implications
regarding relationships between archaebacteria, eubacteria, and eukaryotes. J. Mol. Evol.
37: 573-582.
Gupta, R. S. 1995. Phylogenetic analysis of the 90 kD heat shock family of protein sequences
and an examination of the relationship among animals, plants, and fungi species. Mol
Biol Evol 12: 1063–1073.
Hanzlik, T. N., Hammock, B. D. 1987. Characterization of affinity purified juvenile hormone
esterase from Trichoplusia ni. J. Biol. Chem. 262: 13584–13591.
Harshman, L.G., Ward, V.K., Beetham, J.K., Grant, D.F., Grahan, L.J., Zraket, C.A., Heckel,
D.G., Hammock, B.D. 1994. Cloning, characterization, and genetics of the juvenile
hormone esterase gene from Heliothis virescens. Insect Biochem. Mol. Biol. 24: 671–676.
Hartl, F. U., Hayer-Hartl, M. 2002. Molecular chaperons in the cytosol: from nanscent chain
to folded protein. Science 295: 1852-1266.
Haunerland, N.H. 1996. Insect storage proteins: gene families and receptors. Insect
Biochemistry and Molecular Biology 26: 755–765.
Haunerland, N. H., Bowers, W. S. 1986. Binding of insecticides to lipophorin and arylphorin,
two hemolymph proteins of Heliothis zea. Archives of Insect Biochemistry and
Physiology 3: 87–96.
Hayward, S. A. L., Rinehart, J. P., Denlinger, D. L. 2004. Desiccation and rehydration elicit
distinct heat shock protein transcript responses in flesh fly pupae. Journal of
Experimental biology 207: 963-971.
Hendrick, J. P. and Hartl, F. 1993. Molecular Chaperone Functions of Heat-Shock Proteins.
An Rev Bioch 62: 349-384.
von Heijne, G. 1986. A simple method for predicting signal peptide cleavage sequences.
Nucleic Acids Research 14: 4683–4690.
Hilal, A. 1977. Mise en evidence d’ un etat diapause vrae chez Sesamia nonagrioides Lef.
(Lepidoptera-Noctuidae). C. R. Acad. Sci. Ser. D. Paris 285, 365-367.
Hinton, A. C., Hammock, B. D. 2001. Purification of juvenile hormone esterase and
molecular cloning of the cDNA from Manduca sexta. Insect Biochem. Mol. Biol. 32: 57–
66.
Hirai, M., Kamimura, M., Kikuchi, K., Yasukochi, Y., Kiuchi, M., Shinoda, T., Shiotsuki, T.
2002. cDNA cloning and characterization of Bombyx mori juvenile hormone esterase: an
inducible gene by the imidazole insect growth regulator KK-42. Insect Bioch. Mol. Biol.
32: 627–635.
Hung, J. J., Cheng, T. J., Chang, M. D., Chen, K. D., Huang, H.L. and Lai, Y. K. 1998.
Involvement of heat shock elements and basal transcription elements in the differential
induction of the 70-kDa heat shock protein and its cognate by cadmium chloride in 9L rat
brain tumor cells. J Cell Biochem. 71: 21-35.
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 37

Hwang, S. J., Cheon, H. M., Kim, H. J., Chae, K. S., Chung, D. H., Kim, M. O., Pars, J. S.
Seo, S. J. 2001. cDNA sequence and gene expression of storage protein-2-a juvenile
hormone-supressible hexamerin from the fall webworm, Hyphantria cunea Drury.
Comparative Biochemistry and Physiology Part B 129: 97–107.
Hwang, J. S., Go, H. J., Goo, T. W., Yun, E. Y., Choi, K. H., Seong, S. I., Lee, S. M., Lee, B.
H., Kim, I., Taehoon Chun, T. and Kang, S.W. 2005. The analysis of differentially
expressed novel transcripts in diapausing and diapause-activated eggs of Bombyx mori.
Arch. Insect Biochem. Physiol. 59: 197-201.
Ingolia, T. D., Craig, F. A. 1982. Four small Drosophila heat shock proteins are related to
each other and to mammalian α-crystallin. Proceedings of National Academic Science
USA 79: 2360-2364.
Ireland, R. C., Berger, E. M. 1982. Synthesis of the low molecular weight heat shock proteins
stimulated by 20-hydroxyecdysone in a cultured Drosophila cell line. Proceedings of
National Academic Science USA 79: 855–859.
James, M., Crabbe, M. J. C., Goode, D. 1994. α-Crystallin: chaperoning and aggregation.
Biochemistry Journal 297: 653-654.
Jamroz, R. C., Stam J. J., Bradfield, J. Y. 1996. Aromatic hexamerin subunit fron adult
female cockroaches (Blaberus discoidalis): molecular cloning, suppression by juvenile
hormone, and evolutionary perspectives. J. Insect Physiol. 42: 115-124.
Jones, G., Hammock, B.D. 1983. Prepupal regulation of juvenile hormone esterase through
direct induction by juvenile hormone. J. Insect Physiol. 29: 471-475.
Jones, G., 1985. The role of juvenile hormone esterase in terminating larval feeding and
initiating metamorphic development in Trichoplusia ni. Entomol. Exper. Appl. 39, 170-
175.
Jones, G., Brown, N., Manczak, M., Hiremath, S. and Kafatos, F.C., 1990. Molecular cloning,
regulation, and complete sequence of a hemocyanin-related, juvenile hormone-
suppressible protein from insect hemolymph. Journal of Biological Chemistry 265:
8596–8602.
Jones, G., Manczak, M. and Horn, M. 1993. Hormonal regulation and properties of a new
group of basic hemolymph proteins expressed during insect metamorphosis. Journal of
Biological Chemistry 268: 1284–1291.
Jones, G., Venkataraman, V., Ridley, B., O'Mahony, P., Turner, H. 1994. Structure,
expression and gene sequence of a juvenile hormone esterase-related protein from
metamorphosing larvae of Trichoplusia ni. Biochem. J. 302: 827–835.
Joplin, K. H., Denlinger, D. L. 1990. Developmental and tissue specific control of the heat
shock induced 70 kDa related proteins in the flesh fly Sarcophaga crassipalpis. J. Insect
Physiol 36: 239.
Kanost, M. R., Kawooya, J. K., Ryan, R. D., Van Heusden, M. C. and Ziegler, R. 1990. Insect
hemolymph proteins. Advanced in Insect Physiology 22: 299–366.
Karouna-Renier, N. K., Yang, W. J. and Ranga, R.K. 2003. Cloning and characterization of a
70 kDa heat shock cognate gene (HSC70) from two species of Chironomus. Insect. Mol.
Biol.12: 19–26.
Kethidi, D. R., Xi, Z., Palli, S. R. 2005. Developmental and hormonal regulation of juvenile
hormone esterase gene in Drosophila melanogaster. J. Insect Physiol. 51: 393–400.
Kiang, J. and Tsokos, G. 1998. Heat shock protein 70 kDa: molecular biology, biochemistry,
and physiology. Pharmacol. Ther. 80: 183–201.
38 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

Kontogiannatos D., Michail X. and Kourti A. 2011. Molecular characterization of an

ecdysteroid inducible carboxylesterase with GQSCG motif in the corn borer, Sesamia
nonagrioides. Journal of Insect Physiology 57:1000–1009.
de Kort, C. A. D. 1996. Cosmic influences on the expression of a specific gene in the
Colorando potato beetle: the diapause protein 1 gene. Archives of Insect Biochemistry
and Physiology 32: 567-573.
de Kort, C. A. D., Granger, N. A. 1996. Regulation of JH titers: the relevance of degradative
enzymes and binding proteins. Arch. Insect Biochem. Physiol. 33: 1–26.
Kostal, V. 2006. Eco-physiological phases of insect diapause. J. Insect Physiol. 52: 113-127.
Kregel, K. C. 2002. Heat shock proteins: modifying factors in physiological stress responses
and acquired thermotolerance. J. Appl. Physiol. 92: 2177–2186.
Kudkiewicz, B., Godlewski, J., Grzelak, K., Cymborowski, B. and Lassota, Z. 1996.
Influence of low temperature on the synthesis of some Galleria mellonella larvae
proteins. Acta Biochimica Polonica 43: 639–644.
Kurtz, S., Rossi, J., Petko, L., Lindquist, S. 1986. An ancient developmental induction heat-
shock proteins induced in sporulation and oogenesis. Science 231: 1154–1157.
Landais, I., Pommet, J., Mita, K., Nohata, J., Gimenez, S., Fournier, P., Devauchelle, G.,
Duonor-Cerutti, M. and Ogliastro, M. 2001. Characterization of the cDNA encoding the
90 kDa heat-shock protein in the Lepidoptera, Bombyx mori and Spodoptera frugiperda.
Gene 271: 223–231.
Leclant, F. 1976. Pest control methods for maize in France. Annals of Applied Biology 87:
237-243.
Lee, K. Y., Horodyski, F. M., Valaitis, A. P., Denlinger, D. L. 2002. Molecular
characterization of the insect immune protein hemolin and its induction during embryonic
diapause in the gypsy moth, Lymantria dispar. Insect Biochemistry and Molecular
Biology 32: 1457-1467.
Lemos, M. F. L., Esteves, A. C., Samyn, B., Timperman, I., van Beeumen, J., Correia, A., van
Gestel, C. A. M., Soares, A. M. V. M. 2010. Protein differential expression induced by
endocrine disrupting compounds in a terrestrial isopod. Chemosphere 79: 570–576.
Leroux, M. R., Melki, R., Gordon B., Batelier, G., Candido, E. P. M. 1997. Structure–
function studies on small heat shock protein oligomeric assembly and interaction with
unfolded polypeptides, Journal of Biological Chemisrty 272: 24646–24656.
Linquist, S. 1986. The heat shock response. Ann. Review Biochem. 55: 1151–1191.
Liu, S., Yang, B., Gu, J., Yao, X., Zhang, Y., Song, F., Liu, Z. 2008. Molecular cloning and
characterixation of a juvenile hormone esterase gene from brown planthopper,
Nilaparvata Lugens. J. Insect Physiol. 54: 1495-1502.
MacRae, T. H. 2000. Structure and function of small heat shock/α-crystallin proteins:
established concepts and emerging ideas. Cell Molecular Life Science 57: 899–913.
Mahroof, R., Zhu, K. Y., Neven, L., Subramanyam, B. and Bai, J. 2005. Expression patterns
of three heat shock protein 70 genes among developmental stages of the red flour beetle,
Tribolium castaneum (Coleoptera: Tenebrionidae). Comp. Biochem. Physiol. A 141:
247–256.
Makka, T., Seino, A., Tomita, S., Fujiwara, H., Sonobe, H. 2002. A possible role of 20-
hydroxyecdysone in embryonic development of the silkworm Bombyx mori. Arch. Insect
Bioch. Physiol. 51: 111–120.
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 39

Marcus, J. M. 2001. The development and the evolution of crossveins in insect wings. J Anat
199: 211-216.
Memmel, N. A., Trewitt, B. N., Grzelak, K., Rajaratnam, V. S., Kumaran, A.K. 1994.
Nucleotide sequence, structure and developmental regulation of LHP82, a juvenile
hormone-suppressible hexamerin gene from the wax moth, Galleria mellonella. Insect
Biochemistry and Molecular Biology 24 :132–144.
Mi, C. H., Hwan, H. I., Hwa, C. D. and Hae, S. S. 1998. Sequence analysis and expression of
met-rich storage protein SP-1 of Hyphantria cunea. Molecular Cells 8: 219–225.
Mizoguchi, A. 2001. Effects of juvenile hormone on the secretion of prothoracicotropic
hormone in the last- and penultimate-instar larvae of the silkworm Bombyx mori. J. Insect
Physiol. 47: 767–775.
van Montfort, R., Slingsby, C., Vierling, E. 2002. Structure and function of the small heat
shock protein/α-crystallin family of molecular chaperones. Adv. Protein Chem. 59: 105–
156.
Munyiri, F. N., Ishikawa, Y. 2007. Molecular cloning and developmental expression of the
gene encoding juvenile hormone esterase in the yellow-spotted longicorn beetle,
Psacothea hilaris. Insect Bioch. Mol. Biol. 37: 497–505.
Nathan, D. F., Vos M. H., and Lindquist S. 1997. In vivo functions of the Saccharomyces
cerevisiae Hsp90 chaperone. Proc Natl Acad Sci USA. 94:12949–12956.
Narberhaus, F. 2002. Alpha-crystallin-type heat shock proteins: socializing minichaperones in
the contex of a multichaperone network. Microbiology and Molecular Biology Reviews
66: 64-93.
Nijhout, H. F. 1994. Genes on the wing. Science 265: 44-45.
Nicholas, K. B., Nicholas, H. B. Jr. 1997. GeneDoc: Analysis and Visualization of Genetic
Variation, [Link]/biomed/genedoc/.
Nover, L. And Scharf, K. D. 1997. Heat stress proteins and transcription factors. Cell Mol.
Life Sci. 53: 80-103.
Oakeshott, J. G., Claudianos, C., Campbell, P. M., Newcomb, R. D., Russell, R. J. 2005.
Biochemical genetics and genomics of insect esterases. In: Gilbert, L. I., Iatrou, K., Gill,
S. S., (Eds.), Comprehensive Molecular Insect Science Pharmacology, Elsevier, Oxford,
pp 309-381.
Palli, S. R., Ladd, T. R., Ricci, A. R., Primavera, M., Mungrue, I. N., Pang, A.S.D. and
Retnakaran, A. 1998. Synthesis of the same two proteins prior to larval diapause and
pupation in the spruce budworm, Choristoneura fumiferana. J. Insect Physiol. 44: 509–
524.
Pan, R., Levenbok, L., Bauer, C. 1979. Inhibitory effect of beta-ecdysone on protein by
blowfly fat in vitro. Experientia 35: 1449-1451.
Pan, M. L., Telfer, W. H. 1996. Methionine-rich hexamerin and arylphorin as presursor
reservoirs for reproduction and metamorphosis in female luna moths. Archives of Insect
Biochemistry and Physiology 33: 149-162.
Parsell, D. A. and Lindquist, S. 1993. The function of heat-shock proteins in stress tolerance –
degradation and reactivation of damaged proteins. Annu Rev. Genet. 27: 437–496.
Pérez-Hedo M., Albajes R., Eizaguirre, M. 2011. Modification of hormonal balance in larvae
of the corn borer Sesamia nonagrioides due to Bacillus thuringiensis protein ingestion. J
Econ Entomol 104: 853-861.
40 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

Pérez-Hedo M., Goodman WG, Schafellner C, Martini A, Sehnal F, Eizaguirre M. 2011.

Control of larval–pupal-adult molt in the moth Sesamia nonagrioides by juvenile
hormone and ecdysteroids. J Insect Physiol 57: 602-607.
Persaud, D. R., Haunerland, N. H. 2004. Cloning and expression of the VHDL receptor from
fat body of the corn ear worm, Helicoverpa zea. Journal of Insect Science 4: 1-10.
Peter, M. G., Scheller, K. 1991. Arylphorin and the integument. In: A. Retnakaran and K.
Binnington, Editors, The Physiology of Insect Epidermis, CSIRO, Australia, pp. 113–122.
Picard, D., Khursheed B., Garabedian, M. J., Fortin, M. G., Lindquist, S. and Yamamoto, K.
R. 1990. Reduced levels of hsp90 compromise steroid receptor action in vivo. Nature
348: 166 – 168.
Picard, D. 2002. Heat-shock protein 90, a chaperone for folding and regulation. Cell Mol Life
Sci 59: 1640–1648.
Pla, M., Huguet, G., Verdaguer, D., Puigderrajols, P., Llompart, B., Nadal, A., Molinas, M.
1998. Stress proteins co-expressed in suberized and lignified cells and in apical
meristems. Plant Science 139: 49–57.
Qin, W., Tyshenko, M. G., wu, B. S., Walker, V. K., Robertson, R. M. 2003. Cloning and
characterization of a member of the hsp70 gene family from Locusta migratoria, a highly
thermotolerant insect. Cell Stress Chaperones 8: 144-152.
Ramsey, A. J., Russell, L. C, Whitt, S. R. and Chinkers, M. 2000. Over-lapping sites of
tetratricopeptide repeat protein binding and chaperone activity in heat shock protein 90. J
Biol Chem 275: 17857-17862.
Ranson, H., Claudianos, C., Ortelli, F., Abgrall, C., Heminway. J., Sharakhova, M. V., Unger,
M. F., Collins, F. H. and Fryereisen, R. 2002. Evolution of supergene families associated
with insecticide resistance. Science 298: 179-181.
Riddiford, L. M., Hice, R. H. 1985. Developmental profiles of the mRNAs for Manduca
arylphorin and two other storage proteins during the final larval instar of Manduca sexta.
Insect Biochemistry 15: 489-502.
Riddiford, L. M. 1996. Juvenile hormone: the status of its "status quo” action. Arch. Insect.
Biochem. Physiol. 32: 271-86.
Riddiford, L. M. 1996. Molecular aspects of juvenile hormone action in insect
metamorphosis. In: Gilbert, L. I., Tata, J. R., Atkinson, B. G., (Eds.), Metamorphosis:
Post-embryonic Reprogramming of Gene Expression in Amphibian and Insect Cells.
Academic Press, San Diego, pp. 223–251.
Rinehart, J. P., Yocum, G. D. and Denlinger, D. L. 2000. Developmental upregulation of
inducible hsp70 transcripts, but not the cognate form, during pupal diapause in the flesh
fly, Sarcophaga crassipalpis. Insect Biochem. Mol. Biol. 30: 515–521.
Rinehart, J. P., Li A., Yocum, G. D., Robich, R. M., Hayward, S. A. L., Denlinger, D. L.
2007. Up-regulation of heta shock proteins is potential for cold survival during insect
diapause. PNAS, 104: 11130-11137.
Rubin, D. M., Mehta, A. D., Zhu, J., Shoham, S., Chen, X., Wells, Q. R. and Palter, K. B.
1993. Genomic structure and sequence analysis of Drosophila melanogaster Hsc70
genes. Gene 128: 155-163.
Rutheford, S. L. and Lindquist, S. 1998. Hsp90 as a capacitor for morphological evolution.
Nature 396: 336-342.
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 41

Rybczynski, R. and Gilbert, L. I. 2000. cDNA cloning and expression of a hormone-regulated

heat shock protein (hsc70) from the prothoracic gland of Manduca sexta. Insect Biochem.
Mol. Biol. 30: 579–589.
Ryan, R. O., Kein, P. S., Wells, M. A., Law, J. H. 1985. Purification and properties of a
predominantly female-specific protein from the haemolymph of the larva of tobacco
hornworm, Manduca sexta. Journal of Biological Chemistry 260: 782-787.
vom Saal, F. S., Welshons, W. V. 2006. Large effects from small exposures. II. The
importance of positive controls in low-dose research on bisphenol Environ. Res. 100: 50-
76.
Sakano, D., Li, B., Xia, Q., Yamamoto, K. 2006. Genes encoding small heat shock proteins of
the silkworm, Bombyx mori. Biosc. Biotechno. Biochem. 70: 2443-2450.
Sakurai, H., Fujii, T., Izumi, S., Tomino, S. 1988. Complete nucleotide sequence of gene for
sex-specific storage protein of Bombyx mori. Nuclei Acid Research 16: 7717-7718.
Salama, M., Miller, T. 1992. A diapause associated protein of the pink bollworm
Pectinophora gossypiella. Archives of Insect Biochemistry and Physiology 21: 1–11.
Schafellner, C., Eizaguirre, M., López, C., Sehnal, F. 2008. Juvenile hormone esterase
activity in the pupating and diapausing larvae of Sesamia nonagrioides. J. Insect Physiol.
54: 916-921.
Scheufler, C., Brinker, A., Bourenlov, G., Pegoraro, S., Moroder, L., Bartunik, H., Hartl, F.
U. and Moarefi, I. 2000) Structure of TPR domain-peptide complexes: critical elements
in the assembly of the HSP70-HSP90 multichaperone machine. Cell 101: 199-210.
Scheller, K., Fischer, B., Schenkel, H. 1990. Molecular properties, functions and
developmentally regulated biosynthesis of arylphorin in Calliphora vicina. In: Hagedorn
H. H., Hildebrand J. G., Kidwell M. G. and Law J. H., Editors, Molecular Insect Science,
Plenum Press, New York , pp. 155–162.
Schelling, D. and Jones, G. 1996. Analysis of induction of hsc70, hsp82 and juvenile
hormone esterase genes by heat shock in Trichoplusia ni. J. Insect Physiol. 42: 295-301.
Shue, G., and Kohtz, D.S. (1994) Structural and functional aspects of basic helix-loop-helix
protein folding by heta-shock protein 90. J Biol Chem 269: 2707-2711.
Sonoda, S., Fukumoto, K., Izumi, Y., Ashfaq, M., Yoshida, H., Tsumuki, H. 2006. A small
HSP gene is not responsible for diapause and cold tolerance acquisition in Chilo
suppressalis. Journal of Applied Entomology 130: 309-313.
Spyliotopoulos A., Gkouvitsas, T., Fantinou, A. and Kourti, A. 2007. Expression of a c
cDNA encoding a member of the hexamerin storage proteins from the moth Sesamia
nonagrioides (Lef.) during diapause. Comp. Biochem. Physiol. B, Biochem. Mol. Biol.
148: 44-54.
Stavrakis, G. 1967. Contribution à l’étude des espèces nuisibles au maïs en Grêêce du genre
Sesamia (Lépidoptères-Noctuidae), Ann. Inst. Phytopath. Benaki, N. S., vol. 8, pp. 19–22.
Sula, J., Kodik, D., Socha, R. 1995. Hexameric haemolymph protein related to adult diapause
in the red firebug, Pyrrhocoris apterus. J. Insect Physiol. 41: 793–800.
Tauber C., Tauber M. 1981. Insect seasonal cycles: Genetics and evolution. Ann Rev Ecol
Syst 12:281-308.
Tauber, M. J., Tauber, C. A., and Masaki, S. 1986. Seasonal adaptations of insects. Oxford
Universit PRESS, New York.
42 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

Tachibana, S. I., Numata, H., Goto, S. G. 2005. Gene expression of heat-shock proteins
(Hsp23, Hsp70 and Hsp90) during and after larval diapause in the blow fly Lucilia
sericata. J. Insect Physiol. 51: 641–647.
Tammariello, S. P., Denlinger, D. L., 1998. G0/G1 cell cycle arrest in the brain of Sarcophaga
crassipalpis during pupal diapause and the expression pattern of the cell cycle regulator,
proliferating cell nuclear antigen. Insect Biochemical Molecular Biology 28: 83–89.
Tawfik, A. I., Kellner R., Hoffman K.H., Lorenz, M. W. 2006. Purification, characterization
and titre of the haemolymph juvenile hormone binding proteins from Schistocerca
gregaria and Gryllus bimaculatus. Journal of Insect Physiology 52, 255-268.
Telfer, W. H., Kunkel, J. G. 1991. The function and evolution of insect storage hexamers.
Annual Review of Entomology 36: 205–228.
Teese, M. G., Campbell, P. M., Scott, C., Gordon, K. H. J., Southon, A., Hovan, D., Robin,
C., Russell, R.J., Oakeshott, J.G. 2010. Gene identification and proteomic analysis of the
esterases of the cotton bollworm, Helicoverpa armigera. Insect Biochem. Mol. Biol. 40:
1-16.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., Higgins, D. G. 1997. The
ClustaIX windows interface: flexible strategies for multiple sequence alignment aided by
quality analysis tools. Nucleic Acids Research 25: 4876–4882.
Thomas, H. E. and Lengyel, J. A. 1986. Ecdysteroid-regulated heat-shock gene expression
during Drosophila melanogaster development. Developm Biol 115: 434-438.
Tojio, S., Nagata, M., Kobayachi, M., 1980. Storage proteins in the silkworm, Bombyx mori.
Insect Biochemistry 10: 289-303.
Tojo, S., Yoshiga, T. 1994. Purification and characterization of three storage proteins in the
common cutworm, Spodoptera litura. Insect Biochemistry 24: 729–738.
Torchia, J., Rose, D. W., Inostroza, J., Kamei, Y., Westin, S., Glass, C. K., Rosenfeld, M. G.
1997. The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor
function. Nature 387: 677-684.
Truman, J. W., Riddiford, I. M. 2002. Endocrine insights into the evolution of metamorphosis
in insects. Ann. Rev. Entomol. 47: 467-500.
Tsitsipis, J. A. 1984. Rearing the corn borer Sesamia nonagrioides on artificial media in the
laboratory. In Proceedings, XVII International Congress of Entomology, 16-26 August,
Hamburg, Abstracts, p. 316.
Tsitsipis, J. A. 1990. Contribution toward the development of an integrated control method
for the corn stalk borer. in Pesticides and Alternatives, Casida J. E., Ed., pp. 217–228,
Academic Press, Amsterdam, The Netherlands,
Tungjitwitayakul J., Singtripop T., Nettagul A., Oda Y., Tatum N., Sekimoto T., Sakurai S.,
2007. Identification, characterization, and developmental regulation of two storage
proteins in the bamboo borer Omphisa fuscidentalis. J. Insect Physiol. 54: 62-76.
Vermunt, A. M. W., Koopmanschap, A. B., Vlak, J. M., De Kort, C. A. D. 1997. Cloning and
sequence analysis of cDNA encoding a putative juvenile hormone esterase from the
Colorado potato beetle. Insect Biochem. Mol. Biol. 27: 919–928.
Vontas, J. G., Small, G. J., Hemingway, J. 2000. comparison of esterase gene amplification,
gene expression and esterase activity in insecticide susceptible and resistant strains of the
brown planthopper, Nilaparvata lugens (Stal). Insect Molecular Biology 9: 655-660.
Walter, S., Buchner, J. 2002. Molecular chaperons-cellular machines for protein folding.
Angew Chem Int Ed Engl 41: 1098-1113.
 The Molecular Components of Diapause in the Moth Sesamia Nonagrioides … 43

Wang, X. Y., Frohlich, D. R., Wells, M. A. 1993. Polymorphic cDNAs encode for the
methionine-rich storage protein from Manduca sexta. Insect Molececular Biology 2: 13–
20.
Ward, V. K., Bonning, B. C., Huang, T. L., Shiotsuki, T., Griffeth, V. N., Hammock, B. D.
1992. Analysis of the catalytic mechanism of juvenile hormone esterase by site-directed
mutagenesis. Inter. J. Biochem. 24: 1933–1941.
Webb, B. A., Riddiford, L. M. 1988. Synthesis of two storage proteins during larval
development of the tobacco hornworm, Manduca sexta. Developmental Biology 130:
671–681.
Whitesell, L., Mimnaugh , E. G., de Costa, B., Myers, C. E., and Neckers, L. M.. 1994.
inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by
benzoquinone ansamycins: essential role for stress proteins in ongogenic transformation.
Proc nat acad Sci USA 91: 8324-8328.
Willott, E., Wang, X. Y., Wells, M. A 1989. cDNA and gene sequence of Manduca sexta
arylphorin, an aromatic amino acid-rich larval serum protein: homology to arthropod
hemocyanins. Journal of Biological Chemistry 264: 19 052–19 059.
Wyatt, G. R. [Link] biochemistry of insect hemolymph. Ann Rev Entomol 6:75-102.
Xu Y., and Lindquist, S. 1993. Heat-shock proteins 90 geverns the activity of pp60v-80rc
kinase. Proc Natl Acad Sci USA 90: 7074-7078.
Yiangou, M., Tsapogas, P., Nikolaidis, N. and Scouras, Z. G. 1997. Heat-shock gene
expression during recovery after transient cold shock in Drosophila auraria (Diptera:
Drosophillidae). Cytobios 92: 91-98.
Yin, C.-M., Chippendale, G. M. 1979. Diapause of the southwestern corn borer, Diatrea
grandiosella: further evidence showing juvenile hormone to be the regulator. J. Insect
Physiol. 25: 513–523.
Yocum, G. D, Joplin, K. H, Denlinger, D. L. 1991. Expression of heat shock proteins in
response to high and low temperature extremes in diapausing pharate larvae of the gypsy
moth, Lymantria dispar. Archives of Insect Biochemistry and Physiology 18: 239–249.
Yocum, G. D., Joplin, K. H., Denlinger, D. L. 1998. Upregulation of a 23 kDa small heat
shock protein transcript during pupal diapause in the flesh fly, Sarcophaga crassipalpis.
Insect Biochemistry and Molecular Biology 28: 677-682.
Yocum, G. D. 2001. Differential expression of two HSP70 transcripts in response to cold
shock, thermoperiod, and adult diapause in the Colorado potato beetle. J. Insect Physiol.
47: 1139–1145.
Yocum, G. D., Kemp, W. P., Bosch, J. and Knoblett, J. N. 2005. Temporal variation in
overwintering gene expression and respiration in the solitary bee Megachile rotundata. J.
Insect. Physiol. 51: 621-9.
Young, J. C., Moarefi, I. and Hartl, F. U. 2001. Hsp90: a specialized but essential protein-
folding tool. J Cell Biol 154: 267-273.
Yu, Q. Y., Lu, C., Li, W. L., Xiang, Z. H., Zhang, Z. 2009. Annotation and expression of
carboxylesterases in the silkworm Bombyx mori. BMC Genomics 10: 553.
Zheng, Y., Yoshiga, T., Tojo, S. 2000. cDNA cloning and deduced amino acid sequences of
three storage proteins in the common cutworm, Spodoptera litura. Applied Entomological
Zoology 35: 31–39.
44 Anna Kourti, Theodoros Gkouvitsas and Dimitrios Kontogiannatos

Zhu, Y. C., Muthukrishman, S., Kramer, K. J. 2002. cDNA sequences and mRNA levels of
two hexamerin storage proteins PinSP1 and PinSP2 from the Indianmeal moth, Plodia
interpunctella. Insect Biochemistry and Molecular Biology 32: 525-536.
In: Lepidoptera ISBN: 978-1-62417-248-9
Editors: Elia Guerritore and Johannes DeSare © 2013 Nova Science Publishers, Inc.

Chapter 2

CURRENT ISSUES IN INTEGRATED PEST

MANAGEMENT OF LEPIDOPTERA PEST THREATS
IN INDUSTRIAL CROP MODELS

Petros T. Damos
Aristotle University of Thessaloniki,
Faculty of Agriculture, Department of Plant Protection,
Laboratory of Applied Zoology and Parasitology, Thessaloniki, Greece

ABSTRACT
The sweeping intensification of agriculture using industrial models that grows
annual crops in monoculture is characterized by strong dependency of influxes. Pest
management in particular relies heavily upon the use of synthetic chemical compounds
and related energy inputs. Common side effects of pesticides include not only resistance
development but also environmental impacts on the diversity, composition and
functioning of natural ecosystems and rural areas throughout the world. This chapter
explores current Integrated Pest Management (IPM) strategies to control Lepidoptera
pests-threats that are damaging industrial crops models including sugar beet (Beta
vulgaris), potato (Solanum tuberosum), cotton (Gossypium hirsutum) and corn (Zea
mays). The information is in particular designed to encourage the development alternative
control options for rational management of moths with high economic significance for
crop production, including: Agrotis sp. (Lepidoptera: Noctuidae), Phthorimaea
operculella (Lepidoptera: Gelechiidae), Helicoverpa armigera (Lepidoptera: Noctuidae)
and Sesamia nonagrioides (Lepidoptera: Noctuidae). Representative examples of
decision making tools, utile in IPM, are discussed such as monitoring and scouting
procedures, the use of phenological models and the development of economic injury
levels and thresholds. Moreover, efforts are made to integrate most of the available-
alternative pest control options on a combined manner. The significance of cultivation
measures and biological control option, as well as the use of semiochemicals and
pheromones are emphasized. Additionally, since pesticides rotation is essential for IPM,
to avoid resistance development, the major pesticides groups are classified according to


E-mail: damos@[Link].
46 Petros T. Damos

their mode of action and chemical structure. The IPM compatibility of biorational
products is outlined, while the sustainable use of pesticides is discussed according to the
latest directives of the European Parliament upon their future use in Agriculture.
Although we address the subject primarily from a perspective to manage Lepidoptera
pests, most IPM principles of the current chapter are presented on a holistic manner in the
view to be wider adopted in several industrial crop models to control several pests.

1. INTRODUCTION
By definition Agricultural Ecosystems (Agro-ecosystems) are spatially and functionally
coherent units of agricultural activity, and include the living and nonliving components
involved in that unit as well as their interactions [4] [5] [6] [38] [123]. However, most
agricultural ecosystems are actively managed by humans to optimize the provision of food,
fibres, and fuels and in most cases annual-industrial crop models play an important role in the
base of countries productivity in the developed and developing world [98]. Generally,
covering over a third of total global land area FAOSTAT, 1999)1 [47] [48], agriculture
represents humankind's largest engineered ecosystem. However, productivity of agro
ecosystems rely heavily on the use energy influxes, while the sustainability depends on how
agro-ecosystems are managed at the site scale as well as on the diversity, composition, and
functioning of the surrounding landscape and related energy influxes [106] [107]. The
development of current industrial crop models was the result of a rapid identification in crop
production in the late 1960s that followed a series of research, development, and technology
transfer initiatives. This resulted to a significant increase in the world yields production and
referred as the first Green Revolution.
The radical intensification in crop production can be summarised as the result of the
following actions ([38] after modification):

 progressively enlargement of field land

 the use of monoculture (as a result of the development of genetic uniform crops)
 development of high-yielding varieties of cereal grains and distribution of hybridized
seeds
 the increase in plant density
 the increase in all kind of energy influxes (synthetic fertilisers, pesticides -
agrochemicals)
 expansion of irrigation infrastructure and availability of water supply
 specialisation and mechanisation
 globalisation and increase of international exchange in all aspects (i.e. plant material,
products, capital)
 improvement of agricultural science

However, by following an holistic perspective, the process of intensification in crop

production should be considered as the result of all above actions in an combined manners

1
Agriculture land use statistics compiled by FAO for 1992-1993.
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 47

and is proper to state that the annual-industrial crop models have evolved from the
contribution of all branches of agricultural and biological sciences [38] [120].
Figure 1 and 2 are some typical examples displaying the global trends in population
history and yield production concerning a typical monoculture (i.e. wheat). Since figures are
given on a comparative time scale, the correlation between agricultural development and
demographic change is obvious [60].

Figure 1. world population history (source: United States Census Bureau (USCB).

Figure 2. Yearly global trends in a typical monoculture - industrial crop model.

48 Petros T. Damos

Source: USDA Forest Service, [Link]

Figure 3. Overall increase in use of chemical environmental contaminants in the USA.

For pest management in particular, development has been driven by the changes in
several pest problems that farmers are regularly facing. In this topic research knowledge and
scientific innovations are offering new options in pest management science, in which the
development of novel products by the chemical industry have an important role.
However, the green revolution did not only increase agricultural productivity, but cause
significant problems because it involved a significant increase in the use of agrochemicals.
Figure 3 is a typical example which depicts the overall increase in the use of chemical
contaminants, including that used in agriculture and during the past decades. It is notable that
overall use of chemical was up dramatically from 1960-1980, and then has leveled off [97].
As far as plant protection concerned the traditional use of non-selective insecticides is
associated to a variety of problems including: environmental degradation, insecticide
resistance, negative impacts on natural enemies and safety for pesticide applicators and the
food supply [5] [7] [21] [127]. Concerns about these consequences have increased the interest
in the development of alternative means for pest control that have little or no impact on
humans, beneficial organisms and sensitive ecosystems [33] [34] [35]. Moreover, a part of the
gradual intensification that agriculture has experienced that last century now continues to
change in response to the society needs and progressively moves to the direction of rational
managing of agro-ecosystems to provide sufficient supporting in sustainable agriculture [5]
[7] [21].
Sustainable agriculture enrolls the recognition of site-specific differences within fields
and adjusts management actions accordingly and these variations can further traced to
management practices, soil properties and/or environmental characteristics [5] [7]. Hence, it
is not an exaggeration to state that sustainability is attainable only due the integration of all
available new research information and technologies on a complementary manner [34].
Actually the idea is to go beyond the use of alternative practices and to develop Agro-
Ecosystem with minimal dependence on high agrochemical and energy inputs. Moreover, a
great deal of research attention has been devoted by the United Nations Food and Agricultural
Organization (FAO) to the sustainability of industrial farm systems [46]. The adoption of
sustainable farming practices allows small farmers of cotton in developing countries for
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 49

instance to increase their yields and incomes, reduce their use of toxic pesticides and protect
the environment [5] [7] [33] [34] [72].
Current need of ecological concepts and principles to the design and management of
sustainable Agro- Ecosystems seems to be the aftermath of the complex socio-environmental
problems of our modern times and mostly aim to regulate agricultural productivity on a
rational manner. In this context, the current chapter extensively explores the use of Integrated
Pest Management (IPM) strategies as a decision-based process. The scope is to involve the
coordinated use of multiple tactics for optimizing the control of moth key-pests2 that are
regularly damaging industrial crop models and to provide means for ecologically and
economically sound manner of control. Thus, building on previous works and reviews, we
discus first the economic significance of some representative industrial crop models, as well
as the life cycles of crop-related moths threats. In the next sections we summarize some latest
concerted research efforts seeking to develop alternatives to pesticides and their final
integration to IPM systems. Moreover, since the last decade has seen considerable advances
in the development and adoption of IPM systems in certain crops, representative examples
upon the use of decision tolls and alternative to pesticide management options are given.
Finally, efforts are made to conceptualize the foundation of integrated pest management
strategies in industrial crop models and to emphasize on the sustainable use of pesticides in
the line of the latest EU directives for IPM adoption by member states.

1.1. Some Representative Industrial Crop Models

Cultivated throughout the World

Since industrial crops are an important source of income not only on the ‘developed
world’ but also for many small landholders in developing countries, some representative
industrial crops will be here in short discussed. Throughout the chapter the term ‘industrial’
refers upon extensive use of monocultures and their products which mostly include oils,
sugars, starches, proteins, fibers, gums, food additives, elastomers, medical substances and
stimulants.

a. Beta vulgaris
The sugar Beet Beta vulgaris L. (Chenopodiaceae) is hardy biennial plant grown
commercially in wide variety of temperate climates for its root which contains high
concentration of sucrose. Sugar beet is cultivated as annual plan for its storage root which is
harvested during the first growing season and whose dry mass is 14-18% sucrose by weight.
However, sugar beet was initially grown for its leaves in the eastern Mediterranean and as
such is also mentioned in Mesopotamian literature in the 9th century BC, while the
exploitation of the thickened root is of much more recent date. Additionally, the root is a
source of dietary fiber, vitamin C, tryptophan, phosphorus, potasium and trace elements such
as manganese, iron, and copper. Moreover, sugar is also used for bioethanol production, while
residues of sugar beet (dried pulp and leaves) are also of value and is mostly used as animal
feed and for biogas production.

2
IPM in principle includes the rational control of all classes of pests, including not only insects and mites, but also,
pathogens, vertebrates and weeds [31] [34] [43].
50 Petros T. Damos

Sugar beet is the most important sugar crop worldwide after sugar cane and currently the
world’s three largest sugar beet producers are the European Union, the United States, and
Russia. The annual global production of sugar, including sugar cane, from 121 producing
countries is 120 million tons per year, while 70% of it is consumed in the origin countries.
The majority of sugar produced worldwide is originated from sugar cane (~70%), while sugar
produced from beets represents approximately the on third of global production (~30%). Due
to the high susceptibility to pests and diseases and extreme conditions sugar beet in the
tropics3 has been a marginal crop for sugar production in contrast to sugar cane. B. vulgaris
express relatively high tolerance of saline and alkaline soils and has relatively low water use
(50% lower) per unit of sugar (or bioethanol) as compared to sugarcane and is mostly grown
in temperate and drier areas of the world.
The development of varieties that are resistant to diseases4 and the development of crop
protection strategies against pests and diseases are crucial to continue sugar beet production.
For instance crop rotation is probably the most important cultivation measures to prevent
diseases. However, for a good yield, the crop requires sufficient chemical control of pests
such as Agrotis sp., the lined click beetle Agriotes lineatus and the beet beetle Atomaria
linearis [76].

b. Solanum Tuberosum
Solanum tuberosum (Solanaceae) is herbaceous annual crop which is extensively
cultivated. Its tuber, the potato, is reach starch source that ranks as the world’s fourth most
important industrial food crop after maize, wheat and rice [46]. The European potato varieties
belong mostly to the Chilotanum group which has been developed from the Andean cultivars,
although the second group Andigenum, which is adapted to short day conditions, is also
cultivated around the world [64].
Although that Europe5, including former Soviet Union countries, was the undisputed
world leader in potato production for most of the 20th century and until the early 90’s, there is
now a significant increase in potato demand and production in Asia, Africa and Latin
America and the world potato sector (fresh potatoes, potato starch, alcohol and other related
products) is currently undergoing major changes. Many western European countries are
shifting from growing to processing and production of potato seed tubers for export. FAO
data shown in Figure 4 depict the increasing trend in potato production in developing
countries. In fact, in the last decade, for the first time the developing world’s potato
production exceeded that of the developed world, with China being now the biggest potato
producer (Figure 5).

3
Resistant varieties have been recently breaded as well and new tropical sugar beet varieties produce more sugar
per hectare than sugar cane, in about half the time and with less water [40]. It is therefore expected that the
incorporation of new varieties in the tropics will lead to increasing cultivation of sugar beet because of its high
productivity per hectare and incorporation with slide modifications in current sugar candy factories without
high investment.
4
Most common diseases that may occur include leaf diseases caused by Cercospora, powdery mildew, or root
diseases such as rot caused by Rhizoctonia solani and the viral diseas rhizomania.
5
This continent has also the highest level of potato consumption in the world (almost 90 kg per capita per year),
while Germany is the most important potato processor and exporter. In 2005 it processed 6.5 million tonnes of
potatoes; including 3.3 million tonnes transformed into potato starch, and exported 1.3 million tonnes of fresh
potatoes and 2 million tonnes (primary equivalent) of processed products. It is also a leading importer of early
potatoes (nearly 550000 tonnes, mostly from France, Italy and Egypt, in 2005) [47].
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 51

350

300

Production (million tones)

250

200

150

100 Developed
Developing
50
World
0
1991 1993 1995 1997 1999 2001 2003 2005 2007
Year
Source: FAOSTAT 2007[47].

Figure 4. Yearly trend in world potato production and differences between developed and developing
countries during the period 1991-2007.

140000000

120000000
Quantity (tonnes)

100000000

80000000

60000000

40000000

20000000

0
Eu e
a

Be y
G nd
ne
a

pe
s

s
U s
d.

di

c
an

ru
n

nd
e

an
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ro
at

la
ai
hi

In

la
m

rl a
kr

Po
C

St

Fr
er
n

he
ia

d
te
s

et
us

ni

N
R

Top potato producers (2007)

Source: FAOSTAT 2007 [47].

Figure 5. Top ten countries of potato production.

However, potato production faces still significant problems due to the regular presence of
pest such as the Colorado potato beetle Leptinotarsa decemlineata [94], whiles the potato
tuberworm Phthorimaea operculella (Zeller) has been evolved recently as the most important
moth threat.
52 Petros T. Damos

30000

Production of cotton (in thousand balls)

25000

20000

15000

10000

5000

m tan
i
Pa i a

an
na

n
t
ria
G y
Au ce

li a
Tu il

al

e
yp
az

ni
e

bw
d
U

M
st

rk

ra
hi

Sy

s
In

Eg

Tu Be
Br

i
re
ki
C

ek

ba
st

m
rk

Zi
Top cotton Producers worldwide
Source: FAOSTAT 2007.

Figure 6. Top world cotton producer for 2007 [48].

c. Gossypium hirsutum
Cotton is currently leading plan fibre industrial crop worldwide and is commercially
grown in more than 50 countries in the temperate and tropical regions. G. hirsutum
accounting for 90% of world production, while G. barbandence represents 5% of world fibre
production [71] [101]. Additionally, it is estimated that cotton is cultivated on approximately
2.4% of the global arable land [19]. Figure 6 depicts the top cotton producers worldwide.
The cotton plants are used mostly for the production of fabric, but are a source for many
other important products as well. Among the most important is cottonseed, which is pressed
for cottonseed oil that is used in commercial products such as salad oils and snack foods,
soaps, cosmetics, candles, detergents, and paint. The hulls and meal are used for animal feed.
Cotton is also a source for cellulose products, fertilizer, fuel, automobile tire cord, pressed
paper, and cardboard [102].

d. Zea mays
The maize Zea mays L. belong to the tribe Andorpogenae, in the subfamily Panicoideae,
in the family Poaceae. There are 86 recognized genera within the Andopogoneae trip. The
genus Zea has five species which have a chromosome number 2n=20 (except of [Link]:
2n=40). However, Z. mays is the only cultivated corn species, while the other species and
subspecies are wild grasses [59]. The maize plant may be defined as a metabolic system
whose end product is mainly starch deposited in specialized organs, the maize kernels. Corn
is one of the most productive crop species having a global average yield of more than
1tonne/0.1he. The land area planted with maize increased from 105 million ha in 1961 to
about 127 million ha in 1987 [46]. This significant increases in production resulted from
genetic improvement and more efficient technological field practices and fertilizer
applications, as well as from the introduction of new, more highly reproductive varieties [46]
[88].
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 53

The developing countries have more area given to maize cultivation than developed
countries, but yield in the latter is about four times higher, while the US is the leader in corn
production followed by China (Figure 7). Worldwide, corn ranks behind only wheat in total
production. Additionally, approximately 80% of corn production is used for livestock and
poultry feed and the rest is used in food and industrial applications. However in many
developing countries apart of life stock feed corn is also an important food crop [46] [55].
Moreover, corn continuous to be used as basic food source in subsistence agriculture, while in
developed countries more than 60% of the total production is used in compounded feeds for
poultry, pigs and ruminant animals. The by-products of dry milling include the germ and the
seed-coat [20]. Among the pest damaging corn the stem borers Sesamia nonagrioides and
Ostrinia nubilalis are considered as the most important and worldwide efforts are made to
prevent their activity.
Corn Production (thousand metric tonnes)

300,000

250,000

200,000

150,000

100,000

50,000

0
ge o
S

do a
a

U pt
C ia

N ry
H ain
ut da
na

M il

ia

ia
R sia
Ar xic

In f ric
as

in
U

y
d

ga

er
an
So ana
hi

Eg
nt

In

kr
ne
e
Br

hA

ig
un
C

om

Top corn production countries

Source: FAOSTAT [46].

Figure 7. Top corn producers – figures for 2003/2004.

1.2. Representative Lepidoptera Pest Threats of Industrial Crops

Although modern agricultural mechanisation as well as extensively use of chemical

influxes and pesticides, resulted in a rapid expansion and yield increase of industrial crop
models, monocultures still face problems due to a regular presence of economically important
pests threats. For instance, Beta vulgaris is subject to attack by more than 150 species of
insects and mites [76]. In some cases, the impact of different pest’s species is difficult to be
assessed because it varies with the specific situation [13].
Nevertheless, in this section we are mostly interested in those moths that regularly
damaging industrial crops. Additionally, there are cases in which a moth species has
poliphagous habits and can damage more than one crop hosts (e.g. Agrotis sp., Helicoverpa
armigera). In this section the most representative moth species damaging industrial crops are
54 Petros T. Damos

in brief discuses including: Agrotis sp. (Lepidoptera: Noctuidae), which is regularly damaging
most of the affront mentioned industrial crops (especially at the early stages of vegetation).
Phthorimaea operculella (Lepidoptera: Gelechiidae), which is damaging potato either in the
field or during storation. Helicoverpa armigera (Lepidoptera: Noctuidae) which is considered
along with Pectinophora gosypiella (Lepidoptera: Gelechiidae) as the most serious pest threat
in cotton production and Sesamia nonagrioides (Lepidoptera: Noctuidae), which along with
Ostrinia nubilalis are the most serious moths of corn [13].

a. Agrotis sp.
Here are included several species. The black Cutworm Agrotis ipsilon (Hufnagel)
(Lepidoptera: Noctuidae) is probably the most damaging [9] [25]. The species Agrotis
segetum (Denis and Schiff.) is considered as the most damaging in continental Europe [18]
[22], while although Agrotis trux (Hübner 1824) has a circum-Mediterranean distribution
[13]. Agrotis sp. attack over 30 important crops, damaging mostly seedlings, including potato,
cotton, tobacco, several vegetables. Additionally, Agrotis sp. is and occasionally corn pest
that is attracted to no-till fields [24] [30] [95]. The species overwinters at a larval stage at
warmer areas and as pupae in shelters few centimeters above the ground. Pupation occurs
during spring and emerged adults mate. Females have high reproductive potential and the
moths can lay approximately 1100-1400 eggs in clusters. The egg clusters can be distributed
on low densely growing plants and weeds like chickweed - Stellaria media (Caryo-
phyllaceae), curly dock - Rumex crispus (Polygonaceae), mustard Sinapis alba (Brassicaceae)
and Graminae weed species [13]. New hatched larvae feed above ground on weeds and/or
sugar beet leaves. Most of the feeding occurs on nights in which larvae extensively feed and
cutting off plants at the base. During the day larvae hide and arrest beneath plant debris or
few centimeters in loose soil. Since cutworm damage may occur as soon as seedlings occur,
damage can be extremely severed at the early plant stages. Additionally, larvae are devouring
and one individual can damage more than one plant during successive nights [9]. Most
frequently the damage is detected at the field edges, although stand loss can occur also in a
spotty pattern throughout the field. In corn, instars of A. ipsilon feed on the leaves of
seedlings, but the most serious damage results from leaf and stem cutting by late instars [30].
The species have more than 3 flights depending upon prevailing temperatures. The use of a
granular insecticide sown with the seed or later worked into the top 15 cm of soil is
considered among the current conventional methods of controlling these species.

b. Phthorimaea operculella
The potato tuberworm (PTW), Phthorimaea operculella (Zeller) (Lepidopterea:
Gelechiidae) is a pest of many solanaceous crops, although is considered as one of the most
important lepidopterous pest threat for potatoes crops [53] [96].The species is commonly
found in tropical and subtropical regions throughout the world. Besides potato, P. operculella
has been reported to infest other solanaceous plants such as tomato, pepper, eggplant,
tobacco, and nightshade [94]. The adults are very small moths (e.g. micro moths) with a wing
expanse of 0.5 inch (1.2 cm). When at rest, the wings are held close to the body giving the
moth a slender appearance. The general color is gray with darker gray-brown or black
markings. Female adults lay their eggs on foliage, soil, plant debris, or exposed tubers. Moths
can crawl through soil cracks or burrow short distances through loose soil to find tubers on
which to deposit eggs. The damage is provoked by larvae which mine leaves, stems, and
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 55

petioles and excavate tunnels through potato tubers, but the typical damage results from
larvae mining the tubers [13]. Larvae close to pupation drop from infested foliage to the
ground and may burrow into tubers. Larvae usually enter the tuber at the eyes and tunnel
entrances are marked by small deposits of frass and spin webbings. In most cases the larvae
feed just below the potato surface constructing a dark tunnel but bore also occasionally deep
in to the tuber. P. operculella do not tunnel through stems and roots into the tubers. The
species pupate on the soil surface or/and in debris under the plant insight spin silk cocoons.
The damage provoked by larvae continuous also in shelters after harvest. Exposed tubers are
predisposed to tuberworm damage. Injured potatoes are additionally infested by
microorganism and rot in storage. As a result infected production is non-marketable. These
losses are of particular concern in regions where large quantities of potatoes are stored. Lately
integrated pest management (IPM) strategies for potato tuber pests have been developed to be
regular used by various institutions. The IPM system is mainly based on the application of
biopecticides applied on the surface of the tubers in farm storage [128]. In this context
botanical pesticides have received a great deal of attention because of their favorable
ecotoxicological properties [1].

c. Helicoverpa armigera
The cotton bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)
(synonyms: Heliothis armigera, Chloridea obsoleta, Heliothis obsoleta) and the pink
bollworm (Pectinophora gossypiella) are the most serious pest threats of cotton [13].
However, H. armiger is highly polyphagous damaging a broad spectrum of families some of
them include important agricultural crops such, maize, chickpea, pigeon pea, sorghum,
sunflower, soybean and groundnuts and other [54].The species overwinters at a pupal stage in
a cell few cm underground. Adults emerge from April till May depending upon prevailed
temperatures. Females lay eggs on the flowering and fruiting structures crops, where
voracious larval feeding leads to substantial economic loss [32]. Additionally, one of the
major factors contributing to the pest status of this moth, is the ability of ovipositing females
to locate and utilize a wide range of hosts from a number of families is [50] [124] [126].
Larvae of the moth species are capable of dramatically reducing yield. Historically control of
H. armigera with insecticides has led to insecticide resistance [109] [126] and dramatic
reductions in beneficial populations which allows populations of other pests with fast life
cycles such as aphids and spider mites to develop to damaging levels. Helicoverpa armigera
considered as key pest in establishment of IPM systems in cotton [51] (see also section 4).

d. Sesamia nonagrioides
The corn stalk borer Sesamia nonagrioides Lefevre (synonym: S. vuteria, S. hesperica)
(Lepidoptera: Nuctuidae) is a serious pest of corn in the Mediterranean regions and Central
Africa [8] [73] [81]. The species is polyphagous with a range of host plants including not only
corn, but also sorghum, millet, rice, sugar cane, asparagus, melon, palms, banana and
ornamental plants such as Strelitzia reginae. The species has been reported in Portugal ,
Spain, the Canary Islands, France ,Italy, Greece, Cyprus, Turkey, Morocco, Israel, Syria, Iran,
Ethiopia, Ghana , and several other African countries [44 and references given]. In the
circum-Mediteranean areas S. nonagrioides has been designated as the most important moth
of corn. Nevertheless, its active damage potential is difficult to be estimated in precise since
its damage is not distinguishable to that caused by Ostrinia nubilalis (Lepidoptera: Pyralidae)
56 Petros T. Damos

[8]. Actually the damage caused by maize borers could range from 5%to 30% of the yield
depending on the date of sowing and the cultural cycle of maize [10].The species usually
completes three to four generations in Greece [44] [114] and two generations, having a partial
third one per year, in Spain and Portugal [78].
All the above Lepidoptera have a wide distribution in Europe, North America and
northern Asia and thus are considered as regularly important pests threats for industrial crop
production worldwide [13]. The reason that they have been chosen to be described in this
chapter is because, in most cases, farmers face problems with all the above pests which
simultaneously appear in more than one industrial crops.

2. INTEGRATED PEST MANAGEMENT IN INDUSTRIAL CROP MODELS

Among the available alternative pest control strategies, IPM has by far received the most
attention as a comprehensive pest management approach [77]. Historically IPM was
introduced using the term Integrated Control by Barlet (1956) [16] and was further elaborated
by Stern et al. in 1959 [103], as a concept which in principal is integrating the use of
biological and other control measures in complementary ways. In 1962, van den Bosch and
Stern [115] broadened the conceptual framework of IPM to embrace the coordinated use of
all available biological, cultural, and artificial practices. In this context, various authors have
advocated the principle of incorporating all available control methods and measures to
manage pests that are been chosen by environmental, economic and social criteria.
Subsequently, the term IPM incorporates the full array of pest management practices, which
are adopted following certain criteria, included into a total systems approach which ideally
should meet all crop production objectives [35] [38].
At an early stage of development IPM uses current comprehensive information on the life
cycles of key pests6 and how they interacted with their environment. This is a difficult task
considering that detailed information upon the key-pest and how it interacts with its
environment is not always available [111]. This knowledge is further combined with the use
of those control methods that are selected among several available, by economic, social (least
possible hazard to people) and environmental friendly means [34] [35] [43] [112].
However, when it comes to translate IPM in praxis it is primarily based on pest field
monitoring and relative thresholds establishment, in which mostly pesticides are used at a
need basis if alternatives are either non available or non-efficient [77]. Unfortunately, much
less emphasis is given on understanding and promoting inherent strengths within systems to
limit pest populations through the use of approaches such as landscape ecology [77]. Thus,
ideally, the seasonal presence of a particular pest should be regarded as part of the agro
system and not just abstracted. Moreover, external driven actions can affect the systems
behaviour and cause significant feedbacks, which are not always predictable. For instance, the
application of conventional pesticides can cause a temporal eradication of the target pest but
also modifies the systems behaviour which in turn can result to outbreaks of secondary pests.

6
The definition of key pests is essential to be made in Agro Ecosystems since it constitutes the basis for the
development of Integrated Pest Management (IPM) strategies and the development of reliable decision tools
for Integrated Fruit Production (IFP) at the first stages of development [33] [34].
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 57

Hence, conceptually IPM does not focus on a total eradication of a pest, but uses decision
tools to take measures at critical points which modify the system functionality to a direction
accepting the presence of a tolerable pest density.

2.1. Pest Management Options and Principal Decision Making

In most IPM programs the available pest management option can be briefly classified in
the following categories [38]:

i. Cultural and mechanical techniques. In annual crops these techniques can be either
large scaled or depending upon the practice and special applications and mostly
includes techniques such as cultivation selection (resistance, harvesting time), crop
rotation, and cleanliness. Mechanical techniques involve the use of some form of
power, which is based on fossil fuels necessary for the work of relative equipment.
Tillage is a representative example of mechanical technique.
ii. Biological measures. Traditionally is referred to natural occurring or augmented
parasites and predators, although the term is used by some authors under a broadly
sense including also the release of sterile males [39] and in some cases the use of
semiochemicals as well [38]. Additionally, the use of biological control agents such
parasitic nematodes, insect infectious fungi, bacteria and viruses offer new
successfully biological options for IPM.
iii. Biotechnical measures. Several options are here included and most of them are very
promising for IPM such as the use of semiochemicals, including in most cases sex
pheromones used either for moth monitoring or matting disruption. IPM
traditionally makes use of pheromones which are substances which occur in nature
and are used for chemical communication between animals (see section 3.2).
iv. Chemical options. These have been traditionally used to manage pest and are
straightforward at the initial point of application when we compare them to most
other options. However, a discrimination should be made between conventional
pesticides and the use of novel compounds that express high selectivity towards the
target organism (more details upon the mode of action of pesticides and their
compatibility with IPM are given in section 3.4.).

For more details upon the above and/or related classification schemes the reader should
advise references [38] and [74].
Furthermore, a major issue for IPM is which criteria are used to select among the
available control options as well as the definition of accurate application times. For instance
regardless of the available quantity of the novel chemical compounds, their choices in IMP,
are evaluated based on more than one criteria in which application time is essential for their
success [39]. From an economic perspective, extensive annual crop systems are treated as
purely industrial crop production processes which are focusing on maximising profits and
minimising costs [82]. Thus, the crop system has to be either profitable, or have access to
outside capital, in order to stay in business.
58 Petros T. Damos

Additionally, crop protection advisors and farmers should have managerial skills and use
specific decision tolls to maximise the effectiveness of their control actions.
Some principal decision7 options including:

i. Monitoring

By definition monitoring is the process of regularly inspecting the presence of pests that
pose a continual threat throughout a lengthy period such as a growth season [17]. Pest
monitoring is a fundamental component of IPM programs and in most cases pheromone-
baited traps are routinely used to monitor the flight periods of several adult Lepidoptera pests
threats in annual or perennial crops [118] [119]. The discovery and chemical construction of
sex pheromones for most Lepidoptera pests, leaded to the wider adoption of pheromone traps
to monitor adult male populations.
Nevertheless, considering that damage is provoked by immature stages additional
monitoring techniques and scouting procedures should be also performed. Bins et al. [17], for
instance, defines monitoring as an overall strategy which includes setting up of a schedule of
potential sampling occasions providing instantaneous critical information related to each
sample. These samples are further used as decision tool to specify either a positive
management action or to resemble.
At the field level this involves the use of monitoring protocols aiming to direct how
sampling resources are to be allocated based on characteristic operating functions [17].
However, the difficulty in obtaining precise knowledge of the state of a crop-system of
interest will affect the characteristic operation functions and related decision curves. Figure 8
depicts the shape of a typical probability decision curve and provides knowledge on pest
density that should be related to intervention (or non-intervention) of a management tactic.
The range of density over which this curve shapes, reflect the expected range of uncertainty
about crop loss and provide means to estimate the critical pest density (i.e. Economic
Threshold) utile in decision making [17] [33].
Unfortunately, in most cases it’s quite difficult to obtain precise knowledge of the crop-
pest relation and extensive and costing sampling procedures are needed. Therefore most IPM
cases are dealing with a limited content of information that is sufficient to provide a basis for
decision making. One additional problem is that, for pests that pose a continuous threat
during the cultivation seasons, monitoring must be repeated during successive time points
[34].
The problem of accurate estimation of the population threat can be even more
complicated considering that population densities can follow several spatial patterns (i.e.
uniform, random, aggregated) [67] [68] [121]. Thus a simple sampling procedure which
doesn’t takes in to account the spatiotemporal arrangement of the pest stages, often results to
fault density estimations.

7
Most of these decision tools have been proposed for several IPM programs and are regularly used. However, the
reader should advice citations [82] [90] and [91] and references given, for a comprehensive based discussion
for these and related concepts as well as the potential benefits of implementation.
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 59

0.9

0.8

Intervention / non intervention

Probability of decision
0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 5 10 15 20 25 30 35 40 45 50

Pest Density

Figure 8. typical shapes of the probability decision curve and intervention/non-intervention curve for a
hypothetical pest density obtained from random generated sample data. This function represents the
probability of not implementing a management action against a pest and further used in field scouting
to determine the occurrence and seasonal activity of pests. For more details refer to [17].

Figure 9. A hypothetical example illustrating the effect of sample amplitude (n:4,10,20, and for
p=0.05) on the probability of a sampled specimen (i.e. leaf, reproductive organ e.t.c.) not to be injured
(i.e. no presence of larvae or no plant injury) in relation to the actual % crop injury. Probability of
specimen sampled not to be injured is described according to a binomial (no injury – injury) probability
distribution function. Note that for common % injury specimen in population (e.g. say 10% of the total
cotton ball population is registered to be injured) the probability of sampling an injured organ is 5 to 10
times higher when the population sample increases from 4 to 20.
60 Petros T. Damos

Additionally, several other factors can affect the intervention thresholds such as the
amplitudes of the sample (number of specimen collected), and the population of the pest (i.e.
its actual population injury). Figure 8 is an example which depicts the effect of both, sample
amplitude and actual % crop injury, on the probability that a sampled specimen (plant,
reproductive organ e.t.c.) will not be injured. Here we are dealing with an oversimplified
situation in which each specimen is completely independent of the location of the others (e.g.
a random pattern exists) and since the only available information is whether a specimen
(sampling unit) contains a pest (i.e. bollworm), we can describe the probability of being
injured based on a binomial distribution.
Hence if economic injury is related to pest incidence, the binomial distribution can be
used directly to develop sampling plans for decision making in crop protection [17].
However, if economic injury is related to density in which sample data are proportions of
plants containing a pest, the problem arises of relating incidence to density.
Additionally, there are cases in which spatial distribution and sampling is not random
(actually more often in praxis) but is described according to a negative binomial or beta-
binomial distribution, in which an aggregation parameter8 must be first provided [67] [68]
[34]. To estimate the population distributional patterns several kind of regressions9 are
performed by relating for instance the variance to the mean density.
For more details upon the use of decision tools in IPM refer to [82] [85] [89] [90] and
[91]. The basic concepts and statistical theory of sampling in pest management are very well
described in [17]. Applications of formulae, techniques and methods to estimate species
distributions and for a dipper discussion upon the ecological perspectives of species
spatiotemporal arrangements refer to [121] and references given.

ii. Phenology Modelling

Although in most cases sex pheromone traps are used to indicate population emergence
and temporal evolution of Lepidoptera populations, they often result to false estimations due
to several factors affecting insect abundance and moth flight [104] [112]. Weather changes
(e.g. temperature fluctuations and abrupt rain), can cause changes on population dynamics
and is cause of bias [108] [125]. Additionally, several factors can affect spatial dispersion and
emergency patterns of immature stages in field. Thus, in relation with other methods such as

8
The value of the aggregation parameter is either fixed or more precise can vary in relation to a variance-mean or
variance-incidence model. The negative binomial is described by two parameters, the mean and the exponent
k, which is a measure of the amount of aggregation and is often referred to as the dispersion parameter [99].
Values of k are generally in the region of 2, becoming larger as the distribution tends towards the Poisson (i.e.
random). When k = 0, fractional values imply a distribution tending towards the logarithmic series, which
occurs. Aggregation values are not necessary for Poisson or Binomial distributions.
9
Taylor's power law relates variance (s2) to mean density (m) so that: log s2 = b logm + log a, where α parameter is
considered to be a sampling factor, and b the slope is considered as an index of aggregation. This index is
specific and constant for a species. The range of values of b from b<1 corresponds to a uniform distribution,
through b = 1 for random distributions and for b>1 for distributions which are aggregated [104] [105]. Iwao's
and Kuno’s method, [69] [70], uses a regression of mean crowding (m) upon the mean (m) in the linear model
as follows: m = a + b*m, where a and b are regression constants characteristic of respective distributions. The
intercept α, the index of basic contagion, has a value of 0 for distributions where a single individual is the
basic unit, however takes a positive value, where the population exists as a cluster of individuals. The slope
represents, the density-contagiousness coefficient, which describes how such individuals distribute themselves
in the habitat and takes. The slope can take values of β>1, β = 1 and β <1 for individuals arranged in
aggregated, random or uniform pattern, respectively.
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 61

the temperature summation monitoring procedures they are able to describe more precisely
the population dynamics and occurrence of a species for a particular region.
Degree-day phenology models are based upon the assumption that all poikilothermic
organism, including insect as well, are related to a species specific thermal constant. This
constant corresponds to the time heat units that must be accumulated to complete a species
specific developmental event [34] [35] [36]. Although that development accelerates or slows,
according to prevailing temperatures, the final time units to complete a particular
developmental event should be constant.
In most cases phenological models are based on regression functions between
accumulated degree-days and cumulative moth catches (or general cumulative emergence of
any stage). However, it is important to estimate first the vital temperature thresholds of the
species of interest and especially the lower temperature threshold, to start heat summations.
These empirical regression models are most useful in predicting adult moth emergence and
population peak [35] [37].

Insect Phenology

Diapause 1st Generation 2nd Generation 3rd Generation Diapause

Termination Induction

100

80
Moth captures (%)

60

40

20

n Tmax (1)  Tmin(1) Tmax ( 2)  Tmin( 2) Tmax ( n )  Tmin( n )

 DD  [(
1 2
 Tb )  ( DD2 
2
 Tb )        ( DDn 1 
2
 Tb )]

Host phenology and degree –day’s summation

Figure 10. Typical shape of a cumulative moth emergence function in relation to the host (corn)
phenological stages. Note that heat summations are performed using the simple average method with no
upper cut of (calendar Biofix: 1st April). Each function corresponds to a generation observable during
the growth seasons. The shape and the number of curves are strongly affected by region specific
conditions (temperature and photocycle).
62 Petros T. Damos

A typical example of a degree-day model is generated in Figure 10 which depicts the

shape of a cumulative density function for a moth species (i.e. Sesamia nonagrioides) in
relation to the phenological stages of its host (i.e. corn). The shape of the function has the
form of typical cumulative distribution function (cdf) and has been constructed for illustrative
reasons based on some fixed parameters. For more details upon the degree –day phenology
modelling of the corn borer Sessamia nonagrioides and its vital thermal requirements refer to
[78].
These regression lines are empirically fitted curves and should more likely be considered
as the best mathematical fit since they do not possess any clear biological meaning [35] [36].
However, if they adequately describe the relations between temperature and insect phenology
they provide useful insights on the occurrence and seasonal activity of pests and are often
used as decision tool to implement a management action [35] [37].
For more details upon heat summations methods the reader should advice references [2],
and [12]. For emphasis on the degree-day modelling approach in describing insect phenology
refer to [63]. For specific examples in modelling seasonal moth emergency patterns in fruit
crops and IPM refer to [33] [34] [35] [36] [80].

iii. Threshold Values as Basis for Decision-Making

Stern et al. (1959), [103], introduced the ideas of economic injury level (EIL) and
economic threshold (ET) (referred as critical density by Binns et al. 2000, [17]) as way to
assess the pest status based not only on biological characteristics but incorporating economics
as well. In principal the concept of the EIL integrates biology and economics and uses control
actions (unfortunately mostly pesticides) only when economic loss is anticipated [89] [90].
The EIL is cornstone for IPM and Crop Protection (CP) since it provides the practical means
to define how much pest injury can be tolerated.
The EIL is related to five primary variables [35]: the cost of management tactic per
production unit (C), the price of commodity (V), the injury units per pest (I), the damage per
unit injury (D) and the proportionate reduction of injury averted by the application of a tactic
(K). These variables are related as follows: EIL = C/VIDK.
In fact the variables I and D are related to each other and are the biological characteristics
of the function by representing the yield loss associated per pest [90]. The parameters D and I
can be obtained from the slope of the yield or damage function (Y = a + bx), where Y = yield;
b = yield loss/pest; a = 0 and x = number of pests per sampling unit, while the coefficient b
represents the loss per insect, which is equal to I*D or D’. Thus, for the application of EILs it
is necessary to estimate first the mathematical relation between insect pest injury and yield
loss. This relation is called damage function, or damage curve, and consist the biological part
of the EIL concept [23] [33]. Generally in most cases it’s quite difficult to estimate the
expected crop loss for each pest density and related plant injury. Therefore it is often assumed
that the presence of an individual is direct related to yield loss if a crop is damaged. Figure 10
depicts a hypothetical pest-damage function resulted from artificial infestations caused by
different levels of larval population (e.g.: 5 treatments with 1-5 larva (L2 stage), releases at
flowering stage of the crop in which yield loss can be expressed in % as the ratio of infested
and healthy organs per plant [49] [124].
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 63

Figure 11. Typical shape of a hypothetical pest-damage function (y = 7.6742x - 3.3908, R2 = 0.8472,
p<0.05) which associates percent yield loss of a sampling unit (i.e. cotton plant: G. hirsutum) to the
number of larvae observed (i.e. bollworms) at a specific plant phenological stage (i.e. blooming).

Figure 12. Extrapolation of the Economic Injury Level (EIL) model concerning typical moth larvae
damaging an industrial crop. Effects of commodity value (V) and cost of management tactic (C) on the
EIL in respect to a standard insect–yield function (D’) and proportionate reduction of injury averted be
the application of a tactic (K) are generated as surface plot. Note that for a given damage function (D’)
and pesticide efficacy (K) the EIL is strongly governed by the cost of management (i.e. bio-pesticide
price and application costs) (see text for details).
64 Petros T. Damos

To further illustrate the concept of an economic injury level an example of the EIL model
is generated in Figure 12 based on estimates of Figure 11. Such kinds of models are generated
to provide objective guidelines for decision making and especially pesticide use.
It is important to outline the dynamic behaviour of the EIL model and several related
limitation that can exists [58] [92]. For instance, it is evident that for given species with a
specific damage function (i.e. parameter D’) and pesticide efficacy (K), the EIL is strongly
governed by the cost of management (C) and the commodity value (V) [33] [35]. Thus,
different combinations among the four variables result to different EIL levels. It is also
noteworthy to state that several factors exert influence to the damage function (parameter D)
as well. Region specific factors for example, such as the cultivation of cotton varieties that are
tolerant to pest attacks modify the slope of the damage function and can result to region
specific alterations of the EIL.
The EIL is further used to define the Economic Threshold (ET). The ET is an operational
criterion that can be used by plant protection advisors and farmers to define population
density at which control measures should be initiated to prevent an increasing pest population
from reaching the EIL [23] [35]. There are several ways to estimate the ET although for
simplicity reason the ET can be calculated by representing a fixed value of the EIL.
Both, EILs and ETs, are considered as basic theoretical concepts for a successful IPM in
practice. For more comprehensive details refer to [89] and [90].

3. PEST MANAGEMENT TOOLS AND RELATED

STRATEGIES IN IPM
3.1. Cultural Measures

Cultural measures refer to cultivation actions which aim to modify the agricultural
environment in order to be less favourable for pest invasion, reproduction, survival and
dispersal. Most of these measures are variations of standard agronomic practices, which
should be regularly carried out by farmers and among others include: crop rotations, planting
characteristics, crop barriers and plant traps, phytosanitation and irrigation along with
standard cultivation practices. Table 1 list some representative cultivation measures that can
be applied in IPM industrial crop models to control and/or suppress pest population levels.
Cultural measures may not be give alone satisfactory pest control albeit within an IPM
system they provide a framework in which most other tactics are deployed. Moreover,
cultural practices are often help to reduce dependency on chemical pesticides [4] [77].
Moreover, all cultural measures depending on the particular conditions and aim to enhance
vigour and promote plant health are important to tolerate pest injury and therefore cultural
measures are often in the base of biointensive IPM systems. In this framework each IPM
component complements and often augments the effect of others. This action is referred as
ecological synergism [74].
Obviously we do not presume to have offered an exhaustive discussion of cultural pest-
control practices in this section and therefore the reader should advice the references [61],
[84] , [100] and given citations for additional details.
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 65

Table 1. Some representative cultivation measures used in IPM industrial crop models
to control and/or suppress pest population levels

What to do How to do Why to do

Producing -Growing resistant varieties - Increase plant ability to compete pest load
healthy and
vigorous crops
-Reducing environmental stresses - Upper the EIL and ET levels
through better soil and crop management
Support diverse -Include perennial crops: Plant fruit trees, -Serve as habitat for beneficial insects
landscapes and permit trees and shrubs growing along (providing food and shelters)
plant stream banks
communities -Enrich crop system: grassed waterways,
or forage grasses and legumes
Increase -Crop Rotation: Temporally: perform -Pests will encounter confusing patchwork
diversity/reduce regular rotations of three or more crops stress and which decreases abilities to locate
homogeneity - Spatially patchwork quilts: include their hosts in both space and time
different crops on the same farm at - Resistance to control measures will be
various stages and under diverse hampered.
management practices -Ground, diversity a medley of soil
-Scalable: Preserve some undisturbed organisms helps maintain low pest
areas between farms and natural parches populations
Preserve soil -Perform moderate tillage to avoid -Destruction or expose to predation of
properties compacting soil overwintering stages (e.g. pupa bollworm)
-Conservation tillage, preserving soil
structure and complex food webs for ground
beetles and other beneficials
-Encourage on-farm nutrient
cycling and help organic matter accumulate
Sanitation - Destroy and/or remove crop residues - Reduce the carryover of the pest through
- Rewoven of fallen crops during harvest the cultivation seasons.
(infection source) -(e.g. destroying the stubble of cotton, maize,
- Use pest free seed and equipment to destroy and/or remove overwintering
stages boll worms and stem borers)
Create multiple -Placing non-host crop in a rotation -Destabilising habitat and disrupt pest
stresses in pest -Scrutinize the life cycles of pests and populations
beneficial organisms, looking for times -Provoke pest mortality in small increments
and places where small which can eventually pare a big problem to
low population levels
Enhance -Sowing cover crops between rows of - Harboring natural predators, parasitoids and
beneficial cash crops, wildlife in perennial grasses,
organism activity -Keep shrubs and trees on field edges - Providing alternative food sources which
providing permanent refuge are critical to the development of slow-
reproducing predators ([Link] wasps
and predacious depend on a daily supply of
honeydew, nectar and pollen for energy and
reproduction)
Disrupt pest-crop -Timing of planting and harvesting - Early planted crops are in some cases less
phenology palatable to herbivores
synchrony - Early planted crops become more
established before the pest outbreak
- Early maturation alow toescape
infestations by late season pests and
generations
66 Petros T. Damos

Table 2. Major pheromone compounds and relative analogy

in moths damaging industrial crops

Pheromone Moth Species

Agrotissp. Phthorimaea Helicoverpa Sesamia
operculella Armigera nonagrioides
Name Chemical Structure Reference
Gemenoet Ono et al. Picardi et al. Mazomenos,
al. (2000) (1990) [86], (1977), [93] (1989). [79]
[57] Ono et al. Nesbitt et al. Krokos et al.
(1997) [87] (1979) [83] (2002) [75]
(Z)-11 √ √
Hexadecenyl
acetate
(Z)-11 √ √
Hexadecen-1-ol
(Z)-11 √ √
Hexadecenal
Hexadecanal √
Hexadecan-1-ol √
(Z)-9 √
Tetradecenyl
acetate
(Z)-7-
Dodecenyl
acetate
(E,Z,Z)-4,7,10- √
Tridecatrienyl
acetate
(E,Z)-4,7- √
Tridecadienyl
acetate

3.2. Semiochemical and Biotechnical Measures

By definition semiochemicals are chemical compounds emitted by living organisms

(insects, plants, etc) that induce a physiological response and/or behavioural changes in other
individuals. The term has its origin in the Greek word semeio (σημείο) which is translated to
a sign or mark of a chemical signal. Semiochemicals are classified as pheromones when they
act intraspecific and as allelochemicals whether they act as interspecific (Figure 5).
Allelochemicals further include allomones (emitting species benefits), kairomones (receptor
species benefits) and synomones (both species benefit) [62] [75]. There are cases however in
which a single chemical signal may act as both; as pheromone and allelochemical [113]
[118].
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 67

From a chemical point of view pheromones10 and semiochemicals consist of a wide range
of organic molecules which could be volatile or non-volatile. Sex pheromones consist in
individual molecules or specific blend of compounds in a given ratio. Non-volatile
semiochemicals for instance include cuticular hydrocarbons, acting in mate recognition or in
cannibalism regulation of several insect species [62], while volatile compounds (e.g.
pheromones) have small molecular weights (<300) and allow communication at relatively
long distances depending upon environmental conditions [115].
Currently, the number of pheromones identified and their application have greatly
increased since the first announcement of a pheromone chemical structure [11] [56] [65],
while the most of them used in IPM are that emitted by Lepidoptera. From a practical point of
view, pheromone traps are often used in IPM programs to monitor adult moth phenology.
Table 2 lists the major pheromones discovered in Lepidoptera of economic significance for
industrial crops that are cited in this chapter. Note that they are relative analogies concerning
the chemical structures in pheromones among individuals of close related taxa.
Apart of monitoring, semiochemicals have now wider applicability in modifying insect
behaviour and this technology is progressively incorporated in to IPM programs. Identified
semiochemicals can act at an ecological level by expressing the following modes of actions
[35]: Habituation, which is the progressive customization of individual males to the
pheromone. Since pheromone is perceived as a natural part of the chemical environment,
males do not notice female chemical signals.
Camouflage, is the cataclysm of the environment (i.e. orchard) by pheromone camouflages
and signals produced by the females are not distinguishable. Completive attraction is exerted
by the lures rather than the female signals. Males are actively sought to find the lures and
reduce the chance to mate with females. Trapping, lures with pheromones are placed in trap
devices and males are first trapped and then removed.
Additionally, this knowledge has been transformed to major strategy in IPM to change
pest behaviour, or its enemies, and finally is adopted at a crop system level (Figure 13).
Mating disruption for instance is a relatively new commercial strategy in the control of
lepidoptera of economic significance [34]. In principal this technology is based on the use of
semiochemicals and the pest management strategy is using synthetically produced
pheromones through controlled release to confuse males in order to limit their ability to locate
females for mating.
The reduce of the likelihood of successful mating is the main objective of the strategies
and is accomplished through the distribution of pheromone dispensers in the field [27] [28]
[3].The attract and kill strategy is another semiochemical-based approach involving the
combination of a semiochemical lure with an insecticide and this approach is already
successfully applied to the control Lepidoptera in fruit orchards such as the codling moth
[35].

10 10
The most common use of semiochemicals is that of pheromones to monitor insect phenology [110] and the
term pheromone comes from the Greek pherein (φέρειν) = to transfer and hormon (ορμών) = a dash for=to
excite. To date, the first chemical identification of a pheromone took place in the late 1950s, by a team led by
Adolf Butenandt (1959) [26]. The team chose to study the domesticated silk moth, Bombyx mori, which could
be easily reared in the high numbers needed and spent more than 20 years using extracts from ½ million
female silkmoths to accomplish the feat. The silk moth pheromone was identified as (10E,12Z)-hexadecadien-
1-ol, referred as bombykol.
68 Petros T. Damos

Figure 13. Classification scheme of semiochemicals, their action modes and process of integration and
adoption as IPM strategies.

The ‟push-pull‟ strategy uses a combination of behavior-modifying stimuli to manipulate

the distribution and abundance of insect pests and/or natural enemies. Although the push and
pull strategy is a novel tool in IPM it appears very promising especially in managing pests of
industrial crops. In brief, in this strategy the pests are repelled or deterred away from the main
crop (push) by using stimuli that mask host appearance or are repelled, while they are
simultaneously attracted (pull), using highly apparent and attractive stimuli, to other areas
[35]. The attractive stimuli can be either traps or more frequently alternative hosts and crops.
The ‘trap-host’ then serves to concentrate the pest out of the main cultivation and thereby
facilitating their control. For moth species in particular they can be pushed by volatile
chemicals by intercropped plants which attract natural enemies. On the other hand, volatile
chemicals are produced by border trap plants and attract moths to lay their eggs.
Because semiochemicals do not exhibit adverse effects on non target pests, while insect
pest resistance is unlikely to appear, theses strategies are very promising for IPM of industrial
crop models. From an environmental perspective, the comprising properties allowed them to
be characterized as non persistent displaying relatively low toxicity to non target species and
are environmentally safe.

3.3. Biological Control

For entomologist, biological control is by definition the use of living organisms,

excluding host plants, as pest control agents. There are several types of biological control
strategies, including [38]:
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 69

 Introduction of natural enemies: This typically involves the introduction of a natural

enemy for the control of an exotic pest. In cases in which this kind of biological
control is successful, it offers permanent levels of control having few associated risks
although in most cases it provides a very cost-effective solution.
 Augmentative control: This approach is mostly used in cases where long term
stability is not major but more likely in cases where pest numbers are needed to
reduce below economic thresholds. This involves a number of releases throughout
the season and performs temporal control of pests when needed.
 Inoculation: Here, pest management throughout the seasons may be achieved
through regular use of inoculations. The inoculation release take place at areas where
the pest is absent at an initial crop stage, to colonize it and thereby prevent the pest
build-up.
 Inundation: This procedure involves the inundation with a large numbers of native or
introduced natural enemies on a regular basis in like pesticides applications. The use
of Bacilus thuringiensis (Bt) to manage lepidoptera pests and/or the use of other
natural pathogens are typical examples of inoculation using conventional pesticides
applicators. Mixtures of different d-endotoxins11 are usually present in the inclusion
and individual toxin proteins are designated with the prefix species specific toxic cry
protein. The toxin proteins contain a few hundred to over 1000 amino acids. After
they are ingested, the d-endotoxins are cleaved to an active form by proteases within
the midgut. Referred as bio-pesticides these commercial12 developments have
focused on using Bt or nematodes and viruses in the same manner as chemical
insecticides and not as a living biological control agent. This simple means that they
are not adapted to degree that allows them having a continuous impact on pests in
crops systems, which can cause continuing outbreaks (epizootics) which suppress
pests under natural conditions. Moreover, since they are considered as natural
mortality agents and environmentally safe, there is worldwide interest in the use and
manipulation of entomopathogenic fungi for biological control of insects and other
arthropod pests. In particular, the asexual phases of Ascomycota (Beauveria spp.,
Lecanicillium spp., Paecilumyces spp., Metarhisium spp. and others) are under
intense scrutiny due to the traits favouring their use as bio-insecticides.
 Conservation: This technique involves measures that encourage and conserve the
natural populations of natural enemies in the crop system to be able to control
inhabitant or immigrant populations of pest. Recently this area has receives attention
in IPM and focuses on the enhancement of already adaptive native natural enemies
by minimizing conventional non selective pesticides. Reduces in pesticides
applications may be sufficient in some cases to encourage population growth to
levels where they may exert sufficient levels of suppression to pest populations (i.e.

11
Where biotechnology has been used, engineering genes for Bt toxins into plants is an ingenious method of
delivering these toxins to pests by feeding which might naturally. These engineered crops are promising a high
level of vertical resistance although in most cases it is not necessarily desirable since it brings the risk of
resistance development by the pest, which in contrary to other forms of resistance like horizontal, which is
built on the quantitative effect of many genes, can be effective and sustainable [109] [116] [117]. Thus from
an IPM perspective, this technology has more similarities to plant resistance breeding than biopesticide
development and therefore is not covered in details in this chapter.
12
Insect-resistant transgenic (Bt) cotton, maize and potato were commercialized in the USA on 1996 [71].
70 Petros T. Damos

cultivation measures presented in Table 1 section 3.1). The approach also involves
the application of successfully developed techniques such as the development of
plant barriers and flowering plant in field margins to provide for source for adult
parasitoids or predators.

Table 3. Variety of predatory and parasitic insects, spiders and diseases

that have been referred to attack Helicoverpa at different
stages of its life cycle

Natural Enemy
Type Common name Scientific names Host
Predators Shield bugs Dictyotus caenosus Larvae
Oechalia schellenbergii
Predatory Ladybird beetles Coccinella transversalis Eggs and small
beetles Coccinella semptenpuncata larvae
Micraspis frenata
Harmonia octomaculata
Diomus notescens
Stethorus spp
Harmonia conformis
Coelophora inaequalis,
Hippodamia variegata
Carab beetles Calosoma schayeri Larvae
Agrypnus spp.
Spiders Flower spiders or family Thomisidae Several stages
crab spiders
Wolf spiders Genus Lycoa
Nightstalking Cheiracanthium spp.
spiders
Orbweavers Genera Araneus and Agriope Families
Araneidae and Tetragnathidae
Tangle web spiders Achaeranea veruculata
Jumping spiders - Family: Salticidae
Lynx spiders - Oxyopes spp.
Lacewings Family: Chrysopidae Eggs small larvae
Parasitoids Minute wasps Trichogramma spp., Eggs
Telenomus spp.
Braconidae wasps Microplitis demolitor Larvae
Cotesia spp.
Large parasitoid Netelia, Heteropelma, Ichneumon Larvae
wasps
Flyes Carcelia spp., Goniophthalmus spp., Caterpilars
Argyrophylax proclinata, Chaetophthalmus
spp., Exorista spp., Winthemia spp.
Pathogens Nomuraea rileyi, Beauveria bassiana, Several stages
Metarhizium anisopliae, Entomopthorales,
Ascovirus
(transmited by micropolitis)
Bacteria Bacillus thuringiensis var. kurstaki Larvae
Viral Nucleopolyhedrovirus Larvae
deseases
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 71

Table 3 is an example which presented some currently observed natural enemies of H.

armigera (mostly recorded in southern hemisphere) [14]. Some of the species presented, such
as many predators are opportunity feeders that also feed on prey other than Helicoverpa
armigera. Most predators found commonly in crops will not feed on helicoverpa at all and
some may only feed on certain stages (for example, larvae of a particular size, or only eggs).
Additionally, many of these and/or taxonomic related arthropods are known as native natural
enemies observed in annual crops and express polyphagus habits throughout the world.
Development of management methods for preserving functional biodiversity of native natural
enemies is major task in IPM.

3.4. Target-specificity and Minimization of Pesticides Side Effects

The use of several pesticides and the relate target specificity, is an important constrain of
IPM establishment and improvement. In the European Union (EU) the evaluation of side
effects of pesticides on terrestrial non-target arthropods for the registration of plant protection
products are referred to the first SETA/ESCORT13 guidance document on regulatory testing
procedures for pesticides with non-target organisms [15]. These assessment schemes where
lunched in the view to provide a simplified and better tuned tiered testing and assessment
procedures which provide realistic decision criteria and trigger values for both in field and off
field risk assessments [29].
The length of the available classes and insecticides compounds encouraged to be used in
IPM varies greatly and is to certain degrees subjectively defined depending on the number of
compounds involved, their chemical structures and related modes of action, as well as the
amount of available information upon side effect and persistence to non-target organisms and
environment.
Recently novel bio rational compounds are used in IPM including systemic insecticides,
insect growth regulators and products containing Bacillus thuringiensis or its components
which are referred as bio-pesticides. Table 4 for instance presents several classes of
insecticides that have been traditionally used in industrial crops pest management as well as
novel compounds. The list includes a number of new insecticides in new chemical classes
which have been recently become available, or will likely become available, in the near
future. Additionally, their compatibility classification to IPM programs has been made based
on their biorational nature and target specificity.
The term biorationality corresponds to a compound which express relative toxicity to
non-target organism compared to other classes and do not defines a particular category having
specific chemical properties or mode of action [34]. Traditionally the term referred to
soaps/detergents, oils and botanicals but now includes, systemic insecticides, insect growth
regulators and products containing B. thuringiensis and other. Additionally, the biorationality
nature of a compound depends not only by its mode of action but also the application mode.
The nicotinoids for instance are a relatively new class of compounds (although imidacloprid
has been available for use on tomatoes since 1994) and are in several cases compatible in IPM
programs because of their highly systemic action.

13
Society of Environmental Toxicology and Chemistry-Europe/European Standard Characteristics of Beneficial
Regulatory Testing. Updated by the commission directive 96/12/EC.
72 Petros T. Damos

Table 4. List of representative chemical compounds used in crop protection

and related classification according to their mode of action

Insecticides classification and related compatibility to IPM

Mode of Chemical group Chemical Compounds IPM
Action Configuration Compatibility
Acetylcholine Carbamates aldicarb, Very low
sterase carbaryl,
(AChE) carbuforan,
inhibitors carbusulfan,
methomyl,
oxamyl,
pyrimicarb,
propoxur
Organophosphates acephate, Very low
azynphos
methyl,
chloropyriphos,
diazinon,
Parathion,
Dichlorvos
GABA-gated Cyclodienes and aldrin, dieldrin, Very low
chloride other organo- chlordane,
channel chlorines (OCs) Endosulfan
antagonists

Phenylpyrazoles Fibronil Very low

(Fiproles)

Sodium Pyrethrum and pyrethrum, Extremely low

Chanel Pyrethrins resmethrin,
modulators bioallethrin,
tetramethrin

Pyrethroids cypermethrin, Extremely low

pertmethrin
deltamethrin,
etofenprox,
ensfelerate,
fenvalerate
Fluvalinate
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 73

Insecticides classification and related compatibility to IPM

Mode of Chemical group Chemical Compounds IPM
Action Configuration Compatibility
Acetylcholine Nicotine Nicotine
receptors
(nAChR)
agonists
Neonicotinoids imidacloprid, Mediate
acetamiprid, (e.g. depending
clothianidin, upon time and
nitenpyram, application
thiacloprid, mode)
thiamethoxam
nAChR Macrocyclic Spinosad mediate - high
alosteric lactones
agonists

Chloride Avemectins, abamectin, mediate - high

channel Milbemectins emamectin
activators benzoate,

Milbemectins Milbemectin mediate - high

nAChR Nereistoxin cartap, high

channel analogues thiosultap,
blockers (Lumbriconereis thiocyclam
heteropoda)
Voltage Oxadiazine indoxacarb high
dependent (Steward)
sodium
channel
blockers
74 Petros T. Damos

Table 4. (Continued)

Insecticides classification and related compatibility to IPM

Mode of Chemical group Chemical Compounds IPM
Action Configuration Compatibility
Selective Anthranilic rynaxapir, high
activator of diamides Coragen
insect (novel
ryanodine compound)
receptors
Microbial or Bacillus B. thuringiensis Very high
derived – thuringiensis subsp. aizawai
membrane (B.t. spays or Cry (against Lep.:
disruptors of proteins14) Noctuidae)
insect mitgut B. thuringiensis
subsp. hurstaki
(against Lep. e.g.
[Link])

Other Entomopathogenic Beauveria Very high

microbial fungi bassiana,
Metharhizum
anisopliae,
Paecilomyces
fumusoroseus
Nematodes Heterorhabditis Very high
megidis,
Steinernema spp.
Nuclear Poly- e.g. Helicoverpa against Very high
hedrosis viruses nucleopolyherdovirus (NPV) Lepidoptera
and
Granuloviruses
Insect Growth Juvenile hormone Fenoxycarb, Very high
Regulators mimics Methoprene,
(IGRs) Pyriproxyfen

Inhibitors of Diflubenzuron, high

chitin Bistrifluron,
biosynthesis Flucloxuron,
(benzoylureas) Flufenoxuron,
novaluron
(against
lepidoptera),
buprofezin
(against
Hemiptera),
cyromazine
(against
Diptera)

14
d-entotoxin 3D structure - source: [Link]
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 75

Insecticides classification and related compatibility to IPM

Mode of Chemical group Chemical Compounds IPM
Action Configuration Compatibility
Ecdysone agonist methoxyfenozide high
– (against Lep.),
diacylhydrazines tebufenozide
(against Lep.),
chromafenozide
molting Azadirachtin high
disruptors

Mitochondrial rotenone high

electron
transport chain
inhibitors
(METI)

phenoxypyrazole phenpyromate high

(acaricide and
some insects)

The given chemical configurations are constructed according to the IUPAC nomenclature and
correspond to chemical groups or compound given in bold scripts, while IPM compatibility
classification is made according to target specificity and biorational nature of the compounds.

Neonicotinoids actually represent the most effective chemical class for the control of
sucking insects such as aphids, whiteflies, leafhoppers, and planthoppers, as well as thrips,
some microlepidoptera, and a number of coleopteran insect species [41]. When applied to the
roots they further distributed through the plant and are translaminar applied to new growth
and thereby can express toxicity only to plant feeding organism. Moreover, the impact of soil
applications (e.g. seed coating) on natural enemies would be expected to be less than that of
foliar applications because most natural enemies would not be exposed directly to the
compounds. Seed coating for instance is commonly used technique in industrial crop models
to protect seedlings at the early stages of development.
Note that the IPM compatibility in Table 4 is based on the target specificity and
biorational nature. Unfortunately, in most cases very little or nothing is known about pest
target specificity and the relative toxicity of several compounds of biorational nature to
natural enemies of interest. Thus for insect growth regulators, although they are characterized
by high specificity and tebufenozide, methoxyfenozide and novaluron would be expected to
have minimal impact on natural enemies; this has not been demonstrated. Additionally,
although most of these compounds are considered as nontoxic to honeybees they are
considered moderately to highly toxic to fish, highly toxic to the aquatic invertebrate and
76 Petros T. Damos

affect growth and reproduction after chronic exposures. On the other hand although novel
compounds such as spinosad shows low toxicity when ingested by mammals and no adverse
chronic exposure effects, it is highly toxic to bees, oyster and other marine molluscs and
Trichogrammatidae and Braconidae species as well.
For additional information upon the compatability of biorational pecticides with IPM
refer to [35] and references given.

4. CONCLUSIONS AND FUTURE PERSPECTIVES OF IPM

There is increasing evidence that agricultural industrialization will keep going on the
developing and developed countries to support current demand for food, fibre, fuel and other
products for human consumption and processing. On the other hand, there are concerns that
warn the growing push toward extensive monocultures cultivations which heavy relies on
agrochemicals are negatively impacting ecosystem integrity, public health, food quality and
nourishment, traditional rural livelihoods [4] [5].
With the growing interest in sustaining agriculture, IPM is expected to play a prominent
role in sustainable agriculture as part of Integrated Crop Production systems (ICP) [122].
Based on the same principles as IPM, the ICP system is treated more as special case of agro-
ecosystem in which plant – animal communities interacting with their physical and chemical
environments that have been specially modified to sustain crops. By following an agro
ecological perspective the idea is that by understanding relationships and processes the
system can be manipulated to improve production and to produce more sustainably, with
fewer negative environmental or social impacts and fewer external inputs [4] [6] [77].
IPM arose as response to concerns about impacts15 of pesticides on the early 60s,
environment by providing alternative strategy to conventional agriculture. However, although
that IPM, as broader strategy, uses a deeper understanding of the insect-crop system in which
all available control tactics and measures are integrated on a complementary manner, relayed
upon the use of pesticides for many years. This could be explained, to some extent, by the fact
that the available solutions for successfully management of pest and outbreaks were quite
limited and also due to lack of detailed knowledge upon pest-crop systems interactions.
Nevertheless after the first introduction of IPM it shifted the emphasis in pest control from a
single tactic (chemically based) to a multitactic agro-ecologically based. Thus IPM promotes
a more diversified approach which will limit overreliance on any specific technology and the
consequences of this, such as resistance development. Ideally, IPM promotes greater reliance
on exploiting living, self-renewing processes in pest control, such as the action of natural
enemies of pests.
Figure 14 is a graphic representation of a biointensive IPM strategy to manage cotton
key-pest threats (i.e. H. armigera). It conceptualizes in particular the foundation of integrated
pest management and illustrates some of the positive interactions with other pest management
actions. The contribution of each action is represented as a proportion of the triangle. Cultural
measures include most of the techniques presented in table 1 (section 3.1) and provide a
balanced environment for biological mediated soil fertility and sustainable yields.

15
Rachel Carson outlined the impact of the misuse and overuse of pesticides in the environment on book Silent
Spring.
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 77

Additionally, the use of semiochemical and pheromones in particular enhance natural pest
regulation and further low-energy input (sections 3.2 and 3.3). Since IPM combines several
practices to manage pests, such as tillage, rotation, and variety selection, field scouting and
specific decision tools it promotes greater reliance on exploiting living, self-renewing
processes in pest control and reduce pesticide dependency. Furthermore, once the decision
tools have been developed (i.e. adult phenology models, thresholds of immature sages,
section 2.1) they can be used to define the critical time points in which bio-rational
insecticides and/or biopecticides should be applied (section 3.4).

Figure 14. Conceptualized foundation of integrated pest management in industrial crop models.
Triangle represents a biointensive IPM system in which the contribution of each control tactic is
represented as a proportion of the area of the triangle sector. IPM promotes a more diversified approach
which will limit overreliance on any specific technology and the consequences of this, such as
resistance development. It promotes greater reliance on exploiting living, self-renewing processes in
pest control, such as the action of natural enemies of pests. The available management options are
selected according to the principles of IPM and are progressively integrated. Note that chemical
measures are on the top of the triangle and used only under specific criteria (i.e. DD: degree-days, ET:
Economic Threshold).

At this point, and for a successfully long established IPM strategy, the basic criterion
(e.g. model or/and population threshold) should be taken in to account in taking chemical
measures, including non-extensive use of pesticides and rotation of different products during
the growth season. To avoid resistance the application of products expressing different modes
of action must encouraged. Rotation, for instance, of Bt and IGR’s with other compounds
such as emamectin and dibenzoylhydrazine IGRs can decrease the possibility of resistance
development. Additionally, most of these compounds are effective when applied only on
particular developmental stages of the target pest. Phenology models are therefore once again
78 Petros T. Damos

an essential part of an IPM program and forecasting models provide manners to introduce
control methods at the time in which they can express their maximal activity on the target pest
and the lowest side effects to beneficial species.
One other important issue in IPM is related to the particular legislation frame that
determines the production, registration of ingredients and final availability of insecticides that
have permission to be used only for certain pests and crops. Moreover, since the status of
pesticide approvals in the EU is continually changing the registration of newer products, such
as Biorational and Bio insecticides , the use of alternative control methods are extremely
important for realistic implementation of IPM [33] [34].
The framework in which these products can be used in IPM programs is also rapidly
changing [66]. At the end of November 2009 for instance, the European Commission
officially adopted and published the Regulation concerning the placing of plant protection
products on the market, the directive establishing a framework for Community action for the
sustainable use of pesticides [34] [45]. Another part, of the directive on the sustainable use of
pesticides, emphasizes the importance of IPM, which will have to be encouraged by Member
States as an alternative to the use of pesticides. Table 5 presents the major key points of the
EU directive concerning the sustainable use of pesticides and how they should be translated
by the member states into National Action Plans [34] [45].
In addition, a new legislation is also being passed on the collection of statistics on
pesticides and an amendment is being made to the directive dealing with machinery for
pesticide application. The new legislation will only gradually supersede existing EU law and
available pesticides which can be placed on the market under current legislation will remain
available until their existing authorization expires.

Table 5. The key points of the Directive on the sustainable use of pesticides (to be
translated into National Action Plans by Member States by December 2012)

ENDURE16's position on European pesticide policy

1. ‟The principle of integrated pest management (IPM) is laid down, i.e. the promotion of non-
chemical pest control methods such as crop rotation, to be used wherever possible as alternatives
to pesticides‟.
2. ‟Aerial crop spraying will in general be banned, albeit with exceptions subject to approval by
the authorities. No spraying will be allowed in close proximity to residential areas‟.
3. ‟Member States must take appropriate measures to protect the aquatic environment and drinking
water supplies from the impact of pesticides. These are to include buffer zones around bodies of
water and safeguard zones for any surface and groundwater used for drinking water‟
4. ‟The use of pesticides must be minimised or prohibited in specific areas used by the general
public or by vulnerable groups, such as parks, schools, sports grounds and close to hospitals‟.
5. ‟New rules are being introduced on the training of pesticide users and salespeople, on handling
and storage, on information and awareness-raising and on the inspection of pesticide application
equipment‟.

16
European Network for the Durable Exploitation of Crop Protection Strategies, [Link]
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 79

Obviously the IPM systems will, of necessity, be more complex than the conventional
pesticide based system approachof the past, and will require greater effort to be adopted on a
regular basis. Additionally there remains a need for ongoing research to develop a suite of
management tactics and progressively incorporate them them into IPM systems to meet
shortcoming future demands. In this context it is commonly accepted that traditional insect
management will change in the forthcoming years as a result of higher environmental
concerns and pressures exhibited by all parts, including not only consumers and growers but
also political authorities, industry and international organizations. The strict stance taken by
the EU on pesticide legislation means that there must be parallel, sustained and equally
determined action to promote the design and implementation of new solutions [42]. The aim
is to develop integrated pest management schemes that contribute to sustainable development
in order to preserve the competitiveness of European agriculture. Hence, the development of
alternatives on a realistic basis that will reduce our dependency on conventional
agrochemicals and traditional pest management in industrial crop models is essential to
permit IPM to be feasible on a real basis.

REFERENCES
[1] Abd El-Aziz, M. F., 2011. Bioactivities and Biochemical Effects of Marjoram Essential
Oil used against Potato Tuber Moth Phthorimaea operculella Zeller (Lepidoptera:
Gelechiidae). Life Science Journal, 8, 288-297.
[2] Allen, J., C., 1976.A modified sine wave method for calculating degree- days,”
Environmental Entomology, 5, 388–396, 1976.
[3] Albajes, R., M. Konstantopoulou, O., Etchepare, M. et al., 2002. Mating disruption of
the corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae) using sprayable
formulations of pheromone. Crop Protection, 21, 217-225.
[4] Altieri, M., A., 1987, Agroecology: the scientific basis of alternative agriculture.
Westview Press, New York, 185pp.
[5] Altieri, M. A., Nicholls, C. I. 2000. Agroecology in action. Indigenous and modern
approaches to IPM in Latin America. ESPM Division of Insect Biology, University of
California, Berkeley, USA. [Link]
modern_approaches.html
[6] Altieri, M.A., C. I. Nicholls. 2005. Agroecology and the Search for a Truly Sustainable
Agriculture. United Nations Environment Programme Environmental Training Network
for Latin America and the Caribbean, ISBN 968-7913-35-5.
[7] Amano, H., 2001. Species structure and abundance of invertebrate natural enemies in
sustainable Agroecosystems. In: Structure and Function in Agroecosystem Design and
Management, Shiyomi M. andH. Koizumi (Eds.). CRC Press, New York, U.S.A., pp.
167-182.
[8] Anglade, P., 1972. “Les sesamie,” in Entomologie Appliqu´ee `a l’Agriculture. Tomme
II Lepidopt`eres, A. S. Balachoswsky, Ed., vol. 2, Masson, Paris, France.
[9] Archer, T. L., G. L. Musick, R. L. Murray, 1980. Influence of temperature and moisture
on black cutworm (Lepidoptera: Noctuidae) development. Can. Entomol. 112:665–673.
80 Petros T. Damos

[10] Arias, A., C. Alvez, 1973. “Ciclos biológicos de los taladros del maız (Sesamia
nonagrioides Lef. y Pyrausta nubilalis Hb.) durante 1971 en las Vegas del Guadiana
(Badajoz), Boletin Informativo Plagas, 103, 9–87.
[11] Arn, H.,M. Toth, E Presner (2000). List of sex pheromones Lepidoptera and related
male attractants, Internet Edition: [Link]
[12] Arnold, C. Y., 1959. “The determination and significance of the base temperature in a
linear heat unit system,” Proceedings of the American Society of Horticultural Science,
vol. 74, 430– 445.
[13] Balachosky, A.S., 1972. Entomologie appliquée à l'agriculture: Lépidoptères. 2 v,
Masson et cie.
[14] Bailey P.T., [Link] of field crops and pastures: identification and control.
Collingwood, Vic. CSIRO Publishing,
[15] Barrett, K.L., Granfy, N., Harrison, E.G., Hassan, S. and Oomen, P. (eds.), 1994:
Guidance document on regulatory testing procedures for pecticides with non-target
arthropods. SETAC Europe, Brussels.
[16] Barlett, B. R., 1956. Natural predators: Can selective insecticides help to preserve biotic
control? Agric. Chem. 11, 42–44.
[17] Binns, M.R., Nyrop, J.P. and van der Werf, W. (2000) Sampling and monitoring in crop
protection. The theoretical basis for developing practical decision guides. 284 pp.
Wallingford, Oxon, CAB International.
[18] Blair R.,W, 1976. Comparison of the development of Agrotis ipsilon Hufn. and Agrotis
segetum Denisand Schiff. (Lepidoptera: Noctuidae) at constant temperatures. Journal of
the Entomological Society of Southern Africa 39, 27 1-277.
[19] Blaise, D., 2006. Yield, Boll Distribution and Fibre Quality of Hybrit Cotton
(Gossypium hirsutum L.) as influenced by organic and modern methods of cultivation.
Journal of Agronomy and Crop Science, 192, 248-256.
[20] Bressani, R., 1972. The importance of maize for human nutrition in Latin America and
other countries. In Nutritional improvement of maize. INCAP publication L-4, p. 5-29.
Guatemala, INCAP.
[21] Boller, E. F., J. Avilla, E. Jörg, C. Malavolta, F. Wijnands, P. Esbjerg, 2004. Integrated
Production: Principles and Technical Guidelines, 3rd edition 2004. [Link]
iobc_bas.pdf.
[22] Bowden, J., [Link], B.J. Emmet, T.E. Minall, P. L. Sherlock. 1983. A survey of
cutworm attacks in England and Wales, and a descriptive population model for Agrotis
segetum (Lepidoptera:Noctuidae). Ann. appl. Biol.,102, 29-47.
[23] Buntin G. D. 1996. Economic Thresholds for Insect Management. In: Economic
thresholds for integrated pest management. Edited by L. G. Higley and L. P. Pedigo.
University of Nebraska Press. Lincoln and London, pp. 128-147.
[24] Busching, M. K., F. T. Turpin. 1976. Oviposition preferences of black cutworm moth
among various crop plants, weeds, and plant debris. J. Econ. Entomol., 69, 587–590.
[25] Busching, M. K. and F. T. Turpin, 1977. Survival and development of black cutworm
(Agrotis ipsilon) larvae on various species of crop plants and weeds. Environ. Entomol.
6, 63–65.
[26] Butenandt ,A., R. Beckmann, D. Stamm, E. Hecker, 1959. Über den Sexuallockstoff
den Seidenspinners Bombyx mori. Reindarstellung und Konstitution. Z. Naturforsch
14b: 283-284.
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 81

[27] Cardé R.T., A.K. Minks,1995. Control of moth pests by mating disruption. Annu. Rev.
Entomol., 40, 559–585.
[28] Chamberlain, D. J, N.J. Brown, [Link], E. Casagrande, 2000. Field evaluation of a
slow release pheromone formulation to control the American bollworm, Helicoverpa
armigera (Lepidoptera: Noctuidae) in Pakistan. Bull. Entomol. Res.,90,183-90.
[29] Candolfi, M. P., S. Blümel, R. Forster et al. 2000. Guidelines to evaluate side-effects of
plant protection products to non target arhtropodes, IOBC, BART and EPPO Joint
Initiative.
[30] Clement, S. L., D. A. McCartney, 1982. Black cutworm (Lepidoptera: Noctuidae):
Measurement of larval feeding parameters on field corn in the greenhouse. J. Econ.
Entomol. 75,1005-1008.
[31] Cross, J. V., E. Dickler, 1994. Guidelines for Integrated Production of pome fruits in
Europe: IOBC Technical Guideline III. IOBC/wprs Bulletin 17, 1–8. International
Organization for Biological and Integrated Control of Noxious Animals and Plants,
Montfavet, France.
[32] Cunningham J.P, Zalucki M.P., S.A. West, 1999. Learning in Helicoverpa armigera
(Lepidoptera: Noctuidae): a new look at the behaviour and control of a polyphagous
pest. Bulletin of Entomological Research, 89, 201–207.
[33] Damos, P. and Savopoulou-Soultani, M. (2010a). Population dynamics of Anarsia
lineatella (Lep:Gelechiidae) in relation to crop damage and development of Economic
Injury Levels. J. Appl. Entomol., 134, 105-115.
[34] Damos P.T., M. Savopoulou-Soultani, (2010b). Development and statistical evaluation
of models in forecasting moth phenology of major lepidopterous peach pest complex
for Integrated Pest Management programs, Crop Protection, 29, 1190–1199.
[35] Damos P. and M. Savopoulou-Soultani, (2011a). “Microlepidoptera of Economic
Significance in Fruit Production: Challenges, Constrains and future Perspectives for
integrated pest management,” in Moths: Types, Ecological Significance and Control
Methods, Nova Science, NY.
[36] Damos, P. and M. Savopoulou-Soultani (2011b). Temperature-driven models for insect
development and vital thermal requirements, Psyche, Vol. 2012, 1-13.
[37] Damos P. and S. Karabatakis (2012). Real time pest modeling through the World Wide
Web: decision making from theory to praxis, International Congress in Integrated Crop
Production, Izmyr, 7-12 October, 2012. Submitted IOBC/wprs.
[38] Dent, D. 1994. Integrated Pest Management. Chapman and Hall, NY.
[39] Duffy, M.D. 1996. Alternative approaches in decision making. In: Economic thresholds
for integrated pest management. Edited by L. G. Higley and L. P. Pedigo. University of
Nebraska Press. Lincoln and London, pp. 74-85.
[40] Elbersen W., L. Oyen. 2009. Nieuwe Grondstoffen voor Biobrandstoffen. Alternatieve
1e Generatie Energiegewassen. Senternovem number: Report GAVE-09-01 AFSG
number: 1075, ISBN-number: 978-90-8585-563-7.
[41] Elbert, A., M. Haas, B. Springer, W. Thielert, and R. Nauen.2008. Applied aspects of
neonicotinoid uses in crop protection. Pest Manag. Sci., 64, 1099-1110.
[42] Endure 2008. ENDURE's position on European pesticide policy [pdf - 43.96 kB].
[43] Ehler. L. E., 2006. Perspective Integrated pest management (IPM): definition, historical
development and implementation, and the other IPM. Pest Manag. Sci., 62,787–789.
82 Petros T. Damos

[44] Eizaguirre, M., A. A. Fantinou, 2012. Abundance of Sesamia nonagrioides (Lef.)

(Lepidoptera: Noctuidae) on the Edges of the Mediterranean Basin. Psyche, Vol. 2012,
1-7.
[45] EU parliament, 2009. Directive 2009 128 EC framework sustainable use of pesticides
[pdf - 806.06 kB].
[46] FAO, 1992. Maize in Human Nutrition. Food and Agriculture Organisation of the
United Nations, available online at [Link]
Contents Rome.
[47] FAO. 2009. "International Year of the Potato 2008 – The potato".
[48] FAOSTAT, 1999. Available online at: [Link]
[49] Farringtion, J. 1977. Economic threshold of insect pest population in present
agriculture: A question of applicability, Pest Articles and News Summaries, 23,143-
148.
[50] Fitt, G.P., 1989. The ecology of Heliothis in relation to agroecosystems. Annual Review
of Entomology, 34, 17–52.
[51] Fitt, G. P. 2000. An Australian approach to IPM in Cotton: integrating new
technologies to minimize insecticide dependence. Crop. Prot. 19, 793–800.
[52] Flint, M. L., van den Bosch, R.,1981. Introduction to Integrated Pest Management,
Plenum, New York.
[53] Flint, M., 1986. Integrated pest management for potatoes in the western United States.
Publication, 3316. University of California. pp. 1–146.
[54] Firempong, S., Zalucki, M.P., 1990. Host plant preferences of populations of
Helicoverpa armigera (Hübner) (Lepidoptera : Noctuidae) from different geographic
locations. Australian Journal of Zoology, 37, 665–673.
[55] Farnham, D. E., G. O., Benson, R. B. Pearce, 2003. Corn perspective and culture.
Chapter 1. In: P.J. White, L.A. Johnson, eds. Corn: chemistry and technology, 2nd Ed.
American Association of cereal Chemicals, Inc. St. Paul, Minnesota, USA, pp. 1-33.
[56] Frerot, B., Malosse, C., Cain, A.H. 1997. Solid-phase micro extraction (SPME): a new
tool in pheromone identification in Lepidoptera. J. High Res. Chromatogr. 20,340-342.
[57] Gemeno, C., A.F., Lutfallah, Haynes K.F., 2000. Pheromone blend variation and cross-
attraction among populations of the black cutworm moth (Lepidoptera: Noctuidae).
Ann. Entomol. Soc. Am. 93, 1322-1328.
[58] Hammond R. B. 1996. Limitations to EILs and Thresholds. In: Economic thresholds for
integrated pest management. Edited by L. G. Higley and L. P. Pedigo. University of
Nebraska Press. Lincoln and London.
[59] Hassan R. M. Corbett J. D. Njoroge K. 1998. Maize Technology Development and
Transfer: A GIS Application for research Planning in Kenya, p. 43-68. CAB
International, Wallington, Oxon, UK.
[60] Hayami, Y., Otsuka, K., 1994. Beyond the Green Revolution: agricultural development
strategy into the new century, cited in Estudillo, J. [Link] Otsuka, K., 1999. Green
Revolution, human capital, and off-farm employment: changing sources of income
among farm households in central Luzon, 1966-1994., Economic Development and
Cultural Change, 47, 498- 523.
[61] Herzog, D. C., J. E. Funderburk 1986. Ecological bases for habitat management ad pest
cultural control. In: Kogan, M. (ed.) Ecological Theory and Integrated Pest
Management Practice. John Wiley and Sons, New York, pp. 217-250.
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 83

[62] Heuskin, S., F. J. Verheggen, E. Haubruge, J. P. Wathelet, G. Lognay. 2011. The use of
semiochemical slow-release devices in integrated pest management strategies.
Biotechnol. Agron. Soc. Environ., 15, 459-470.
[63] Higley, L., L. Pedigo, K. Ostlie, 1986. Degday: a program for calculating degree-days
and assumptions behind the degree-day approach. Environ. Entomol. 85, 2218-2227.
[64] Hijmans, R.J.; D. M. Spooner, 2001. Geographical distribution of wild potato. Amer.
Journ. Botan., 88, 2101–2112.
[65] Inscoe, M. N., B. A. Leonhardt, R. L. Ridgwey, 1990. Commercial availability of insect
sex pheromones and other attractants, In: Behaviour-Modifying Chemicals for Insect
Management, (eds R. L. Ridgway, R. M. Silverstein and M. N. Inscoe), Narcel Dekker
Inc., New York, pp. 631-715.
[66] IPM Europe, 2000. For the harmonisation of European support to developing countries
in the use of IPM to improve agricultural sustainability.
[67] Ifoulis A, Savopoulou-Soultani M. 2006. Developing the optimum sample size and
multistage sampling plans for Lobesia botrana (Lepidoptera: Tortricidae) larval
infestation and injury in northern Greece. J. Econ. Entomol. 99, 1890–1898.
[68] Ifoulis A., Savopoulou-Soultani M., 2007. Probability distribution, sampling unit, data
transformation and sequential sampling of European vine moth Lobesia botrana
(Lepidoptera: Tortricidae) larval counts from Northern Greece vineyards. Eur. J.
Entomol. 104, 753–761.
[69] Iwao, S., E. Kuno, 1968. Use of the regression of mean crowding on mean density for
estimating sample size and the transformation of data for the analysis of variance. Res.
Popul. Ecol. 10, 210-214.
[70] Iwao, S., E. Kuno,1971. An approach to the analysis of aggregation pattern in
biological populations. pp. 461-513 in Statistical ecology, Patil, G. P., Pielou, E. C. and
Waters, W. E. (Eds.).Vol. [Link] Park, Penn. St. Univ. Press.
[71] Jenkins, J. N., 2003. Cotton. In: Traditional crop breeding practices: an historical
review to serve as a baseline for assessing the role of modern biotechnology. OECD,
pp. 61-70.
[72] Kassam, A., L. NeBambi, T. Friedrich, E. Kueneman, M. Salvatore, M. Bloise, J.
Tschirley, 2012. Natural Resource Assessment for Crop and Land Suitability: An
application for selected bioenergy crops in Southern Africa region, Integrated Crop
Management, Vol.14-2012, ISSN 1020-4555.
[73] Kravchenko V. D., G. Müller, 2008. “Seasonal and spatial distribution of noctuid moths
(Lepidoptera: Noctuidae) in the northern and central Arava Valley, Israel,” Israel
Journal of Entomology, 38, 19–34.
[74] Koul, O., G. S. Dhaliwal, G. W. Cuperus, 2004. Integrated Pest Management.
Potential, Constrains and Challenges. CABI Publishing.
[75] Krokos, F. D., Ameline, A., Bau, J. et al. 2002. Comparative studies of female sex
pheromone components and male response of the corn stalk borer Sesamia
nonagrioides in three different populations. J. Chem. Ecol. 28, 1463-1472.
[76] Lange H. W. 1987. Insect pests of sugar beet. Ann. Rev. Entomol. 32, 341-60.
[77] Lewis, W. J., J. C. van Lentern, S.C. Phatak, J. H. Tumlinson, 1997. A total system
approach to sustainable pest management. Proc. Natl. Acad. Sci. USA, 94, 12243–
12248.
84 Petros T. Damos

[78] López C., A. Sans, L. Asin, and M. Eizaguirre, 2001. Phenological model for Sesamia
nonagrioides (Lepidoptera. Noctuidae), Environmental Entomology, 30, 23–30.
[79] Mazomenos, B.E. 1989. Sex pheromone components of corn stalk borer Sesamia
nonagriodes (Lef.). Isolation, identification, and field tests. J. Chem. Ecol. 15,1241-
1247.
[80] McHardy, W. E., 2000. Current Status of IPM in Apple orchards. Crop Protection, 19,
801-806.
[81] Moyal, P., P. Tokro, A. Bayram et al., 2011. “Origin and taxonomic status of the
Palearctic population of the stem borer Sesamia nonagrioides (Lefèbvre) (Lepidoptera:
Noctuidae), Biological Journal of the Linnean Society, 103, 904–922.
[82] Mumford, J. D., G. A. Norton. 1984. Economics of decision making in pest
management. Annu. Rev. Entomol., 29, 157-174.
[83] Nesbitt, B.F., Beevor, P.S., Hall, D. R., and Lester, R. 1979. Female sex pheromone
components of the cotton bollworm, Heliothis armigera. J. Insect Physiol., 25,535-541.
[84] Norris, R. F., Caswell-Chenn, E. P., M. Kogan, 2003. Concepts of Integrated Pest
Management. Prentice Hall, Upper Saddle River, New Jersey, 586pp.
[85] Norton, G. A., J. D. Mumford (ed.). 1993. Decision tools for pest management. CAB
International, Oxford UK.
[86] Ono, T., R.E Charlton, R.T. Cardé, 1990. Variability in pheromone composition and
periodicity of pheromone titer in potato tuberworm moth, Phthorimaea operculella
(Lepidoptera: Gelechiidae). J. Chem. Ecol., 16,531-542.
[87] Ono, T., P. Chouvalitwongporn, T. Saito, 1997. Comparison of the sex pheromone
system between Japanese and Thai populations of the potato tuberworm moth,
Phthorimaea operculella (Lepidoptera: Gelechiidae). Appl. Entomol. Zool., 32, 514-
517.
[88] Paliwal, R.L., 2000. Introduction to maize and its importance. Chapter 1. In: Tropical
Maize: Improvement and Production. Food and Agriculture Organization of the United
Nations Rome. pp. 105-144.
[89] Pedigo L. P. and L. G. Higley, 1986. Introduction to Pest Management and Thresholds.
In: Economic thresholds for integrated pest management. Edited by L. G. Higley and L.
P. Pedigo. University of Nebraska Press. Lincoln and London, pp. 128-147.
[90] Pedigo, L. P., S. H. Hutchins, L. G. Higley. 1992. The economic Injury Levels in theory
and practice. Annu. Rev. Entomol., 31, 12-21.
[91] Pedigo, L. P. and Buntin, G. D. 1994. Handbook of sampling methods for arthropods in
Agriculture. CRC press, Boca Raton, Florida.
[92] Poston, F. L., L. P. Pedigo, S. M. Welch, 1983. Economic Injury Levels: Reality and
Practicality. Bull. Entomol. Soc. Am., 29, 49-53.
[93] Piccardi, P., Capizzi, A., Cassani, et al. 1977. A sex pheromone component of the old
world bollworm Heliothis armigera. J. Insect Physiol. 23:1443-1445.
[94] Radcliffe, E.B., 1982. Insect pest potato. Ann. [Link]., 27, 173–204.
[95] Rings, R. W., F. J. Arnold, and B. A. Johnson. 1975. Host range of the black cutworm
on vegetables: a bibliography. Bull. Entomol. Soc. Am., 21, 229-234.
[96] Rondon S. I., S. J. DeBano, G. H. Clough, et al. 2007. Biology and Management of the
potato tuberworm in the pacific Nord west. PNW 594 April 2007, 1-8.
 Current Issues in Integrated Pest Management of Lepidoptera Pest Threats … 85

[97] Schmitt, C. J. 1998. Environnemental Contaminants. Pages 131-165 in Mac MJ, Opler
PA, Puckett Haecker CE, Doran PD. Status and trends of the nation's biological
resources. U.S. Department of the Interior, U.S. Geological Survey, Reston, Va.
[98] Silvie P., J.P. Deguine, S. Nibouche, B. Michel, M. Vaissayre, 2001. Potential of
threshold-based interventions for cotton pest control by small farmers in West Africa,
Crop Protection, 20, 297-301.
[99] Southwood, T. R. E., 1978. Ecological methods with particular reference to the study of
insect populations. 2nd eds., 524 pp. London, Chapman and Hall.
[100] Speight, M. R., Hunter, M. D. and Watt, A.D. 1999. Ecology of Insects-Concepts and
Applications. Blackwell Science, London.
[101] Smith, W.C., 1991. Production statistics. Chapter 3.1. In: W.C. Smith, J.T. Cothern,
eds. Cotton: Origin, History, Technology and Production. John Wiley and Sons, Incs.,
pp. 435-449.
[102] Smith, C. W.,1995. Cotton (Gossypium hisrutum L.). Chapter 6. In: Crop Production:
Evolution, History, and Technology. John Wiley and Sons, Inc., New York,pp 287-349.
[103] Stern V. M., R. F. Smith, R. van den Bosch, K. S. Hagen, 1959a. The integration of
chemical and biological control of the spotted alfalfa aphid. The integrated control
concept. Hilgardia, 29, 81–101.
[104] Taylor, L. R., 1961. Aggregation, variance and the mean. Nature, Lond. 189,732-735.
[105] Taylror, L. R., 1963. Analysis of the effect of temperature on insect flight. Jour. Anim.
Ecolo., 32, 99-117.
[106] Tilman, D., 1979. Resources: a graphical –mechanistic approach to competition and
predation. The American Naturalist, 116, 362-393.
[107] Tilman, D., 1999. Global environmental impacts of agricultural expansion: the need for
sustainable and efficient practices. Proceedings of the National Academy of Sciences of
the United States of America, 96, 5995–6000.
[108] Tilman, D., Fargione, J., Wolff, B., et al. 2001. Forecasting agriculturally driven global
environmental change. Science, 292, 281–284.
[109] - -Molina, A. Lacasa-Plasencia, P. Bielza-Lino, 2002.
Crop Protection, 21, 1003–1013.
[110] Toyoshima, G., Kobayashi, S., and Yoshihama, T. 2001. Control of Helicoverpa
armigera (Hubner) by mating disruption using diamolure in lettuce fields. Jap. J. Appl.
Entomol. Zool., 45, 183-188.
[111] Thomas M. B., J. K. Waage, 1996. Integrating Biological Control and Host Plant
Resistance Breeding: A scientific and literature review. Wageningen: Technical Centre
for Agricultural and Rural Cooperation of the European Union (CTA), 99p.
[112] Tilman , D., 1989. Population dynamics and species interactions, in Perspectives in
Ecological Theory, (eds J. Roughgarten, R. M. May and S.A. Levin), Princeton
University Press, NY, pp. 89-117.
[113] Tumlinson, J. H., Lewis, W. J., Vet, L. E. M. 1993. How beneficial parasitic wasps find
plant feeding caterpillars. Sci. Am. 268, 100–106.
[114] Tsitsipis, J. A., 1990. “ Contribution toward the development of an integrated control
method for the corn stalk borer ,” in Pesticides and Alternatives, J. E. Casida, Ed., pp.
217–228,Academic Press, Amsterdam, The Netherlands, 1990.
[115] van den Bosch, R., Stern, V. M. 1962. The integration of chemical and biological
control in arthropod pests. Annu. Rev. Entomol. 7,367–387.
86 Petros T. Damos

[116] Waage, J. K., 1996. "Integrated Pest Management and Biotechnology: An analysis of
their potential for integration". In: G.J. Persley, (ed.). Biotechnology and Integrated
Pest Management. Oxon, UK: CAB International. pp. 3660.
[117] Waage J.K. 1997. "Biopesticides at the Crossroads: IPM products or chemical clones?"
In: Microbial Insecticides: Novelty or necessity? BCPC Proceedings No. 68.
Proceedings for a Symposium, Warwick, UK, 1618 April 1996. Bracknell: BCPC
Publications, pp. 1119.
[118] Wilson E.O., Bossert, W.H., 1963. Chemical communication among animals. Recent
Progr. Hormone Res., 19, 673-716.
[119] Willson, H. and Trammel, K. 1980. Sex pheromone trapping for control of codling
moth, Oriental fruit moth, lesser appleworm, and three totricid leafrollers in a New
York apple orchard. J. Econ. Entomol. 73, 291-295.
[120] Winch. T. 2006. Growing food: A guide to food production, Springer.
[121] Young, L. J., Y. H. Young, 1998. Statistical Ecology, a population perspective. Kluwer
academic publishers Group.
[122] Zadoks, J.C., 1993. Crop protection: why and how, In: Crop Protection and Sustainable
Agriculture, (eds D. J. Chadwick and J. Marsch), John Wiley and Sons, Chichester, pp.
48-60.
[123] Zhang, W., T. H., Ricketts, C. Kremenc, K. Carneyd, S. M. Swintona, 2008. Ecosystem
services and dis-services to agriculture, Ecological Economics.
[124] Zahid, M. A., M.M. Islam, M. H. Reza, M.H.Z. Prodhan, M. R. Begum, 2008.
Determination of economic injury levels of Helicoverpa armigera (Hubner)in Chickpea
Bangladesh J. Agril. Res. 33, 555-563, December.
[125] Zalom, F. W. Barnett, R. Rice, C. Weakey, [Link] associated with flight patterns
of the peach twig borer (Lepidoptera, Gelichiidae) observed using pheromone traps. J.
Econ. Entomol., 85, 1904-1909.
[126] Zalucki, M.P., Daglish, G., Firempong, S., Twine, P.H.,1986. The biology and ecology
of Heliothis armigera (Hübner) and H. punctigera Wallengren (Lepidoptera:
Noctuidae) in Australia: what do we know? Australian Journal of Zoology 34, 779–
814.
[127] Zalucki, M., M. Zhu, D. Zhang, F. Tan, J. Shen, 1982. A study of the resistance of
agricultural pests to insecticides. I. An investigation of H. armigera (Hübner) J.
Nanjing Agric. Coll., 2, pp. 1–7.
[128] Zeddam, J.L., Orbe, K., Léry, X., Dangles, O., Dupas, S. and Jean-François, S., 2008.
An isometric virus of the potato tuber moth Tecia solanivora (Povolny) (Lepidoptera:
Gelechiidae) has a trisegmented RNA genome. Journal of Invertebrate Pathology 99,
204–211.
In: Lepidoptera ISBN: 978-1-62417-248-9
Editors: Elia Guerritore and Johannes DeSare © 2013 Nova Science Publishers, Inc.

Chapter 3

ODOR PLUMES AND ODOR SOURCE INTERACTIONS:

BEHAVIORAL AND PHYSIOLOGICAL
IMPLICATIONS ON PHYTOPHAGOUS INSECTS

Muhammad Binyameen
Division of Chemical Ecology, Department of Plant Protection Biology,
Swedish University of Agricultural Sciences, Alnarp, Sweden

ABSTRACT
In nature, insects live in an olfactory landscape of diverse semiochemicals. When
odor molecules are released from “attractive” and “inhibitory” sources, they can mix
together, and travel in the form of complex plumes similar to those seen in the form of
smoke plumes. To locate and select hosts or conspecifics and to avoid non-hosts or
danger, insects must discriminate between these “positive” and “negative” signals present
in the odor plumes. Various factors affect this discrimination of “positive” and “negative”
signals, of which odor plume structure and odor source interactions are of importance.
Odor dispersion takes place by molecular diffusion and wind turbulence. With molecular
diffusion, random movement of molecules gradually causes them to move away from
each other with time, and creates clines of concentration on very small scales (mm- cm).
However, odor dispersal strongly depends on wind and turbulence, which are the most
frequent odor dispersal features that an insect has to deal with in order to find its odor
source over distance. In an odor plume, odors travel in package or filament laden air
interspersed by clean air. These filaments maintain their highest concentrations near the
odor source and, as they travel downwind, become elongated and torn apart by eddies and
wind turbulence. Consequently, the odor concentration within plumes decreases as they
move downwind, but as odor filaments are interspersed with pockets of clean air and due
to their filamentous nature, compound ratios stay very similar. Insects infrequently or
never utilize time-averaged concentrations and instead use near-instantaneous
concentrations. With distance from the odor source, the plume width and height increase,
while filament intensity and intermittency decrease and distance between filaments
increase. This implies that the interval between filaments and the concentration within


Correspondence to be sent to: [Link]@[Link].
88 Muhammad Binyameen

filaments may both provide important information for insects trying to locate odor
sources. Behavioral studies suggest that odor-induced and wind-guided (anemotaxis)
behaviors are the key components for insects to locate the odor sources. The insect
olfactory system identifies odors of interest in complex plumes for successful selection of
mate or host. Many electro-physiological (extracellular as well as intracellular) studies
also suggest that precise temporal or spatio-temporal patterns of olfactory neurons
underlie the spatio-temporal neural codes for odors. Among these, several studies suggest
that co-localization of disparate olfactory receptor neurons in the same sensillum,
specificity of olfactory receptor proteins, and neuronal interactions in the primary
olfactory centre (antennal lobe) and higher brain centre (mushroom body and calyx) all
play roles in the coding of complex mixture of odors in a plume. Thus, odor plume
structure and interaction between plumes may play a key role in host or mate selection in
phytophagous insects. Plume structure can be analyzed with advanced technologies and
analytical tools such as photo ionization detectors and electroantennography as well as by
those that are simple and less expensive, like soap bubbles generators. However, there is
very little, if any, understanding of interactions of odor plumes and their implications on
insect’s behaviors and sensory physiologies; these areas should be a focus of future
research.

Photo by: M. Binyameen.

GLOSSARY
Anemotaxis: Orientation movement of a free-living organism in response to wind.
Attractnat: A chemical that causes an insect to make oriented towards the source of odor.
Burst return period: Time between the leading edge of one burst and the next.
Chemotaxis: Orientation movement of a free-living organism in response to certain
chemicals in its environment.
Clinotaxis: Movement of an organism in response to a gradient of a stimulus.
Deterrent: A chemical that inhibits feeding or oviposition.
Diffusion: Dispersion of molecules by random molecular movements in an odor plume,
usuallay over very short distance.
 Odor Plumes and Odor Source Interactions 89

Host-selection: Acceptance or rejection of a host.

Intermittency: The dynamic occurrence of the chemical signal that is a proportion of time
in which a signal is present or absent.
Kairomones: Interspecific semiochemicals mediating an interaction that benefits the
receiver to the detriment of the emitter, for example, plant volatiles used for host finding by
herbivorous insects.
Odor plume structure: The pattern of odor distribution within the plume.
Olfactory receptor neuron (ORN): Nerve cell used to detect odors. It is also known as
olfactory sensory neuron (OSN).
Optomotor response: An innate behavior common to insects, it serves for course
stabilization during free locomotion through an involuntary displacement from a straight
course. The purpose of this behavior is to regain the desired course of locomotion.
Phasic response: A response of the cell that diminishes very quickly and then stops. It
conveys information on rapid changes in stimulus intensity and rate.
Pheromones: Intraspecific semiochemicals (used for mate finding, territory marking and
alarm and aggregation behaviors).
Odor plume: Plumes form when turbulent wind or water currents disperse odor molecules
from their source.
Recognition: When an insect can distinguish host from a non-host.
Semiochemicals: Chemical compounds or mixtures that mediate interactions between
organisms.
Turbulence: Three-dimensional mixing of odors in a media.
Tonic response: A response that continues over the duration of the stimulus or longer. It
conveys information about the duration of the stimulus.
Turbulent dispersion: The transport or dispersion of chemicals within a system due to
random and chaotic time dependent motions of the media, usually over longer distance.
Repellent: A chemical that causes an insects to make oriented movements away from the
source.

ABBREVIATIONS
AIR: Active inhibitory range
AL: Antennal lobe
CNS: Central nervous system
EAG: Electroantennography
GC-EAD: Gas chromatography coupled electroantennographic detection
GC-MS: Gas chromatography coupled mass spectrometry
GC-SSR: Gas chromatography coupled single sensillum recording
GLV: Green leaf volatiles
LIF: Laser Induced Fluorescence
LN: Local neuron
MGC: Macro glomerular complex
NHV: Non-host volatile
OBP: Olfactory binding protein
90 Muhammad Binyameen

ODE: Odor degrading enzyme

OR: Olfactory receptor (protein)
Or: Olfactory receptor (gene)
ORN: Olfactory receptor neuron or, OSN: Olfactory sensory neuron
PID: Photo ionization detector
PLIF: Planar Laser Induced Fluorescence
PN: Projection neuron
SSR: Single sensillum recording
VOC: Volatile organic compound

INTRODUCTION
The sense of smell and ability to respond to chemicals exists in many living creatures
(Hildebrand and Shepherd 1997; Mustaparta 2002; Touhara and Vosshall 2009). The sense of
smell (olfaction) involves detection of volatile chemicals. For phytophagous insects, locating
a suitable host-plant is crucial for nutrition and successful reproduction. In nature, insects live
in a highly complex odorant environment where odors are released from various sources and
become complex mixtures of odor plumes. Insects follow odors to perform various
ecologically important activities, such as selection of oviposition site, communication with
conspecifics, avoidance of enemies, and location of food (Hadfield and Scheuer 1985;
Weissburg and Zimmer-Faust 1994). Identifying how insects respond to odors has helped us
to understand the mechanisms that they use to navigate to odor sources (Murlis et al. 1992;
Vickers 2000).
Plant released odors are complex blends of a wide range of substances, which may vary
in composition, blend ratio and overall concentration not only between different species but
also within the same species. To facilitate discrimination of different odors, insects have
hundreds to thousands of sensory hairs (sensilla) over their olfactory organs, mainly on
antennae, but also on the maxillary palp, leg tarsi ovipositor and other tissues. Every sensory
hair house one or more olfactory sensory neurons (OSNs) where each neuronal type is
equipped with a specific olfactory receptor (OR) protein. The ORs are more or less
specialized to respond to specific odors with specific spatio-temporal features (Wilson and
Leon 1988; Schaefer and Margrie 2007). The specificity of ORs enable the activation of
different olfactory receptors (ORs) and OSNs to different degrees, which provides the basis
for neural encoding and further down, processing of odor information in the primary olfactory
centre, (antennal lobe, AL) and higher olfactory centres, the mushroom body (MB) and lateral
horn (LH) (Hildebrand and Shepherd 1997).
Often, insects feed on plants of economic importance and are considered pests when the
damage exceeds the economic threshold level. Prior to the 1970s, pests were mainly
controlled by conventional pesticides. Application of toxic chemicals causes environmental
pollution, health hazards, insect resistance, and death to beneficial insects (Ramakrishnan et
al. 1984; Ahmad et al. 2008). Nowadays various studies have been performed that show
promising results to control insect-pests by alternative environmental friendly strategies, such
as aggregation and sex pheromones for mass trapping and/or mating disruption to control pest
populations (Shorey 1973; Carde and Minks 1995; Raty et al. 1995; James et al. 1996;
 Odor Plumes and Odor Source Interactions 91

Brunner et al. 2002; El-Sayed et al. 2006; Witzgall et al. 2010) and non-host volatiles
(NHVs) for treating host-plant area to protect host-plants (Borden et al. 2001; Zhang and
Schlyter 2004; Jactel et al. 2011). In addition, bio-agents (predators, parasitoids, viruses,
fungi, nematodes etc) are used as a biological control of insect-pests (Mwangi et al. 1991;
OsbOSNe and Landa 1992; Hagler 2000; Faria and Wraight 2001; Hajek et al. 2007; Zehnder
et al. 2007).

Odor Dispersal - Why Plumes Are Important

Odor plumes form when turbulent wind or water currents disperse odor molecules from
their source (Koehl 2006). Airborne odor plumes carry odorant information from an odor
source to an insect’s olfactory sensory hair (sensillum) and then molecular diffusion transport
odorants to sensillum lymph. These odor molecules bind to the olfactory binding proteins
(OBPs) that transport them to the olfactory receptor (OR) proteins expressed on the dendrites
of OSNs (Hildebrand and Shepherd 1997; Zufall and Munger 2001; Koehl 2006). To find the
position of a sex pheromone source, insects have to deal with both pheromone blend
composition and the structure of the odor plumes (Vickers and Baker 1997). On the scale of
an antenna, an odor plume is not a diffuse cloud, but is rather a series of fine filaments of
scent swirling in the odor-free environment. The spatio-temporal patterns of these filaments
change with distance downwind from the odor source (Baker 1990; Mafra-Neto and Carde
1994; Justus et al. 2002). The physical interaction of an antenna with the surrounding fluid
(air) affects the temporal patterns of odor concentration that an insect perceives in a turbulent
odor plume (Koehl 2006); (Hildebrand and Shepherd 1997). It has been of basic interest to
determine the physical dimensions of odor plumes and their interactions for various species
and signal types. Thus, to understand the physical mechanisms responsible for the temporal
patterns of odorants arriving at receptors, we must understand odorant dispersion in turbulent
air (Vickers 2000). Understanding how the structure of odor plumes and their interactions
influence insect behavior and sensory physiology can help us to invent new methodologies to
control insect pests.
The pheromones of many important pest species have been identified, synthesized, and
formulated for use in pest-management schemes as mentioned above, and they are widely
used for population monitoring, mass trapping, and mating disruption (Witzgall et al. 2010).
Chemical Ecologists and Entomologists recognize the need for a more fundamental
understanding of the factors affecting olfactory communication. They seek answers to several
practical questions, for instance, the effective range of attraction of pheromones and
kairomones or the active inhibitory range (AIR) of NHVs, or the strength and positioning of
sources for disrupting olfactory communication (Schwalbe and Mastro 1988; Byers et al.
1989; Howell et al. 1992; Schlyter 1992; Carde and Minks 1995; Byers 1999; Zhang and
Schlyter 2003). The availability of highly sensitive detection technologies nowadays provides
answers to how odor plumes affect the insect’s physiological as well as behavioral responses.
In this review, we have attempted to summarize the current knowledge on the physical
properties of plumes and odor source interactions and how these affect insect behavior and
sensory physiology.
92 Muhammad Binyameen

PHYSICS OF ODOR PLUMES

The complex physical characteristics of a gas or fluid directly affect the spatio-temporal
distribution of odorant molecules dispersed within it, and from that, insect orientation towards
an odor source (Vickers 2000). How are insects able to detect and discriminate odors in order
to locate the odor sources of their interest within complex mixtures of odors in their olfactory
landscape? It is generally assumed that the temporal characteristics of odors and odor plumes
make it possible for the insects to discriminate between odors, either different odors released
from the same source, or the same type of odors released from different sources (Baker et al.
1985; Baker 1990; Byers 1996; Carde 1996; Baker et al. 1998; Fadamiro et al. 1999;
Heinbockel et al. 1999; Bau et al. 2005).

Figure 1. Odor plumes are spatio-temporally dynamic. A) A schematic of a conceptual model for plume
dispersion, modified by Riffell et al. (2008) from (Mylne 1992). I) When the plume width is smaller than the
dominant crosswind eddy size, the plume will begin meandering. II) Once the plume width approaches the
eddy size the plume becomes mixed with clean air. III) The plume will continue to develop and increase in
size until its width exceeds the eddy scale. The plume structure as a whole will depend upon the dominant
eddy scale and the variation in concentration fluctuations of the plume.
B) A mass chromatogram of a cis- 3-hexenyl acetate plume as measured by proton transfer reaction mass
spectrometry (PTRMS). Measurements occurred 4 m downwind from the source at an average wind velocity
of 40 cm/s, dominant eddy size of 2.1 m and frequencies of 10 s. The PTRMS sample rate was 4 Hz. Taken
from Riffell et al. (2008).
 Odor Plumes and Odor Source Interactions 93

The dynamic occurrence of the odor in a plume is called its intermittency (Cardé and
Willis 2008), which is very crucial for the upwind flight of a moth. The integration of
counterturning (casting) and optomotor anemotaxis (see Glossary) allows insects to fly
upwind in odor plumes (Baker et al. 1985; Willis and Arbas 1991; Mafra-Neto and Carde
1994). Several studies have shown that chemical signals are dynamic in both space and time
(Murlis and Jones 1981; Murlis et al. 2000; Riffell et al. 2008). Measurement of physical
atmospheric processes in the field can help us to understand the spatio-temporal features of
the odor cues available to insects, as shown in Figure 1.
The concentration fluctuations also have important behavioral implications, such as when
an insect encounters different chemical stimulus dynamics close to an odor source compared
to further downwind (Riffell et al. 2008). The physical interactions of sensory organs with the
surrounding fluid also affects the temporal pattern of odor concentrations that an animal
experiences in a turbulent plume (Koehl 2006). Characterization of the chemical signal’s
environment makes it possible for us to know when and where the behaviors mediated by
olfaction control ecological interactions (Riffell et al. 2008). In the following sections, I will
describe other characteristics of odor plumes and how they change with distance downwind
from the odor source and also the other factors affecting these temporal properties.

Physical Dispersal Mechanisms of Odors

There are two major physical dispersal mechanisms of odors; molecular diffusion and
turbulent dispersion caused by wind and local turbulence. In molecular diffusion, random
movement of molecules gradually causes them to move away from each other with time.
Molecular diffusion of odor molecules in the media create clines of concentration on very
small scales (mm- cm) (Vogel 1996). However, to create an odor plume traceable over greater
distances (dm- m), movement of the media (air, water) is needed. Turbulent dispersal is
common and the most frequent odor dispersal feature that an insect has to deal with in order
to find its odor source, usually over longer distances (Murlis et al. 1992; Murlis et al. 2000).

Factors Affecting Temporal Characteristics of Odor Plumes

Olfactory cues are strongly affected by abiotic factors such as wind and temperature.
Wind is, more or less, always a factor, dispersing the olfactory signal away (downwind) from
an odor source. The dispersal of odors strongly depends on wind velocity (Zanen and Carde
1996; Campbell and Borden 2006). In an odor plume, odors travel in packages or filaments
interspersed with air (Vickers 2000). These filaments maintain highest concentrations near the
odor source and, as they travel downwind, become elongated and torn apart by eddies and
wind turbulence (Riffell et al. 2008). Consequently, the odor concentration within plumes
decrease as they move downwind (Baker and Haynes 1989; Voskamp et al. 1998; Murlis et
al. 2000), but as odor filaments are interspersed with pockets of clean air and due to its
filamentous nature, compound ratios stay essentially the same within an odor package (Murlis
and Jones 1981; Liu and Haynes 1992; Vickers 2000; Cardé and Willis 2008). The plume
width and height increase while filament intensity and intermittency decrease with distance
from the odor source (Mylne and Mason 1991). The distance between filaments also increases
94 Muhammad Binyameen

with distance from the odor source. This implies that the interval between filaments and the
concentration within filaments may both provide important information for insects trying to
locate odor sources.
A number of different factors can influence plume intermittency. Air turbulence also
effects the structure of the plume (Murlis et al. 1992; Murlis et al. 2000; Vickers 2000; Cardé
and Willis 2008; Riffell et al. 2008). For example, a higher wind speed or turbulence can
increase the filament frequency (Fackrell and Robins 1982). Behavior of an insect itself or of
the odor source releasing the odor can also affect the temporal features of an odor plume. For
example, a female moth can release pheromone into the air by exposing its gland, while a
flower in the wind by oscillating back and forth (Sprayberry and Daniel 2007). The size and
position of an odor source can also affect the plume dynamics. For instance, plume
intermittency is higher for larger sources than smaller ones, but when a small odor source is
close to a larger structure (e.g. a female moth calling from a tree), then the plume will behave
differently (Riffell et al. 2008). Odor source size also depends on the size of the insect's perch
rather than the size of the insect or its odor-emitting gland. For instance, bark beetles
(Scolytidae) can generate a large plume with an effective source, the size of an entire tree
trunk with hundreds of odor-emitting beetles as compared to a female moth, which releases
pheromones while sitting on a plant leaf (Krasnoff and Roelofs 1988), corresponding to the
smallest source sizes (Fackrell and Robins 1982). Such different plume structures may exert
different selective pressures on insect sensory systems during orientation towards an odor
source (Andersson et al. 2011). Burst length increases with distance away from the odor
source (Murlis and Jones 1981; Voskamp et al. 1998; Murlis et al. 2000) while frequency of
bursts decreases (Murlis and Jones 1981; Murlis et al. 2000; Justus et al. 2002).

Why Are Concentration Fluctuations Important?

It is very important to consider the concentration fluctuations of an odor plume, because

relevant concentrations are those only experienced on time scales of a few seconds or less, not
the average of minutes or hours (Mylne 1992). A number of studies have been performed to
develop modeling techniques to measure concentration fluctuations theoretically as well as
experimentally (Hanna 1984; Murlis et al. 1992). A series of tracer experiments were done by
Mylne and Mason (1991) to measure the concentration fluctuations in a plume at ranges of
between 50 m and 1000 m.
Murlis and Jones (1981) concluded that an odor plume appears as a series of bursts of
odor which are variable in strength and duration. They also hypothesized that distribution of
the length of bursts and the time between them does not greatly vary with distance from the
source, although concentration decreases with distance from the source. To the stationary
insect the strength of stimuli varies from burst to burst, whether measured in terms of the total
quantity of stimuli contained in a burst or as the peak concentration in the burst (Murlis et al.
1992). Averaged over time, however, the total quantity of material arriving will depend upon
the insect's position: particularly large changes of position occur in the first few meters
downwind from the source. Of course, the concentration will be very high close to the source
as compared to a few meters downwind from the source. Insects flying or walking rarely, or
never use time-averaged odor information (Elkinton and Carde 1984; Riffell et al. 2008) and
instead use near-instantaneous information present in the odor plume (Mafra-Neto and Carde
 Odor Plumes and Odor Source Interactions 95

1994; Vickers and Baker 1994). Thus, in moving from one point to another point upwind, the
instantaneous concentration would not provide a reliable guide of the mean concentration to
the insects relative to their position from the source. Bartell and Lawrence (1977) were able to
show that the response of a male apple moth, Epiphyas postvittana, to sex pheromone,
presented intermittently, differed from that found with continuous exposure to pheromone.
Farkas et al. (1974) described the effect of sex pheromone concentrations on visual
responses in flying male moths. When the pheromone concentrations in the air stream were
low, males ignored a strong visual cue, but at high concentration they orientated towards it.
Murlis and Bettany (1977) described changes in flight behavior in male Spodoptera littoralis
flying in the field towards different doses of a sex pheromone.
Furthermore, the magnitude of an insect neural response to odor stimulus above the detection
level is proportional to odor concentration (Kaissling 1971; Durbin 1980).

A.

B.

Figure 2. A) Different scales in host selection sequence, adapted from Introductory paper by Andersson
(2007). B) Host selection process and discrimination between negative and positive stimuli, modified
from Renwick (1989).
96 Muhammad Binyameen

Scale Dependent Structure of Odor Plumes

The process of locating and accepting or rejecting a host plant for an insect involves
multiple steps from the large scale of landscape to the fine scale of host-plants or parts thereof
(Renwick 1989; Saint-Germain et al. 2004), but the relative importance of different search
modes varies between scales (Saint-Germain et al. 2007). For example, at the landscape scale,
insects must be able to find suitable habitats (Schooley and Wiens 2003) and then have to
search for the right host-species and a specific site on the host-plant, considering various
factors such as food quality, risk of predation and parasitism as well as the population density
of conspecifics (Renwick 1989) (Figure 2). An understanding of the sequences of scales
requires knowledge of the mechanisms of insect orientation to odors and the sensory inputs
that mediate them (Kennedy 1977).
Insects produce and respond to odor plumes that differ markedly. Insects with differing
phylogenies independently evolve orientation mechanisms to assure multiple solutions to the
location of odor sources (Murlis 1986). At different scales the effects of a plume structure on
insect orientation behavior are different. A study on orientation behavior of Pityogenes
bidentatus indicated an active inhibitory range (AIR) of 0.5-1.0 m from a trap baited with
pheromone and non-host bark (Byers et al. 2000). Another study on I. typographus showed
an AIR of at least 2 m of NHV plus verbenone blends baited on traps around a central
pheromone trap at 1, 2 and 4 m radii (Zhang and Schlyter 2003). Andersson et al. (2011)
showed that moths use lower scale (at least by one order of magnitude) for odor
discrimination compared to beetles, suggesting that moths may use an odor plume of different
structure as compared to beetles.
Murlis et al. (1992) suggested three levels of odor plume structure:

Time-Averaged Plume Structure

This determines the probability that an insect will contact the odor plume at different
locations downwind of the odor source. The Sutton model provides estimates of mean
concentration of odor downwind from a point source. The model developed by Sutton (1953)
assumed normal distribution of mean concentration in all directions perpendicular to the
plume central line, and a simple power law to describe the decrease in the mean concentration
along the plume centre line at increasing distances from the source (Figure 3 a). Similar
methods were used by Bossert and Wilson (1963) and Nakamura (1976) to calculate
concentration gradients and active regions of the plume.

Large-Scale Structure
The shape and average odor strength of the body of the plume affect the orientation
strategy of insects. For example, in a meandering plume (Figure 3 b), an insect encounters
much higher levels of odor than the mean, therefore predicting concentration in the
meandering plume is of great importance. Large-scale smoke-plume field data produce well-
defined spectra but the dominant scales of concentration fluctuations are considerably shorter
than the dominant turbulence scales (Hanna and Insley 1989).
 Odor Plumes and Odor Source Interactions 97

Small-Scale Structure
The fluctuating odor concentration within the plume body affects the input to the insect’s
central nervous system (CNS) and hence their instantaneous response to the plume, as seen
with the filamentous structure of a plume (Figure 3 c). Odors used by insects are difficult to
measure with sufficient time resolution to allow the fine-scale structure of odor plumes to be
assessed directly. Concentrations in a smoke plume can be measured over short averaging
periods with photometers. Hadjitofi and Wilson, (1979) obtained better resolutions of more
than 30 ms using sulphur dioxide and phosphorus pentaflouride tracers with fast flame
photometric detectors. Conversly, a male moth can resolve two odors separated with 1 mm in
space and 1 ms in time (Baker et al. 1998).

Measurement Methods

Various methods have been used to obtain a better understanding of the odor plume
structure. The 1st micro-meteorological model was developed by Sutton (1953), and later it
was used by Wright (1958) to estimate the active space in an odor plume. Various other
models have been developed during the last 50 years that have helped to estimate
concentration fluctuations e.g. meandering plume models (Gifford 1959), a similarity model
(Chatwin and Sullivan 1979), statistical models (Durbin 1980), pdf models (Venkatram
1984), and krypton-85 (Lung et al. 2002). Recently, the spatio-temporal structure of a
turbulent plume was measured using Planar Laser Induced Fluorescence (PLIF) and Laser
Induced Fluorescence (LIF) techniques (Crimaldi and Koseff 2001).
An ionized air current was used by Baker et al. (1985) and Murlis (1986) to understand
the physical structure of pheromone plumes using an ion detector. Many other tracers also
have been used for visibility and measurement of odor plume structure. A stream of ionized
air was used to determine the fine-scale structure of odor plumes in relation to insect
orientation to odor sources (Murlis and Jones 1981). A flame photometric technique was
used to measure fine-scale structure of plumes using the chemical tracers, sulphur dioxide and
phosphorus pentaflouride (Hadjitofi and Wilson 1979). Justus et al. (2002) used a Photo
Ionization Detector (PID) and a tracer gas to measure the odor plume structure in a wind-
tunnel. A PID can quantify the cumulative concentration of all detectable gases and was used
to understand the structure of insect pheromone plumes as mentioned above; a PID thus
provides a powerful means to characterize the physics of odor environment.
Insects antennae are used as biosensors to determine biologically important compounds
(Roelofs and Bjostad 1984). Electro-antennography (EAG) and single sensillum recording
(SSR) techniques coupled with gas-chromatography (GC), and using GC coupled mass
spectrometry (GC-MS), have emerged as sensitive tools for identification of behaviorally
active volatile organic compounds (VOCs) (Schneider 1969; Mustaparta 1975; Hedin et al.
1979; Visser et al. 1979; Zhang et al. 2000; Stranden et al. 2003; Ghaninia et al. 2008;
Bengtsson et al. 2009).Due to the delicacy and high sensity of the recording setup, single-
sensillum recordings on the insect antennae have only been made indoors, wherease EAG has
been used in the field to analyze the structure of odor plumes (Ando et al. 1980; Baker and
Haynes 1989; Sauer et al. 1992; Rumbo et al. 1995; Koch et al. 1997; Murlis et al. 2000).
However, EAG is not neuronally-specific like SSR is. Thus, SSR in field or semi-field
98 Muhammad Binyameen

conditions will be a future task to analyze physical characteristics of odor plumes at greater
levels of detail.

Figure 3. a) Time-average Gaussian plume model, showing the principle axes and the source positioned
at height h. b) A meandering plume model with concentration in each disc distributed normally about
the meandering centre line. c) The filamentous structure of a real plume. Modified from Murlis et al.
(1992).

ODOR SOURCE INTERACTIONS

Insects live in diverse habitats with a great diversity of odors released from host and non-
host as well as dangerous sources. To cope with the environment, insects use their
sophisticated olfactory system to discriminate between the odors of interest from those of
either no-interest or at least less interest. To understand these mechanisms we need to study
how the odors or odor-components released from different ecological sources interact and
how they are used by the insects. An understanding of the odor source interaction is also
 Odor Plumes and Odor Source Interactions 99

necessary if we want to use odors to manipulate insect behaviors and control insect-pests.
There are different types of odor source interactions. I summarize a few of them here.

Pheromone Component Interactions

Most often behaviorally-active pheromones consist of several individual components.
The successful location of the pheromone source normally depends on the composition of the
blend as well as the plume structure. Wind tunnel experiments have been used widely to study
the mechanism used by male moths during orientation towards female sex pheromone
components (Linn and Gaston 1981; Willis and Baker 1984; Linn et al. 1986; Baker 1990;
Mayer and McLaughlin 1991; Mafra-Neto and Carde 1994). The pheromonal studies have
mainly been carried out on moths as they have a very specialized system for the recognition
of pheromone components with correct blends and enantiomeric ratios. For example, in the
European corn borer, Ostrinia nubilalis, males of the two strains (Z and E) prefer opposite
ratios of the two pheromone components (Anglade et al. 1984). The male antennal lobes of
the two strains contain a pheromone sensitive macroglomerular complex (MGC) that is
identical in morphology, but reversed in functional topology (Kárpáti et al. 2008). In a wind
tunnel bioassay examining S. littoralis males, the major pheromone component elicits all
behavioral steps from take-off to landing. Attraction to minor components, when presented
individually, was significantly lower than to the major component alone, while in multi-
component blends presentation of some of the minor components inhibited the attraction to
some extent (Quero et al. 1996). Male moths of Helicoverpa zea responding to female sex
pheromone blend cease their upwind flight when additional compounds (behavioral
antagonists) were added to the blend (Baker et al. 1998). In contrast, only at 1mm spacing
between pheromone and antagonist, the moths were able to reach the pheromone source. Such
fine scale resolution of odorants may have importance in mate finding, as males should fly
only to the correct blend.

Pheromone and Plant Odor Interactions

In general, the pheromone OSNs are very specific in their response to pheromone
components (Ljungberg et al. 1993), while OSNs tuned to plant-associated volatiles have
been found to be specialist as well as generalist (Hansson 1995; Bruce et al. 2005; Ignell and
Hansson 2005). In extracellular recordings of Helicoverpa zea males it was found that host
plant volatiles synergize responses of pheromone specific OSNs when binary mixtures of
pheromone and plant compounds were used, while there was no effect on spontaneous
activity when OSNs were stimulated with only the host plant volatiles (Ochieng et al. 2002).
From these results, the authors proposed that a male moth might use plant odors as
background signals to find the calling female sitting on or very close to the host-plant.
Conifer inhabiting bark beetles are mainly attracted by secondary attractants, the aggregation
pheromones that often are synergized by host compounds (Byers 1995). In another study on
Spodoptera littoralis males, the interaction of pheromone and plant compounds were also
found in the primary olfactory centre, the antennal lobe (AL) (Anton and Hansson 1995).
Both, local neurons (LN) and projection neurons (PN) responding to both sex pheromone and
100 Muhammad Binyameen

plant odors arborized into ordinary glomeruli (defined on pg. 22) as well as within the macro
glomerular complex (MGC), a compartment for pheromone processing present only in male
moths. A mass trapping study on corn earworm and codling moth also showed that addition
of host-plant green-leaf volatiles can increase the capture in pheromonal traps (Light et al.
1993). Plant odors play an important role in reproductive isolation of Yponomeuta spp, each
species living on specific host-plants. The interaction between pheromone and plant odors
were found also at the receptor level using electroantennography (EAG) and single sensillum
recordings (SSR) (Van der Pers et al. 1980). Drosophila melanogaster prefer fermented fruit
to feed, mate and oviposit on. Both male and female flies are attracted to aggregation
pheromones but attraction is dependent on the presence of a suitable food source (Bartell et
al. 1985). These studies suggest that pheromone and plant odor interaction take place at
different levels, from brain to behavior.

Host and Non-host Odor Interactions

In general, at large distances host odors are the primary signals used by phytophagous
insects during host-selection. Behavioral studies have shown that plant volatiles are involved
in all behavioral steps performed by a female moth to select a host-plant for oviposition or
nectar feeding (Renwick 1989; Bernays and Chapman 1994). The NHVs are odor cues
released from sources other than hosts, e.g. non-host plants, predators, parasitoids, pathogens
etc. NHVs generally inhibit insect attraction towards the attractive odor source. Many studies
have been performed to identify behaviorally active NHVs and their potential to protect hosts
by inhibiting host location. However, this strategy has until now been limited to forestry
(Zhang and Schlyter 2003; 2004; Andersson et al. 2009; Jactel et al. 2011). In a recent study
on the bark beetle I. typographus, it was shown that almost 25 % of the responding OSNs are
dedicated to NHVs signals, which indicate the importance of non-host odors for a specialist
insect in a complex olfactory landscape (Andersson et al. 2009).

Tri-trophic Interactions

There are three essential components of tri-trophic interactions, plants (primary host),
herbivores (insect-pest or secondary host) and carnivores (predator or parasitoid). Usually
plants release volatile organic compounds (VOCs) in the surrounding environment that attract
beneficial insects (pollinators) as well as herbivores (insect-pest). Herbivores use nectars from
these host-plants to feed themselves and then lay eggs to produce their progeny. After
hatching from the eggs, larvae normally eat on the same plant because they are often
immobile, particularly in the early instar stages. When plants are damaged by feeding larvae,
or even solely by oviposition, they can release induced host compounds to defend themselves.
These defensive compounds may either affect the herbivore directly (toxic compounds) or
indirectly by attracting predators and parasitoids. In a study of mass trapping of bark beetles
with their pheromone components ipsenol and ipsdienol, the predatory beetles Thanasimus
spp. were also captured, indicating that the emitted pheromone signals were used by this
predator as a kairomone (Schlyter et al. 1987). Some species of predators and parasitoids
track their hosts by the host’s aggregation- or sex pheromones since these cues usually are
 Odor Plumes and Odor Source Interactions 101

emitted in high quantities and have a potential to disperse over long distances (Vet and Dicke
1992). There are examples of parasitoids responding to host feces, see review by Turlings et
al. (1993) and to odors from the host larva’s host plant (Turlings et al. 1995). In another study
it also has been suggested that parasitoids track an odor blend from the host organism and the
host organism’s host plant (Murlis et al. 1992). So, a multi-trophic approach is required to
understand the mechanisms used by the plants to defend themselves from herbivory, and also
the mechanism used by phytophagous insect during host-selection considering the risk of
parasitism or predation. Co-localization of OSNs for plant or insect derived attractants and/or
inhibitors (Baker et al. 1998; Bruce et al. 2005; Andersson et al. 2010) could explain how
insects discriminate between attractive and non-attractive sources.

INSECT OLFACTION AND ODOR PLUMES

Insects Olfactory System and Odor Coding

In nature, an insect is exposed to complex mixtures of odors from the surrounding

environment; the insect´s olfactory system must detect and sort out odors of importance from
background noise. Odor detection is performed by OSNs housed in the sensory hairs, called
sensilla, which cover the surface of olfactory appendages, such as the antennae. An olfactory
sensillum unit consists of a multiporous cuticular structure, neurons and accessory cells
(Figure 4). Typically, there are 2 - 5 OSNs in each sensillum, but in honeybees Apis mellifera
up to 30 neurons and in locusts Schistocerca gregaria up to 50 neurons can innervate a single
sensillum (Schoonhoven et al. 2005). Sensilla are classified according to their morphological
form. They include typical trichoid sensilla (typically associated with perception of
pheromones), pegs and cones (basiconic and coeloconic sensilla, respectively, often involved
in plant odor perception and small polar compounds) and pore-plate organs (sensilla
placodea, which might be involved in detection of plant volatiles) (Schoonhoven et al. 2005).
Odor molecules striking an antenna are absorbed to the waxy surfaces of its sensilla and
diffuse through narrow pores in the cuticular walls into the lumen. The odor molecules enter
through pores in the sensillum and bind to olfactory binding proteins (OBPs) present in the
sensillum lymph, which transfer them to ORs present on the dendrites of OSNs (Hildebrand
and Shepherd 1997).
The OSNs transform the chemical signals into electrical and send them to the primary
olfactory centre (AL, Figure 5) by terminating their axons in the glomeruli. Glomeruli are
spherical neuropil structures in the AL where the axon termini of OSNs meet the dendrites of
the projection neurons (PNs), along with branches of the local neurons (LNs). Glomeruli in
the AL are often interconnected by these LNs. LNs can be inhibitory or excitatory, releasing
gamma-amino butyric acid (GABA) or probably acetylcholine (Masse et al., 2009),
respectively. Both excitatory and inhibitory neurons form extensive connections throughout
the AL and make inter- and intra-glomerular connections. After primary processing in the
AL, the information is transferred to the higher brain centres in the protocerebrum, the
mushroom body (MB) and the lateral horn (LH), through PNs, of which dendrites innervate
the glomeruli and axons terminate in the MB and/or LH (Figure 5).
102 Muhammad Binyameen

Figure 4. Schematic drawing of insect olfactory sensillum innervated by two olfactory sensory neurons
(OSNs), cell bodies of OSNs are surrounded by the accessory cells (Drawing courtesy of Prof. Dr. R.
A. Steinbrecht, Max Planck Institute, Seewiesen, Germany).

Figure 5. Olfactory system: A schematic of the organization of the Drosophila olfactory system, with
mammalian counterparts in parentheses. Olfactory sensory neurons expressing the same receptor
project their axons to the same glomeruli in the antennal lobe. Projection neurons send their dendrites to
glomeruli in the antennal lobe and axons to the higher olfactory centres, the mushroom body and the
lateral horn. Taken from Komiyama et al. (2003).

Since the olfactory system of the insects must accomplish several tasks, there are
different types of olfactory receptors with different functions. A variety of approaches,
including single-cell electrophysiology, multi-unit recording and population level studies
(optical imaging) have demonstrated that an animal’s olfactory system is capable of detecting
and discriminating behaviorally meaningful odor mixtures with astounding sensitivity and
specificity against noisy irrelevant odor mixtures (De Bruyne et al., 2008). Depending on the
type of response breadth, OSNs that respond to plant odorants can be classified into two
 Odor Plumes and Odor Source Interactions 103

categories, narrowly tuned and broadly tuned (Lie et al., 2008). Depending on the chemical
identity, some plant odorants may activate a few specialized types of OSNs, whereas others
may activate a large group of OSN types (Lei et al., 2008).
There are two major ways an insect can sort out odor signals from background noise.
First, responses to single odorants, such as pheromones, can be processed through specific
neural channels (e.g., the MGC in the AL of male moths), which mean that receptors and
neurons respond specifically to certain compounds (Ljungberg et al. 1993). Secondly,
responses to more complex odor blends, such as plant odors, can operate across large groups
of receptors. This happens in two ways: single receptor neurons may respond to several
different odors, or a single odor can elicit responses in different receptor neurons as in the
case of female moths (Anderson et al. 1995; Anderson et al. 1996; Shields and Hildebrand
2001) and bark beetles (Andersson et al. 2009).

Figure 6. Coincidence detection in peripheral receptors: Paired olfactory sensory neurons (OSNs)
specific to compounds A and B respond when each compound is detected. Recordings from two OSNs
are shown in two different scenarios: in (a), OSNs for compound A and compound B are stimulated at
the same time, whereas in (b), they are stimulated at different times. Because of these temporal effects,
the central nervous system (CNS) can determine whether A and B were emitted from a host plant or
from the non-host plants. Taken from Bruce et al. (2005).
104 Muhammad Binyameen

Bark beetles have OSNs that respond to NHVs in addition to OSNs that respond to
aggregation pheromone and host compounds (Andersson et al. 2009). Thus, an important
odor mixture for the insect will be characterized by a specific spatial and temporal activity
pattern over the activated receptor groups and will subsequently activate the neurons that
express the responsive receptors (Lemon and Getz 1997; Baker et al. 1998). However, odor
discrimination is not a simple task that is made only by the OSNs or ORs but is actually
complex, involving other types of olfactory neurons (LN, PN) present in the AL and further
downstream processing of odor signals in the higher brain centres (mushroom body and
lateral horn) through PNs (Hildebrand and Shepherd 1997).
Bruce et al. (2005) hypothesized that host-plant selection depends on ratios and blends of
plant volatiles and not on just detection of presence or absence of a particular compound.
Bruce et al. (2005) explained two hypotheses proposed by Visser (1986): species-specific
recognition and ratio-specific recognition of host plants. The authors supported the hypothesis
of ratio-specific recognition of host plants because of the co-localization of OSNs in the same
sensillum. Response of co-localized OSNs to different compounds with different spatio-
temporal patterns (Baker et al. 1998; Stensmyr et al. 2003; Andersson et al. 2010) makes it
possible for the insect to discriminate a host from a non-host (Figure 6). Recently, in an
electrophysiological study on locusts examining extracellular and intracellular recordings,
followed by modeling, it was also hypothesized that temporal firing pattern of OSNs, LNs
and PNs underlie spatio-temporal neural codes for odors (Raman et al. 2010). These studies
indicate how insects recognize their host in natural environments where thousands of odors
are present including signals from the non-host or background noise.
Taxonomic characteristics of odors are highly specific and consist of compounds not
found in unrelated plant species. Taxonomical characteristics of volatile components arise
from the breakdown of secondary plant substances, as in the Brassicacea family (one of the
most extensively studied plant families due to its economic importance), where non-volatile
glucosinolates are decomposed to produce volatile isothiocyanates, thiocyanates and nitriles,
particularly when the plant tissue is damaged, but also at a much lower rate during normal
catabolism (Schoonhoven et al. 2005). These volatile allylisothiocayantes give cruciferous
plants their characteristic pungent smell, which several insect species that feed preferentially
on Cruciferae use to locate the host plant.
Several phytophagous insects species have shown specific behavioral responses to odors
that are characteristic of their host plants (Table 1), for example Crucifer feeding insects,
Delia brassicae, pollen beetles Meligethes aeneus and flea beetles Phyllotreta cruciferae are
attracted to allylisothiocyanate compounds (Visser, 1986). In the diamondback moth, Plutella
xylostella, isothiocyanates play a very crucial role in host-plant finding when plants are
damaged and stimulate oviposition (Renwick 1989). Taxon-specific odors of certain plants
can also play an important role in making host plant volatiles less attractive by masking host
odors or by repelling insects away from the host plant. For example, electrophysiological
recordings showed the presence of specific OSNs in Brevicoryne brassicae (cabbage aphid),
Lipaphis erysimi (mustard aphid) and Aphis fabae (black bean aphid) that responded to
isothiocyanates (3-butenyl isothiocyanate). The cabbage seed weevil, Ceutorhynchus
assimilis, which feeds on seed rape, also uses isothiocyanates as an important host compound.
 Odor Plumes and Odor Source Interactions 105

Odor Plumes Structure and Its Influence on Insect Behavior

Various studies have been performed to understand the behavioral mechanisms used by
insects to discriminate different odors in a plume, although most work has been done under
laboratory conditions (e.g. in wind tunnels), and only very few behavioral experiments have
been performed under natural conditions to evaluate plume structures. To see how an odor is
presented in nature it is necessary to determine the odor plume structure, that is, the pattern of
odor distribution within the plume.
Characterization of the chemical signal’s environment helps us to determine when and
where olfactory-mediated behaviors may control ecological interactions (Riffell et al. 2008).
Many attempts have been made to analyze the structure of odor plumes using different
methods and technologies as well as mathematical and statistical models, and considerable
progress has been made in understanding the fine scale structure of dispersing plumes in the
atmosphere (Murlis and Jones 1981; Elkinton and Carde 1984; Murlis 1986; Dinar et al.
1988; Moore and Atema 1991; Mylne and Mason 1991; Mafra-Neto and Carde 1994; Carde
1996; Justus et al. 2002; Moore and Crimaldi 2004; Thistle et al. 2004; Koehl 2006; Vetter et
al. 2006; Cardé and Willis 2008; Riffell et al. 2008; Strand et al. 2009). It is possible to
characterize an odor plume with the physics of dispersion (Murlis et al. 1992). However, with
respect to animal behavior, the link between cause and effect is very important but difficult to
determine. For example, male moth pheromone detection has some limitations, such as the
ability of odor degrading enzymes (ODEs) to degrade pheromone compounds (Kaissling
1974). This might significantly affect the receptor’s ability to send sufficiently fluctuating
signals to the CNS. Baker et al, (1985) suggested that the insects' receptors and pheromone
blends (that can initiate or shut-off upwind flight) should be used to find correlations between
behavior and fluctuating signals.
As flying insects approach an odor source, their flight patterns typically change, and
ultimately landing may occur. The effect of plume structure on flight orientation of insects
has been mainly studied in moths (Lepidopteran). The plume structure shows a strong impact
on insect behavior (Murlis and Jones 1981; Murlis 1986; Linn Jr et al. 1987; Baker 1990;
Mafra-Neto and Carde 1994; Fadamiro et al. 1999; Justus et al. 2002).
It has been hypothesized that as male moths fly upwind in a pheromone plume, sensory
adaptation of their receptors may be a significant factor in their ability to locate the
pheromone source (Shorey 1973; Baker et al. 1988). Lewis and Macaulay, (1976) described
differences in the flight pattern made by moths towards a pheromone in correspondence to the
shape and structure of a plume issuing from the same source. An air-borne plume of a sex
pheromone from a female moth or a synthetic source has a fine, filamentous structure that
creates rapid fluctuations in concentration for a male moth flying upwind (Baker et al. 1988).
Male moths exposed to turbulent or mechanically pulsed plumes fly faster and straighter
upwind, and locate sources more frequently than males exposed to continuous and narrow
plumes (Willis and Baker 1984). According to these hypotheses, the moths’ sensory system
adequately registers the fine structure of the odor plume and insect may respond to such
fluctuating signals by generating an upwind flight behavior (Murlis 1986; Baker et al. 1988).
To investigate this type of hypothesis, it is necessary to measure how insect’s olfactory
receptors perceive and discriminate different odors in an odor plume. A high resolution ion-
detector was used to measure discreet signals in time and space (Murlis et al. 1990). The
106 Muhammad Binyameen

authors also found a compareable response pattern from gypsy moth antennae using EAG as
the ion-sensor, Figure 7.

Table 1. Species-specific plant kairomones used by phytophagous insects,

from Bruce et al. (2005)

Insects Host plants Kairomone

Lepidoptera
Codling moth, Cydia pomonella
Rosaceae Ethyl (2E, 4Z)-2,4-decadienoate
Arctid moths, Cisseps fulvicollis,
Asteraceae (S)-(+)-Hydrioxydanaidal;
Ctenucha virginica and Halysidota
(R)-(+)-Hydrioxydanaidal
tessellaris

Dimondback moth, Plutella xylostella Brassicaceae Allyl isothiocyanate

Hemiptera Isothiocyanates (3-butenyl and 4-

Brassicaceae
Cabbage aphid. Brevicoryne brassicae pentenyl

Birdcherry aphids, Rhopalosiphum

Rosaceae Benzaldehyde
padi

Diptera
Apiaceae (E)-Asarone
Carrot fly, Psila rosae
Alliaceae Dipropyl disulfide
Onion fly, Delia antiqua
Oleracea Allylisothiocyannate
Cabbage root fly, Delia brassicae

Coleoptera
Corn rootworm, Diabrotica vergifera Cucurbitaceae 6-Methoxy-2-benzoxazolinone
virgifera Isothiocyanates (3-butenyl, 4-
Cabbage seed weevil. Ceutorhynchus Brassicaceae pentenyl. 2-phenylethyl)
assimilis

An EAG was also used by Conner et al. (1980), to measure the fluctuations in pheromone
concentration on Utethesia ornatrix male’s antenna at 60 cm downwind in low wind speeds,
but they did not find any correlation between concentration fluctuations and behavior.
However, Baker et al. (1985) found that lack of upwind flight in a uniform plume was
correlated with a continuous high level of EAG activity.
Murlis (1981) described an experimental method for simulating an odor plume in the
field for the determination of its fine-scale characteristics. He found that the ‘odor’ arrived up
to 15 m from the source in a series of discrete bundles, which were widely distributed in time
but were typically 0.1 s long and 0.5 s apart. The strength of the bundles was also determined
and found to be widely distributed and some contained considerable fluctuations. A PID was
used to monitor odor concentration at a particular point with high resolution (Justus et al.
2002). It has been found that plume structure has a significant effect on orientation of Carda
cautella males (Mafra-Neto and Carde 1994). It has also been shown that the successful
 Odor Plumes and Odor Source Interactions 107

location of a sex pheromone source by a male moth is dependent on blend composition, as

well as plume structure (Vickers and Baker 1997). Linn and Gaston (1981) showed that there
was no effect on upwind orientation of cabbage looper moths by separating the pheromone
components 12 cm apart and releasing moths 85 cm downwind from the odor source, but
when the separation was only 8 cm between components and moths were released 35 cm
downwind, significantly fewer numbers of moths approached the sources. However, one of
the components was attractive alone while the other was not. The males of Helicoverpa zea,
can distinguish pheromone strands from those of a behavioral antagonist separated by only 1
mm in distance and 1 ms in time (Baker et al. 1998). This means that male moths have the
ability to resolve closely spaced filaments, which is consistent with other studies (Fadamiro et
al. 1999). It has also been shown in the field that the response of beetles was dependent on
spacing distance and dose as beetles showed discrimination to different strengths and spacing
distances between pheromone components (Byers 1987; Schlyter et al. 1987). The complete
blend has the lowest concentration threshold for eliciting all behaviors observed in
Argyrotaenia velutinana, Tricoplusia ni, G. molesta and S. littoralis (Dunkelblum et al. 1982;
Linn et al. 1986), thus the entire blend governed the behaviorally active space. Plant volatiles
in some cases can also improve pheromone attraction (Dickens et al. 1993; Light et al. 1993;
Landolt and Phillips 1997; Deng et al. 2004).

Figure 7. Fluctuating signals from an ion concentration sensor showing burst length and burst return
period. EAG signal recorded from a gypsy moth antenna responding to pheromone at the same time and
in the same position as the ion sensor. Modified from Murlis (1992).

In open fields, moving upwind, while in a plume, should aim the insect toward the odor's
source, particularly within tens of meters, but because the plume meanders, an insect heading
upwind often will exit the plume. Casting enhances the likelihood of directly re-contacting the
plume, as the cast widens the insects position, or provides a station-keeping maneuver until
the plume shifts back to the insect's position (Baker 1990). Odor-induced anemotaxis is the
108 Muhammad Binyameen

primary mechanism for locating a distant odor source; various other forms of chemotaxis
have also been hypothesized to be used by insects in orientation behavior. Anemotaxis
behavior allows insects to fly upwind in odor plumes. The upwind anemotaxis behavior is
also used by walking insects. The anemotaxis behavior has been studied in the beetle
Leptinotarsa decemlineata (Schoonhoven et al. 2005). Odor-guided walking towards host
plants using has also been observed in the field for the beetle Trirhabda canadensis (Puttick
et al. 1988). The upwind walking orientation has also been studied in the bark beetle Ips
typographus (Schlyter and Löfqvist 1986) using aggregation pheromones, and in the moth
Spodoptera littoralis (Anderson et al. 2007; Binyameen 2009) using sex pheromones. Such a
navigational system might rely upon directional cues extracted from the plume’s structure or
temporally monitored changes in the signal intensity.
Wright (1958) pointed out for the first time that the filamentous nature of odor plumes
might provide directional cues. He proposed that flying insects could measure frequency of
bursts to determine sources’ direction, but he later abandoned this idea in favor of
anemotaxis. In wind-tunnel trials with two tortricid moths, continued upwind progress did not
occur in a homogeneous cloud of pheromone, rather a pulsed plume was required (Baker et
al. 1985). They proposed that cessation of upwind progress, which occurs near the pheromone
source, might be triggered by fusion of the receptors’ output when the receptor system could
no longer resolve the individual odor pulses.

Odor Plumes Structure and Its Influence on Insect Sensory Physiology

The ability of an insect to resolve individual bursts of odor is based on the characteristics
of the odor-detection system. The OSNs themselves, at least in the case of moth pheromones,
appear to be very specific and respond to single or closely related components. Among
receptors tuned to plant volatiles both specialized and general responses have been found
(Shields and Hildebrand 2001). The receptor potentials arising from the dendrite are
transduced into action potentials and send information to the CNS. The frequency of such
action potentials is proportional to the odor intensity (Reynolds 2005). The odor molecules
are rapidly degraded by enzymes, so, that receptors are available to respond to a new odor
pulse. Most receptors exhibit a phasic/tonic response to an odor puff of a second or longer
duration. A strong odor causes an initial high-frequency burst of action potentials (the phasic
response), followed by a period of reduced frequency (the tonic response) that persists for the
duration of the odor pulse. At low odor concentrations, only the tonic phase occurs (Baker
1990). In Ips typographus, two OSNs present in the same sensilla show different patterns. In
this case, one responds to an aggregation pheromone component cV, and the second to a
behavioral inhibitory host volatile, 1,8-cineole. The binary blend of these compounds inhibits
the activity of the OSN responding to cV (Andersson et al. 2010). Another study on
Antheraea polyphemus showed that two cells had different response patterns, where one OSN
type could resolve 20-ms pulses at 5 or more stimuli, while the other could only distinguish 2
stimuli/s. Thus the structure of the odor plume might be registered in a different pattern by
each receptor type (Lemon and Getz 1997). The fine scale temporal dynamics and odor
intensity can change the firing pattern of antennal lobe neurons’ output and reflect ongoing
changes in the odor plume (Vickers et al. 2001).
 Odor Plumes and Odor Source Interactions 109

CONCLUSIONS AND FUTURE DIRECTIONS

Insects using their sense of smell are capable of detecting and discriminating between
behaviorally relevant and irrelevant odors in the environment. In consideration of the
complex olfactory environment, insects have relatively few odorant receptors. Nevertheless,
most OSNs respond to a broad range of odorants. Therefore odor coding is done in a
combinatorial fashion; odor identity is encoded by the activation of specific combinations of
OSNs that in turn elicit characteristic activity patterns across glomeruli. In a broad ecological
context spanning diverse taxa of insectsm, commonality among olfactory stimuli and the
OSNs that respond to such common olfactory stimuli in spite of having biases toward
different hosts suggests that complex, higher-order processing of peripheral signals are
necessary for ultimate behavioral decisions. Measurement of chemical signals in time and
space is critical for determining how chemical signaling processes determine ecological and
evolutionary interactions. A multi-level approach is required to investigate and understand the
olfactory environment that insects inhabit and the olfactory neural mechanisms involved in
their host-seeking behaviors. Advances in analytical tools provide exciting opportunities to
characterize the physico-chemical environment and determine olfactory mechanisms, and also
when and where these olfactory-mediated behaviors control ecological interactions (Riffell et
al. 2008). Different physical environments, such as a forest or a crop field, size of the odor
source and the size of an insect itself, can affect the availability of chemical stimuli and may
exert a different selective pressure on the sensory system. This means that different “olfactory
landscapes” will set constraints on insects’ olfactory system and odor-tracking navigational
behavior. Do different insect types (e.g. a moth and a bark beetle) living in different physical
environments (forest and cropping systems, respectively) but inhabiting similar fluid (air)
regimes utilize same scales and equivalent behavioral strategies to locate odor sources? To
answer these questions, studies on insects of different phylogenies (e.g. a small bark beetle
and a large moth) are needed in the laboratory as well as in the field. A further goal will be
the invention and improvement of analytical technologies that will not only increase
understanding of the physico-chemical environment but will also enable scientists to
determine how olfactory landscape shape insect behaviors and sensory systems.

ACKNOWLEDGMENT
I thank Prof. Fredrik Schlyter for his sound recommendations and valuable comments
during the preparation of this chapter. I also thank to my colleagues; Dr. W. Walker III, Dr.
M. N. Andersson and Dr. C. Schiebe, for their linguistic support and valuable comments on
an earlier version of this chapter.

REFERENCES
Ahmad M, Sayyed AH, Saleem MA, Ahmad M. 2008. Evidence for field evolved resistance
to newer insecticides in Spodoptera litura (Lepidoptera: Noctuidae) from Pakistan. Crop
Protect. 27: 1367-1372.
110 Muhammad Binyameen

Anderson P, Hansson BS, Löfqvist J. 1995. Plant-odour-specific receptor neurones on the

antennae of female and male Spodoptera littoralis. Physiol. Entomol. 20: 189-198.
Anderson P, Hansson BS, Nilsson U, Han Q, Sjoholm M, Skals N, Anton S. 2007. Increased
behavioral and neuronal sensitivity to sex pheromone after brief odor experience in a
moth. Chem. Senses 32: 483-491.
Anderson P, Larsson MC, Löfqvist J, Hansson BS. 1996. Plant odour receptor neurones on
the antennae of the two moths Spodoptera littoralis and Agrotis segetum. Entomol. Exp.
Appl. 80: 32-34.
Andersson M. 2007. The effects of non-host volatiles on habitat location by phytophagous
insects. Introductory paper at the Faculty of Landscap Planning, Horticulture and
Agricultural Sciences, SLU, Sweden.
Andersson MN, Binyameen M, Sadek MM, Schlyter F. 2011. Attraction modulated by
spacing of pheromone components and anti-attractants in a bark beetle and a moth. J.
Chem. Ecol. 37: 899-911.
Andersson MN, Larsson MC, Blazenec M, Jakus R, Zhang QH, Schlyter F. 2010. Peripheral
modulation of pheromone response by inhibitory host compound in a beetle. J. Exp. Biol.
213: 3332-3339.
Andersson MN, Larsson MC, Schlyter F. 2009. Specificity and redundancy in the olfactory
system of the bark beetle Ips typographus: Single-cell responses to ecologically relevant
odors. J. Insect Physiol. 55: 556-567.
Ando T, Kishino K, Tatsuki S, Takahashi N. 1980. Sex pheromone of the rice green
caterpillar: chemical identification of three components and field tests with synthetic
pheromones. Agric. Biol. Chem. 44: 765-775.
Anglade P, Stockel J, Cooperators I. 1984. Intraspecific sex-pheromone variability in the
European cOSN borer, Ostrinia nubilalis Hbn.(Lepidoptera, Pyralidae). Agron. J. 4:
183-187.
Anton S, Hansson BS. 1995. Sex pheromone and plant-associated odour processing in
antennal lobe interneurons of male Spodoptera littoralis (Lepidoptera: Noctuidae). J.
Comp. Physiol., A 176: 773-789.
Baker TC. 1990. Upwind flight and casting flight: complimentary phasic and tonic systems
used for location of sex pheromone sources by male moths. In Proceedings of the Tenth
International Symposium on Olfaction and Taste, K. B. Døving, ed. Graphic
Communication Systems Oslo: 18-25.
Baker TC, Fadamiro HY, Cosse AA. 1998. Moth uses fine tuning for odour resolution.
Nature 393: 530-530.
Baker TC, Hansson BS, Löfstedt C, Löfqvist J. 1988. Adaptation of antennal neurons in
moths is associated with cessation of pheromone-mediated upwind flight. Proc. Natl.
Acad. Sci. USA 85: 9826-9830.
Baker TC, Haynes KF. 1989. Field and laboratory electroantennographic measurements of
pheromone plume structure correlated with oriental fruit moth behaviour. Physiol.
Entomol. 14: 1-12.
Baker TC, Willis MA, Haynes KF, Phelan PL. 1985. A pulsed cloud of sex pheromone elicits
upwind flight in male moths. Physiol. Entomol. 10: 257-265.
Bartell RJ, Lawrence LA. 1977. Reduction in responsiveness of male light-brown apple
moths, Epiphyas postvittana, to sex pheromone following pulsed pre-exposure to
pheromone components. Physiol. Entomol. 2: 89-95.
 Odor Plumes and Odor Source Interactions 111

Bartell RJ, Schaner AM, Jackson LL. 1985. cis-Vaccenyl acetate as an aggregation
pheromone in Drosophila melanogaster. J. Chem. Ecol. 11: 1747-1756.
Bau J, Justus KA, Loudon C, Carde RT. 2005. Electroantennographic resolution of pulsed
pheromone plumes in two species of moths with bipectinate antennae. Chem. Senses 30:
771-780.
Bengtsson JM, Wolde-Hawariat Y, Khbaish H, Negash M, Jembere B, Seyoum E, Hansson
BS, Larsson MC, Hillbur Y. 2009. Field attractants for Pachnoda interrupta selected by
means of GC-EAD and single sensillum screening. J. Chem. Ecol. 35: 1063-1076.
Bernays EA, Chapman RF. 1994. Host-plant selection by phytophagous insects. Springer.
Binyameen M. 2009. Plume interaction and odour source spacing of pheromone and non-host
volatiles (NHV). Department of Plant Protection Biology, Division of Chemical Ecology,
Swedish University of Agricultural Sciences, Sweden, Master thesis.
Borden JH, Chong LJ, Gries G, Gries R, Huber DPW, Pierce HDJ, Wilson LM. 2001.
Non-host volatiles as repellents for conifer-infesting bark beetles. US Patent No.
6217891. 26 p.
Bossert WH, Wilson EO. 1963. The analysis of olfactory communication among animals. J.
Theor. Biol. 5: 443-469.
Bruce TJA, Wadhams LJ, Woodcock CM. 2005. Insect host location: a volatile situation.
Trends Plant Sci. 10: 269-274.
Brunner J, Welter S, Calkins C, Hilton R, Beers E, Dunley J, Unruh T, Knight A, Van
Steenwyk R, Van Buskirk P. 2002. Mating disruption of codling moth: a perspective
from the Western United States. IOBC wprs Bull. 25: 11-20.
Byers JA. 1987. Interactions of pheromone component odor plumes of western pine beetle. J.
Chem. Ecol. 13: 2143-2157.
Byers JA. 1995. Host-tree chemistry affecting colonization in bark beetles. Chem. Ecolo.
Insects 154-212.
Byers JA. 1996. Temporal clumping of bark beetle arrival at pheromone traps: Modeling
anemotaxis in chaotic plumes. J. Chem. Ecol. 22: 2133-2155.
Byers JA. 1999. Effects of attraction radius and flight paths on catch of Scolytid beetles
dispersing outward through rings of pheromone traps. J. Chem. Ecol. 25: 985-1005.
Byers JA, Anderbrant O, Löqvist J. 1989. Effective attraction radius. J. Chem. Ecol. 15:
749-765.
Byers JA, Zhang QH, Birgersson G. 2000. Strategies of a bark beetle, Pityogenes bidentatus,
in an olfactory landscape. Naturwiss. 87: 503-507.
Campbell SA, Borden JH. 2006. Integration of visual and olfactory cues of hosts and non-
hosts by three bark beetles (Coleoptera : Scolytidae). Ecol. Entomol. 31: 437-449.
Carde RT. 1996. Odour plumes and odour-mediated flight in insects. Wiley Online Library. p.
54-66.
Carde RT, Minks AK. 1995. Control of moth pests by mating disruption: successes and
constraints. Annu. Rev. Entomol. 40: 559-585.
Cardé RT, Willis MA. 2008. Navigational strategies used by insects to find distant, wind-
bOSNe sources of odor. J. Chem. Ecol. 34: 854-866.
Chatwin PC, Sullivan PJ. 1979. The basic structure of clouds of diffusing contaminant. In
Mathematical Modelling of Turbulent Diffusion in the Environment (edited by Harris
CJ). Academic Press, New York, pp. 3-32.
112 Muhammad Binyameen

Conner WE, Eisner T, Meer RK, Guerrero A, Ghiringelli D, Meinwald J. 1980. Sex attractant
of an arctiid moth (Utetheisa OSNatrix): a pulsed chemical signal. Behav. Ecol.
Sociobiol. 7: 55-63.
Crimaldi J, Koseff J. 2001. High-resolution measurements of the spatial and temporal scalar
structure of a turbulent plume. Exp. Fluids 31: 90-102.
de Bruyne, M. and Baker, T. (2008) Odor Detection in Insects: Volatile Codes. J. Chem.
Ecol., 34, 882-897.
Deng JY, Wei HY, Huang YP, Du JW. 2004. Enhancement of attraction to sex pheromones
of Spodoptera exigua by volatile compounds produced by host plants. J. Chem. Ecol. 30:
2037-2045.
Dickens JC, Smith JW, Light DM. 1993. Green leaf volatiles enhance sex attractant
pheromone of the tobacco budworm, Heliothis virescens (Lep.: Noctuidae). Chemoecol.
4: 175-177.
Dinar N, Kaplan H, Kleiman M. 1988. Characterization of concentration fluctuations of a
surface plume in a neutral boundary layer. Bound-Lay Meteorol. 45: 157-175.
Dunkelblum E, Kehat M, Gothilf S, Greenberg S, Sklarsz B. 1982. Optimized mixture of sex
pheromonal components for trapping of male Spodoptera littoralis in Israel.
Phytoparasitica 10: 21-26.
Durbin P. 1980. A stochastic model of two-particle dispersion and concentration fluctuations
in homogeneous turbulence. J. Fluid Mech. 100: 279-302.
El-Sayed A, Suckling D, Wearing C, Byers J. 2006. Potential of mass trapping for long-term
pest management and eradication of invasive species. J. Econ. Entomol. 99: 1550-1564.
Elkinton JS, Carde RT. 1984. Odor dispersion. In WJ Bell and RT Carde, Eds. Sunderland,
MA: Sinauer: 73-91.
Fackrell JE, Robins AG. 1982. Concentration fluctuations and fluxes in plumes from point
sources in a turbulent boundary layer. J. Fluid Mech. 117: 1-26.
Fadamiro HY, Cosse AA, Baker TC. 1999. Fine-scale resolution of closely spaced
pheromone and antagonist filaments by flying male Helicoverpa zea. J. Comp. Physiol.,
A 185: 131-141.
Faria M, Wraight SP. 2001. Biological control of Bemisia tabaci with fungi. Crop Protect.
20: 767-778.
Farkas SR, Shorey HH, GASTON LK. 1974. Sex pheromones of Lepidoptera. Influence of
pheromone concentration and visual cues on aerial odor-trail following by males of
Pectinophora gossypiella. Ann. Entomol. Soc. Am. 67: 633-638.
Ghaninia M, Larsson M, Hansson BS, Ignell R. 2008. Natural odor ligands for olfactory
sensory neurons of the female mosquito Aedes aegypti: use of gas chromatography-linked
single sensillum recordings. J. Exp. Biol. 211: 3020-3027.
Gifford F. 1959. Statistical properties of a fluctuating plume dispersion model. Adv. Geophys.
6: 117–137.
Hadfield MG, Scheuer D. 1985. Evidence for a soluble metamorphic inducer in Phestilla:
ecological, chemical and biological data. Bull. Mar. Sci. 37: 556-566.
Hadjitofi A, Wilson MJG. 1979. Fast-response measurements of air pollution. Atmos.
Environ. 13: 755-760.
Hagler JR. 2000. Biological control of insects. In: Rechcigl JR, Rechcigl NA, editors. Insect
Pest Management: Techniques for Environmental Protection. New York, NY: Lewis
Publishers: 207-241.
 Odor Plumes and Odor Source Interactions 113

Hajek AE, McManus ML, Delalibera Junior I. 2007. A review of introductions of pathogens
and nematodes for classical biological control of insects and mites. Biol. Control 41:
1-13.
Hanna SR. 1984. The exponential probability density function and concentration fluctuations
in smoke plumes. Bound-Lay Meteorol. 29: 361-375.
Hanna SR, Insley EM. 1989. Time series analyses of concentration and wind fluctuations.
Bound-Lay Meteorol. 47: 131-147.
Hansson BS. 1995. Olfaction in Lepidoptera. Cell. Mol. Life Sci. 51: 1003-1027.
Hedin PA, Mckibben GH, Mitchell EB, Johnson WL. 1979. Identification and field
evaluation of the compounds comprising the sex pheromone of the female boll weevil. J.
Chem. Ecol. 5: 617-627.
Heinbockel T, Christensen TA, Hildebrand JG. 1999. Temporal tuning of odor responses in
pheromone-responsive projection neurons in the brain of the sphinx moth Manduca sexta.
J. Comp. Neurol. 409: 1-12.
Hildebrand JG, Shepherd GM. 1997. Mechanisms of olfactory discrimination: Converging
evidence for common principles across phyla. Annu. Rev. Neurosci. 20: 595-631.
Howell J, Knight AL, Unruh TR, Brown DF, Krysan J, Sell CR, Kirsch PA. 1992. Control of
codling moth in apple and pear with sex pheromone-mediated mating disruption. J. Econ.
Entomol. 85: 918-925.
Ignell R, Hansson B. 2005. Insect olfactory neuroethology - an electrophysiological
perspective. Methods in insect sensory neuroscience. Boca Raton, FL: CRC Press. p:
319–347.
Jactel H, Birgersson G, Andersson S, Schlyter F. 2011. Non-host volatiles mediate
associational resistance to the pine processionary moth. Oecologia 166: 703-711.
James DG, Bartelt RJ, Moore CJ. 1996. Mass-trapping of Carpophilus spp.(Coleoptera:
Nitidulidae) in stone fruit orchards using synthetic aggregation pheromones and a
coattractant: Development of a strategy for population suppression. J. Chem. Ecol. 22:
1541-1556.
Justus KA, Murlis J, Jones C, Cardé RT. 2002. Measurement of odor-plume structure in a
wind tunnel using a photoionization detector and a tracer gas. Environ. Fluid Mech. 2:
115-142.
Kaissling KE. 1971. Insect olfaction. Handb. Sens. Physiol. 4: 351-431.
Kaissling KE. 1974. Sensory transduction in insect olfactory receptors. In Jaenicke, L. (ed.),
Biochemistry of Sensory Functions, 25. Springer, Berlin.: 243-273.
Kárpáti Z, Dekker T, Hansson BS. 2008. Reversed functional topology in the antennal lobe of
the male European cOSN borer. J. Exp. Biol. 211: 2841-2848.
Kennedy JS. 1977. Olfactory responses to distant plants and other odor sources. Chem.
Control Insect Behav. Wiley-Intersci. New York: 67-91.
Koch UT, Lüder W, Clemenz S, Cichon LI. 1997. Pheromone measurements by field EAG in
apple orchards. IOBC wprs Bull. 20: 181-190.
Koehl MAR. 2006. The fluid mechanics of Arthropod sniffing in turbulent odor plumes.
Chem. Senses 31: 93-105.
Komiyama T, Johnson WA, Luo L, Jefferis GSXE. 2003. From lineage to wiring specificity:
POU domain transcription factors control precise connections of Drosophila olfactory
projection neurons. Cell 112: 157-167.
114 Muhammad Binyameen

Krasnoff SB, Roelofs WL. 1988. Sex pheromone released as an aerosol by the moth
Pyrrharctia isabella. Nature 333: 263-265.
Landolt PJ, Phillips TW. 1997. Host plant influences on sex pheromone behavior of
phytophagous insects. Annu. Rev. Entomol. 42: 371-391.
Lei, H., and Vickers, N. 2008. Central processing of natural odor mixtures in insects. Journal
of Chemical Ecology 34: 915-927.
Lemon WC, Getz WM. 1997. Temporal resolution of general odor pulses by olfactory
sensory neurons in American cockroaches. J. Exp. Biol. 200: 1809-1819.
Lewis T, Macaulay EDM. 1976. Design and elevation of sex-attractant traps for pea moth,
Cydia nigricana (Steph.) and the effect of plume shape on catches. Ecol. Entomol. 1:
175-187.
Light DM, Flath RA, Buttery RG, Zalom FG, Rice RE, Dickens JC, Jang EB. 1993. Host-
plant green-leaf volatiles synergize the synthetic sex pheromones of the cOSN earworm
and codling moth (Lepidoptera). Chemoecol. 4: 145-152.
Linn CE, Campbell MG, Roelofs WL. 1986. Male moth sensitivity to multicomponent
pheromones: Critical role of female-released blend in determining the functional role of
components and active space of the pheromone. J. Chem. Ecol. 12: 659-668.
Linn CE, Gaston LK. 1981. Behavioural function of the components and the blend of the sex-
pheromone of the cabbage-looper, Trichoplusia ni (Lepidoptera: Noctuidae). Environ.
Entomol. 10: 751-755.
Linn Jr CE, Campbell MG, Roelofs WL. 1987. Pheromone components and active spaces:
what do moths smell and where do they smell it? Science 237: 650-652.
Liu YB, Haynes KF. 1992. Filamentous nature of pheromone plumes protects integrity of
signal from background chemical noise in cabbage looper moth, Trichoplusia ni. J.
Chem. Ecol. 18: 299-307.
Ljungberg H, Anderson P, Hansson BS. 1993. Physiology and morphology of pheromone-
specific sensilla on the antennae of male and female Spodoptera littoralis (Lepidoptera:
Noctuidae). J. Insect Physiol. 39: 253-260.
Lung T, Müller HJ, Gläser M, Möller B. 2002. Measurements and modelling of full-scale
concentration fluctuations. Agrartech. Forsch-Agr. 8: 5-15.
Mafra-Neto A, Carde RT. 1994. Fine-scale structure of pheromone plumes modulates upwind
orientation of flying moths. Nature 369: 142-144.
Mayer MS, McLaughlin JR. 1991. Discrimination of female sex pheromone by male
Trichoplusia ni (Hubner). Chem. Senses 16: 699-710.
Moore P, Crimaldi J. 2004. Odor landscapes and animal behavior: tracking odor plumes in
different physical worlds. J. Mar. Syst. 49: 55-64.
Moore PA, Atema J. 1991. Spatial information in the three-dimensional fine structure of an
aquatic odor plume. Biol. Bull. Mar. Biol. Lab. Woods Hole 181: 408-418.
Murlis J. 1986. The structure of odour plumes. In Mechanisms in Insect Olfaction, T. L.
Payne, C. E. J. Kennedy, and M. C. Birch, eds. Clarendon Press, Oxford, U K.: 27–38.
Murlis J, Bettany BW. 1977. Night flight towards a sex pheromone source by male
Spodoptera littoralis (Boisd.) (Lepidoptera, Noctuidae). Nature 268: 433-435.
Murlis J, Elkinton JS, Carde RT. 1992. Odor plumes and how insects use them. Annu. Rev.
Entomol. 37: 505-532.
Murlis J, Jones CD. 1981. Fine-scale structure of odour plumes in relation to insect
orientation to distant pheromone and other attractant sources. Physiol. Entomol. 6: 71-86.
 Odor Plumes and Odor Source Interactions 115

Murlis J, Willis MA, Cardé RT. 1990. Odour signals: patterns in time and [Link]
Proceedings of the Tenth International Symposium on Olfaction and Taste, K. B. Døving,
ed. Graphic Communication Systems, Oslo, Norway. p 6–17.
Murlis J, Willis MA, Cardé RT. 2000. Spatial and temporal structures of pheromone plumes
in fields and forests. Physiol. Entomol. 25: 211-222.
Mustaparta H. 1975. Behavioural responses of the pine weevil Hylobius abietis L.(Col.:
Curculionidae) to odours activating different groups of receptor cells. J. Comp. Physiol.,
A 102: 57-63.
Mustaparta H. 2002. Encoding of plant odour information in insects: peripheral and central
mechanisms. Entomol. Exp. Appl. 104: 1-13.
Mwangi EN, Dipeolu OO, Newson RM, Kaaya GP, Hassan SM. 1991. Predators, parasitoids
and pathogens of ticks: a review. Biocontrol Sci. Technol. 1: 147-156.
Mylne K, Mason P. 1991. Concentration fluctuation measurements in a dispersing plume at a
range of up to 1000 m. J. Royal Meteorol. Society 117: 177-206.
Mylne KR. 1992. Concentration fluctuation measurements in a plume dispersing in a stable
surface layer. Bound-Lay Meteorol. 60: 15-48.
Nakamura K. 1976. The effect of wind velocity on the diffusion of Spodoptera litura (F.) sex
pheromone. Appl. Entomol. Zool. 11: 312-319.
Ochieng SA, Park KC, Baker TC. 2002. Host plant volatiles synergize responses of sex
pheromone-specific olfactory sensory neurons in male Helicoverpa zea. J. Comp.
Physiol., A 188: 325-333.
Osbosne L, Landa Z. 1992. Biological control of whiteflies with entomopathogenic fungi.
Fla. Entomol.: 456-471.
Puttick GM, Morrow PA, Lequesne PW. 1988. Trirhabda canadensis (Coleoptera:
Chrysomelidae) responses to plant odors. J. Chem. Ecol. 14: 1671-1686.
Quero C, Lucas P, Renou M, Guerrero A. 1996. Behavioral responses of Spodoptera littoralis
males to sex pheromone components and virgin females in wind tunnel. J. Chem. Ecol.
22: 1087-1102.
Ramakrishnan N, Saxena VS, Dhingra S. 1984. Insecticide resistance in the population of
Spodoptera litura (F.) in Andhra Pradesh. Pesticides 18: 23-27.
Raman B, Joseph J, Tang J, Stopfer M. 2010. Temporally diverse firing patterns in olfactory
sensory neurons underlie spatiotemporal neural codes for odors. Eur. J. Neurosci. 30:
1994-2006.
Raty L, Drumont A, De Windt N, Grégoire JC. 1995. Mass trapping of the spruce bark beetle
Ips typographus L.: traps or trap trees? For. Ecol. Manage. 78: 191-205.
Renwick JAA. 1989. Chemical ecology of oviposition in phytophagous insects. Cell. Mol.
Life Sci. 45: 223-228.
Reynolds AM. 2005. Scale-free movement patterns arising from olfactory-driven foraging.
Physical Rev. 72: 41928.
Riffell J, Abrell L, Hildebrand JG. 2008. Physical processes and real-time chemical
measurement of the insect olfactory environment. J. Chem. Ecol. 34: 837-853.
Roelofs WL, Bjostad L. 1984. Biosynthesis of Lepidopteran pheromones. Bioorg. Chem. 12:
279-298.
Rumbo ER, Suckling DM, Karg G. 1995. Measurement of airbOSNe pheromone
concentrations using electroantennograms: Interactions between environmental volatiles
and pheromone. J. Insect Physiol. 41: 465-471.
116 Muhammad Binyameen

Saint-Germain M, Buddle CM, Drapeau BP. 2007. Primary attraction and random landing in
host selection by wood feeding insects: a matter of scale? Agric. For. Entomol. 9: 227-
235.
Saint-Germain M, Drapeau BP, Hébert C. 2004. Comparison of Coleoptera assemblages from
a recently burned and unburned black spruce forests of northeastern North America. Biol.
Conserv. 118: 583-592.
Sauer AE, Karg G, Koch UT, De Kramer JJ, Milli R. 1992. A portable EAG system for the
measurement of pheromone concentrations in the field. Chem. Senses 17: 543-553.
Schaefer AT, Margrie TW. 2007. Spatiotemporal representations in the olfactory system.
Trends Neurosci. 30: 92-100.
Schlyter F. 1992. Sampling range, attraction range, and effective attraction radius: Estimates
of trap efficiency and communication distance in coleopteran pheromone and host
attractant systems1. J. Appl. Entomol. 114: 439-454.
Schlyter F, Birgersson G, Byers JA, Löfqvist J, Bergström G. 1987. Field response of spruce
bark beetle, Ips typographus, to aggregation pheromone candidates. J. Chem. Ecol. 13:
701-716.
Schlyter F, Löfqvist J. 1986. Response of walking spruce bark beetles Ips typographus to
pheromone produced in different attack phases. Entomol. Exp. Appl. 41: 219-230.
Schneider D. 1969. Insect olfaction: deciphering system for chemical messages. Science 163:
1031-1037.
Schooley RL, Wiens JA. 2003. Finding habitat patches and directional connectivity. Oikos
102: 559-570.
Schoonhoven LM, Van Loon JJA, Dicke M. 2005. Insect-plant biology. Oxford University
Press, USA.
Schwalbe CP, Mastro VC. 1988. Gypsy moth mating disruption: dosage effects. J. Chem.
Ecol. 14: 581-588.
Shields VDC, Hildebrand JG. 2001. Responses of a population of antennal olfactory receptor
cells in the female moth Manduca sexta to plant-associated volatile organic compounds.
J. Comp. Physiol., A 186: 1135-1151.
Shorey HH. 1973. Behavioral responses to insect pheromones. Annu. Rev. Entomol. 18: 349-
380.
Sprayberry JDH, Daniel TL. 2007. Flower tracking in hawk moths: behavior and energetics.
J. Exp. Biol. 210: 37-45.
Stensmyr MC, Giordano E, Balloi A, Angioy AM, Hansson BS. 2003. Novel natural ligands
for Drosophila olfactory sensory neurones. J. Exp. Biol. 206: 715-724.
Strand T, Lamb B, Thistle H, Allwine E, Peterson H. 2009. A simple model for simulation of
insect pheromone dispersion within forest canopies. Ecol. Model. 220: 640-656.
Stranden M, Røstelien T, Liblikas I, Almaas TJ, Borg-Karlson AK, Mustaparta H. 2003.
Receptor neurones in three heliothine moths responding to floral and inducible plant
volatiles. Chemoecol. 13: 143-154.
Sutton OG. 1953. Micrometeorology. McGraw-Hill, New York.
Thistle HW, Peterson H, Allwine G, Lamb B, Strand T, Holsten EH, Shea PJ. 2004.
Surrogate pheromone plumes in three forest trunk spaces: composite statistics and case
studies. For. Sci. 50: 610-625.
Touhara K, Vosshall LB. 2009. Sensing odorants and pheromones with chemosensory
receptors. Annu. Rev. Physiol. 71: 307-332.
 Odor Plumes and Odor Source Interactions 117

Turlings TC, Loughrin JH, McCall PJ, Röse US, Lewis WJ, Tumlinson JH. 1995. How
caterpillar-damaged plants protect themselves by attracting parasitic wasps. Proc. Natl.
Acad. Sci. USA 92: 4169-4174.
Turlings TCJ, Wäckers FL, Vet LEM, Lewis WJ, Tumlinson JH. 1993. Learning of host-
finding cues by hymenopterous parasitoids. In insect learning: Ecological and
evolutionary perspectives, D.R. Papaj, A.C. Lewis, Ed. Chapman and Hall, pp. 51–78.
Van der Pers JNC, Thomas G, Den Otter CJ. 1980. Interactions between plant odours and
pheromone reception in small ermine moths (Lepidoptera: Yponomeutidae). Chem.
Senses 5: 367-371.
Weissburg MJ, Zimmer-Faust RK. 1994. Odor plumes and how blue crabs use them in
finding prey. J. Exp. Biol. 197: 349.
Venkatram A. 1984. The uncertainty in estimating dispersion in the convective boundary
layer. Atmos. Environ. 18: 307-310.
Vet LEM, Dicke M. 1992. Ecology of infochemical use by natural enemies in a tritrophic
context. Annu. Rev. Entomol. 37: 141-172.
Vetter RS, Sage AE, Justus KA, Carde RT, Galizia CG. 2006. Temporal Integrity of an
airbOSNe odor stimulus is greatly affected by physical aspects of the odor delivery
system. Chem. Senses 31: 359-369.
Vickers NJ. 2000. Mechanisms of animal navigation in odor plumes. Biol. Bull. Mar. Biol.
Lab. Woods Hole 198: 203-212.
Vickers NJ, Baker TC. 1994. Reiterative responses to single strands of odor promote
sustained upwind flight and odor source location by moths. Proc. Natl. Acad. Sci. USA
91: 5756-5760.
Vickers NJ, Baker TC. 1997. Chemical communication in heliothine moths. Correlation
between diminished responses to point-source plumes and single filaments similarly
tainted with a behavioral antagonist. J. Comp. Physiol., A 180: 523-536.
Vickers NJ, Christensen TA, Baker TC, Hildebrand JG. 2001. Odour-plume dynamics
influence the brain's olfactory code. Nature 410: 466-470.
Willis MA, Arbas EA. 1991. Odor-modulated upwind flight of the sphinx moth, Manduca
sexta L. J. Comp. Physiol., A 169: 427-440.
Willis MA, Baker TC. 1984. Effects of intermittent and continuous pheromone stimulation on
the flight behaviour of the oriental fruit moth, Grapholita molesta. Physiol. Entomol. 9:
341-358.
Wilson D, Leon M. 1988. Spatial patterns of olfactory bulb single-unit responses to learned
olfactory cues in young rats. J. Neurophysiol. 59: 1770.
Visser JH. 1986. Host odor perception in phytophagous insects. Annu. Rev. Entomol. 31: 121-
144.
Visser JH, Straten S, Maarse H. 1979. Isolation and identification of volatiles in the foliage of
potato, Solanum tuberosum, a host plant of the Colorado beetle, Leptinotarsa
decemlineata. J. Insect Physiol. 5: 13-25.
Witzgall P, Kirsch P, Cork A. 2010. Sex pheromones and their impact on pest management.
J. Chem. Ecol. 36: 80-100.
Vogel S. 1996. Life in moving fluids: the physical biology of flow. Princeton Univ Pr NJ.
Voskamp K, Den Otter CJ, Noorman N. 1998. Electroantennogram responses of tsetse flies
(Glossina pallidipes) to host odours in an open field and riverine woodland. Physiol.
Entomol. 23: 176-183.
118 Muhammad Binyameen

Wright RH. 1958. The olfactory guidance of flying insects. Can. Entomol. 90: 81-89.
Zanen PO, Carde RT. 1996. Effects of host-odour plume altitude and changing wind velocity
on upwind flight manoeuvres of a specialist braconid parasitoid. Physiol. Entomol. 21:
329-338.
Zehnder G, Gurr GM, Kühne S, Wade MR, Wratten SD, Wyss E. 2007. Arthropod pest
management in organic crops. Annu. Rev. Entomol. 52: 57-80.
Zhang QH, Schlyter F. 2003. Redundancy, synergism, and active inhibitory range of non-host
volatiles in reducing pheromone attraction in European spruce bark beetle Ips
typographus. Oikos 101: 299-310.
Zhang QH, Schlyter F. 2004. Olfactory recognition and behavioural avoidance of angiosperm
nonhost volatiles by conifer-inhabiting bark beetles. Agric. For. Entomol. 6: 1-19.
Zhang QH, Schlyter F, Birgersson G. 2000. Bark volatiles from nonhost angiosperm trees of
spruce bark beetle, Ips typographus (L.) (Coleoptera: Scolytidae): Chemical and
electrophysiological analysis. Chemoecol. 10: 69-80.
Zufall F, Munger SD. 2001. From odor and pheromone transduction to the organization of the
sense of smell. Trends Neurosci. 24: 191-193.
In: Lepidoptera ISBN: 978-1-62417-248-9
Editors: Elia Guerritore and Johannes DeSare © 2013 Nova Science Publishers, Inc.

Chapter 4

IMMUNE RESPONSE IN LEPIDOPTERA

1 2 2,
Eileen Uribe-Querol , Nancy Mora and Carlos Rosales
1
División de Estudios de Posgrado e Investigación, Facultad de Odontología,
Universidad Nacional Autónoma de México, Mexico City, Mexico
2
Immunology Department, Instituto de Investigaciones Biomédicas,
Universidad Nacional Autónoma de México, Mexico City, Mexico

ABSTRACT
Insects like many other organisms are exposed to a wide range of infectious agents.
Defense against these agents is provided by innate immune systems, which include
physical barriers, humoral responses, and cellular responses. Much of what we know
about insect immune systems comes from studies on Lepidopteran insects. Caterpillars
are also among the most important agricultural pests, and understanding their immune
systems has enormous implications. Upon infection, Lepidopteran larvae activate cellular
and humoral defense responses. The cellular responses include nodulation, encapsulation,
melanization, and phagocytosis, while the humoral responses lead to the production of
antimicrobial peptides. An early response includes hemocyte adhesion, leading to
phagocytosis, nodule formation, or encapsulation. Microorganisms are recognized by
binding of hemocytes or hemolymph proteins to microbial surface components. This
“pattern recognition” initiates phagocytosis, activates prophenoloxidase and
melanization, and also triggers Serine proteinase cascade pathways that lead to activation
of the cytokine Spätzle, which in turn initiates the Toll pathway leading to expression of
antimicrobial peptides. This chapter describes Lepidoptera innate immune functions with
emphasis on recent advances on our understanding of humoral and cellular responses and
the receptors involved in the recognition of microorganisms.

Keywords: insect; hemocyte; innate immunity; phagocytosis; phenoloxidase; Toll pathway


To whom correspondence should be addressed: Departamento de Inmunología, Instituto de Investigaciones
Biomédicas - UNAM, Apdo. Postal 70228, Cd. Universitaria, México D.F. - 04510, Mexico. Phone: 52 - 55 -
5622 – 8945, FAX: 52 - 55 - 5550 – 3982. E-mail: carosal@[Link].
120 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

INTRODUCTION
Lepidoptera is a large order of insects that includes moths and butterflies. It is one of the
largest insect orders in the world. The name comes from the Greek words “lepido” for scale
and “ptera” for wing, and refers to the scales covering the body and wings of these insects.
The scales are modified, flattened "hairs", and give butterflies and moths their great variety of
colors and patterns. Butterflies and moths are holometabolous insects, meaning they undergo
complete metamorphosis. From eggs, larvae commonly called caterpillars develop. They are
completely different from their adult moth or butterfly form, having a cylindrical body with a
head, mandible mouth parts, and usually eight pairs of prolegs. Larvae will grow through a
series of stages called instars, until matured, and then develop into a pupa, referred to as a
chrysalis in the case of butterflies. Some butterflies and many moth species spin a silk case or
cocoon prior to pupating [1].
Many moth and butterfly species are of economic interest because a great number of
them act as pest species, others produce silk, and some are even food items. The larvae of
many Lepidopteran species are major pests for agricultural crops and forest trees. The larvae
of the Noctuidae genus Spodoptera (armyworms) and Helicoverpa (corn earworm) can cause
extensive damage to certain crops [1]. For silk production, the best example is the
domesticated silkworm moth (Bombyx mori), the larvae of which make their cocoons out of
silk, which can be spun into cloth [2]. As food items, the Maguey worm is considered a
delicacy in Mexico [3], and in the Carnia region of Italy, children catch and eat Zygaena
moths in early summer [4]. In addition, moth larvae have become useful experimental models
for studies on the innate immune system of insects. Many hemolymph proteins with immune
functions were first detected in caterpillars by biochemical methods [5], and Lepidopteran
larvae have also been important for describing the functions of insect hemocytes [6].
Research on Lepidopteran immunity has used large moth species such as the tobacco
hornworm, Manduca sexta, wild silkmoths such as Hyalophora cecropia, the domestic
silkmoth, Bombyx mori, and the wax moth, Galleria mellonella. Also research on immunity
of insects is conducted with moths whose larvae cause extensive damage to agricultural crops
[1], including species in the family Noctuidae [5].
Upon infection, Lepidopteran larvae activate cellular and humoral defense responses.
Early cellular responses include hemocyte adhesion, leading to phagocytosis, nodule
formation, encapsulation, and melanization; while humoral responses lead to the production
of antimicrobial peptides. Microorganisms are recognized by binding of hemocytes or
hemolymph proteins to microbial surface components. This “pattern recognition” initiates
phagocytosis, activates prophenoloxidase and melanization, and also triggers Serine
proteinase cascade pathways that lead to activation of the cytokine Spätzle, which in turn
initiates the Toll pathway leading to expression of antimicrobial peptides. Here, we describe
Lepidoptera innate immune functions with emphasis on recent advances on our understanding
of humoral and cellular responses and the receptors involved in the recognition of
microorganisms.
 Immune Response in Lepidoptera 121

INSECT IMMUNITY
Millions of insect species live in practically every known habitat and ecologicalniche on
land. This diversity exposes insects to all sorts of infectious agents, such as viruses, bacteria,
fungi, and protozoa. Insects have evolved an effective innate immune system that permits
rapid and efficient responses against infectious agents. Their innate immune system consists
of physical barriers, humoral responses, and cellular responses [6, 7].Physical barriers include
the integument and the peritrophic membrane. Integument, the outer surface of an insect, is
formed by a single layer of cells covered by a multi-layered cuticle [8]. The peritrophic
membrane or peritrophic matrix is a chitin and glycoprotein layer that lines the insect midgut.
It is functionally similar to the mucous secretions of the vertebrate digestive tract and hence it
acts as a physical barrier, protecting the midgut epitheliumfrom abrasive food particles,
digestive enzymes, and some digestive pathogens [9, 10]. Together these structures form the
first line of protection for the hemocel (the insect body cavity) and the midgut epithelium
from invading microorganisms. In the case thesebarriers are breached, the humoral and
cellular immune responses are activated. Humoral immune responses include biosynthesis of
antimicrobial peptides, activation of enzymes,such as lysozyme and the prophenoloxidase
(proPO) system, to regulate coagulation of hemolymph, and production of reactive oxygen
species [11, 12]. Cellular immune responses include nodulation, encapsulation, and
phagocytosis [12, 13].
Hemolymph, the liquid that fills the hemocel of an insect, has an analogousfunction to
both blood and lymph in mammals. It is involved in transport of nutrients and waste products,
although not transport of respiratory gases. In addition, it containsseveral types of free-
moving cells or hemocytes. Hemocytes originate from mesodermally derived stem cells that
differentiate into specific lineages. The most common types of hemocytes are granulocytes,
plasmatocytes, spherulocytes, and oenocytoids [6, 14].However, it is important to emphasize
that not all these hemocyte types exist in all insect species [15-17].
At the onset of an infection there are early systems, such as constitutive expression of
antimicrobial peptides, recognition of microorganisms by pattern-recognition proteins, and
activation of phagocytic cells, involved in detecting and eliminating pathogens. These
responses are the innate immune system. For insects, the view is that they, like other
invertebrates, depend only on its innate immune response to fight invading microorganisms;
by definition, innate immunity lacks adaptive characteristics. However, there are some reports
showing that priming M. sexta caterpillars with the non-pathogenic bacterium Escherichia
coli, protects against an otherwise-lethal second challenge of the highly virulent insect
pathogen Photorhabdus [18]. And pre-exposure of larvae of G. mellonella to Saccharomyces
cerevisiae protects against a subsequent infection with Candida albicans cells [19]. These
results point out that insect immune responses can indeed adapt, and suggest that insect
hemocytes may also present an activation response similar to the one known in mammalian
leukocytes [20].
122 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

CELLULAR IMMUNE RESPONSES

Cellular immune responses are immediate after an invasion of the hemocel, while
humoral responses appear several hours after an infection. Hemocytes are responsible for a
variety of defense responses in insects. There are various types of hemocytes described in
Lepidoptera, including granular cells and plasmatocytes. These hemocytes are capable of
adhesion and phagocytosis [6]. Other types of hemocytes are oenocytoids, which can produce
prophenoloxidase (proPO) and small spherule cells, whose functions are not clear. This
classification of hemocytes, based on morphology, does not always correlate well with cell
function. Thus other attempts have been made to classify hemocyte types. By flow cytometry
three major types of hemocytes can be separated: large granular cells, small semigranular
cells, and small hyaline cells [21]. Also, there are some monoclonal antibodies that can
distinguish hemocytes based on antigenicity rather than morphology [22-25]. In Pseudoplusia
includens two types of hemocytes were found [24], while in M. sexta four types of hemocytes
were distinguished in one report [23], and two subpopulations of plasmacytoids were detected
in another report [25]. This variation may reflect different stages of differentiation or
functional phenotypes of cells. A number of monoclonal antibodies could also inhibit some
cellular responses [26, 27]. In addition, the number and types of hemocytes vary through
hematopoiesis and larval development [28, 29]. Clearly, a better classification of insect
hemocytes, based on molecular markers and function, is needed. Thus, these results underline
the importance of monoclonal antibodies for classifying hemocyte types and for describing
particular cellular functions. New and better-characterized monoclonal antibodies against
insect hemocytes will certainly become available in the future.
Insect immune responses initiate with adhesion of granular hemocytes and plasmatocytes
to foreign surfaces or to other cells [30]. Adhesion of both types of hemocytes leads to
phagocytosis, and also to nodule formation and encapsulation. Adhesion of hemocytes to
injured body wall probably helps to seal wounds and prevent bleeding as well [30].

Phagocytosis

Phagocytosis is the process by which cells recognize, bind, and ingest relatively large
particles [31]. In insects, phagocytosis is performed by a subset of hemocytes in the
hemolymph [13]. Professional phagocytes in Diptera and Lepidoptera have also been
described as plasmatocytes and granular hemocytes, respectively [6, 32]. In agreement with
this, plasmatocytes or granulocytes are the main phagocytic cells in most insects [16, 21, 33-
35]. Interestingly, a hyperphagocytic cell type, large hemocytes capable of ingesting up to
500 bacteria, has been described in M. sexta [36]. Phagocytic cells are also capable of
stimulating other hemocytes to increase phagocytosis. In G. mellonella phagocytosis-
stimulating factors have been reported [37, 38], but their molecular nature remains to be
elucidated. It seems however, that they could be small peptides or members of the eicosanoid
family [37]. Cytokines capable of activating hemocyte functions have also been reported in
Lepidoptera.
 Immune Response in Lepidoptera 123

A hemocyte chemotactic peptide from Pseudaletia separata induces migration and

aggregation of hemocytes [39]. This peptide belongs to a group of Lepidopteran cytokines
called ENF peptides, which have various biological activities, including plasmatocyte
adhesion and spreading, and release of proPO activation [40-43].

Nodulation
When the initial phagocytic immune response is not sufficient, hemocytes activate other
mechanisms to control infections. Nodulation is a predominant cellular response in insects to
large bacterial infections. It consists of formation of multicellular hemocyte aggregates that
entrap large numbers of bacteria. It begins when hemocytes, together with antimicrobial
molecules in the hemolymph, surround bacteria. Hemocytes with their entrapped bacteria
form small aggregates that grow by joining with additional hemocytes to form large nodules.
The nodule is completed by covering it with layers of flattened hemocytes. In some cases, but
not always, nodules are also melanized. These melanin-covered nodules are an effective way
to isolate the invading bacteria from the hemolymph, since they have an impermeable wall
between the nodule and the rest of the insect organism. The process of nodule formation is
not completely characterized, but clearly eicosanoids are likely to be important for nodule
formation in many insect species [44-48], and prophenoloxidase (proPO) and dopa
decarboxylase (Ddc) are also involved in this process. In addition, screenings for novel
immune genes from an Indian saturniid silkmoth (Antheraea mylitta) larvae, and from the
silkworm (B. mori) larvae, identified two proteins, Noduler [49] and Reeler1 [50]
respectively, with a characteristic reeler domain in the fat-bodies of these insects. These
proteins were essential in mediating nodulation response against E. coli K12 and Bacillus
subtilis bacteria challenge.

Encapsulation

Encapsulation is the response of hemocytes to large targets such as parasites, protozoa,

and nematodes. Hemocytes bind to the target in multiple cell layers until they form a capsule
around the invader. The capsule is normally melanized at the end [51]. Inside the capsule the
invading organism is killed by reactive cytotoxic products or by asphyxia [52, 53]. Insect
hemocytes aggregate in multiple layers during encapsulation and bind to microorganisms
during phagocytosis. These functions can be mediated by integrins [54] and indeed various
integrins have been found in insect hemocytes [32].  integrins are also relevant for
encapsulation by M. sexta hemocytes [55, 56]. Various  and  integrins are required for
microbial recognition by granulocytes and plasmatocytes of P. includens [57].

Melanization

Melanization is the process of melanin formation. It is activated during wound healing

and also in nodule and capsule formation against large pathogens or parasites in some
Lepidopteran and Dipteran insects, such as Manduca, Pseudolusia, and Drosophila [6, 7, 58,
124 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

59]. The enzyme phenoloxidase (PO) is key in this process. Activation of proPO to PO [60,
61] is mediated by a Serine proteinase cascade [62], and requires pattern-recognition proteins
such as peptidoglycan recognition protein (PGRP) [63, 64] or -1,3-glucan recognition
protein (GRP) [65, 66]. Interaction of these recognition proteins with microbial peptide-
glycan or -1,3-glucan molecules initiates the activation of the proPO cascade. Then PO
binds to foreign surfaces including hemocyte membranes [67], where it initiates melanin
formation. PO acts on tyrosine and converts it to dopa [68]. Dopa can then be decarboxylated
by Ddc to dopamine or further oxidized by PO to dopaquinone. Both products are then further
metabolized to eumelanin and finally melanin [68].

RECOGNITION OF MICROORGANISMS
Phagocytosis and other innate immune responses of insects can be triggered through the
interaction of phagocyte membrane receptors or plasma proteins with specific molecules,
such as lipids or sugars, which form part of the cell wall of many microorganisms [32, 69].
Some of these pattern-recognition proteins were first identified in silkworms, and seem also
to be present in other insect groups, whereas some other proteins seem to be unique to
Lepidoptera [7]. Pattern-recognition proteins can be grouped in various types including
peptidoglycan recognition protein (PGRP) [63, 64], -1,3-glucan recognition protein (GRP),
hemolin, C-type lectins, lipophorins, and immune-stimulatory peptides.

Peptidoglycan Recognition Proteins (PGRPs)

Peptidoglycan recognition proteins (PGRPs) are innate immunity proteins, conserved

from insects to mammals, which recognize bacterial peptidoglycan, and function in
antibacterial immunity and inflammation. PGRPs have at least one carboxy-terminal PGRP
domain (approximately 165 amino acids long), which is homologous to bacteriophage and
bacterial type 2 amidases. Mammals have four PGRPs [70, 71]. They are secreted proteins
expressed in polymorphonuclear leukocytes (PGRP1), in liver (PGRP2), or on body surfaces
and in secretions (PGRP3 and PGRP4). All PGRPs recognize bacterial peptidoglycan and
three of them (PGRP1, PGRP3, and PGRP4 are directly bactericidal for both Gram-positive
and Gram-negative bacteria [71]. PGRPs were first discovered in Lepidoptera. B. mori
PGRP-S1 was isolated from hemolymph of silkwork larvae as a 19 kDa proteins that binds to
Micrococcus luteus peptidoglycan and activates the proPO system [64]. Insects have up to 19
PGRPs, classified into short (S) and long (L) forms. The short forms are present in the
hemolymph, cuticle, and fat-body cells, whereas the long forms are mainly expressed in
hemocytes [72, 73]. Lepidoptera PGRPs have been cloned from B. mori [63], M. sexta [74,
75], G. mellonella [76], Samia cynthia [77], Trichoplusia ni [78], Plutella xylostella [79], and
Ostrinia nubilalis [80, 81]. The expression of insect PGRPs is often upregulated by exposure
to bacteria. In B. mori larvae, PGRP-S1 is upregulated after injection of Enterobacter cloacae
[63]. M. sexta PGRP can stimulate proPO activation [82]. Insect PGRPs activate the Toll or
Immune deficiency (Imd) signal transduction pathways (described later) or induce proteolytic
cascades that generate antimicrobial products [83, 84].
 Immune Response in Lepidoptera 125

Beta-1,3-glucan Recognition Proteins (GRPs)

Insect -1,3-glucan recognition proteins (GRPs) and Gram-negative bacteria binding

proteins (GNBPs) are a family of plasma proteins with an amino-terminal glucan-binding
domain and a carboxyl-terminal region similar to -1,3-glucanases. GRP1 from B. mori
[63], GRP1 and GRP2 from M. sexta [85, 86], and GRP from Plodia interpunctella [87],
all bind to -1,3-glucans and to bacteria and can activate the proPO cascade. M. sexta GRP1
is constitutively expressed in fat-body, whereas GRP2 gene expression is increased during
the early wandering stage prior to pupation or after and immune challenge [85, 86]. Binding
of these GRPs to hemolymph proteinase-14 precursor (proHP14) (see later) induces
autoactivation of HP14 to initiate a proteinase cascade leading to proPO activation [88, 89].
A GRP with glucanase activity was isolated from midgut extract of Helicoverpa armigera
larvae. This enzyme hydrolyzes -1,3-glucan but not -1,4-glucan, and it probably functions
more as a digestive enzyme than an immune activator [90]. A GNBP from B. mori binds E.
cloacae but not to Bacillus licheniformis most likely via recognition of lipopolysaccharide
[91].

Hemolin

Hemolin is a plasma protein first identified in H. cecropia [92, 93] and M. sexta [94, 95].
It is a 48 kDa protein with four immunoglobulin (Ig) domains commonly found in adhesion
molecules of vertebrates [96]. Hemolin is a common protein in several Lepidopteran species,
including B. mori [97], Hyphantria cunea [98], Lymantria dispar [99], Antheraea pernyi
[100], A. mylitta [101], P. xylostella [79], S. cynthia [102], but it has not been identified in
insects from other orders. Hemolin binds to bacterial lipopolysaccharide (LPS) and
lipoteichoic acid [103, 104]. Its expression is induced by bacteria or by LPS injected into the
hemocel. In H. cecropia this transcriptional activation is controlled by an intronic enhancer
that contains a B motif [105]. Hemolin also associates with hemocytes [106], thus serving as
a bridge between microorganisms and hemocytes, and inducing phagocytosis or nodulation.
Reducing hemolin expression with RNAi in M. sexta larvae resulted in much less
phagocytosis and nodulation of E. coli [107]. In an attempt to reduce hemolin expression with
RNAi in the Chinese oak silk moth A. pernyi, it was found that hemolin was induced by
double-stranded RNA per se [108]. In line with this report, Baculovirus infection of the
noctuid moths Cotton bollworm (Helicoverpa zea) and Tobacco budworm (Heliothis
virescens), induced high expression of hemolin [109]. This enhanced expression was not
induced by bacterial infections. These reports suggest that hemolin has an important role
during viral infections of Lepidoptera [110].

C-type Lectins (CTLs)

C-type lectins (CTLs) from animals are a large group of carbohydrate-recognition

molecules that bind ligands in a calcium-dependent manner [111]. B. mori LPS-binding
protein (LBP or CTL20) [112, 113], M. sexta immulectin-1 [114], and H. cunea Hdd15 [115]
126 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

are some of the first identified C-type lectins in Lepidopteran larvae. Other C-type lectins
from Lepidoptera include immulectins-2, -3, and -4 from M. sexta [114, 116, 117], MBP
(CTL10) [118], CTL11, CTL19, CTL21 [97], LEL-1, and LEL-2 [119], from B. mori, and
Ha-lectin from H. armigera [120]. All these lectins have two carbohydrate-recognition
domains, and their genes suggest that these type of lectins are rather unique to Lepidoptera,
since they have not been found in other insect species [5], except for one in the beetle
Tribolium castaneum [121].
Most Lepidopteran CTLs bind to bacterial LPS and some also to lipoteichoic acid [113,
115-117], inducing agglutination of bacteria and yeast [114, 117, 118], probably because each
of the two carbohydrate-binding domains bind to sugar residues on the surface of adjacent
microbial cells [5]. This microbial aggregation may help hemocytes eliminate pathogens via
phagocytosis and nodule formation. Indeed, M. sexta immulectin-2 increases clearance of
Serratia marcesens [122] and inhibition of its expression by RNAi reduced larval survival
after Photorhabdus infection [18]. In addition, the immulectins can promote proPO activation
and melanization of microorganism encapsulated by hemocytes [51, 114, 116, 123], leading
to more efficient elimination of pathogens.

Lipophorins

Lipophorins are hemolymph proteins that transport lipids between insect tissues [124,
125]. Two protein subunits, apolipophorin-I and apolipophorin-II bind lipids to form a
lipophorin particle. In addition, Apolipophorin III (apoLp-III) is a low molecular weight
lipoprotein that plays an important role in the enhanced neutral lipid transport during insect
flight [126]. The protein exists in lipid-free and lipid-bound states. The lipid-bound state is
the active form of the protein and occurs when apoLp-III associates with lipid-enriched
lipophorins [126]. ApoLp-III also associated with low-density lipophorin (LDLp) during
embryogenesis, presumably to provide lipids for organogenesis [127]. Lipophorin seems also
to be involved in hemolymph clotting [128], and in several defense mechanisms [30],
including binding to LPS [129] and to fungal conidia and -1,3-glucan [130], therefore acting
as a pattern recognition molecule for multiple microbial pathogens. The lipopolysaccharide-
binding capacity of lipophorins also contributes to detoxification of hemolymph [131].

Immune-stimulatory Peptides

Microbial pathogens produce proteinases to degrade host proteins and use them as
nutrients, or to block host defense actions. Thermolysin metalloproteinases from bacteria and
fungi are important virulent factors [132]. Fortunately, these microbial proteinases can also be
recognized by the innate immune system of insects. Such a sensing mechanism has been
described in G. mellonella [133, 134]. Thermolysin is also a potent activator of the proPO
system [135]. In addition, microbial proteinases can in G. mellonella, also generate peptide
fragments that are themselves strong inducers of antimicrobial peptide production [136]. The
stimulatory peptides were identified as collagen IV fragments containing the RGD motif,
which bind to integrins on hemocytes [137]. In addition, G. mellonella expresses in
 Immune Response in Lepidoptera 127

hemolymph an inducible metalloproteinase inhibitor (IMPI). This is the only specific peptide
inhibitor of thermolysin-like metalloproteinases reported from any animal [138]. The IMPI
gene codes for two distinct inhibitors [139], those surely contribute to control microbial
proteinases [132, 140].

INSECT HUMORAL RESPONSES

Prophenoloxidase (proPO)

The prophenoloxidase (proPO) system is an important component of the innate immune

system in insects. It is activated by bacteria infections and participates in several humoral and
cellular responses including melanization, wound healing, encapsulation, nodulation, and
phagocytosis [60]. After bacterial challenge, cell-free and membrane-bound proPO is
converted to active PO (Figure 1) through limited proteolysis by Serine proteinases [141,
142]. Active PO then uses Tyrosine, dopa, and dopamine to catalyze the formation of
quinones that are reactive intermediates for melanization, and nodulation [11, 67]. PO-
mediated oxidation of dopamine results in production of 5,6-dihydroxyindole, which presents
antimicrobial activity against bacteria and fungi [143]. PO is produced as a proenzyme,
proPO, which requires a specific proteolitic cleavege to be converted into active PO. This
mechanism ensures protection to the insect tissues from the harmful effects of the reactive
chemicals produced by PO. Thus, proPO activation is tightly regulated by a Serine proteinase
cascade that gets triggered only during infection or tissue damage. In Lepidoptera insects such
as B. mori and M. sexta, proPO is present in the hemolymph as a heterodimer of two 80-kDa
subunits [144, 145]. Activation of proPO involves cleavage of a conserved Arg-Phe bond
about 50 amino-acid residues from the amino-terminal [7]. Serine proteinase cascades found
in animals, control rapid responses to pathogen infection, tissue damage, or physiological
signals [62]. In insects, many of the Serine proteinases involved in the proPO activation
system, present a carboxy-terminal Serine proteinase domain similar to trypsin or
chymotrypsin and one or two amino-terminal clip-domains, which seem to have regulatory
functions [146]. Most of these Serine proteinases responsible for proPO activation are clip-
domain proteinases, and many of them are expressed in fat-body or hemocytes [147]. In the
B. mori genome 15 clip-domain proteinases have been identified [97].
In Lepidoptera, proPO-activating proteinases (PAPs) have been described in great detail
in B. mori [148-150] and M. sexta [151]. The PAPs are present in hemolymph as proenzymes
at low concentrations in naïve larvae, but their expression is upregulated in fat-body after a
bacterial infection. Purified PAP1, PAP2, and PAP3 from M. sexta did not activate proPO
efficiently [152]. This led to the identification of protein cofactors in the hemolymph that
allowed complete proPO activation. These cofactors are Serine proteinase homologs (SPHs)
that contain clip-domains, but lack proteinase activity due to substitution of a Ser in the active
site for Gly (214-215). SPH1 and ShP2 from M. sexta are also produced as proenzymes that
require cleavage to achieve function [152, 153]. It is not clear however what are the
molecules responsible for the initial activation of these proSPHs, although PAP1 may be one
of them [154] (Figure 1).
128 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

Figure 1. Prophenoloxidase (proPO) activation system in Lepidoptera. Model of Serine Proteinase

Cascades in M. sexta, leading to phenoloxidase activation. Detection of microorganisms by
peptidoglycan recognition protein (PGRP) or -1,3-glucan recognition protein (GRP) [not shown] and
binding of these pattern-recognition receptors to hemolymph proteinase-14 precursor (proHP14)
induces autoactivation of HP14 to initiate a proteinase cascade. Active HP14 turns hemolymph
proteinase-21 precursor (proHP21) into active HP21, which in turn can activate proPO-activating
proteinases 2 and 3 (proPAP2 and proPAP3) into their active forms. Active PAP2 and PAP3, then
convert cell-free and hemocyte membrane-bound prophenoloxidase (proPO) into active phenoloxidase
(PO). Active PO then catalyzes tyrosine into dopa. Another enzyme, dopa decarboxyilase (Ddc) is also
expressed on the hemocyte surface and it transforms dopa into dopamine. PO can also use dopa and
dopamine as substrates to form quinones that are reactive intermediates for melanization, nodulation,
and phagocytosis. Activation of proPO can also be accomplished by PAP1, when the cofactors Serine
proteinase homologs (SPHs) are present in a high molecular complex. HP6, whose direct activator is
not known, activates proPAP1. Active PAP1 can also activate proSPH1 and proSPH2 and indirectly
proHP6, thus generating a positive feedback loop.

Complete activation of the M. sexta proPO system also requires other serine proteinases.
Hemolymph proteinase 14 (HP14) functions as an initiator of the proteolytic cascade after its
interaction with -1,3-glucagon and GRP [88]. The recognition of pathogen molecular
patterns through the amino-terminal regulatory domain of HP14 [89], induces HP14
autoactivation by cleavage after Leu387 [155]. Once activated, HP14 activates another clip-
domain proteinase HP21, which in turn can activate proPAP2 and proPAP3 [155, 156],
leading finally to proPO activation. ProPAP1 gets activated by another proteinase HP6 [157].
Active PAP1 can then activate SPH1 and SPH2, and also HP6 itself, and HP8 [154]. This
forms a positive feedback loop that results in high levels of PO activity (Figure 1).
 Immune Response in Lepidoptera 129

Negative Regulation of Prophenoloxidase

Serine proteinase cascades result in proPO activation, but PO products are not only
microbicidal, but also can be harmful to insect own tissues. To control the proteinase activity,
there are proteinase inhibitors in the hemolymph of insects [158]. Serpins are one group of
these inhibitors. They are approximately 50-kDa proteins found in the plasma of vertebrates
and hemolymph of invertebrates and are irreversible inhibitors of proteinase cascades [159].
Serpins have been reported in many Lepidopteran species including B. mori [160, 161], M.
sexta [162-165], H. cunea [98], Mythimna unipuncta [166], and Mamestra configurata [167].
Serpin genes use alternative splicing that results in several serpin isoforms. In M. sexta,
serpin-1 gene present 12 different versions [168], originating several serpin molecules with
diverse inhibitory activity [169]. In B. mori, serpin has three isoforms [170], and in M.
configurata serpin-1 has 9 isoforms [169].
Serpins from M. sexta are the best studied. Serpin-1A, -1E, and -1J can inhibit HP8 [171].
Serpin-1J also inhibits the three PAPs to control proPO activation [150, 151, 153, 172].
Serpin-1I can inhibit HP14 [88, 89]. Serpin-4 inhibits HP1, HP6, and HP21, which are
upstream of PAPs, thus controlling proPO activation [164]. Serpin-6 inhibits PAP3 to control
proPO activation, and also inhibits HP8 to control the Toll pathway [173].

Antimicrobial Peptides (AMPs)

Phagocytosis is the earliest and arguably the most important immune response in insects
[32]. Phagocytosis efficiently clears infection when numbers of bacteria are relatively low.
Larger numbers of microorganism induce other cellular responses such as aggregation of
hemocytes and nodule formation. Other mechanisms to deal with invading pathogens also
exist in insects. Upon microbial infection a series of antimicrobial peptides (AMPs) and
proteins are produced and released into the hemolymph [11]. Expression of these AMPs
comes mainly from fat-body although hemocytes also contribute to their production [68].
AMPs are also found in the midgut of Lepidopteran prepupae, where they probably have a
protective role against infection during metamorphosis [174]. The major groups of AMPs are
listed next.
Lysozyme from G. mellonella was the first antimicrobial protein described in insects and
it is structurally similar to C-type lysozyme (from chicken) [175]. Lysozyme has anti Gram-
positive bacterial activity due to its capacity to degrade bacterial cell wall peptidoglycans. It
also presents modest activity against Gram-negative bacteria [175-177] and against some
fungi [178]. Recently, a regulatory role of M. sexta lysozyme over proPO activation has been
reported [179].
Amphipathic peptides present antimicrobial activity due to their capacity to damage
pathogen cell membranes. The first amphipatic antimicrobial peptide from insects was
identified in hemolymph of the silkworm H. cecropia and was named cecropin [180]. Now,
several cecropin family genes from many lepidoptera species are known. In B. mori, 13
cecropin genes were found [97]. Cecropins are aproximatelly 4-kDa basic peptides with an
amphipatic -helix conformation [181]. Moricins are another group of amphipatic -helical
antimicrobial peptides [182] found first in B. mori [183]. In the B. mori genome nine moricin
130 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

genes were found [97], and in G. mellonella eight moricin homologs are reported to have
activity against bacterial as well as against yeast and filamentous fungi [184].
Attacins are glycine-rich AMPs found in lepidoptera [185]. Two attacin isoforms, one
acid and one basic, have been cloned from H. cecropia [186] and they induce an increase of
permeability of the outer-membrane of bacteria, binding mainly to LPS. This explains why
the basic attacin is more effective against E. coli than the acid attacin [186]. Attacins have
also been cloned from other Lepidoptera such as the beet armyworm, Spodoptera exigua
[187].
Gloverins and lebocins are proline-rich AMPs also found in Lepidoptera. These peptides
also inhibit bacterial growth by blocking outer-membrane protein sytnthesis [188]. Gloverins
have been reported in many Lepidopteran species [17, 76, 189-191]. Gloverins present
antibacterial and antifungal activity [190, 191], but recently it has also been reported that they
may have antiviral activity [192]. Similarly, lebocins have been reported in several
Lepidopteran species [193-197].
Insect defensins are another group of AMPs, characterized by having three or four
stabilizing intramolecular disulfide bonds. The name comes from their molecular similarity to
mammalian  and  defensins [181]. Insect defensins form two groups, one with peptides
presenting -helix/-sheet mixed structure and peptides presenting triple-stranded antiparallel
-sheets. Defensins with antibacterial and antifungal activity have been reported in many
Lepidopteran species [198-202]. Particularly, two cysteine-rich defensin-like peptides from
G. mellonella are reported to selectively reduce growth of filamentous fungi [200, 201].

SIGNALING PATHWAYS INDUCING

ANTIMICROBIAL PEPTIDE SYNTHESIS
Once a microorganism is detected by an immune cell, a series of signaling molecules are
activated inside the cell to instruct it for different responses. These molecules follow
particular signaling pathways that determine the final cellular response. In insects, the
signaling pathways involved in humoral immune responses are best described in the fruit fly
Drosophila melanogaster [35] (Figure 2). The humoral immune responses mainly involve the
release of antimicrobial peptides by the fat-body, via the Toll [203] and the Immune
deficiency (Imd) pathways [204]. Gram-positive bacteria and fungi predominantly induce the
Toll signaling pathway, whereas Gram-negative bacteria activate the Imd pathway.

Toll and Imd Signaling Pathways in Lepidoptera

D. melanogaster Toll pathway was initially identified as a developmental pathway.

It involves NF-κB signaling and is essential for embryonic development and immunity [203,
205]. From there, the subsequent characterization of Toll-like receptors (TLRs) has changed
our understanding of the mammalian immune system [206]. Activation of the transmembrane
receptor Toll requires a proteolytically cleaved form of an extracellular cytokine-like
polypeptide, Spätzle [207], suggesting that Toll requires cooperation of other pattern-
recognition receptors. This idea is supported by the fact that a mutation in a peptidoglycan
 Immune Response in Lepidoptera 131

recognition protein (PGRP-SA) blocks Toll activation by Gram-positive bacteria

(Micrococcus luteus, Streptococcus faecalis, Bacillus thuringiensis) and significantly
decreases resistance to this type of infection [208]. Toll activation is not only mediated by
PGRPs, but it requires GNBP1 (Gram-negative binding protein 1) for Gram-positive
(Micrococcus luteus or Enterococcus faecalis) bacterial infections [209], and GNBP3 for
fungal (Beauveria bassiana, M. anisopliae, C. albicans, Candida glabrata, Saccharomyces
cerevisiae, and Aspergillus fumigatus) infections [210, 211]. In addition, the Drosophila
Persephone proteinase activates the Toll pathway when proteolytically matured by secreted
fungal virulence factors [210]. Toll activation initiates the signaling pathway
Myd88/Pelle/Tube that leads to degradation of Cactus (an IB inhibitor) and translocation to
the nucleus of the NF-B transcription factors Dorsal and Dif [203, 212, 213] (Figure 2A).

Figure 2. Toll or Immune deficiency (Imd) signal transduction pathways in Drosophila. A) Activation
of the transmembrane receptor Toll requires a proteolytically cleaved form of an extracellular cytokine-
like polypeptide, Spätzle. After a complex of 4-Spätzle:2-Toll is formed the signaling pathway
Myd88/Pelle/Tube leads to phosphorylation and degradation of Cactus (an IB inhibitor) and
translocation to the nucleus of the NF-B transcription factors Dorsal and Dif. These factors in turn
activate transcription of antimicrobial peptides (AMP). B) The Imd pathway is activated by binding of
peptidoglycan (PGN) to the PGRP-LC receptor. This initiates signaling to the transcription factor
Relish. Imd is connected to the caspase DREDD via the adaptor protein Fas-associated DD protein
(FADD). DREDD is believed to have two functions: upstream, it activates the TAK1/IKK complex,
which in turn phosphorylates Relish; downstream, DREDD cleaves phosphorylated Relish. The N-
terminal NF-B module of Relish then translocates into the nucleus where it activates transcription of
antimicrobial peptides (AMP).
132 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

D. melanogaster Imd pathway is activated by the PGRP-LC receptor [214] and initiates
signaling to the transcription factor Relish [215], via the pathway FADD/DREDD and the
pathway TAK1/IKK [204, 216, 217] (Figure 2B).
In Lepidoptera, the existence of functional Toll and Imd pathways has recently been
demonstrated [218]. Genomic analysis of B. mori indicates that there are 14 Toll-like
receptors in its genome [97]. BmToll and BmToll2 genes are upregulated in fat-body after
injection of microorganisms [219]. In M. sexta, a Toll-like receptor is present in hemocytes,
fat-body, epidermis, and midgut. Its expression is also upregulated in hemocytes, but not in
fat-body, after injection of microorganisms [220].
Spätzle, the ligand for Toll has been described in B. mori and M. sexta [221, 222]. An
inactive precursor, proSpätzle requires proteolytic processing to release the active form
Spätzle. In M. sexta, proSpätzle expression is greater in hemocytes than fat-body [221], and in
B. mori expression was reported in fat-body, but hemocytes were not tested [222]. Purified
Spätzle from both species, when injected into larvae, induced the production of AMPs,
including attacins, cecropins, and moricin, from the fat-body, [221, 222]. In contrast,
proSpätzle had little effect; confirming with this the need for proteolytic activation of this
cytokine-like polypeptide.
Hemolymph proteinase-8 (HP8) is a clip-domain proteinase shown to activate proSpätzle
in M. sexta [157]. HP8 in turn is also expressed as a proenzyme in hemolymph. Activation of
proHP8 into active HP8 is achieved by another clip-domain proteinase, HP6, an apparent
ortholog of Drosophila Persephone [157]. Injection of active HP8, or active HP6 into larvae
induces the expression of the AMPs cecropin, gloverin, and morecin [157]. ProHP6 is
activated in hemolymph exposed to bacteria or to the -1,3-glucan curdlan. Because HP6 also
activates proPAP1 (see Figure 1), it becomes a point of convergence for the Serine proteinase
cascades that activate proPO activation and that induce the production of AMPs via the
Toll/Spätzle pathway. However, the proteinase responsible for proHP6 activation remains
unknown (Figure 3).
As mentioned above, induction of the Drosophila Toll and Imd pathways leads to
activation of NF-B transcription factors (Dorsal, Relish). In B. mori two sets of genes also
code for NF-B transcription factors. BmRel codes, by alternative splicing, for RelA and
RelB, which are orthologs of Drosophila Dorsal. Similarly, BmRelish, also codes for Relish1
and Relish2. RelA activated the lebocin-4 gene, while RelB induced the attacin gene [223].
When BmRel expression was knocked down in transgenic silkworms, antimicrobial peptides
were not induced by the bacteria M. luteus. Similarly, knock down of BmRelish resulted in no
production of antimicrobial peptides in response to E. coli [224]. In addition, Rel1 and Rel2
were shown to be negatively regulated by B. mori Cactus (IB) [225]. Together, these reports
indicate that the Toll (Figure 3) and Imd pathways are functional in the silkworm to regulate
the expression of antimicrobial peptides. Very recently co-immunoprecipitation assays
showed that MsToll-(ecto) (the ecto-domain of M. sexta Toll) could interact with MsSpz-
C108 (the active C-terminal C108 domain of M. sexta Spätzle) but not with full-length MsSpz
(M. sexta Spätzle) [218]. Moreover, In vivo assays showed that activation of AMP genes,
including cecropin, attacin, moricin and lebocin, in M. sexta larvae by purified recombinant
MsSpz-C108 could be blocked by pre-injection of antibody to MsToll [218]. These data have
further confirmed that a functional Toll-Spätzle pathway is present in M. sexta.
 Immune Response in Lepidoptera 133

Figure 3. Toll signal transduction pathway in M. sexta (Lepidoptera). Activation of the transmembrane
receptor Toll is initiated, after exposure to microorganisms, by activation of hemolymph proteinase-6
precursor (proHP6) by an unknown inducer. Active HP6 then cleaves hemolymph proteinase-8
precursor (proHP8) into active HP8, which in turn can cleave the cytokine-like polypeptide,
proMsSpätzle into its active form MsSpätzle (Manduca sexta Spätzle). MsSpätzle then binds to the
MsToll receptor inducing degradation of MsCactus (an IB inhibitor) and translocation to the nucleus
of the NF-B transcription factors RelA and RelB. These factors in turn activate transcription of
antimicrobial peptides (AMP).

CONCLUSION
Lepidoptera insects have a great economical impact and therefore the interest for
understanding their immune response continues to be enormous. Much has been learned
about hemocytes and antimicrobial proteins in hemolymph, in part due to the availability of
large volumes of hemolymph from insect larvae. The cellular and molecular mechanisms for
innate immunity are relatively well described for B. mori, and M. sexta. However, important
variations in immunity exist among different species. New techniques such as RNAi and
transgene silkworms, flow cytometry, together with system-based approaches such as
genomics and proteomics will certainly generate exciting advances in our understanding of
134 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

insect immunity. Recently, the complete genome of B. mori has been reported and it is
expected that similar information for other Lepidoptera species will also become available.
This will help the new proteomics approach for identifying and studying novel insect genes.
The signaling pathways for activation of phagocytosis and antimicrobial peptide production
are only partially known. Several signaling molecules have been reported as relevant, but we
do not have information yet to place them in a particular signaling pathway. These gaps will
certainly be filled as we continue studying how Lepidoptera cope with the many pathogenic
microorganisms. Ultimately, a better understanding of Lepidoptera innate immune responses
will improve our tactics for insect pest control and will even provide novel possible uses of
antimicrobial peptides at the clinic.

ACKNOWLEDGMENTS
Research work at the authors’ laboratories was supported by grant 168098 (to CR) from
Consejo Nacional de Ciencia y Tecnología, Mexico; and by grants IN205311-2 (to CR) and
IACOD IB200811 (to EUQ) from Dirección General de Asuntos del Personal Académico,
Universidad Nacional Autónoma de México, Mexico.

REFERENCES
[1] Meyer, J. (2009) Lepidoptera. General Entomology. In: Resource Library. NC State
University. [Link]
html. Date accessed: 27-July-2012.
[2] Goldsmith, M.R., Shimada, T., Abe, H. (2005) The genetics and genomics of the
silkworm, Bombyx mori. Annu. Rev. Entomol. 50, 71-100.
[3] Acuña, A.M., Caso, L., Aliphat, M.M., Vergara, C.H. (2011) Edible insects as part of
the traditional food system of the Popoloca town of Los Reyes Metzontla, Mexico. J.
Ethnobiol. 31, 150–169.
[4] Zagrobelny, M., Dreon, A.L., Gomiero, T., Marcazzan, G.L., Glaring, M.A., Møller,
B.L., Paoletti, M.G. (2009) Toxic moths: source of a truly safe delicacy. J. Ethnobiol.
29, 64-76.
[5] Jiang, H., Vilcinskas, A., Kanost, M.R. (2010) Immunity in lepidopteran insects. Adv.
Exp. Med. Biol. 708, 181-204.
[6] Lavine, M.D., Strand, M.R. (2002) Insect hemocytes and their role in immunity. Insect
Biochem. Mol. Biol. 32, 1295-1309.
[7] Kanost, M.R., Jiang, H., Yu, X.Q. (2004) Innate immune responses of a lepidopteran
insect, Manduca sexta. Immunol. Rev. 198, 97-105.
[8] Ashida, M., Brey, P.T. (1995) Role of the integument in insect defense: pro-phenol
oxidase cascade in the cuticular matrix. Proc. Natl. Acad. Sci. USA 92, 10698-10702.
[9] Hegedus, D., Erlandson, M., Gillott, C., Toprak, U. (2009) New insights into
peritrophic matrix synthesis, architecture, and function. Annu. Rev. Entomol. 54,285-
302.
 Immune Response in Lepidoptera 135

[10] Lehane, M.J. (1997) Peritrophic matrix structure and function. Annu. Rev. Entomol. 43,
525-550.
[11] Jiang, H. (2008) The biochemical basis of antimicrobial responses in Manduca sexta.
Insect Science 15, 53-66.
[12] Tsakas, S., Marmaras, V.J. (2010) Insect immunity and its signalling: an overview.
Invertebrate Surv. J. 7, 228-238.
[13] Strand, M.R. (2008) The insect cellular immune response. Insect Science 15, 1-14.
[14] Meister, M., Lagueux, M. (2003) Drosophila blood cells. Cell Microbiol. 5, 573-580.
[15] Manachini, B., Arizza, V., Parrinello, D., Parrinello, N. (2010) Hemocytes of
Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to
Saccharomyces cerevisiae and Bacillus thuringiensis. J. Invertebr. Pathol. [Epub ahead
of print].
[16] Meister, M. (2004) Blood cells of Drosophila: cell lineages and role in host defence.
Curr. Opin. Immunol. 16, 10-15.
[17] Wang, Q., Liu, Y., He, H.J., Zhao, X.F., Wang, J.X. (2010) Immune responses of
Helicoverpa armigera to different kinds of pathogens. BMC Immunol. 11, 9.
[18] Eleftherianos, I., Marokhazi, J., Millichap, P.J., Hodgkinson, A.J., Sriboonlert, A.,
Ffrench-Constant, R.H., Reynolds, S.E. (2006) Prior infection of Manduca sexta with
non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus
luminescens: roles of immune-related proteins shown by RNA interference. Insect
Biochem. Mol. Biol. 36, 517-525.
[19] Bergin, D., Murphy, L., Keenan, J., Clynes, M., Kavanagh, K. (2006) Pre-exposure to
yeast protects larvae of Galleria mellonella from a subsequent lethal infection by
Candida albicans and is mediated by the increased expression of antimicrobial
peptides. Microbes Infect. 8, 2105-2112.
[20] Müller, U., Vogel, P., Alber, G., Schaub, G.A. (2008) The innate immune system of
mammals and insects. In Trends in Innate Immunity (A. Egesten, A. Schmidt and H.
Herwald, eds), Karger, Basel 21-44.
[21] Garcia-Garcia, E., Garcia-Garcia, P.L., Rosales, C. (2009) An fMLP receptor is
involved in activation of phagocytosis by hemocytes from specific insect species. Dev.
Comp. Immunol. 33, 728-739.
[22] Willott, E., Lowenberger, C., Christensen, B.M., Kanost, M.R. (1995) Monoclonal
antibodies against Manduca sexta hemocytes bind Aedes aegypti hemocytes:
characterization of six monoclonal antibodies that bind hemocytes from both species.
Dev. Comp. Immunol. 19, 451-461.
[23] Willott, E., Trenczek, T., Thrower, L.W., Kanost, M.R. (1994) Immunochemical
identification of insect hemocyte populations: monoclonal antibodies distinguish four
major hemocyte types in Manduca sexta. Eur. J. Cell Biol. 65, 417-423.
[24] Gardiner, E.M., Strand, M.R. (1999) Monoclonal antibodies bind distinct classes of
hemocytes in the moth Pseudoplusia includens. J. Insect Physiol. 45, 113-126.
[25] Nardi, J.B., Pilas, B., Bee, C.M., Zhuang, S., Garsha, K., Kanost, M.R. (2006)
Neuroglian-positive plasmatocytes of Manduca sexta and the initiation of hemocyte
attachment to foreign surfaces. Dev. Comp. Immunol. 30, 447-462.
[26] Wiegand, C., Levin, D., Gillespie, J., Willott, E., Kanost, M., Trenczek, T. (2000)
Monoclonal antibody MS13 identifies a plasmatocyte membrane protein and inhibits
136 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

encapsulation and spreading reactions of Manduca sexta hemocytes. Arch. Insect

Biochem. Physiol. 45, 95-108.
[27] Zhang, S., Clark, K.D., Strand, M.R. (2011) The protein P23 identifies capsule-forming
plasmatocytes in the moth Pseudoplusia includens. Dev. Comp. Immunol. 35, 501-510.
[28] Nardi, J.B. (2004) Embryonic origins of the two main classes of hemocytes--granular
cells and plasmatocytes--in Manduca sexta. Dev. Genes Evol. 214, 19-28.
[29] Nardi, J.B., Pilas, B., Ujhelyi, E., Garsha, K., Kanost, M.R. (2003) Hematopoietic
organs of Manduca sexta and hemocyte lineages. Dev. Genes Evol. 213, 477-491.
[30] Schmidt, O., Söderhäll, K., Theopold, U., Faye, I. (2010) Role of adhesion in arthropod
immune recognition. Annu. Rev. Entomol. 55, 485-504.
[31] Rosales, C. (2005) Molecular Mechanisms of Phagocytosis. Landes Bioscience/
Springer Science, Georgetown, Texas 154.
[32] Rosales, C. (2011) Phagocytosis, a cellular immune response in insects. Inv. Surv. J. 8,
109-131.
[33] Castillo, J.C., Robertson, A.E., Strand, M.R. (2006) Characterization of hemocytes
from the mosquitoes Anopheles gambiae and Aedes aegypti. Insect Biochem. Mol. Biol.
36, 891-903.
[34] Lamprou, I., Mamali, I., Dallas, K., Fertakis, V., Lampropoulou, M., Marmaras, V.J.
(2007) Distinct signalling pathways promote phagocytosis of bacteria, latex beads and
lipopolysaccharide in medfly haemocytes. Immunology 121, 314-327.
[35] Lemaitre, B., Hoffmann, J.A. (2007) The host defense of Drosophila melanogaster.
Annu. Rev. Immunol. 25, 697-743.
[36] Dean, P., Potter, U., Richards, E.H., Edwards, J.P., Charnley, A.K., Reynolds, S.E.
(2004) Hyperphagocytic haemocytes in Manduca sexta. J. Insect Physiol. 50,1027-
1036.
[37] Wiesner, A., Wittwer, D., Goetz, P. (1996) A small phagocytosis stimulating factor is
released by and acts on phagocytosing Galleria mellonella hemocytes in vitro. J. Insect
Physiol. 42, 829-835.
[38] Mohrig, W., Schittek, D. (1979) Phagocytosis-stimulating mediators in insects. Act.
Biol. Med. Germ. 38, 953-958.
[39] Nakatogawa, S., Oda, Y., Kamiya, M., Kamijima, T., Aizawa, T., Clark, K.D., Demura,
M., Kawano, K., Strand, M.R., Hayakawa, Y. (2009) A novel peptide mediates
aggregation and migration of hemocytes from an insect. Curr. Biol. 19, 779-785.
[40] Clark, K.D., Pech, L.L., Strand, M.R. (1997) Isolation and identification of a
plasmatocyte-spreading peptide from the hemolymph of the lepidopteran insect
Pseudoplusia includens. J. Biol. Chem. 272, 23440-23447.
[41] Strand, M.R., Hayakawa, Y., Clark, K.D. (2000) Plasmatocyte spreading peptide
(PSP1) and growth blocking peptide (GBP) are multifunctional homologs. J. Insect
Physiol. 46, 817-824.
[42] Volkman, B.F., Anderson, M.E., Clark, K.D., Hayakawa, Y., Strand, M.R., Markley,
J.L. (1999) Structure of the insect cytokine peptide plasmatocyte-spreading peptide 1
from Pseudoplusia includens. J. Biol. Chem. 274, 4493-4496.
[43] Wang, Y., Jiang, H., Kanost, M.R. (1999) Biological activity of Manduca sexta
paralytic and plasmatocyte spreading peptide and primary structure of its hemolymph
precursor. Insect Biochem. Mol. Biol. 29, 1075-1086.
 Immune Response in Lepidoptera 137

[44] Miller, J.S., Howard, R.W., Rana, R.L., Tunaz, H., Stanley, D.W. (1999) Eicosanoids
mediate nodulation reactions to bacterial infections in adults of the cricket, Gryllus
assimilis. J. Insect Physiol. 45, 75-83.
[45] Shrestha, S., Kim, Y.J. (2009) Various eicosanoids modulate the cellular and humoral
immune responses of the beet armyworm, Spodoptera exigua. Biosci. Biotechnol.
Biochem. 73, 2077-2084.
[46] Shrestha, S., Park, Y., Stanley, D., Kim, Y. (2010) Genes encoding phospholipases A2
mediate insect nodulation reactions to bacterial challenge. J. Insect Physiol. 56,324-
332.
[47] Stanley, D., Miller, J., Tunaz, H. (2009) Eicosanoid actions in insect immunity. J.
Innate Immun. 1, 282-290.
[48] Zhao, F., Stanley, D., Wang, Y., Zhu, F., Lei, C.L. (2009) Eicosanoids mediate
nodulation reactions to a mollicute bacterium in larvae of the blowfly, Chrysomya
megacephala. J. Insect Physiol. 55, 192-196.
[49] Gandhe, A.S., John, S.H., Nagaraju, J. (2007) Noduler, a novel immune up-regulated
protein mediates nodulation response in insects. J. Immunol. 179, 6943-6951.
[50] Bao, Y.Y., Xue, J., Wu, W.J., Wang, Y., Lv, Z.Y., Zhang, C.X. (2011) An immune-
induced Reeler protein is involved in the Bombyx mori melanization cascade. Insect
Biochem. Mol. Biol. May 23. [Epub ahead of print].
[51] Ling, E., Yu, X.Q. (2006) Cellular encapsulation and melanization are enhanced by
immulectins, pattern recognition receptors from the tobacco hornworm Manduca sexta.
Dev. Comp. Immunol. 30, 289-299.
[52] Carton, Y., Frey, F., Nappi, A.J. (2009) Parasite-induced changes in nitric oxide levels
in Drosophila paramelanica. J. Parasitol. 95, 1134-1141.
[53] Nappi, A.J., Christensen, B.M. (2005) Melanogenesis and associated cytotoxic
reactions: applications to insect innate immunity. Insect Biochem. Mol. Biol. 35,443-
459.
[54] Rosales, C. (2007) Fc receptor and integrin signaling in phagocytes. Signal
Transduction 7, 386-401.
[55] Zhuang, S., Kelo, L., Nardi, J.B. (2008) Multiple α subunits of integrin are involved in
cell-mediated responses of the Manduca immune system. Dev. Comp. Immunol. 32,
365-379.
[56] Levin, D.M., Breuer, L.N., Zhuang, S., Anderson, S.A., Nardi, J.B., Kanost, M.R.
(2005) A hemocyte-specific integrin required for hemocytic encapsulation in the
tobacco hornworm, Manduca sexta. Insect Biochem. Mol. Biol. 35, 369-380.
[57] Lavine, M.D., Strand, M.R. (2003) Haemocytes from Pseudoplusia includens express
multiple alpha and beta integrin subunits. Insect Mol. Biol. 12, 441-452.
[58] Lavine, M.D., Strand, M.R. (2001) Surface characteristics of foreign targets that elicit
an encapsulation response by the moth Pseudoplusia includens. J. Insect Physiol. 47,
965-974.
[59] Nappi, A., Poirié, M., Carton, Y. (2009) The role of melanization and cytotoxic by-
products in the cellular immune responses of Drosophila against parasitic wasps. Adv.
Parasitol. 70, 99-121.
[60] Cerenius, L., Lee, B.L., Söderhäll, K. (2008) The proPO-system: pros and cons for its
role in invertebrate immunity. Trends Immunol. 29, 263-271.
138 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

[61] Eleftherianos, I., Revenis, C. (2011) Role and importance of phenoloxidase in insect
hemostasis. J. Innate Immun. 3, 28-33.
[62] Krem, M.M., Di Cera, E. (2002) Evolution of enzyme cascades from embryonic
development to blood coagulation. Trends Biochem. Sci. 27, 67-74.
[63] Ochiai, M., Ashida, M. (1999) A pattern recognition protein for peptidoglycan. Cloning
the cDNA and the gene of the silkworm, Bombyx mori. J. Biol. Chem. 274, 11854-
11858.
[64] Yoshida, H., Kinoshita, K., Ashida, M. (1996) Purification of a peptidoglycan
recognition protein from hemolymph of the silkworm, Bombyx mori. J. Biol. Chem.
271, 13854-13860.
[65] Ochiai, M., Ashida, M. (1988) Purification of a beta-1,3-glucan recognition protein in
the prophenoloxidase activating system from hemolymph of the silkworm, Bombyx
mori. J. Biol. Chem. 263, 12056-12062.
[66] Ochiai, M., Ashida, M. (2000) A pattern-recognition protein for beta-1,3-glucan. The
binding domain and the cDNA cloning of beta-1,3-glucan recognition protein from the
silkworm, Bombyx mori. J. Biol. Chem. 275, 4995-5002.
[67] Ling, E., Yu, X.Q. (2005) Prophenoloxidase binds to the surface of hemocytes and is
involved in hemocyte melanization in Manduca sexta. Insect Biochem. Mol. Biol. 35,
1356-1366.
[68] Marmaras, V.J., Lampropoulou, M. (2009) Regulators and signalling in insect
haemocyte immunity. Cell Signal. 21, 186-195.
[69] Stuart, L.M., Ezekowitz, A.B. (2005) Phagocytosis: Elegant complexity. Immunity 22,
539-550.
[70] Dziarski, R., Gupta, D. (2006) Mammalian PGRPs: novel antibacterial proteins. Cell
Microbiol. 8, 1059-1069.
[71] Dziarski, R., Gupta, D. (2010) Review: Mammalian peptidoglycan recognition proteins
(PGRPs) in innate immunity. Innate Immun. 16, 168-174.
[72] Charroux, B., Rival, T., Narbonne-Reveau, K., Royet, J. (2009) Bacterial detection by
Drosophila peptidoglycan recognition proteins. Microbes Infect. 11, 631-636.
[73] Royet, J., Dziarski, R. (2007) Peptidoglycan recognition proteins: pleiotropic sensors
and effectors of antimicrobial defences. Nat. Rev. Microbiol. 5, 264-277.
[74] Yu, X.Q., Zhu, Y.F., Ma, C., Fabrick, J.A., Kanost, M.R. (2002) Pattern recognition
proteins in Manduca sexta plasma. Insect Biochem. Mol. Biol. 32, 1287-1293.
[75] Zou, Z., Najar, F., Wang, Y., Roe, B., Jiang, H. (2008) Pyrosequence analysis of
expressed sequence tags for Manduca sexta hemolymph proteins involved in immune
responses. Insect Biochem. Mol. Biol. 38, 677-682.
[76] Seitz, V., Clermont, A., Wedde, M., Hummel, M., Vilcinskas, A., Schlatterer, K.,
Podsiadlowski, L. (2003) Identification of immunorelevant genes from greater wax
moth (Galleria mellonella) by a subtractive hybridization approach. Dev. Comp.
Immunol. 27, 207-215.
[77] Onoe, H., Matsumoto, A., Hashimoto, K., Yamano, Y., Morishima, I. (2007)
Peptidoglycan recognition protein (PGRP) from eri-silkworm, Samia cynthia ricini;
protein purification and induction of the gene expression. Comp. Biochem. Physiol.
Part B. Biochem. Mol. Biol. 147, 512-519.
 Immune Response in Lepidoptera 139

[78] Kang, D., Liu, G., Lundström, A., Gelius, E., Steiner, H. (1998) A peptidoglycan
recognition protein in innate immunity conserved from insects to humans. Proc. Natl.
Acad. Sci. U S A 95, 10078-10082.
[79] Eum, J.H., Seo, Y.R., Yoe, S.M., Kang, S.W., Han, S.S. (2007) Analysis of the
immune-inducible genes of Plutella xylostella using expressed sequence tags and
cDNA microarray. Dev. Comp. Immunol. 31, 1107-1120.
[80] Coates, B.S., Bayles, D.O., Wanner, K.W., Robertson, H.M., Hellmich, R.L.,
Sappington, T.W. (2011) The application and performance of single nucleotide
polymorphism markers for population genetic analyses of lepidoptera. Front. Genet.2,
38.
[81] Coates, B.S., Sumerford, D.V., Hellmich, R.L., Lewis, L.C. (2008) Mining an Ostrinia
nubilalis midgut expressed sequence tag (EST) library for candidate genes and single
nucleotide polymorphisms (SNPs). Insect Mol. Biol. 17, 607-620.
[82] Sumathipala, N., Jiang, H. (2010) Involvement of Manduca sexta peptidoglycan
recognition protein-1 in the recognition of bacteria and activation of prophenoloxidase
system. Insect Biochem. Mol. Biol. 40, 487-495.
[83] Dziarski, R., Gupta, D. (2006) The peptidoglycan recognition proteins (PGRPs).
Genome Biol. 7.
[84] Steiner, H. (2004) Peptidoglycan recognition proteins: on and off switches for innate
immunity. Immunol. Rev. 198, 83-96.
[85] Jiang, H., Ma, C., Lu, Z.Q., Kanost, M.R. (2004) Beta-1,3-glucan recognition protein-2
(betaGRP-2)from Manduca sexta; an acute-phase protein that binds beta-1,3-glucan and
lipoteichoic acid to aggregate fungi and bacteria and stimulate prophenoloxidase
activation. Insect Biochem. Mol. Biol. 34, 89-100.
[86] Ma, C., Kanost, M.R. (2000) A beta1,3-glucan recognition protein from an insect,
Manduca sexta, agglutinates microorganisms and activates the phenoloxidase cascade.
J. Biol. Chem. 275, 7505-7514.
[87] Fabrick, J.A., Baker, J.E., Kanost, M.R. (2004) Innate immunity in a pyralid moth:
functional evaluation of domains from a beta-1,3-glucan recognition protein. J. Biol.
Chem. 279, 26605-26611.
[88] Wang, Y., Jiang, H. (2006) Interaction of β-1,3-Glucan with Its recognition protein
activates hemolymph proteinase 14, an initiation enzyme of the prophenoloxidase
activation system in Manduca sexta. J. Biol. Chem. 281, 9271-9278.
[89] Wang, Y., Jiang, H. (2010) Binding properties of the regulatory domains in Manduca
sexta hemolymph proteinase-14, an initiation enzyme of the prophenoloxidase
activation system. Dev. Comp. Immunol. 34, 316-322.
[90] Pauchet, Y., Freitak, D., Heidel-Fischer, H.M., Heckel, D.G., Vogel, H. (2009)
Immunity or Digestion: Glucanase activity in a glucan-binding protein family from
Lepidoptera. J. Biol. Chem. 284, 2214-2224.
[91] Lee, W.J., Lee, J.D., Kravchenko, V.V., Ulevitch, R.J., Brey, P.T. (1996) Purification
and molecular cloning of an inducible gram-negative bacteria-binding protein from the
silkworm, Bombyx mori. Proc. Natl. Acad. Sci. U S A 93, 7888-7893.
[92] Andersson, K., Steiner, H. (1987) Structure and properties of protein P4, the major
bacteria-inducible protein in pupae of Hyalophora cecropia. Insect Biochem. 17,133-
140.
140 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

[93] Sun, S.C., Lindström, I., Boman, H.G., Faye, I., Schmidt, O. (1990) Hemolin: an insect-
immune protein belonging to the immunoglobulin superfamily. Science 250,1729-1732.
[94] Ladendorff, N.E., Kanost, M.R. (1990) Isolation and characterization of bacteria-
induced protein P4 from hemolymph of Manduca sexta. Arch. Insect Biochem. Physiol.
15, 33-41.
[95] Ladendorff, N.E., Kanost, M.R. (1991) Bacteria-induced protein P4 (hemolin) from
Manduca sexta: a member of the immunoglobulin superfamily which can inhibit
hemocyte aggregation. Arch. Insect Biochem. Physiol. 18, 285-300.
[96] Rosales, C., O'Brien, V., Kornberg, L., Juliano, R.L. (1995) Signal transduction by cell
adhesion receptors. Biochim. Biophys. Acta 1242, 77-98.
[97] Tanaka, H., Ishibashi, J., Fujita, K., Nakajima, Y., Sagisaka, A., Tomimoto, K.,Suzuki,
N., Yoshiyama, M., Kaneko, Y., Iwasaki, T., Sunagawa, T., Yamaji, K., Asaoka, A.,
Mita, K., Yamakawa, M. (2008) A genome-wide analysis of genes and gene families
involved in innate immunity of Bombyx mori. Insect Biochem. Mol. Biol. 38, 1087-
1110.
[98] Shin, S.W., Park, S.-S., Park, D.-S., Kim, M.G., Kim, S.C., Brey, P.T., Park, H.-Y.
(1998) Isolation and characterization of immune-related genes from the fall webworm,
Hyphantria cunea, using PCR-based differential display and subtractive cloning. Insect
Biochem. Mol. Biol. 28, 827-837.
[99] Lee, K.Y., Horodyski, F.M., Valaitis, A.P., Denlinger, D.L. (2002) Molecular
characterization of the insect immune protein hemolin and its high induction during
embryonic diapause in the gypsy moth, Lymantria dispar. Insect Biochem. Mol. Biol.
32, 1457-1467.
[100] Li, W., Terenius, O., Hirai, M., Nilsson, A.S., Faye, I. (2005) Cloning, expression and
phylogenetic analysis of Hemolin, from the Chinese oak silkmoth, Antheraea pernyi.
Dev. Comp. Immunol. 29, 853-864.
[101] Gandhe, A.S., Arunkumar, K.P., John, S.H., Nagaraju, J. (2006) Analysis of bacteria-
challenged wild silkmoth, Antheraea mylitta (lepidoptera) transcriptome reveals
potential immune genes. BMC Genomics. 7, 184.
[102] Bao, Y., Yamano, Y., Morishima, I. (2007) Induction of hemolin gene expression by
bacterial cell wall components in eri-silkworm, Samia cynthia ricini. Comp. Biochem.
Physiol. Part B. Biochem. Mol. Biol. 146, 147-151.
[103] Daffre, S., Faye, I. (1997) Lipopolysaccharide interaction with hemolin, an insect
member of the Ig-superfamily. FEBS Lett. 408, 127-130.
[104] Yu, X.Q., Kanost, M.R. (2002) Binding of hemolin to bacterial lipopolysaccharide and
lipoteichoic acid. An immunoglobulin superfamily member from insects as a pattern-
recognition receptor. Eur. J. Biochem. 269, 1827-1834.
[105] Roxström-Lindquist, K., Lindström-Dinnetz, I., Olesen, J., Engström, Y., Faye, I.
(2002) An intron enhancer activates the immunoglobulin-related Hemolin gene in
Hyalophora cecropia. Insect Mol. Biol. 11, 505-515.
[106] Bettencourt, R., Lanz-Mendoza, H., Lindquist, K.R., Faye, I. (1997) Cell adhesion
properties of hemolin, an insect immune protein in the Ig superfamily. Eur. J. Biochem.
250, 630-637.
 Immune Response in Lepidoptera 141

[107] Eleftherianos, I., Gökçen, F., Felföldi, G., Millichap, P.J., Trenczek, T.E., ffrench-
Constant, R.H., Reynolds, S.E. (2007) The immunoglobulin family protein Hemolin
mediates cellular immune responses to bacteria in the insect Manduca sexta. Cell.
Microbiol. 9, 1137-1147.
[108] Hirai, M., Terenius, O., Li, W., Faye, I. (2004) Baculovirus and dsRNA induce
Hemolin, but no antibacterial activity, in Antheraea pernyi. Insect Mol. Biol. 13,399-
405.
[109] Terenius, O., Popham, H.J., Shelby, K.S. (2009) Bacterial, but not baculoviral
infections stimulate Hemolin expression in noctuid moths. Dev. Comp. Immunol. 33,
1176-1185.
[110] Terenius, O. (2008) Hemolin-A lepidopteran anti-viral defense factor? Dev. Comp.
Immunol. 32, 311-316.
[111] Vasta, G.R., Quesenberry, M., Ahmed, H., O'Leary, N. (1999) C-type lectins and
galectins mediate innate and adaptive immune functions: their roles in the complement
activation pathway. Dev. Comp. Immunol. 23, 401-420.
[112] Koizumi, N., Imai, Y., Morozumi, A., Imamura, M., Kadotani, T., Yaoi, K.,Iwahana,
H., Sato, R. (1999) Lipopolysaccharide-binding protein of Bombyx mori participates in
a hemocyte-mediated defense reaction against Gram-negative bacteria. J. Insect
Physiol. 45, 853-859.
[113] Koizumi, N., Morozumi, A., Imamura, M., Tanaka, E., Iwahana, H., Sato, R. (1997)
Lipopolysaccharide-binding proteins and their involvement in the bacterial clearance
from the hemolymph of the silkworm Bombyx mori. Eur. J. Biochem. 248, 217-224.
[114] Yu, X.Q., Gan, H., Kanost, M.R. (1999) Immulectin, an inducible C-type lectin from an
insect, Manduca sexta, stimulates activation of plasma prophenol oxidase. Insect
Biochem. Mol. Biol. 29, 585-597.
[115] Shin, S.W., Park, D.S., Kim, S.C., Park, H.Y. (2000) Two carbohydrate recognition
domains of Hyphantria cunea lectin bind to bacterial lipopolysaccharides through O-
specific chain. FEBS Lett. 467, 70-74.
[116] Yu, X.-Q., Kanost, M.R. (2000) Immulectin-2, a Lipopolysaccharide-specific lectin
from an insect, Manduca sexta, is induced in response to Gram-negative bacteria. J.
Biol. Chem. 275, 37373-37381.
[117] Yu, X.Q., Ling, E., Tracy, M.E., Zhu, Y. (2006) Immulectin-4 from the tobacco
hornworm Manduca sexta binds to lipopolysaccharide and lipoteichoic acid. Insect
Mol. Biol. 15, 119-128.
[118] Watanabe, A., Miyazawa, S., Kitami, M., Tabunoki, H., Ueda, K., Sato, R. (2006)
Characterization of a novel C-type lectin, Bombyx mori multibinding protein, from the
B. mori hemolymph: mechanism of wide-range microorganism recognition and role in
immunity. J. Immunol. 177, 4594-4604.
[119] Takase, H., Watanabe, A., Yoshizawa, Y., Kitami, M., Sato, R. (2009) Identification
and comparative analysis of three novel C-type lectins from the silkworm with
functional implications in pathogen recognition. Dev. Comp. Immunol. 33, 789-800.
[120] Chai, L.Q., Tian, Y.Y., Yang, D.T., Wang, J.X., Zhao, X.F. (2008) Molecular cloning
and characterization of a C-type lectin from the cotton bollworm, Helicoverpa
armigera. Dev. Comp. Immunol. 32, 71-83.
142 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

[121] Zou, Z., Evans, J.D., Lu, Z., Zhao, P., Williams, M., Sumathipala, N., Hetru, C.,
Hultmark, D., Jiang, H. (2007) Comparative genomic analysis of the Tribolium immune
system. Genome Biol. 8, R177.
[122] Yu, X.Q., Kanost, M.R. (2003) Manduca sexta lipopolysaccharide-specific immulectin-
2 protects larvae from bacterial infection. Dev. Comp. Immunol. 27, 189-196.
[123] Yu, X.Q., Kanost, M.R. (2004) Immulectin-2, a pattern recognition receptor that
stimulates hemocyte encapsulation and melanization in the tobacco hornworm,
Manduca sexta. Dev. Comp. Immunol. 28, 891-900.
[124] Van der Horst, D.J., Roosendaal, S.D., Rodenburg, K.W. (2009) Circulatory lipid
transport: lipoprotein assembly and function from an evolutionary perspective. Mol.
Cell. Biochem. 326, 105-119.
[125] van der Horst, D.J., D., v.H., van Marrewijk, W.J., Rodenburg, K.W. (2002) Alternative
lipid mobilization: the insect shuttle system. Mol. Cell. Biochem. 239, 113-119.
[126] Weers, P.M., Ryan, R.O. (2003) Apolipophorin III: a lipid-triggered molecular switch.
Insect Biochem. Mol. Biol. 33, 1249-1260.
[127] Tsuchida, K., Yokoyama, T., Sakudoh, T., Katagiri, C., Tsurumaru, S., Takada, N.,
Fujimoto, H., Ziegler, R., Iwano, H., Hamano, K., Yaginuma, T. (2010) Apolipophorin-
III expression and low density lipophorin formation during embryonic development of
the silkworm, Bombyx mori. Comp. Biochem. Physiol. Part B. Biochem. Mol. Biol. 155,
363-370.
[128] Dushay, M.S. (2009) Insect hemolymph clotting. Cell. Mol. Life Sci. 66, 2643-2650.
[129] Ma, G., Hay, D., Li, D., Asgari, S., Schmidt, O. (2006) Recognition and inactivation of
LPS by lipophorin particles. Dev. Comp. Immunol. 30, 619-626.
[130] Whitten, M.M., Tew, I.F., Lee, B.L., Ratcliffe, N.A. (2004) A novel role for an insect
apolipoprotein (apolipophorin III) in beta-1,3-glucan pattern recognition and cellular
encapsulation reactions. J. Immunol. 172, 2177-2185.
[131] Kato, Y., Motoi, Y., Taniai, K., Kadono-Okuda, K., Yamamoto, M., Higashino, Y.,
Shimabukuro, M., Chowdhury, S., Xu, J., Sugiyama, M., et., a. (1994)
Lipopolysaccharide-lipophorin complex formation in insect hemolymph: a common
pathway of lipopolysaccharide detoxification both in insects and in mammals. Insect
Biochem. Mol. Biol. 24, 547-555.
[132] Vilcinskas, A. (2010) Coevolution between pathogen-derived proteinases and
proteinase inhibitors of host insects. Virulence 1, 206-214.
[133] Griesch, J., Wedde, M., Vilcinskas, A. (2000) Recognition and regulation of
metalloproteinase activity in the haemolymph of Galleria mellonella: a new pathway
mediating induction of humoral immune responses. Insect Biochem. Mol. Biol. 30,461-
472.
[134] Fröbius, A.C., Kanost, M.R., Götz, P., Vilcinskas, A. (2000) Isolation and
characterization of novel inducible serine protease inhibitors from larval hemolymph of
the greater wax moth Galleria mellonella. Eur. J. Biochem. 267, 2046-2053.
[135] Vilcinskas, A., Wedde, M. (2002) Insect inhibitors of metalloproteinases. IUBMB Life.
54, 339-343.
[136] Altincicek, B., Linder, M., Linder, D., Preissner, K.T., Vilcinskas, A. (2007) Microbial
metalloproteinases mediate sensing of invading pathogens and activate innate immune
responses in the lepidopteran model host Galleria mellonella. Infect. Immun. 75,175-
183.
 Immune Response in Lepidoptera 143

[137] Altincicek, B., Berisha, A., Mukherjee, K., Spengler, B., Römpp, A., Vilcinskas, A.
(2009) Identification of collagen IV derived danger/alarm signals in insect immunity by
nanoLC-FTICR MS. Biol. Chem. 390, 1303-1311.
[138] Clermont, A., Wedde, M., Seitz, V., Podsiadlowski, L., Lenze, D., Hummel, M.,
Vilcinskas, A. (2004) Cloning and expression of an inhibitor of microbial
metalloproteinases from insects contributing to innate immunity. Biochem. J. 382,315-
322.
[139] Wedde, M., Weise, C., Nuck, R., Altincicek, B., Vilcinskas, A. (2007) The insect
metalloproteinase inhibitor gene of the lepidopteran Galleria mellonella encodes two
distinct inhibitors. Biol. Chem. 388, 119-127.
[140] Mukherjee, K., Altincicek, B., Hain, T., Domann, E., Vilcinskas, A., Chakraborty, T.
(2010) Galleria mellonella as a model system for studying Listeria pathogenesis. Appl.
Environ. Microbiol. 76, 310-317.
[141] Cerenius, L., Soederhaell, K. (2004) The prophenoloxidase-activating system in
invertebrates. Immunol. Rev. 198, 116-126.
[142] Mavrouli, M.D., Tsakas, S., Theodorou, G.L., Lampropoulou, M., Marmaras, V.J.
(2005) MAP kinases mediate phagocytosis and melanization via prophenoloxidase
activation in medfly hemocytes. Biochim. Biophys. Acta. 1744, 145-156.
[143] Zhao, P., Li, J., Wang, Y., Jiang, H. (2007) Broad-spectrum antimicrobial activity of
the reactive compounds generated in vitro by Manduca sexta phenoloxidase. Insect
Biochem. Mol. Biol. 37, 952-959.
[144] Jiang, H., Wang, Y., Ma, C., Kanost, M.R. (1997) Subunit composition of pro-phenol
oxidase from Manduca sexta: molecular cloning of subunit ProPO-P1. Insect Biochem.
Mol. Biol. 27, 835-850.
[145] Yasuhara, Y., Koizumi, Y., Katagiri, C., Ashida, M. (1995) Reexamination of
properties of prophenoloxidase isolated from larval hemolymph of the silkworm
Bombyx mori. Arch. Biochem. Biophys. 320, 14-23.
[146] Jiang, H., Kanost, M.R. (2000) The clip-domain family of serine proteinases in
arthropods. Insect Biochem. Mol. Biol. 30, 95-105.
[147] Jiang, H., Wang, Y., Gu, Y., Guo, X., Zou, Z., Scholz, F., Trenczek, T.E., Kanost, M.R.
(2005) Molecular identification of a bevy of serine proteinases in Manduca sexta
hemolymph. Insect Biochem. Mol. Biol. 35, 931-943.
[148] Jiang, H., Wang, Y., Kanost, M.R. (1998) Pro-phenol oxidase activating proteinase
from an insect, Manduca sexta: a bacteria-inducible protein similar to Drosophila
easter. Proc. Natl. Acad. Sci. USA 95, 12220-12225.
[149] Jiang, H., Wang, Y., Yu, X.Q., Kanost, M.R. (2003) Prophenoloxidase-activating
proteinase-2 from hemolymph of Manduca sexta. A bacteria-inducible serine proteinase
containing two clip domains. J. Biol. Chem. 278, 3552-3561.
[150] Jiang, H., Wang, Y., Yu, X.Q., Zhu, Y., Kanost, M. (2003) Prophenoloxidase-activating
proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase
regulated by serpin-1J and serine proteinase homologs. Insect Biochem. Mol. Biol. 33,
1049-1060.
[151] Gupta, S., Wang, Y., Jiang, H. (2005) Purification and characterization of Manduca
sexta prophenoloxidase-activating proteinase-1, an enzyme involved in insect immune
responses. Protein Expr. Purif. 39, 261-268.
144 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

[152] Gupta, S., Wang, Y., Jiang, H. (2005) Manduca sexta prophenoloxidase (proPO)
activation requires proPO-activating proteinase (PAP) and serine proteinase homologs
(SPHs) simultaneously. Insect Biochem. Mol. Biol. 35, 241-248.
[153] Wang, Y., Jiang, H. (2004) Prophenoloxidase (proPO) activation in Manduca sexta: an
analysis of molecular interactions among proPO, proPO-activating proteinase-3, and a
cofactor. Insect Biochem. Mol. Biol. 34, 731-742.
[154] Wang, Y., Jiang, H. (2008) A positive feedback mechanism in the Manduca sexta
prophenoloxidase activation system. Insect Biochem. Mol. Biol. 38, 763-769.
[155] Wang, Y., Jiang, H. (2007) Reconstitution of a branch of the Manduca sexta
prophenoloxidase activation cascade in vitro: snake-like hemolymph proteinase 21
(HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor.
Insect Biochem. Mol. Biol. 37, 1015-1025.
[156] Gorman, M.J., Wang, Y., Jiang, H., Kanost, M.R. (2007) Manduca sexta hemolymph
proteinase 21 activates prophenoloxidase-activating proteinase 3 in an insect innate
immune response proteinase cascad. J. Biol. Chem. 282, 11742-11749.
[157] An, C., Ishibashi, J., Ragan, E.J., Jiang, H., Kanost, M.R. (2009) Functions of Manduca
sexta hemolymph proteinases HP6 and HP8 in two innate immune pathways. J. Biol.
Chem. 284, 19716-19726.
[158] Kanost, M.R. (1999) Serine proteinase inhibitors in arthropod immunity. Dev. Comp.
Immunol. 23, 291-301.
[159] Gettins, P.G. (2002) Serpin structure, mechanism, and function. Chem. Rev. 1024751-
4804.
[160] Sasaki, T. (1991) Patchwork-structure serpins from silkworm (Bombyx mori) larval
hemolymph. Eur. J. Biochem. 202, 255-261.
[161] Sasaki, T., Kobayashi, K. (1984) Isolation of two novel proteinase inhibitors from
hemolymph of silkworm larva, Bombyx mori. Comparison with human serum
proteinase inhibitors. J. Biochem. 95, 1009-1017.
[162] Gan, H., Wang, Y., Jiang, H., Mita, K., Kanost, M.R. (2001) A bacteria-induced,
intracellular serpin in granular hemocytes of Manduca sexta. Insect Biochem. Mol. Biol.
31, 887-998.
[163] Jiang, H., Kanost, M.R. (1997) Characterization and functional analysis of 12 naturally
occurring reactive site variants of serpin-1 from Manduca sexta. J. Biol. Chem. 272,
1082-1087.
[164] Tong, Y., Jiang, H., Kanost, M.R. (2005) Identification of plasma proteases inhibited by
Manduca sexta serpin-4 and serpin-5 and their association with components of the
prophenol oxidase activation pathway. J. Biol. Chem. 280, 14932-14942.
[165] Wang, Y., Jiang, H. (2004) Purification and characterization of Manduca sexta serpin-
6: a serine proteinase inhibitor that selectively inhibits prophenoloxidase-activating
proteinase-3. Insect Biochem. Mol. Biol. 34, 387-395.
[166] Cherqui, A., Cruz, N., Simões, N. (2001) Purification and characterization of two serine
protease inhibitors from the hemolymph of Mythimna unipuncta. Insect Biochem. Mol.
Biol. 31, 761-769.
[167] Chamankhah, M., Braun, L., Visal-Shah, S., O'Grady, M., Baldwin, D., Shi, X.,
Hemmingsen, S.M., Alting-Mees, M., Hegedus, D.D. (2003) Mamestra configurata
serpin-1 homologues: cloning, localization and developmental regulation. Insect
Biochem. Mol. Biol. 33, 355-369.
 Immune Response in Lepidoptera 145

[168] Jiang, H., Wang, Y., Kanost, M.R. (1994) Mutually exclusive exon use and reactive
center diversity in insect serpins. J. Biol. Chem. 269, 55-58.
[169] Hegedus, D.D., Erlandson, M., Baldwin, D., Hou, X., Chamankhah, M. (2008)
Differential expansion and evolution of the exon family encoding the Serpin-1 reactive
centre loop has resulted in divergent serpin repertoires among the Lepidoptera. Gene
418, 15-21.
[170] Zou, Z., Picheng, Z., Weng, H., Mita, K., Jiang, H. (2009) A comparative analysis of
serpin genes in the silkworm genome. Genomics 93, 367-375.
[171] An, C., Ragan, E.J., Kanost, M.R. (2011) Serpin-1 splicing isoform J inhibits the
proSpätzle-activating proteinase HP8 to regulate expression of antimicrobial
hemolymph proteins in Manduca sexta. Dev. Comp. Immunol. 35, 135-141.
[172] Huang, R., Lu, Z., Dai, H., Velde, D.V., Prakash, O., Jiang, H. (2007) The solution
structure of clip domains from Manduca sexta prophenoloxidase activating proteinase-
2. Biochemistry 46, 11431-11439.
[173] Zou, Z., Jiang, H. (2005) Manduca sexta Serpin-6 regulates immune serine proteinases
PAP-3 and HP8: cDNA cloning, protein expression, inhibition kinetics, and functional
elucidation. J. Biol. Chem. 280, 14341-14348.
[174] Dunn, P.E., Bohnert, T.J., Russell, V. (1994) Regulation of antibacterial protein
synthesis following infection and during metamorphosis of Manduca sexta. Ann. N. Y.
Acad. Sci. 712, 117-130.
[175] Yu, K.H., Kim, K.N., Lee, J.H., Lee, H.S., Kim, S.H., Cho, K.Y., Nam, M.H., Lee, I.H.
(2002) Comparative study on characteristics of lysozymes from the hemolymph of three
lepidopteran larvae, Galleria mellonella, Bombyx mori, Agrius convolvuli. Dev. Comp.
Immunol. 26, 707-713.
[176] Chapelle, M., Girard, P.A., Cousserans, F., Volkoff, N.A., Duvic, B. (2009) Lysozymes
and lysozyme-like proteins from the fall armyworm, Spodoptera frugiperda. Mol.
Immunol. 47, 261-269.
[177] Wang, W.X., Wang, Y.P., Deng, X.J., Dang, X.L., Tian, J.H., Yi, H.Y., Li, Y.F., He,
X.F., Cao, Y., Xia, Q.Y., Lai, R., Wen, S.Y., Paskowitz, S. (2009) Molecular and
functional characterization of a c-type lysozyme from the Asian corn borer, Ostrinia
furnacalis. J. Insect Sci. 9, 17.
[178] Vilcinskas, A., Matha, V. (1997) Effect of the entomopathogenic fungus Beauveria
bassiana on the humoral immune response of Galleria mellonella larvae (Lepidoptera:
Pyralidae). Eur. J. Entomol. 94, 461-472.
[179] Rao, X.J., Ling, E., Yu, X.Q. (2010) The role of lysozyme in the prophenoloxidase
activation system of Manduca sexta: an in vitro approach. Dev. Comp. Immunol. 34,
264-271.
[180] Steiner, H., Hultmark, D., Engström, A., Bennich, H., Boman, H.G. (1981) Sequence
and specificity of two antibacterial proteins involved in insect immunity. Nature 292,
246-248.
[181] Bulet, P., Stöcklin, R. (2005) Insect antimicrobial peptides: structures, properties and
gene regulation. Protein Pept. Lett. 12, 3-11.
[182] Dai, H., Rayaprolu, S., Gong, Y., Huang, R., Prakash, O., Jiang, H. (2008) Solution
structure, antibacterial activity, and expression profile of Manduca sexta moricin. J.
Pept. Sci. 14, 855-863.
146 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

[183] Hara, S., Yamakawa, M. (1995) Moricin, a novel type of antibacterial peptide isolated
from the silkworm, Bombyx mori. J. Biol. Chem. 270, 29923-29927.
[184] Brown, S.E., Howard, A., Kasprzak, A.B., Gordon, K.H., East, P.D. (2008) The
discovery and analysis of a diverged family of novel antifungal moricin-like peptides in
the wax moth Galleria mellonella. Insect Biochem. Mol. Biol. 38, 201-212.
[185] Hultmark, D., Engström, A., Andersson, K., Steiner, H., Bennich, H., Boman, H.G.
(1983) Insect immunity. Attacins, a family of antibacterial proteins from Hyalophora
cecropia. EMBO J. 2, 571-576.
[186] Engström, P., Carlsson, A., Engström, A., Tao, Z.J., Bennich, H. (1984) The
antibacterial effect of attacins from the silk moth Hyalophora cecropia is directed
against the outer membrane of Escherichia coli. EMBO J. 3, 3347-3351.
[187] Bang, K., Park, S., Yoo, J.Y., Cho, S. (2012) Characterization and expression of attacin,
an antibacterial protein-encoding gene, from the beet armyworm, Spodoptera exigua
(Hübner) (Insecta: Lepidoptera: Noctuidae). Mol. Biol. Rep. 39, 5151-5159.
[188] Axén, A., Carlsson, A., Engström, A., Bennich, H. (1997) Gloverin, an antibacterial
protein from the immune hemolymph of Hyalophora pupae. Eur. J. Biochem. 247614-
619.
[189] Kaneko, Y., Furukawa, S., Tanaka, H., Yamakawa, M. (2007) Expression of
antimicrobial peptide genes encoding Enbocin and Gloverin isoforms in the silkworm,
Bombyx mori. Biosci. Biotechnol. Biochem. 71, 2233-2241.
[190] Hwang, J., Kim, Y. (2011) RNA interference of an antimicrobial peptide, gloverin, of
the beet armyworm, Spodoptera exigua, enhances susceptibility to Bacillus
thuringiensis. J. Invertebr. Pathol. 108, 194-200.
[191] Xu, X.X., Zhong, X., Yi, H.Y., Yu, X.Q. (2012) Manduca sexta Gloverin binds
microbial components and is active against bacteria and fungi. Dev. Comp. Immunol.
Jul 30, [Epub ahead of print].
[192] Moreno-Habel, D.A., Biglang-awa, I.M. Dulce, A., Luu, D.D., Garcia, P., Weers, P.M.,
Haas-Stapleton, E.J. (2012) Inactivation of the budded virus of Autographa californica
M nucleopolyhedrovirus by gloverin. J. Invertebr. Pathol. 110, 92-101.
[193] Bao, Y., Yamano, Y., Morishima, I. (2005) A novel lebocin-like gene from eri-
silkworm, Samia cynthia ricini, that does not encode the antibacterial peptide lebocin.
Comp. Biochem. Physiol. Part B. Biochem. Mol. Biol. 140, 127-131.
[194] Furukawa, S., Taniai, K., Ishibashi, J., Hara, S., Shono, T., Yamakawa, M. (1997) A
novel member of lebocin gene family from the silkworm, Bombyx mori. Biochem.
Biophys. Res. Commun. 238, 769-774.
[195] Hara, S., Yamakawa, M. (1995) A novel antibacterial peptide family isolated from the
silkworm, Bombyx mori. Biochem. J. 310, 651-656.
[196] Lavine, M.D., Chen, G., Strand, M.R. (2005) Immune challenge differentially affects
transcript abundance of three antimicrobial peptides in hemocytes from the moth
Pseudoplusia includens. Insect Biochem. Mol. Biol. 35, 1335-1346.
[197] Liu, G., Kang, D., Steiner, H. (2000) Trichoplusia ni lebocin, an inducible immune
gene with a downstream insertion element. Biochem. Biophys. Res. Commun. 269,803-
807.
 Immune Response in Lepidoptera 147

[198] Lamberty, M., Ades, S., Uttenweiler-Joseph, S., Brookhart, G., Bushey, D., Hoffmann,
J.A., Bulet, P. (1999) Insect immunity. Isolation from the lepidopteran Heliothis
virescens of a novel insect defensin with potent antifungal activity. J. Biol. Chem. 274,
9320-9326.
[199] Lamberty, M., Caille, A., Landon, C., Tassin-Moindrot, S., Hetru, C., Bulet, P.,
Vovelle, F. (2001) Solution structures of the antifungal heliomicin and a selected
variant with both antibacterial and antifungal activities. Biochemistry 40, 11995-12003.
[200] Lee, Y.S., Yun, E.K., Jang, W.S., Kim, I., Lee, J.H., Park, S.Y., Ryu, K.S., Seo, S.J.,
Kim, C.H., Lee, I.H. (2004) Purification, cDNA cloning and expression of an insect
defensin from the great wax moth, Galleria mellonella. Insect Mol. Biol. 13, 65-72.
[201] Schuhmann, B., Seitz, V., Vilcinskas, A., Podsiadlowski, L. (2003) Cloning and
expression of gallerimycin, an antifungal peptide expressed in immune response of
greater wax moth larvae, Galleria mellonella. Arch. Insect Biochem. Physiol. 53125-
133.
[202] Volkoff, A.N., Rocher, J., d'Alençon, E., Bouton, M., Landais, I., Quesada-Moraga, E.,
Vey, A., Fournier, P., Mita, K., Devauchelle, G. (2003) Characterization and
transcriptional profiles of three Spodoptera frugiperda genes encoding cysteine-rich
peptides. A new class of defensin-like genes from lepidopteran insects? Gene 319,43-
53.
[203] Valanne, S., Wang, J.H., Rämet, M. (2011) The Drosophila Toll signaling pathway. J.
Immunol. 186, 649-656.
[204] Kaneko, T., N., S. (2005) Bacterial recognition and signalling by the Drosophila IMD
pathway. Cell Microbiol. 7, 461-469.
[205] Ashok, Y. (2009) Drosophila toll pathway: the new model. Sci. Signal. 2, jc1.
[206] Kawai, T., Akira, S. (2011) Toll-like receptors and their crosstalk with other innate
receptors in infection and immunity. Immunity 34, 637-650.
[207] Shia, A.K., Glittenberg, M., Thompson, G., Weber, A.N., Reichhart, J.M., Ligoxygakis,
P. (2009) Toll-dependent antimicrobial responses in Drosophila larval fat body require
Spätzle secreted by haemocytes. J. Cell Sci. 122, 4505-4515.
[208] Michel, T., Reichhart, J.M., Hoffmann, J.A., Royet, J. (2001) Drosophila Toll is
activated by Gram-positive bacteria through a circulating peptidoglycan recognition
protein. Nature 414, 756-759.
[209] Wang, L., Weber, A.N., Atilano, M.L., Filipe, S.R., Gay, N.J., Ligoxygakis, P. (2006)
Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and
present peptidoglycan to PGRP-SA. EMBO J. 25, 5005-5014.
[210] Gottar, M., Gobert, V., Matskevich, A.A., Reichhart, J.M., Wang, C., Butt, T.M.,
Belvin, M., Hoffmann, J.A., Ferrandon, D. (2006) Dual detection of fungal infections in
Drosophila via recognition of glucans and sensing of virulence factors. Cell 127,1425-
1437.
[211] Mishima, Y., Quintin, J., Aimanianda, V., Kellenberger, C., Coste, F., Clavaud, C.,
Hetru, C., Hoffmann, J.A., Latgé, J.P., Ferrandon, D., Roussel, A. (2009) The N-
terminal domain of Drosophila Gram-negative binding protein 3 (GNBP3) defines a
novel family of fungal pattern recognition receptors. J. Biol. Chem. 284, 28687-28697.
[212] Imler, J.L., Hoffmann, J.A. (2002) Toll receptors in Drosophila: a family of molecules
regulating development and immunity. Curr. Top. Microbiol. Immunol. 270, 63-79.
148 Eileen Uribe-Querol, Nancy Mora and Carlos Rosales

[213] Tauszig-Delamasure, S., Bilak, H., Capovilla, M., Hoffmann, J.A., Imler, J.L. (2002)
Drosophila MyD88 is required for the response to fungal and Gram-positive bacterial
infections. Nat. Immunol. 3, 91-97.
[214] Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, J.A., Ferrandon,
D., J., R. (2002) The Drosophila immune response against Gram-negative bacteria is
mediated by a peptidoglycan recognition protein. Nature 416, 640-644.
[215] Choe, K.M., Werner, T., Stöven, S., Hultmark, D., Anderson, K.V. (2002) Requirement
for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial
immune responses in Drosophila. Science 296, 359-362.
[216] Aggarwal, K., Silverman, N. (2008) Positive and negative regulation of the Drosophila
immune response. BMB Rep. 41, 267-277.
[217] Silverman, N., Paquette, N., Aggarwal, K. (2009) Specificity and signaling in the
Drosophila immune response. Invertebrate Surviv. J. 6, 163-174.
[218] Zhong, X., Xu, X.X., Yi, H.Y., Lin, C. (2012) A Toll-Spätzle pathway in the tobacco
hornworm, Manduca sexta. Insect Biochem. Mol. Biol. 42, 514-524.
[219] Cheng, T.C., Zhang, Y.L., Liu, C., Xu, P.Z., Gao, Z.H., Xia, Q.Y., Xiang, Z.H. (2008)
Identification and analysis of Toll-related genes in the domesticated silkworm, Bombyx
mori. Dev. Comp. Immunol. 32, 464-475.
[220] Ao, J.Q., Ling, E., Yu, X.Q. (2008) A Toll receptor from Manduca sexta is in response
to Escherichia coli infection. Mol. Immunol. 45, 543-552.
[221] An, C., Jiang, H., Kanost, M.R. (2010) Proteolytic activation and function of the
cytokine Spätzle in the innate immune response of a lepidopteran insect, Manduca
sexta. FEBS J. 277, 148-162.
[222] Wang, Y., Cheng, T., Rayaprolu, S., Zou, Z., Xia, Q., Xiang, Z., Jiang, H. (2007)
Proteolytic activation of pro-spätzle is required for the induced transcription of
antimicrobial peptide genes in lepidopteran insects. Dev. Comp. Immunol. 31,1002-
1012.
[223] Tanaka, H., Yamamoto, M., Moriyama, Y., Yamao, M., Furukawa, S., Sagisaka, A.,
Nakazawa, H., Mori, H., Yamakawa, M. (2005) A novel Rel protein and shortened
isoform that differentially regulate antibacterial peptide genes in the silkworm Bombyx
mori. Biochim. Biophys. Acta. 1730, 10-21.
[224] Tanaka, H., Matsuki, H., Furukawa, S., Sagisaka, A., Kotani, E., Mori, H., Yamakawa,
M. (2007) Identification and functional analysis of Relish homologs in the silkworm,
Bombyx mori. Biochim. Biophys. Acta. 1769, 559-568.
[225] Furukawa, S., Tanaka, H., Ishibashi, J., Imanishi, S., Yamakawa, M. (2009) Functional
characterization of a cactus homolog from the silkworm Bombyx mori. Biosci.
Biotechnol. Biochem. 73, 2665-2670.
In: Lepidoptera ISBN: 978-1-62417-248-9
Editors: Elia Guerritore and Johannes DeSare © 2013 Nova Science Publishers, Inc.

Chapter 5

GRAPE VINE MOTH, LOBESIA BOTRANA:

CLASSIFICATION, BEHAVIOR AND ECOLOGY

Vanesa Ortega-López1, Mariano Amo-Salas2

and Belén Alonso3
1
Civil Engineering Department, EPS University of Burgos. Burgos, Spain.
2
Mathematics Department, University of Castilla-La Mancha, Ciudad Real, Spain
3
Chemistry Department, EPS University of Burgos. Burgos, Spain

INTRODUCTION
The grape vine moth, Lobesia botrana (Denis and Schiffermüller, 1776), a
microlepidoptera member of the family Tortricidae, is one of the most important pests that
attacks vineyards in Southern Europe and Northern Africa. It is a polyvoltine species with
three or four generations per year depending on the latitude. The number of generations is
mainly modulated by the temperature and photoperiod but other factors including relative
humidity also have an important influence on its development. Temperature and photoperiod
operate on the growth rate and the induction of diapause, respectively. L. botrana passes
through eight development stages in its cycle: egg, five larval stages, pupa and adult.
The efficiency of the control methods (mating disruption with pheromones and chemical
treatments) depends on the treatment of pest populations at their most susceptible stages, the
prediction of the moth’s development cycle would therefore greatly help in determining an
optimal treatment schedule (Moravie et al., 2006). Pheromone traps provide useful
information on male moth activity in vineyards, but by themselves, do not provide a reliable
basis for timing control methods. However, together with the method of temperature
summation, trap samples are important components of phenological models (Riedl et al.,
1976) which could be helpful in identifying the peak flight of [Link], achieving better
timing of treatments. Considering the increasing interest for biorational insecticides where
precise timing of treatments is important, daily temperature models could be a useful tool in
improving their efficiency (Schmid et al., 1977).

E-mail: vortega@[Link]
150 Vanesa Ortega-López, Mariano Amo-Salas and Belén Alonso

GEOGRAPHIC DISTRIBUTION
Lobesia botrana, native to Southern Italy, it was first described from Austria and is now
found throughout Europe, North and West Africa, the Middle East, and eastern Russia. The
first introduction to the western hemisphere was reported in Chile in 2008. Subsequently, this
species was found in California in 2009 (Roehrich and Schmid, 1979). The importance of the
plague depends on the zone. Some zones have usually the plague and in others occasionally,
therefore they do not always need the plague’s treatment.

TAXONOMIC POSITION
The systematic position of L. botrana has suffered repeated modifications. Some of the
adopted names have been: Tortrix botrana (Schiffermüller, 1776); Tortrix vitisana (Jacquin,
1788); Tinea premixtana (Hübner, 1796); Tinea reliquana (Hübner, 1816); Cochylis
reliquana (Treitschke, 1833-1835); Tortrix reliquana (Treitschke, 1835); Cochylis vitisana
(Audouin, 1842); Cochyli sbotrana (Herrich-Schäffer 1843-1856), Panthina vitivorana
(Packard, 1860); Grapholita botrana (Heinemann, 1863); Eudemis rosmarinana (Millière,
1864-1868); Coccyx botrana (Praun, 1869); Eudemis botrana (Frey 1880, Mayet 1890)
(Chkubianishili and Malaniya, 1986; Rossi, 1993).
L. botrana belongs to the family Tortricidae, subfamily Olethreutinae. Its complete
taxonomic position is shown in Table 1 (Buser et al., 1974; Roelofs et al., 1973; Zerny and
Beier, 1936-1938). The wide distribution of L. botrana implies several common names
depending on the location. In Europe, some of the common names are eudémis (France);
tignolleta della vite (Italy); bekreuzter traubenwickle (Germany); polilla del racimo (Spain);
and European grape berry moth, grape leafroller, European vine moth (English-language
literature). Other tortricid species are similar in appearance to L. botrana and cause very
comparable damage to, for example Endopiza viteana or Eupoecilia ambiguella, but these
species should not be confused. They differ in many ways, including life cycle, host range,
pheromone composition, and natural enemies. In other regions of the world, including
Europe, numerous species are commonly referred to as berry and vine moths, thus it is
important to verify the scientific name Lobesia botrana when searching the literature for
information on this pest (Venette et al., 2003).

Table 1. Taxonomic position of L. Botrana

Order Lepidoptera
Suborder Heteroneura
Section Ditrysia
Super family Tortricoidea
Family Tortricidae
Subfamily Olethreutinae
Tribe Olethreutini
Gender LobesiaGuenée
Species L. botrana, Denis andSchiffermüller
 Grape Vine Moth, Lobesia Botrana 151

Grape (Vitis vinifera) and spurge laurel (Daphne gnidium) are preferred hosts (Roehrich
and Boller, 1991), but it has also been reported on blackberry (Rubus fruticosus), gooseberry
(Ribes sp.), black and red currant (Ribes nigurm), olive (Olea europaea), cherry (Prunus
avium), prune (Prunus domestica), persimmon (Diospyrus kakis), kiwi (Actinidia chinensis),
pomegranate (Punica granatum), carnation (Dianthus spp.), and a number of other wild hosts,
approximately 27 plant families (Bradley et al., 1979; Gabel et al., 1992; Moleas, 1988;
Savopoulou-Soultani and Tzanakakis, 1988; Stavridis and Savopoulou-Saultani, 1998).

LIFE CYCLE AND DEVELOPMENT STAGES

L. botrana is a polyvoltine species with two, three or four generations per year depending
on the latitude. The number of generations is mainly modulated by the temperature and the
photoperiod but other factors including relative humidity also have an important influence on
its development. They operate respectively on the growth speed and the induction of the
diapause. L. botrana passes through eight development stages in its cycle: egg, five larval
stages, pupa and adult. The adults of L. botrana (Figure 1) emerge from pupation and mate.
Moths are active at dusk (the period between the sunset and dark). The females normally mate
once while males are capable of mating multiple times (Torres-Vila et al., 1997).
The body length of the adult or imago is around 6-8 mm and the wingspan around 10-13
mm. The first pair of wings has mosaic figures (black-brown-white-blue-red) and the second
pair of wings is gray with a fringed border. The wings are held in a bell shape over the
abdomen when at rest. The males are smaller than the females and they have much more
smart and nervous movements when they are disturbed.

Figure 1. Adults of L. botrana.

152 Vanesa Ortega-López, Mariano Amo-Salas and Belén Alonso

Figure 2. Eggs of [Link].

Figure 3. Larvae and pupae of L. botrana.

Three days after mating, eggs are laid isolated or in groups of 2-3 on host reproductive
organs (Figure 2). Every female lays around 50-80 eggs and later dies. The adults live
approximately 7-10 days after the laying. The eggs are flat and elliptically shaped. Their
dimensions are approximately 0.65-0.90 mm long by 0.45-0.75 mm wide.
Initially they are iridescent creamy white, turning yellow as the embryo develops and
later black when the head of the developing larva is formed. In 7-10 days, the larva emerges
from the edge of the egg and leaves the translucent, iridescent chorion (outer shell) attached.
 Grape Vine Moth, Lobesia Botrana 153

Newly hatched larvae are extremely mobile and invade the host inflorescences. Older
larvae will tie some inflorescences together using silk for protection. The structures
(glomerules) are clearly visible and can be used for detection. When the larva matures, it
moves from the inflorescences to the berries to feed. Initial feeding occurs on the surface of
the berries. Later, the larva penetrates the fruit and feeds on the pulp of the grapes. The larva
passes through five development stages. Its color changes from light yellow to dun. The
larvae size is from 0.95-1.0 mm long to 10-15 mm, just before pupation. Larval growth
requires around 19 to 30 days (Figure 3).
Nondiapausing pupation occurs on the leaves and requires 12 to 14 days (Touzeau,
1981). The chrysalis or pupa presents a milky, blueish or greenish color when newly formed,
passing to a brownish or dark dun in just a few hours. The size and weight are higher in the
female (male 4-7 mm; female 5-9 mm), and in this phase it presents a clear sexual
dimorphism. The pupa perforates the cocoon through abdominal movements and frees the
imago.

Figure 4. Diagram of the life cycle of L. botrana during a typical growing season in Northern Spain
(Baggiolini, 1952; Roehrich, 1968; Schmid et al., 1977;Torres-Vila et al., 1995).
154 Vanesa Ortega-López, Mariano Amo-Salas and Belén Alonso

Diapausing pupation occurs under the bark of the host or in surrounding wood crevices.
Larvae and eggs are capable of remaining dormant under extreme temperature conditions
(Tzanakakis et al., 1988).
The first generation occurs in May-June and appears during the flower bud stage of its
host (phenological stage H), when it damages the inflorescences (Baggiolini, 1952). The
second generation (July) is observed during the closing of the grape clusters (phenological
stage L) and it damages the green berries; the third generation (September) affects mature
grapes (phenological stage N) which can be seriously damaged (Armendáriz et al., 2010;
Ortega-López and Alonso-González, 2008; Vassiliou, 2009). The maturity stage of grape
berries exerts an influence on the development of L. botrana (Savopoulou-Soultani et al.,
1999). Additionally, feeding damage to berries after veraison exposes them to infection by
Botrytis and other secondary fungi. Secondary pests such as raisin moth (Cadra figulilella),
fruit flies, and ants may also be attracted to damaged berries. L. botrana remains dormant as a
pupa from September-October (in the north hemisphere) until the following spring.
Figure 4 shows the life cycle of L. botrana through the year in Northern Spain. The
inhibition of diapause is decisively influenced by the average temperature at the end of winter
and the beginning of spring (Coscollá, 1997; Gabel and Roehrich, 1990).
A rapid development of the larva can ensure that the third generation does not enter
dormancy. The development speed of the first generation is the origin of a partial or total
fourth generation, which becomes almost completely lost before winter. This fact could be
attributable to global warming (Martín-Vertedor et al., 2010).

REGULATORY FACTORS ON THE DYNAMICS

OF POPULATIONS

Factors that influence the cycle of L. botrana can be classified in non-biotic and biotic
factors.
Temperature: This is the variable that most affects the growth of L. botrana, being related
positively to the speed of development of the preimago and negatively to the longevity of the
adult. In addition, in females, it regulates the development of the ovum and the production of
eggs, which reaches its maximum at a temperature around 22ºC (Bergougmoux, 1988). High
temperatures acting on the egg and/or on the larva can induce the pupa to the diapause
(Tzanakakis et al., 1988). High temperatures, due to the fact that they increase the larval
growth speed, lead to smaller adults. It might be negative to the reproductive potential of L.
botrana (Torres-Vila et al., 1996a). The dusk temperature regulates the activity of the adults.
Relative humidity: If it is extreme, it can cause the death of eggs (Coscollá et al., 1986)
and pupae, which are specially sensitive because of their immobility. The larvae do not seem
to be too sensitive to the hygrometric level, taking into account their habitat and ethology. On
adults, the relative humidity determines their activity and longevity, especially if they run out
of water (Bovey, 1966; Torres-Vila et al., 1996b). Temperature and relative humidity are
regularly changing in an antagonistic way in natural conditions, and show a synergic effect on
L. botrana.
 Grape Vine Moth, Lobesia Botrana 155

Figure 5. Climate-graph of Stellwaag for L. botrana (A means the limit of the activity zone and I means
zone of maximum activity and laying) (Stellwaag, 1929).

Figure 5 shows the climate-graph of Stellwaag (Bovey, 1966), that shows the combined
action of both variables on the activity of the adult. It can be observed that they prefer high
temperatures (superior to 20ºC) and the ideal humidity for the L. botrana is between 40% and
70%.
Photoperiod (daily duration of lighting): This factor provokes the entry in diapause of the
pupae coming from eggs incubated with a photoperiod shorter than 15h 40m. The imbalance
of both factors (temperature and photoperiod) in a season can be the origin of a dramatic
mortality. Likewise the water loss of the pupae during the diapause can also play a role in the
insect reproductive potential (Torres-Vila et al., 1996a). As a result of the photoperiod, the
dusk determines the daily cycle of the adults activity.
Wind and rain: They affect adversely the flight and consequently the activity of adults, as
well as the larvae installation and survival. Especially rain, when wet the fruitful organs,
disables the female’s egg laying.
The water condensation of dew on the foliage, as a consequence of inverted curves of
temperature and humidity, is specially important. In certain occasions, it constitutes the only
source of water for the adults and, without drinking water, the reproductive potential of both
sexes diminishes drastically (Torres-Vila et al., 1996b).
Some of the most important regulating biotic factors of L. botrana populations are: the
variety, morphology and phenological stage of the fruitful organs, affecting the installation
and larval mortality (Gabel and Roehrich, 1995), the feeding quality of the fruitful organs
according to the vineyard phenological stage (Torres-Vila et al., 1999) and the larval
competition (Torres-Vila et al., 1997a). The principal natural enemies, that can have a
bounding action on Lobesia botrana, are:
Predators: Bats, birds and a great number of arthropods are the predators of L. botrana.
Coscollá (Coscollá, 1998) quantifies them in 10 species of spiders and 21 of insects.
156 Vanesa Ortega-López, Mariano Amo-Salas and Belén Alonso

Parasites: There are around a hundred of insects that parasitize L. botrana by Coscollá
(Coscollá, 1998). Most of them are Hymenopterae and the rest are different species of
Dipterae, Tachinidae (i.e., Phytomyptera).
Entomopathogens: viruses, bacteria, fungi and protozoa that cause diseases to L. botrana,
well related to non-biotic factor such as temperature and relative humidity (Poinar, 1991).

SYMPTOMS AND DAMAGES

The damages on the grapevine are mainly related to its phenological stage. In the first
generation, the moth feeds on inflorescences and the most of the experimental works show
that the plant has a great capacity to compensate the moth attack (Coscollá et al., 1982). This
explains the trend of not treatment in the first generation. Exceptions to this generalization are
the varieties with very small inflorescences (Basler and Boller, 1976) and the vineyard from
northern regions, where the climate favours the early attacks of gray rot (ACTA-ITV, 1980).
In summer generations, the harvest loss, caused by the destruction of berries in weak or
medium attacks, may not seem very important if it is evaluated just as yield. The severe
damages correspond to the loss of quality, derived from the development of diverse fungi
(Botrytis, Aspergillus, Alternaria, Rhizopus, Cladosporium, Penicillium, etc.) into the wounds
of the berries. The gray rot produced by Botrytis cinerea (Figure 6) is the most serious
infection (Fernaud, 1990). The presence of residual fungal causes bad smells and flavours in
the wine and causes technical problems in the clarification.
The third larval generation coincides with the ripe clusters. In this stage the apparent
defence of green berries disappear almost completely and there is a decrease of the acidity
and an increase of the concentration of sugar, which help the fungi attack.

Figure 6. Damages produced by Botrytis cinerea.

 Grape Vine Moth, Lobesia Botrana 157

The larva perforates the berries in the surface several times, because the inner pulp has
too much water which is not good for a quick penetration, especially during the first larval
stages. This fact greatly increases the intensity of the fungi attack due to the increased number
of penetration routes. On the other hand, the juice lost by the wounds is rich in sugar which
attracts Drosophila sp., whose larvae favour the secondary attack of bacterial acid rot.
When the berry dries up and the larva reaches a suitable size, it usually looks for shelter
inside the husk, assuring it with silk threads. From this protected emplacement it feeds on
closer berries, being able to change its location one or more times.

CONTROL METHODS
Cultural control. The cultural practices in the vineyard may indirectly affect the
population of L. botrana, affecting their development stages or modifying their behavior
(Voukassovitch, 1924). The grapevine pruning and the conduction allow an aeration flow of
the fruitful organs. The irrigation may be unfavorable in certain conditions because the adults
are attracted by the humidity. The harvest can also reduce the plague when the clusters
content larvae from the third generation. The destruction of weeds by burning or extraction,
especially if they are alternative hosts, is a practice to follow, although its effectiveness is
questionable (García López, 1929).
In conclusion, the outlined practices may have importance to assess an impact on the
populations of L. botrana, but in no case have been definitive means of control.
Chemical Control. In the case that the attack reaches certain thresholds, the use of
insecticides is unavoidable. These systems lead the control methods because of their
efficiency and low cost. The available formulations today for the chemical control of L.
botrana are numerous and fall into several chemical families. The chemical control of L.
botrana in the first generation is often unnecessary. However, in the vineyards where the
populations are always very high, can be a preventive treatment from May to early June (in
the north hemisphere). Curative treatments are also possible if the decision to intervene is
taken later and if the population monitoring indicates the need. In the second generation, the
treatment schedule is primarily preventive, applying before larvae enter the berries. The
curative fight is also possible with penetrating products, though often less effective, because
the larvae are protected inside the clusters. For the efficiency of the treatment, we have to
think on the action, persistence, effect on the auxiliary fauna, resistances and other collateral
effects, technological and time of application (preventive or curative). Fixing the damage
thresholds, estimated by the monitoring of the populations and/or continuing the agricultural
tips from the forecast and warning station, the systematic interventions are stopped and the
unnecessary chemical treatments suppressed. In this way a reasonable chemical control is
achieved, which is the first step towards Integrated Pest Management (IPM).
Biological Control. These include control methods based on the rational and directed
natural enemies used against L. botrana. Currently, the use of Bacillus thuringiensis is the
only satisfactory and available method. The first test applications, both under laboratory and
field conditions, had demonstrated the effectiveness of the bacteria and/or their toxins in
reducing the population of L. botrana (Roehrich, 1964; Roehrich, 1967; Roehrich, 1968;
Roehrich, 1970). This effectiveness could be increased by adding 1% sugar to the formula,
158 Vanesa Ortega-López, Mariano Amo-Salas and Belén Alonso

which would act as phagostimulant and increasing persistence by reducing the washing of the
product (Roehrich and Schmid, 1979; Schmid et al., 1977). With current obtained
formulations, the effectiveness is between 75 and 90%, and sometimes even higher. Under
favorable weather conditions, it is as effective as conventional insecticides (Roehrich and
Boller, 1991).
Control by virus has not been the subject of systematic research, so their interest charged
remains obscure. In the former Soviet Union, test applications under field conditions of
granulosis virus, Baculovirus orana, good results were obtained, however, with an efficiency
of 60-100% (Chkubianishili and Malaniya, 1986). The use of parasitic wasp pupa Dibrachys
affinis Masi has also being investigated for biological control of L. botrana, but technical
limitations are extensive to this species (Rossi, 1993).
Biotechnical Control. There are technologies based on the use of chemical mediators and
pheromones, for direct pest control. The pheromones have been defined by the same authors
as chemicals released by an organism, often through a specialized gland, and after received by
an individual of the same species, cause a specific reaction, a particular behavior or an
evolutionary process. They are involved in intraspecific communication, as opposed to
allelochemicals, that act at the interspecific level. From the ecological point of view, they are
usually divided, in alomones and kairomones, according to if provoking an advantage in the
producer or receptor of the signal. According to the induced behavior, pheromones can be
classified into aggregation, alarm, dispersion, and sexual labeling.
Sex pheromones are the most thoroughly studied and the most interesting from the point
of view applied to L. botrana. They can be produced by both sexes, but in L. botrana have
been described only in the female, although there is evidence indicating its existence in the
male. They facilitate the long-range orientation of male and female location, mating and
coupling. It was not until 1973, when the molecule cis-7- trans-9-acetoxy-1-dodecadiene (=
E7 Z9 dodecadienyl acetate or abbreviated E7Z9 DDA) was first described as the main
attractive component for males, by electroantennography (Roelofs et al., 1973) and
subsequently identified in the female gland (Buser et al., 1974). Synthesis of other
compounds similar in nature, named parapheromones (Payne et al., 1973) have been
described as mimetics (Z9- dodecen 7-inyl acetate) (Bruckner et al., 1988), or synergistic
(methyl (Z) -8 - (1-cyclopentenyl)-7-octenoato) (Ujvary et al., 1988) of the natural
pheromone of L. botrana. Among the utilities of sex pheromones in biotech crop protection,
can be distinguished its use for purposes of mass trapping and mating disruption.
Mass trapping. The purpose of the method is the concentration of the plague, by using
pheromones, in some locations where the insects are removed directly through insecticides or
captured in traps of different designs. It is especially indicated for the control of widely
dispersed species, against which chemical control is irrelevant. The results compared to
traditional insect control are poor. One reason for the lack of effectiveness is the impossibility
of having a high density of traps in the surface to be protected, since the unit cost. Also, the
high reproductive potential of males helps to explain the lack of efficacy of the method in L.
botrana (Torres-Vila et al., 1995).
Sexual confusion. The sexual confusion may be defined as the disruption of sexual
attraction of males to females, as a consequence of the air impregnation with a specific
pheromone along the vineyard. The result is a reduction in the mating population rate, which
is iterated in following generations with an accumulative effect in a strong decrease of the
population, which keeps the damages under an economically acceptable threshold. This
 Grape Vine Moth, Lobesia Botrana 159

control method has been described as hyper-preventive, to act not only before the year with
production, but even before its genesis. It has the advantages of safety regarding the natural
enemies, given its high specificity of action and elimination of insecticide residues. However,
requires a reestablishment of a proper ecological balance, so that the natural enemies are able
to control secondary pests and the use of insecticides may be stopped. The development of
mating disruption method with L. botrana began practically after synthesis of the pheromone
and its production in sufficient quantity. The first experiments in the laboratory (Roehrich and
Carles, 1977) and field (Roehrich and Carles, 1980; Roehrich and Carles, 1982), although
sometimes provided mixed results, set the basic points to achieve increase effectiveness,
mainly as regards the effect of design, density and dispensing nozzles, edge zones, movement
of adults population levels to control and minimum area to be protected. The technical
improvements and accumulated knowledge in the following years, along with a better
understanding of insect reproductive behavior (Schmitz, 1992), have led to the current
satisfactory effectiveness and at least similar to that achieved with chemical control (Stockel
et al., 1994; Stockel et al., 1992) also observed after a prolonged period of field application,
their cumulative effect over time.
It should be noted that, currently, there are important limitations to overcome for the
widespread sexual confusion. These are both economic, restricting its use only to regions
whose products have a high added value and operational, as the successful implementation of
the method requires a collective organization, which enables the simultaneous control large
areas and a certain skill of the winemakers.
Plague monitoring. The presence of the plague in the vineyard can be detected by several
methods. The traps for monitoring the flight activity of adults can be food traps, light traps or
pheromone traps. The last ones are the most common in L. botrana. Cardboard delta traps
with sticky inserts baited with synthetic sex pheromone is used to monitor the flight activity
of male L. botrana (Figure 7). Traps are hung at a height of 1-1.5 m above ground level. In all
vineyards, traps must be hung before the first flight and be maintained to the end of the
season. Traps are checked once a week. Sticky inserts are changed as needed and the
pheromone lures are normally refreshed once every 6 weeks, depend on the pheromone lure
(Amo-Salas et al., 2011). Two traps are placed in each of the vineyards, consequently, the
information obtained correspond to the catches in each trap, the average of both traps and the
average per trap per day (PTD), that is, the ratio between the average of both traps and the
number of days between two inspections. Using this information, flight curves, which express
the captures per trap and day throughout the year, are plotted. This methodology enables the
estimation of the actual time of maximum flight in each generation, once these maximum
flights had taken place; therefore a method to predict these times is necessary.
At the same time, it is recommended to carry out a phenological culture study, a weather
monitoring (Figure 8) and a visual control of damages (glomerules, focus), eggs and larvae
presence in the culture. In general, when the 20-30% of the clusters have some infection
focus, it is necessary to apply some method control, except in first generation where it is not
recommended.
Pheromone trap observations and daily temperature forecasts could be helpful in
identifying the peak flight of L. botrana, achieving better timing of treatments. Considering
the increasing interest for biorational insecticides where precise timing of treatments is
important, daily temperature models could be a useful tool in improving their efficiency.
160 Vanesa Ortega-López, Mariano Amo-Salas and Belén Alonso

Figure 7. Sex pheromonetrap.

Figure 8. Station for weather monitoring.

Predictive methods. It is widely known that models based on data collected by

monitoring are used to forecast the phenological development of insects. Predictive methods
have been developed to forecast the appearance of L. botrana in the field and to monitor its
 Grape Vine Moth, Lobesia Botrana 161

flight activity. One of first more important models, and that is used nowadays to forecast the
cycle of L. botrana, was developed by J. Touzeau in 1981 (Touzeau 1981). This model is
based on a function which relates the day-to-day development of the moth to the average
daily temperatures. The values that quantify the degree of development are accumulated only
if the average daily temperature exceeded 10 ºC. This value establishes the lower
developmental threshold but this model does not establish a higher one.
Table 2 shows the different stages of the development of L. botrana, their durations and
the daily accumulated temperatures necessary to achieve them, expressed in degree-days (DD
> 10 ºC), according to Touzeau (Touzeau, 1981). Each developmental stage of L. botrana has
its own total heat requirement. Development can be estimated by accumulating degree-days
between the temperature thresholds throughout the season. The accumulated degree-days
from a starting point can help to predict when developmental stages will bereached.
Nevertheless, only the maximum flight times can be ascertained with monitoring methods.
However this model doesn’t have a biological form and its predictions are not applicable in
zones with different climate conditions relative to France, where the model was first
developed.
From 1981 to present a large number of studies about L. botrana has been published, but
many of them have been written in the native language of the researchers or it is difficult to
find the publication complicating the access to the work (Baumgärtner and Baronio, 1988;
Briolini et al., 1997; Caffarelli and Vita, 1988; Gabel and Mocko, 1984; Gabel and Mocko,
1986). In this chapter recent works published in English and in journals with an important
impact have been considered.

Table 2. Steps of development and accumulated temperatures sum necessary

according to the Touzeau model (Touzeau, 1981)

Temperatures Accumulated
Average duration of different steps
sum (ºC) temperature sum (ºC)
Step (days)
DD > 10 ºC DD > 10 ºC
Maximum flight 1st 125 125 The adult lives 10-15 days
G
Egg eclosion 1st G 75 200 7-10 days after the laying

Larval development 170 370 5 larval stages: 21-30 days

Pupal formation and 130 500 Pupa: 12-14 days before the adults
adult appearance of emerge (adult life: 10 days)
2nd G
Egg eclosion 2nd G 65 565 7-10 daysafter the laying

Larval development 255 820 5 larval stages: 19-25 days

Chrysalis formation 130 950 Pupa: 12-14 days before the adults
and adult emerge (adult life: 10 days)
appearance of 3rd G
Egg eclosion 3rd G 75 1025 7-10 days after the laying
hibernated larvae
162 Vanesa Ortega-López, Mariano Amo-Salas and Belén Alonso

In chronological order, in 2001 Milonas, Savopoulou-Soultani and Stavridis (Milonas et

al., 2001) developed a nonlinear model with three parameters for predicting the cumulative
percentage of captured moths relative to the sum of the effective temperatures reached at the
date of the trap inspection. The model started on March first and the lower threshold
temperature considered in its work was 6.45 ºC. The model was developed in Greece and a
different model is computed for each generation of the moth.
In 2006, Moravie, Davison, Pasquier and Charmillot (Moravie et al., 2006) proposed a
stochastic model using a Bayesian approach for forecasting the emergence times of the L.
botrana in Switzerland. The daily average temperatures were summed from January first and
the lower threshold temperature considered was 10 ºC.
A different point of view was presented by Delbac, Lecharpentier and Thiery (Delbac et
al., 2010). In this work, performed over data from France, a statistical model for describing
the distribution of each larval stage inside each generation based on the head-capsule width
was developed. These distributions were used for estimating the timing of emergence for
adult populations.
More complex models are presented from a mathematical perspective in the works of
Aylaj and Noussair (Aylaj and Noussair, 2010) and Picard, Ainseba and Milner (Picart et al.,
2011). Both models are explained by ordinary differential equations describing the population
dynamics. The first work considered four stages (i.e., egg, larval, pupal and adult) and
established five differential equations, while the adult stage is divided in male and female. In
the second work three differential equations were established, for the stages egg, larval and
adult female.
The work of Sciarreta, Zinni and Trematerra (Sciarretta et al., 2011) describes an
Integrated Pest Management (IPM) where spatial variability is incorporated. They studied the
spatio-temporal distribution of L. botrana eggs, larvae and adult populations in the examined
agro-ecosystem and developed a site-specific IPM for a Mediterranean agricultural landscape
in Central Italy.
Finally, in 2011 the work of Amo-Salas, Ortega-López, Harman and Alonso-González
(Amo-Salas et al., 2011) developed models based on temperature and relative humidity. The
models were based on the logistic function and the sums starts on January first where the
lower temperature threshold is not necessary to take into account. The model had three
parameters and established the thresholds for each generation so it was possible to predict the
dates of maximum number of males with the same equation. This model was developed for a
central region of Spain, includes a biological form and has a simply implementation.

REFERENCES
ACTA-ITV. (1980) Protection intégrée. Contrôles périodiques au vignoble II, Laboreur et
[Link]. pp. 35-38.
Amo-Salas M., Ortega-López V., Harman R., Alonso-González A. (2011) A new model for
predicting the flight activity of Lobesia botrana (Lepidoptera: Tortricidae). Crop
Protection 30:1586-1593.
 Grape Vine Moth, Lobesia Botrana 163

Armendáriz I., Pérez-Sanz A., Miranda L. (2010) Predicción de la polilla del racimo de la vid
(Lobesia botrana) en seis Denominaciones de Origen de Castilla y León. Boletín de
Sanidad Vegetal. Plagas 36:11-22.
Aylaj B., Noussair A. (2010) Global weak solution for a multistage physiologically structured
population model with resource interaction. Nonlinear Analysis-Real World Applications
11:1670-1684. DOI: 10.1016/[Link].2009.03.021.
Baggiolini M. (1952) Les stades repères dans le développement annuel de la vigne et leur
utilisation pratique. Revue romande d'Agriculture et d'Arboriculture 8:4-6.
Basler P., Boller E. (1976) Der traubenwickler in der Ostschweiz: II, Zur bedeutung des
heuwurms. Schweiz. Z. Obst-Weinbau 112:74-79.
Baumgärtner J., Baronio P. (1988) Modello fenologico di volo dei Lobesia botrana Den. et
Schiff (Lep. Tortricidae) relativo alla situazione ambientale della Emilia-Romagna.
Bollettino dell'Instituto di Entomologia "Guido Grandi" della Universita degli Studi di
Bologna 43:157-170.
Bergougmoux P. (1988) Dynamique de la population de Lobesia botrana Schiff. dans un
vignoble de Beumes de Venise (Vaucluse). Etude cmparative deu facteur thermique sur
l'émergence et la ponte dans ne population d'élevage et une poppulation naturelle., Univ.
Marseille, Marseille. pp. 32.
Bovey P. (1966) Superfamille des Tortricoidea, Entomologie Appliquée à l'Agriculture,
Balachowsky A.S., Mason et Cie, Paris.
Bradley J.D., Tremewan W.G., Smith A. (1979) Lobesia botrana (DenisandSchiffermüller)
The Ray Society, London, England.
Briolini G., Di Cola G., Giliolo G. (1997) Stochastic model for population development of
Lobesia botrana (Den. er Schiff.). International Organization for Biological and
Integrated Control of Noxious Animals and Plants-West Palaearctic Regional Section
(IOBC-WPRS Bulletin) 21:79-81.
Bruckner C., Buschmann E., Becker R., Seufert W. (1988) A new highly effective synthetic
pheromone mimic for Lobesia botrana (Lepidoptera: Tortricidae). Zeitschrift für
Naturforschung 43:315-318.
Buser H.R., Rauscher S., Arn H. (1974) Sex pheromones of Lobesia botrana. E7 Z9
dodecadienyl-acetate in the female grape vine moth. Zeitschrift für Naturforschung B
29:731-783.
Caffarelli V., Vita G. (1988) Heat accumulation for timing grapevine moth control measures.
Organisation Internationale de Lutte Biologique et Intégrée contre les Animaux et les
Plantes Nuisibles- Section Régionale Ouest Paléarctique (OILB-SROP Bulletin) 11:24-
26.
Coscollá R. (1997) La polilla del racimo de la vid (Lobesia botrana Den Y Schiff). 1ª edición
ed. Generalitat Valenciana. Consellería de Agricultura, Pesca y Alimentación.
Coscollá R. (1998) Polillas del racimo (Lobesia botrana Den. y Schiff.), Los parásitos de la
Vid. Estrategias de protección razonada, 4ª Edición. Ministerio de Agricultura, Pesca y
Alimentación. Ediciones Mundi-Prensa, Madrid.
Coscollá R., Arias A., Cortés J.A., Esteve R., Martínez-Morga F., Nieto J., Pérez-Marín J.L.,
Rodríguez-Pérez M., Sánchez-García J., Toledo J. (1982) Estudio de los daños
producidos por la primera generación de la polilla del racimo (Lobesia botrana Den. and
Schiff.). Boletin de Sanidad Vegetal de Plagas 8:215-223.
164 Vanesa Ortega-López, Mariano Amo-Salas and Belén Alonso

Coscollá R., Arias A., Cortés J.A., Esteve R., Martínez-Morga F., Nieto J., Pérez-Marín J.L.,
Rodríguez-Pérez M., Sánchez-García J., Toledo J. (1986) Estudio preliminar sobre la
mortalidad de huevos de Lobesia botrana Den. and Schiff. por efecto de altas
temperaturas y bajas humedades relativas en laboratorio. Boletin de Sanidad Vegetal de
Plagas 12:3-7.
Chkubianishili T.A., Malaniya I.G. (1986) Granulosis virus against the european grape moth
5:33.
Delbac L., Lecharpentier P., Thiery D. (2010) Larval instars determination for the European
Grapevine Moth (Lepidoptera: Tortricidae) based on the frequency distribution of head-
capsule widths. Crop Protection 29:623-630.
Fernaud M. (1990) Incidence des attaques larvaires d'Eudémis Lobesia botrana sur le
développement de la Pourriture Grise Botrytis cinerea chez la vigne: rô des facteurs du
milieu et mécanismes mis en jeu, INA PG, INRA, París. pp. 104.
Gabel B., Mocko V. (1984) Forecasting the cyclical timing of the grape vine moth, Lobesia
botrana (Lepidoptera: Tortricidae). Acta Entomologica Bohemoslovaca 81:1-14.
Gabel B., Mocko V. (1986) A functional simulation of european vine moth Lobesia botrana
Den. et Schiff. (Lep.: Tortricidae) population development. Zeitschrift fur Angewandte
Entomologie-Journal of Applied Entomology. 101:121-127.
Gabel B., Roehrich R. (1990) Results of exposure to varyng temepratures of diapausal
nymphs of Lobesia botrana Den. and Schiff. Regulation of Seasonal Cycles in
Invertebrate 52:57-60.
Gabel B., Roehrich R. (1995) Sensitivity of grapevine phenological stages to larvae of
European Grapevine Moth, Lobesia botrana Den. and Schiff. ([Link]). Journal
of Applied Entomology-Zeitschrift Fur Angewandte Entomologie 119:127-130.
Gabel B., Thiéry D., Suchy F., Marion-Poll F., Hradsky P., Farkas P. (1992) Floral volatiles
of Tanacetum vulgare L. atractive to Lobesia botrana Den. et Schiff. females. Journal of
Chemical Ecology 18:693-701.
García López A. (1929) Insectos que atacan a la vid. I, Piral, Cochylis, Eudemis. Ministerio
de Economía Nacional.
Martín-Vertedor D., Ferrero-García J.J., Torres-Vila L.M. (2010) Global warming affects
phenology and voltinism of Lobesia botrana in Spain. Agricultural and Forest
Entomology 12:169-176.
Milonas P.G., Savopoulou-Soultani M., Stavridis D.G. (2001) Day-degree models for
predicting the generation time and flight activity of local populations of Lobesia botrana
(Den. and Schiff.) (Lep., Tortricidae) in Greece. Journal of Applied Entomology-
Zeitschrift Fur Angewandte Entomologie 125:515-518.
Moleas T. (1988) Lobesia botrana [Link] Schiff (Tortricidae-Lepidoptera), a potetntial
danger for kiwi (Actinidae chinensis Planchon). Informatore Fitopatologico 12:71-73.
Moravie M.A., Davison A.C., Pasquier D., Charmillot P.J. (2006) Bayesian forecasting of
grape moth emergence. Ecological Modelling 197:478-489.
Ortega-López V., Alonso-González A. (2008) Descripción, comportamiento y monitorización
de la polilla del racimo (Lobesia botrana, Den y Schiff.) en la Ribera del Duero. Tierras
de Castilla y León 148:50-66.
Payne T.L., Shorey H.M., Gaston L.K. (1973) Sex pheromones of Lepidoptera.
Electroantennogram responses in Autographa californica to cis-7 dodecenyl-acetate and
related compounds. Annals of the entomological society of America 66:703-704.
 Grape Vine Moth, Lobesia Botrana 165

Picart D., Ainseba B.E., Milner F. (2011) Optimal control problem ion insect pest
populations. Applied Mathematics Letters 24:1160-1164.
Poinar G.O. (1991) Nematode parasites. In Tortricids pest: their biology, natural enmies and
control Elsevier, Amsterdam.
Riedl H., Croft B.A., Hoeiy A.J. (1976) Forecasting colding moth phenology based on
pheromone trap catches and physiological time models. Canadian Entomologist 108:449-
460.
Roehrich R. (1964) A comparative study of the sensitivity of three Lepidoptera (Tortricoidea)
to Bacillus thuringiensis Berliner. Journal of Insect Pathology 6:186-197.
Roehrich R. (1967) Sensibilité des chenilles de Tortricidae a l'action des cristaux associés a la
toxine thermostable de Bacillus thuringiensis Berliner, Sérotype I. phytiatrie
phytopharmacie 16:91-95.
Roehrich R. (1968) Essais sur vigne de divers produits à base de Bacillus thuringiensis
Berliner contre l'Eudémis de la Vigne (Lobesia botrana Schiff). Revue de zoologie
agricole appliquée 67:63-68.
Roehrich R. (1970) Essais de deux produits commerciaux à base de Bacillus thuringiensis
pour la protection de la vigne contre l'Eudémis (Lobesia botrana Schiff). Revue de
Zoologie Agricole et de Pathologie Végétale 69:73-78.
Roehrich R., Boller E. (1991) Tortricids in vineyards Elsevier, Amsterdam.
Roehrich R., Carles J.P. (1977) Perturbations dans la reproduction de Lobesia botrana Schiff.
(Lepidoptera: Tortricidae) induites par la présence de la phéromone sexuelle de synthèse.
Comptes rendus des séances de l'Académie des Sciences de Paris 285:237-239.
Roehrich R., Carles J.P. (1980) Essais en vignoble de confusion sexuelle sur les populations
de Lobesia botrana Schiff. et essais d'automarquage, Colloq. INRA, Colmar. pp. 177-
179.
Roehrich R., Carles J.P. (1982) Essai de confusion sexuelle en vignoble contre l'Eudémis de
la Vigne, Lobesia botrana Schiff, Colloq. INRA, Colmar. pp. 365-371.
Roehrich R., Schmid A. (1979) Tordeuses de la grappe: evaluation du risque, détermination
des périodes d'intervention et recherche des méthodes de lutte biologique, Proc. Int.
Symp. IOBC/WPRS, on Integrated Plant Protection in Agriculture and Forestry, Vienna.
pp. 245-254.
Roelofs W., Kochansky J., Cardé R., Arn H., Rauscher S. (1973) Mitteilungen der
Schweizerische Entomologische Gesellschaft. Sex attractant of the grape vine moth,
Lobesia botrana 46:72-73.
Rossi M.M. (1993) Etudes bioécologiques des parasitoïdes oopphages Trichogramma
cacoeciae Marchal et T. evanescens West (Hym.: Trichogrammatidae) et du parasitoïde
nymphal, Dibrachys affinis Masi (Hym.: Pteromalidae) utilisés en lutte biologique contre
Lobesia botrana Den. et Schiff. (Lep.: Tortricidae), Thèse Doctoral, University of Aix-
Marseille, Marseille.
Savopoulou-Soultani M., Nikolaou N., Milonas P.G. (1999) Influence of maturity stage of
grape berries on the development of Lobesia botrana (Lepidoptera: Tortricidae) larvae.
Journal of Economic Entomology 92:551-556.
Savopoulou-Soultani M., Tzanakakis M.E. (1988) Development of Lobesia botrana
(Lepidoptera: Tortricidae) on grapes and apples infected with the fungus Botrytis cinerea.
Environmental Entomology 17:1-6.
166 Vanesa Ortega-López, Mariano Amo-Salas and Belén Alonso

Sciarretta A., Zinni A., Trematerra P. (2011) Development of site-specific IPM against
European grapevine moth Lobesia botrana (DandS) in vineyards. Crop Protection
30:1469-1477.
Schmid A., Antonin P., Guignard E., Caccia R., Raymond J.C. (1977) Bacillus thuringiensis
dans la lutte contre les vers de la grappe, Eudémis (Lobesia botrana) et Cochylis (Clysia
ambiguella) en Suisse Romande. Revue suisse de viticulture arboriculture horticulture
9:119-126.
Schmitz V. (1992) Contribution à l'étude du mécanisme de la confusion sexuelle des mâles
chez l'insecte. Application à la mise au point du procédé biotechnique de lutte contre
l'Eudémis de la Vigne Lobesia botrana Den. et Schiff. (Lep.: Tortricidae), Thèse ENSA,
Rennes. pp. 139.
Stavridis D.G., Savopoulou-Saultani M. (1998) Larval performance on and oviposition
preference for known and potential hosts by Lobesia botrana (Lepidoptera: Tortricidae).
European Journal of Entomology 95:55-63.
Stellwaag F. (1929) Neuere Erfahrungen in der biologischen Bekämpfung schädlicher
Insekten, Verhandlungen deutsch. Ges. angew. Ent., 7. Mitgliederversamml. Munchen.
pp. 15-32 pp. .
Stockel J., Schmitz V., Lecharpentier P., Roehrich R., Neumann U. (1994) La confusion
sexuelle chez l'Eudémis Lobesia botrana (Lepidoptera Tortricidae). Bilan de 5 années
d'expérimentation dans un vignoble bordelais. Agronomie 14:71-82.
Stockel J., Schmitz V., Lecharpentier P., Roehrich R., Neumann U., Torres-Vila L.M. (1992)
Three years experience in the control of the grape moth Lobesia botrana using mating
disruption in a Bordeaux vineyard. Organisation Internationale de Lutte Biologique et
Intégrée contre les Animaux et les Plantes Nuisibles- Section Régionale Ouest
Paléarctique (OILB-SROP Bulletin) 15:117-120.
Torres-Vila L.M., Rodríguez-Molina M.C., Roehrich R., Stockel J. (1999) Vine phenological
stage during larval feeding affects male and female reproductive output of Lobesia
botrana (Lepidoptera: Tortricidae). Bulletin of Entomological Research 89:549-556.
Torres-Vila L.M., Stockel J., Bielza P., Lacasa A. (1996a) Efecto de la diapausa y del capullo
sobre el potencial biótico de la polilla del racimo Lobesia botrana Den. y Schiff.
(Lepidoptera: Tortricidae). Boletín de Sanidad Vegetal. Plagas 22:27-36.
Torres-Vila L.M., Stockel J., Rodríguez-Molina M.C. (1996b) Efecto de la indisponibilidad
de agua sobre el potencial biótico de la polilla del racimo Lobesia botrana Den. y Schiff.
(Lepidoptera: Tortricidae). Boletín de Sanidad Vegetal. Plagas 22:443-449.
Torres-Vila L.M., Stockel J., Rodríguez-Molina M.C. (1997) Physiological factors regulating
polyandry in Lobesia botrana (Lepidoptera: Tortricidae). Physiological Entomology
22:387-393.
Torres-Vila L.M., Stockel J., Roehrich R. (1995) Le potential reproducteur et ses variables
biotiques associees chez le male d l'Eudemis de la vigne Lobesia botrana. Entomologia
Experimentalis et Applicata 77:105-119.
Torres-Vila L.M., Stockel J., Roehrich R., Rodríguez-Molina M.C. (1997a) The relation
between dispersal and survival of Lobesia botrana larvae and their density in vine
inflorescences. Entomologia Experimentalis et Applicata 84:109-114.
Touzeau J. (1981) Modélisation de l'évolution de l'Eudémis de la Vigne por la région MIdi
Pyrénées. Bollettino di Zoologia Agraria e di Bachicoltura , ser. II 16:26-28.
 Grape Vine Moth, Lobesia Botrana 167

Tzanakakis M.E., Savopoulou-Soultani M., Oustapassidis C.S., Verras S.C., Hatziemmanuel

H. (1988) Induction of dormancy in Lobesia botrana by long day and high temperature
conditions Entomologica Hellenica 6:7-10.
Ujvary I., Voigt E., Csutak J., Kis-Tamas A., Novak L. (1988) Synthesis and bioactivity of
cyclic analogues of the sex pheromone of the european grapevine moth, Lobesia botrana
(Den. et Schiff.). Acta phytopathologica et entomologica hungarica 23: 235-242.
Vassiliou V.A. (2009) Control of Lobesia botrana (Lepidoptera: Tortricidae) in vineyards in
Cyprus using the Mating Disruption Technique. Crop Protection 28:145-150.
Venette R.C., Davis E.E., DaCosta M., Heisler H., Larson M. (2003) Mini Risk Assessment,
Grape berry moth, Lobesia botrana (Denis and Schiffermuller) (Lepidoptera:
Tortricidae), Department of Entomology, University of Minnesota.
Voukassovitch P. (1924) Contribution à l'étude de l'Eudémis (Polychrosis botrana Schiff.) de
la Pyrale de la Vigne (Œnophthira pilleriana Schiff.) et de leurs parasites, Thèse Doct.
Fac. Sci. , Toulouse, Toulouse. pp. 248.
Zerny H., Beier M. (1936-1938) Lepidoptera, Handbuch der Zoologie., Kükenthal W. and
Krumbach T., Berlín.
In: Lepidoptera ISBN: 978-1-62417-248-9
Editors: Elia Guerritore and Johannes DeSare © 2013 Nova Science Publishers, Inc.

Chapter 6

SOUTH AMERICAN LONOMIA OBLIQUA

CATERPILLARS: MORPHOLOGICAL
ASPECTS AND VENOM BIOCHEMISTRY

Ana Marisa Chudzinski-Tavassi

and Miryam Paola Alvarez-Flores
Laboratory of Biochemistry and Biophysics, Butantan Institute, Sao Paulo, Brazil

ABSTRACT
Lepidoptera species are widely distributed around the world and the majority of
medically important envenoming occurs after the contact of human skin with the hairs or
bristles of the larva form (caterpillars). In most occasions, the adverse effects are self-
limited and the treatment is based on the use of antipruritics and oral antihistamines.
However, envenomation caused by the South American Lonomia obliqua caterpillar
(family: Saturniidae), can lead to a hemorrhagic syndrome characterized by coagulation
disorders. A specific anti-serum had to be produced for an effective treatment against the
caterpillar venom and for re-establishing the coagulation parameters in poisoned patients.
The biological cycle of L. obliqua is composed by 4 phases with distinct durations (egg,
larva with 6 instars, pupa, and adult). Human envenoming usually occurs with larvae
between instars 4 to 6. After the contact with the human skin, caterpillar bristles are
broken and the toxic principle quickly reaches the bloodstream, affecting mainly the
hemostatic system. Recent studies have demonstrated that the mechanisms triggered by
L. obliqua toxins are complex and some multifunctional toxins have been related to the
pathophysiology of envenoming but also related to the development process of the
animal, such as regulation of the cell cycle. The main toxins isolated from this venom (a
prothrombin activator and a factor X activator) act at different levels on the coagulation
cascade. This chapter reviews the information currently available about distribution,
morphological aspects, adverse effects in humans and venomous components related to
the Lonomia obliqua caterpillar that have been described so far.

*
To whom correspondence to be addressed: Ana Marisa Chudzinski-Tavassi, Laboratório de Bioquímica e
Biofísica, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, SP, Brazil, CEP 05503-900. Phone: +55-11-
3726-7222 ext. 2043; Fax: +55-11-3726-1505; E-mail: amchudzinski@[Link]; miryam_paolaa@hot-
[Link].
170 Ana Marisa Chudzinski-Tavassi and Miryam Paola Alvarez-Flores

Keywords: Lonomia, erucism, consumption coagulopathy, hemorrhagic syndrome, venom

biochemistry

ACCIDENTS INVOLVING LEPIDOPTERA

The lepidopter Order, belonging to the Insect Class, presents more than 250.000 species
distributed in the planet. This order consists of 124 families and 30 superfamilies [1]. Its adult
phase is known as butterfly and moth, and their larvae are known as caterpillars. In the
development phase of larvae, some species are considered a plague for agriculture and some
have commercial importance, such as the silkworm (Bombix mori) [2]. Many adult and larvae
are considered of medical importance, as they inflict serious human injuries after the contact
with poisonous bristles or hairs [3]. Erucism (erucae = larva) is the term used to describe
adverse reaction caused by skin contact with caterpillar hairs, bristles, hemolymph or cocoons
[3-5]. On the other hand, envenoming by adult moths (well-known as lepidopterism) is rare or
poorly documented [2, 3]. For example, some species of the genus Hylesia in South America
(Saturniidae family) are responsible for lepidopterism [6-10].
Clinical effects caused by caterpillar species of moths or butterflies range from severe
pain, urticarial dermatitis (papules or wheals in the skin), respiratory problems to
osteochondritis, consumption coagulopathy, renal failure, and intracerebral hemorrhage [3,
11, 12]. Some species provoke serious lesions, sometimes irreversible, that can lead to death.
The severity of the symptoms is highly variable depending on the caterpillar species or the
structures involved in the contact [6, 12-16]. For example, stinging caterpillars inject the
venom parenterally through their hollow spines causing moderate to severe local effects,
characterized by severe pain, and less commonly systemic effects. On the other hand, itchy
caterpillars possess non-venomous hairs able to cause irritation, dermatitis and/or foreign
body reactions after contact [14, 16, 17].
Rural workers are most susceptible to suffer erucism (because they are frequently
exposed to caterpillars) being considered an occupational disease. This is especially common
in plantation of South America and Southeast Asia [12]. In China, it is frequently observed
that individuals who work in sericulture (silkworm industry) became sensitized to silk
proteins developing allergic reactions [12, 18]. Even the contact with silk products (quilts and
clothes) can also cause skin irritation, conjunctivitis, allergic rhinitis, and asthma [12, 19].
Besides this, silkworm pupa is used in Chinese cuisine and traditional Chinese medicine for
the treatment of hypertension and fatty liver being able to cause hypersensitivity [20, 21].
Less common are the oropharyngeal manifestations caused by caterpillar ingestion or by
contact with the cocoons whose common symptoms are dysphagia and erythema at the site of
contact, followed by pain, edema, drooling, pruritus, and shortness of breath [14, 22, 23].
General recommendation for the treatment of erucism consists primarily of removal of the
caterpillar or cocoon spines followed by symptomatic treatment such as ice packs and
antihistamines and administration of steroid (dexamethasone or prednisone) and antibiotics
[2, 6, 14, 22, 24]. An exception is the envenomation by the South American caterpillar
Lonomia obliqua (family Saturniidae), whose systemic effects are very serious. This species
causes a hemorrhagic diathesis associated to a strong fibrinolysis [25, 26]. This condition is
known as lonomism. A specific anti-serum (anti-lonomic antivenom) had to be produced by
 South American Lonomia Obliqua Caterpillars 171

the Butantan Institute in Brazil for an effective treatment against the caterpillar venom and for
re-establishing the coagulation parameters in poisoned patients [27].
Besides Lonomia obliqua, other species of caterpillars in Brazil are responsible for
inducing some deleterious effect in human health and belong to the families Arctiidae,
Megalopygidae and Saturniidae [2]. In southern Brazil, Premolis semirufa (family Arctiidae)
or pararama (as commonly named) is responsible for causing chronic inflammation in
interphalangeal joints in rubber tappers after multiple contacts with the caterpillar or cocoon
hairs, finally leading to deforming arthritis [2, 3]. Although the bristle extract of pararama is
not lethal in vivo, it can contribute to the severe inflammation observed in patients because of
the high immunogenicity of the toxic contents [28]. However, the most important is the
pathology caused by the caterpillars of the Lonomia genus (family Saturniidae) because they
are capable of drastically altering the physiological parameters of blood coagulation,
changing a patient from a consumption coagulopathy to a hemorragic syndrome, and, in the
absence of the correct treatment, the patient may die. The hemorrhagic syndrome is
characterized by coagulation disorders, bleeding from scars and mucous membranes,
intracerebral bleeding and acute renal failure, sometimes progressing to death [26, 29-39].
Many in vitro and in vivo studies have been carried out to understand the pathophysio-
logical mechanism of the envenoming by L. obliqua [26, 37-40]. While in vivo studies with
bristles extract reported both antithrombotic and thrombolytic actions, most in vitro studies
report procoagulant agents [41-49]. Other toxic principles were also found in the bristle’s
extract and the hemolymph, probably related to the envenoming [37]. In the last few years,
new approaches have been introduced, including cellular and molecular methods such as
cDNA libraries and microarray [37, 44, 50, 51]. This chapter reviews the information
currently available about distribution, behavior, morphology and adverse effects in humans,
and venomous components produced by Lonomia obliqua caterpillar that have been described
so far.

SOUTH AMERICAN LONOMIA OBLIQUA CATERPILLAR

The first report of a human accident involving the Lonomia genus dates from 1914,
where symptoms such as hematuria and blood in saliva were observed in the patient [52]. In
Venezuela, such type of erucism was first described in the late 1960s by Arocha-Piñango and
co-workers, where it was described as a hemorrhagic syndrome in five patients [53, 54].
Since then, more than 1000 cases of lonomism have been reported in Venezuela, Brazil,
French Guyana, Colombia, Peru, Paraguay and Argentina [29, 55-64]. Lemarie identified the
caterpillars from Venezuela as the larvae of Lonomia achelous (Cramer) [61]. In northern
Brazil (Marajó Island, State of Amapá) similar cases were also caused by Lonomia achelous
with a mortality rate of 38% [65]. On the other hand, the caterpillars from southern Brazil
were classified as Lonomia obliqua (Walker) (Figure 1). Although 26 species of the genus
Lonomia are spread on the American Continent only L. obliqua and L. achelous were reported
to provoke hemorrhagic effects in human beings [26, 33, 36, 55, 60-62, 65, 66]. Both species
may lead to a consumption coagulopathy, resembling a disseminated intravascular
coagulation (DIC) in combination with a hyperfibrinolytic state [26, 37-39, 67, 68].
172 Ana Marisa Chudzinski-Tavassi and Miryam Paola Alvarez-Flores

Figure 1. Lonomia obliqua caterpillar. Photograph: personal collection Miryam P. Alvarez-Flores.

Envenoming by Lonomia obliqua caterpillars were considered rare until the late 1980s,
when a burst of accidents with hemorrhagic manifestations were reported in the States of Rio
Grande do Sul, Santa Catarina and Paraná [26, 31, 60, 63, 66, 69]. From 1997 to 2005, 1009
accidents resulting in seven deaths were registered in the state of Rio Grande do Sul [70],
while in the state of Paraná, 354 cases were reported between 1989 and 2005 [38, 63].
Between 1998 and 2000, 201 cases were registered in the Emergency Department of the
Regional Hospital of Chapecó, State of Santa Catarina [26]. Nowadays, envenomation due to
Lonomia obliqua caterpillars is considered a serious problem to public health in Brazil [29,
37].

CLINICAL PROFILE OF ENVENOMATION

BY LONOMIA OBLIQUA CATERPILLARS

Similar to the accidents caused by contact with Lonomia achelous caterpillars,

envenomation caused by Lonomia obliqua caterpillar is characterized by a disseminated
intravascular coagulation and a consumptive coagulopathy, which can lead to a hemorrhagic
syndrome [26, 32, 33, 36, 37, 65, 71]. The hemorrhagic syndrome takes place between 12 and
24 hours after envenomation. Bleeding may appear spontaneously or as result of mild injuries
[66]. The clinical profile resulting from L. obliqua accidents varies depending on the species
involved and on the physical conditions of the victim, e.g. health, age, and body weight [6,
11, 38].
The initial signs and symptoms are burning pain, reddening, and slight local swelling;
followed by nausea, vomiting, headache, dizziness and weakness [26]. Few hours after the
accident, patients develop alterations in blood coagulation, with or without bleeding from
 South American Lonomia Obliqua Caterpillars 173

recent wounds, mucous membranes (gingival and nasal hemorrhage), and hematemesis. If not
treated with the anti-lonomic serum within the first few hours from the accident, the patient
may develop edema, ecchymotic lesions, hematomas, hematuria, acute renal failure, and
abdominal, pulmonary, glandular and intracerebral hemorrhage that may lead to death. The
main complications resulting from envenomation are central nervous system hemorrhage and
acute renal failure. Prior to the develop and distribution of the anti-lonomic serum, a few
deaths due to intracranial hemorrhage and acute renal failure were reported [26, 29-33, 35, 69,
71].
Clinical studies showed that hemostasis alteration in this kind of accident could be severe
as soon as within the first 6 hours, with intense fibrinogen reduction [26]. The most sensible
diagnostic tests are those measuring thrombin time (TT) and fibrinogen (Fg) concentration,
followed by prothrombin time (PT), activated partial thromboplastin time (APTT), and whole
blood clotting time (WBCT). The patients showing altered coagulation parameters in
diagnostic tests must be hospitalized to receive treatment with the anti-lonomic serum [26, 27,
29]. Several studies using experimental animals have shown the efficacy of the anti-lonomic
serum in solving the hemorrhagic syndrome and promoting a rapid recovery of the fibrinogen
levels [26, 27, 38, 39, 72, 73].
The hemorrhagic syndrome is probably a combination of events which lead to this
pathology [38]: (1) a disseminated intravascular coagulation due to thrombin generation; (2)
an intravascular fibrin formation; and (3) a secondary activation of the fibrinolytic system
[26]. In accordance with the intensity of the hemostatic disorders, the accident can be
classified as: a) Mild: local manifestations with no alteration in coagulation, until 12 hours
after the accident. b) Moderate: local manifestations, general symptoms (dizziness, headache,
nausea, and vomiting), and alterations in global coagulation tests, with or without skin or
mucous hemorrhage, and c) Severe: hemostatic disorders, systemic hemorrhage,
hemodynamic alterations and/or multiple system failure.

BEHAVIOR AND ECOLOGY

Human accidents involving contact with L. obliqua caterpillars occur more frequently in
summer months, similar to accidents with Bothrops serpents [16, 38]. The seasonal aspect of
these accidents is related to the beginning of the yearly hot and rainy period. The climatic
aspect, with relation to warmth and the occurrence of rain, has been considered a determining
factor to the proliferation of these caterpillars in a determined period of the year. Therefore,
the predominance of accidents during warmer months would be linked to the beginning of the
hot, rainy season, which is ideal for the hatching of the eggs and subsequent development of
the caterpillars [74].
In the last years, the number of human accidents has been increasing in other Brazilian
states such as São Paulo [35], Rio de Janeiro [75] and Minas Gerais [76]. The high incidence
of L. obliqua caterpillar accidents in other regions throughout Brazil could be facilitated by
several factors affecting the larvae or other stages: a) environmental changes: reduction by
natural phenomena of parasites (Diptera, Tachinidae and Hymenoptera, Ichneumonidae)
which promote the natural control of the population; b) human intervention: intensive use of
pesticides, destruction of natural forests causing this species to move to urban areas
174 Ana Marisa Chudzinski-Tavassi and Miryam Paola Alvarez-Flores

facilitating their contact with humans; and c) climatic (warmth and the occurrence of rain).
Even the characteristics of the caterpillar, such as gregarious habits, may increase the number
of direct contacts and outbreaks [2, 6, 13, 16, 38, 74, 76-79].
Most victims are young men working in fields, and lesions are especially common on
upper extremities. This kind of accident is common among field workers and is characterized
as a potentially severe work risk. Gregarious caterpillars use to be common in wild trees such
as cedar, araticum, rubber, Ficus enormis ("figueira do mato") and Tabebuia ssp. ("ipê"), but,
due to deforestation, nowadays they are usually founded in fruit trees such as avocado, pear,
plum, peach, banana, blackberry and maple, thus, favoring human exposition to the larvae
[16, 29, 80].

MORPHOLOGICAL ASPECTS AND LIFE PHASES

OF LONOMIA OBLIQUA CATERPILLARS

In Brazil, L. obliqua caterpillars are generally known as taturanas (from the American-
Indian-Tupi-Guarani tatá and raná which means “similar to fire” [79]. The biological cycle of
L. obliqua is composed by 4 stages with distinct durations (Figure 2) [2, 79].

Figure 2. Life phases of Lonomia obliqua. A) eggs; B) caterpillar at 6th instar; C) gregarious habits; D)
Pupas; E) Male adult. Photographs A, E and F are from Roberto H. P. Moraes; the others are from
Miryam P. Alvarez-Flores personal collection.
 South American Lonomia Obliqua Caterpillars 175

Figure 3. Structures of Lonomia obliqua caterpillar bristles. The bristles are prominent structures named
Scoli [16]. Each Scoli consists of multiple setae of different sizes and the tip (t) is linked to the base (b)
of the setae by a weak articulation (art) easily broken by accident. Photographs personal collection of
Miryam P. Alvarez-Flores.

Eggs

Duration of the egg stage is approximately 17 days. The ellipsoidal eggs measure about
1.98 mm mean height, 1.45 mm width and 1.61 mm long. Females commonly deposit clusters
of 70 eggs in plant leaves. The values of the incubation period and egg viability are 31.8 ± 5.8
days and 80.9 ± 20.97%, respectively [81]. Initially, the eggs have a clear green color with
dark micropyles, small cavities in the top of the eggs. Afterwards, they became translucent,
making possible the observation of the embryonic development [79].

Larva

Duration of the larvae stage is about 90 days. There are 6 phases or instars. Upon
hatching, the larvae have a dark brown head covered by few hairs and a light brown
measuring about 5 mm. Larvae development attain a length of up to 6 cm at maturity. By the
end of the last instar, larvae are covered by green-brownish bristles with different sizes (setae)
forming a structure similar to tree branches (scoli) with strong and dark heads (Figure 3) [11,
16, 79].
The bristles are formed by a non-porous tegument from the caterpillar cuticle and have a
hollow canal that stores the venom. When the bristle is broken, the venom is released from
the internal canal [16]. The venom is transported through a bristle channel and introduced into
the skin [16]. The venomous spicules and spines of moth caterpillars contain non-protein and
protein components. The venom is produced by a secretory epithelium above the tegument,
both tissues outgrowths as a continuous evagination of the body to form the bristles [16].
The larvae have gregarious habits, even in laboratory conditions [80]; they live in
colonies showing mimetic characteristics in host trees. They are considered polyphagous, and
176 Ana Marisa Chudzinski-Tavassi and Miryam Paola Alvarez-Flores

have been frequently found in fruit trees, feeding at night (communal feeder). Before pupa
stage (at last instar), larvae stop feeding [80]. During the day, dozens of caterpillars descend
until the tree trunk and lower branches, facilitating the contact with human's arms and legs
[11, 12, 16, 79]. Accidents usually occur with larvae at instars 4 to 6 [26, 37]. Many larvae
are crushed in the accident making possible the release of the toxic principle from the broken
setae, penetrating the human skin and finally the blood system [16]. In this way, size and
larval stage seem to be an important factor in the clinical evolution of envenoming patients.
Furthermore, the anatomical extension of the contact, the number of caterpillars involved, the
deepness of the injury (smashing them or not) and the amount of venom injected, also seem to
contribute to the severity of the accidents [16, 37, 38]. Most accidents occur in rural areas,
during field activities [26]. Unlike accidents with serpents in which 70.8% of the bites occur
on the lower members [29], human contact with the caterpillars occur most frequently in
upper members (88%), being young males (69%) the most affected [38]. Accidents with L.
obliqua caterpillars may be considered an occupational hazard, in rural areas, with social and
economic repercussions.

Pupa

The larva is encased in a chrysalis and undergoes metamorphosis. Depending on climatic

conditions, it can remain 30 to 100 days in the ground. In laboratory conditions, the mean
time of the pupal period is between 31-34 days at 25°C of temperature [80]. Statistically,
female pupas are larger than males (mean lengths of 28.73 mm for males and 30.23 mm for
females) [79]. Young pupae are yellowish in color, and become reddish-brown after a few
hours maintain this color until the end of the pupal stage (Figure 2D) [80].

Moth

Life span lasts for about 8 days. Adults live for a short period of time because they do not
feed, the primary purpose of adult life is the reproduction (mating and ovipositing) [80]. The
mean life period is 5.9 ± 1.59 days for males and 7.7 ± 2.27 days for females [80]. L. obliqua
adults present sexual dimorphism. A male is about 6 cm long presenting yellow-orange dorsal
wings with transversal black lines; a female is usually bigger and has brown-gray dorsal
wings [79].

STUDIES RELATED WITH LONOMIA OBLIQUA

VENOM AND ENVENOMATION
Although the clinical symptoms of the envenomation by L. achelous and L. obliqua are
similar, the mechanism that induces haemostatic disorders can be different. The differences
are related to the diversity of venom components on each species [37]. In patients poisoned
by L. achelous, the factor XIII (FXIII) of blood coagulation is drastically reduced due to a
FXIII degradation factor present in that venom [55]. It contrasts a slight decreased level of
 South American Lonomia Obliqua Caterpillars 177

plasma FXIII zymogen in treated rats showed to be similar when compared to human
accidents with L. obliqua caterpillars [26, 41, 82]. However, in vitro studies have shown
normal levels of FXIII in defibrinogenated plasma previously incubated with L. obliqua
bristle extract [82] indicating that the slight reduction of FXIII observed in patients
envenomed by L. obliqua may be related to its consumption during the coagulation activation
process [41]. Similar observations can be made in DIC (disseminated intravascular
coagulation) associated with other clinical situations [38].
Several in vitro studies have demonstrated that L. obliqua bristle extract presents
procoagulant activity, is unable to activate plasminogen and does not present t-PA- or u-PA-
like activities or direct lysis of fibrin [37, 82]. Moreover, bristle extract neither is able to
directly induce FXIII degradation nor possesses the ability to form a complex with FXIII
[82]. FXIII is a transglutaminase that stabilizes the clot by covalent cross-linking of fibrin
[83].
Some studies have shown that L. obliqua bristles secretions possess fibrinogenolytic
activity [82, 84]. Bristle extract has direct fibrinogenolytic activity, but this activity is unable
to affect cross-linked fibrin [82]. Moreover, the fibrinogenolytic activity hardly occurs with
high concentrations of the bristles extract or after a long time incubation with the fibrinogen
molecule, inducing proteolytic activity on the A fibrinogen chain and generating products
different from those induced by the plasmin [82]. When the plasma clot is formed in vitro by
the procoagulant activity of the venom, no clot lyses was observed before a 24-hour period.
Despite all this, a 35-kDa fibrin(ogen)olytic protein (Lonofibrase) was purified from L.
obliqua venom [85]. This enzyme cleaves preferentially the A chain fibrinogen, presenting
smaller efficiency by B chain. It is quite probable that the direct fibrinogenolytic activity of
the venom is not directly responsible for the L. obliqua hemorrhagic syndrome. That is
because anti-fibrinolytic drugs such as aprotinin and -aminocaproic acid used for the
treatment of envenomed animals with bristle extract failed at reversing the clinical
manifestations and indeed, instead of reversing them, it really exacerbates them [32].
Several studies shown that Lonomia obliqua venom is able to induce pain, edema
formation, erythema and fall of arterial pressure, probably due to the presence of several
activities such as nociceptive and edematogenic (91), hyaluronidase (95) and kallikrein-kinin
system activation activity (93). On the other hand, clinical and experimental studies suggest a
combination of toxins which lead to systemic effects such as intravascular hemolysis,
inflammation, platelet aggregation and adhesion dysfunction, consumption coagulopathy and
incoagulability [37-39, 86-89].
Two molecules present in bristle extract are related to the pathophysiology of the
envenoming and, apparently, to the development process of the animal such as regulation of
the cell cycle [37]. Those molecules are the prothrombin and factor X activators named
Lopap and Losac, respectively [43, 49]. Interestingly, both molecules are no similar to any
well-known procoagulant molecules from human or any other species [37].
Lopap (Lonomia obliqua Prothrombin Activator Protease) is a 69-kDa protein purified
from the bristle extract [49]. When administered in vivo, Lopap induces blood clotting into
microvessels, resulting in fibrinogen consumption and blood incoagulability [90]. These
effects resemble the consumption coagulopathy triggered by the whole venom, indicating the
involvement of this prothrombin activator as a key toxin in envenoming [47]. This protein
showed no similarity with other prothrombin activators or serine proteases, but was similar to
178 Ana Marisa Chudzinski-Tavassi and Miryam Paola Alvarez-Flores

lipocalin family members, either from insects or mammals [91]. Lopap is the first lipocalin
described that displays proteolytic activity. The recombinant protein (rLopap) was expressed
in the bacteria E. coli BL21(DE3) as monomer of 21 kDa [44, 91]. Moreover, a synthetic
peptide designed from Lopap’s primary structure has been proposed as a sequence signature
among lipocalins, sharing a common role in cell protection and development process [92].
Interestingly, other lipocalins that have been described with antiapoptotic activity have
conformations in their tridimensional structures similar to Lopap [92]. Lopap and its synthetic
peptide (known as survicalin) are able to modulate endothelial responses [86, 93, 94]. Lopap
promotes cell survival, IL-8, nitric oxide and prostaglandin release and expression of the cell
adhesion molecules ICAM-1 and E-selectin [86, 93, 95]; while survicalin induces fibroblast
responses: enhanced production of extracellular matrix collagen, fibronectin, tenascin and
laminin, decreasing of caspase-3 and increasing of Bcl-2, Ki-67, IL-1β and IL-8 and IL-6
receptors [96].
On the other hand, Losac (Lonomia obliqua Stuart-factor activator) is a factor X
activator purified from bristle extract as a 45-kDa protein [43]. The recombinant form of this
molecule was expressed in bacteria [97]. Studies using the native or recombinant form of
Losac (rLosac) revealed specificity toward factor X [43, 44, 97]. This protein was recognized
by the anti-lonomic serum produced in the Butantan Institute [97]. Thus, it is plausible that
this protein participates in the consumption coagulopathy observed in patients. Biochemical
characterization of Losac has shown that, Losac activity can be accelerated in the presence of
calcium and phospholipids, two important cofactors in the assembling of blood coagulation
complexes [98]. Its sequence did not show similarity with other factor X activators,
nevertheless Losac possesses a mechanism of action similar to RVV-X, a factor X activator
purified from Russell’s viper venom Daboia russelli [97, 99, 100]. Unlike RVV-X, Losac can
activate factor X independently of the presence of calcium and phospholipids. However, both
activators require a stable conformation of factor X and the presence of the Gla-domain of
factor X for proper activity. Interestingly, the cleavage fragments of factor X generated by
both activators were quite similar. Although there are strong functional similarities, the major
difference is in the structure of both activators. Apparently, Losac activates factor X through a
serine protease-like activity, while RVV-X has a typical metalloproteinase structure [44, 97,
101]. In the same way as Lopap, Losac is also capable of inducing proliferation and inhibiting
endothelial cell death [102, 103]. The production/expression of some important molecules
involved in inflammation and coagulation systems such as ICAM-1, PGI2, DAF, IL-8, vWF
and tissue factor were not affected by Losac [43].
Besides Lopap and Losac, several studies have shown that hemolymph from some insects
can increase cell longevity by inhibiting apoptosis [104, 105]. The increase of Spodoptera
frugiperda Sf-9 cell growth in almost 3-fold was reported after supplementation with L.
obliqua hemolymph [106]. This effect was attributed to the presence of three factors with
different activities: a potential antiapoptotic factor, a growth-promoting factor, and an enzyme
that hydrolyzes sucrose. Furthermore, an antiapoptotic protein of 51 kDa was purified from L.
obliqua hemolymph [107]. This protein was able to prevent apoptosis in Sf-9 cell culture
induced by nutrient deprivation and by Actinomycin D. Later reports [108, 109] described in
the hemolymph a potent antiviral activity against human virus.
 South American Lonomia Obliqua Caterpillars 179

CONCLUSION
Most lepidopterans may cause dermatologic or systemic symptoms in humans. However,
among all caterpillars of medical interest in the world, Lonomia sp. caterpillars (family:
Saturniidae) is the only genus that causes dramatic damages in human blood coagulation.
Several studies suggest that L. obliqua venom exerts mainly procoagulant activity causing a
consumption coagulopathy leading to a hemorrhagic state induced by fibrinogen depletion
and secondary fibrinolysis. Due to this, a specific anti-serum was produced by the Butantan
Institute for an effective treatment against the venom and for re-establishing the coagulation
parameters in poisoned patients. Toxins produced by the caterpillar of Lonomia obliqua are
diverse and some of them feature unusual multifunctional properties, being Lopap and Losac
the most studied toxins. Several factors could affect the distribution and behavior of this
species increasing the incidence of new cases of hemorrhagic syndrome. Thus, this chapter
reinforces the extreme importance of more studies about the venom of the Lonomia obliqua
caterpillars given the medical significance that this species represent in public health.

ACKNOWLEDGMENTS

This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de
Sao Paulo (1998/14307-9 CAT-CEPID and 2010/52669-3) and by grants from Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq/INCTTOX) and CAPES.

REFERENCES
[1] Heppner, J. B., Keys to families of Lepidoptera. Tropical Lepidoptera, 1993. 4(3): p. 1-
28.
[2] Cardoso, A. E. and J. V. Haddad, Accidents caused by lepidopterans (moth larvae and
adult): study on the epidemiological, clinical and therapeutic aspects. An Bras
Dermatol., 2005. 80(6): p. 573-8.
[3] Hossler, E. W., Caterpillars and moths: Part II. Dermatologic manifestations of
encounters with Lepidoptera. J Am Acad Dermatol, 2010. 62(1): p. 13-28; quiz 29-30.
[4] Mullen, G. R., Moths and Butterflies (Lepidoptera), in Medical and Veterinary
Entomology G.R. Mullen and L. Durden, Editors. 2002, Academic Press: Boston. p.
363-381.
[5] Rezende, J. M. D., Erucismo. Revista da Sociedade Brasileira de Medicina Tropical,
2004. 37: p. 424-424.
[6] Hossler, E. W., Caterpillars and moths. Dermatol Ther, 2009. 22(4): p. 353-66.
[7] Glasser, C. M., et al., Surtos epidêmicos de dermatite causada por mariposas do gênero
Hylesia (Lepidóptera: Hemileucidae) no Estado de São Paulo, Brasil. Rev. Saúde
Pública, 1993. 27(3): p. 217-20.
[8] Benvenuti, L. A., Cardoso J. L., and. Moraes R. H, Cutaneous leucocytoclastic
vasculitis from contact with Hylesia moths (Lepidoptera: Saturniidae). Trans R Soc
Trop Med Hyg., 1998. 92: p. 428-429.
180 Ana Marisa Chudzinski-Tavassi and Miryam Paola Alvarez-Flores

[9] Rodriguez-Morales, A. J., et al., Lepidopterism due to exposure to the moth Hylesia
metabus in northeastern Venezuela. Am J Trop Med Hyg., 2005. 73(5): p. 991-3.
[10] Hassing, R. J. and A. G. Bauer, Pruritic dermatitis on an oil tanker after a visit to
French Guyana. J Travel Med. , 2008. 15(6): p. 464-5.
[11] Diaz, J. H., The evolving global epidemiology, syndromic classification, management,
and prevention of caterpillar envenoming. Am J Trop Med Hyg, 2005. 72(3): p. 347-57.
[12] Mullen, G. R., Moth and Butterflies (Lepidoptera), in Medical and veterinary
entomology, Mullen G. R. and Durden L., Editors. 2002, Academic Press: Boston. p.
363-381.
[13] Hossler, E. W., Caterpillars and moths: Part I. Dermatologic manifestations of
encounters with Lepidoptera. J Am Acad Dermatol, 2010. 62(1): p. 1-10; quiz 11-2.
[14] Balit, C. R., et al., Prospective study of definite caterpillar exposures. Toxicon, 2003.
42(6): p. 657-62.
[15] Balit, C. R., et al., Outbreak of caterpillar dermatitis caused by airborne hairs of the
mistletoe browntail moth (Euproctis edwardsii). Med J Aust Dent J, 2001. 175(641-43).
[16] Veiga, A. B., Blochtein B., and Guimaraes J. A., Structures involved in production,
secretion and injection of the venom produced by the caterpillar Lonomia obliqua
(Lepidoptera, Saturniidae). Toxicon, 2001. 39(9): p. 1343-51.
[17] Kawamoto, F. and Kumada N., Biology and venoms of Lepidoptera, in Handbook of
Natural Toxins. Vol. 2. Insect Poisons, Allergens, and Other Invertebrate Venoms., A.
T. Tu, Editor 1984, Marcel Dekker, Inc.: New York. p. 291-330.
[18] Liu, Z., et al., Identification and characterization of an arginine kinase as a major
allergen from silkworm (Bombyx mori) larvae. Int Arch Allergy Immunol. , 2009.
150(1): p. 8-14.
[19] Ji, K. M., et al., Anaphylactic shock caused by silkworm pupa consumption in China.
Allergy, 2008. 63: p. 1407-8.
[20] Cheng, W. and Tam P. K., Foreign-body ingestion in children: experience with 1,265
cases. J Pediatr Surg, 1999. 34: p. 1472-6.
[21] Chen, W., Xiao G., and Liao S., Advances in the study and development of medical
uses of sericulture and mulberry resources (in Chinese). Zhong Yao Cai, 1999. 22: p.
481-4.
[22] Lee, D., Pitetti R. D., and Casselbrant M. L., Oropharyngeal manifestations of
lepidopterism. Arch Otolaryngol Head Neck Surg., 1999. 125(1): p. 50-2.
[23] Tripi, P. A., et al., An unusual case of ingestion of a moth cocoon in a 14-month-old
girl. Am J Otolaryngol. , 2010. 31(2): p. 123-126.
[24] Diaz, J. H., The epidemiology, diagnosis, and management of caterpillar envenoming
in the southern US. J La State Med Soc, 2005. 157(3): p. 153-7.
[25] Arocha-Pinango, C. L. and Guerrero B., [Hemorrhagic syndrome induced by
caterpillars. Clinical and experimental studies. Review]. Invest Clin, 2003. 44(2): p.
155-63.
[26] Zannin, M., et al., Blood coagulation and fibrinolytic factors in 105 patients with
hemorrhagic syndrome caused by accidental contact with Lonomia obliqua caterpillar
in Santa Catarina, southern Brazil. Thromb Haemost, 2003. 89(2): p. 355-64.
[27] da Silva, W. D., et al., Development of an antivenom against toxins of Lonomia
obliqua caterpillars. Toxicon, 1996. 34(9): p. 1045-9.
 South American Lonomia Obliqua Caterpillars 181

[28] Villas-Boas, I. M., et al., Premolis semirufa (Walker, 1856) Envenomation, Disease
Affecting Rubber Tappers of the Amazon: Searching for Caterpillar-Bristles Toxic
Components. Plos Neglected Tropical Diseases, 2012. 6(2).
[29] Ministério-da-Saúde, Acidentes por Lepidópteros, in Manual de diagnóstico e
tratamento de acidentes por animais peçonhentos, FUNASA, Editor 2001, Fundação
Nacional de Saúde (FUNASA): Brasil. p. 120.
[30] Kowacs, P. A., et al., Fatal intracerebral hemorrhage secondary to Lonomia obliqua
caterpillar envenoming: case report. Arq Neuropsiquiatr, 2006. 64(4): p. 1030-2.
[31] Duarte, A. C., et al., Insuficiência renal aguda por acidentes com lagartas. Journal
Brasileiro de Nefrologia, 1990. 12: p. 3.
[32] Duarte, A. C., et al., Intracerebral haemorrhage after contact with Lonomia caterpillars.
Lancet, 1996. 348(9033): p. 1033-1033.
[33] Kelen, E. M. A., Picarelli Z. P., and Duarte A. C., Hemorrhagic syndrome induced by
contact with caterpillars of the genus Lonomia (saturniidae, hemileucinae). Journal of
Toxicology-Toxin Reviews, 1995. 14(3): p. 283-308.
[34] Fan, H. W. and Duarte A. C., Acidentes por Lonomia, in Animais peçonhentos no
Brasil: biologia, clínica e terapêutica dos acidentes, J. L. Cardoso, et al., Editors.
2003, Sarvier: Sao Paulo. p. 224-232.
[35] Fan, H. W., et al., Hemorrhagic syndrome and acute renal failure in a pregnant woman
after contact with Lonomia caterpillars: a case report. Rev Inst Med Trop Sao Paulo,
1998. 40(2): p. 119-20.
[36] Arocha-Pinango, C. L. and Guerrero B., Lonomia obliqua and haemorrhagic syndrome.
Lancet, 1999. 354(9186): p. 1304.
[37] Alvarez Flores, M. P., Zannin M., and Chudzinski-Tavassi A. M., New insight into the
mechanism of Lonomia obliqua envenoming: toxin involvement and molecular
approach. Pathophysiol Haemost Thromb, 2010. 37(1): p. 1-16.
[38] Chudzinski Tavassi, A. M. and Zannin M., Aspectos clínicos y epidemiológicos del
envenenamento por Lonomia obliqua, in Emergencias por animales ponzoñosos en las
Américas, D'Suze G., G. A. Corzo Burguete, and Paniagua J. F. Solis, Editors. 2011,
Instituto Bioclon: México, D.F. p. 303-321.
[39] Chudzinski Tavassi, A. M. and Alvarez-Flores M.P., Lonomia obliqua brasileña:
bioquímica y fisiopatologia del veneno, in Emergencias por animales ponzoñosos en
las Américas, D'Suze G., Corzo Burguete G. A., and Paniagua J. F. Solis, Editors.
2011, Instituto Bioclon: México, D. F. p. 323-369.
[40] Carrijo-Carvalho, L. C. and Chudzinski-Tavassi A. M., The venom of the Lonomia
caterpillar: an overview. Toxicon, 2007. 49(6): p. 741-57.
[41] Prezoto, B. C., et al., Antithrombotic effect of Lonomia obliqua caterpillar bristle
extract on experimental venous thrombosis. Braz J Med Biol Res, 2002. 35(6): p. 703-
12.
[42] Donato, J. L., et al., Lonomia obliqua caterpillar spicules trigger human blood
coagulation via activation of factor X and prothrombin. Thrombosis and Haemostasis,
1998. 79(3): p. 539-542.
[43] Alvarez Flores, M. P., et al., Losac, a factor X activator from Lonomia obliqua bristle
extract: its role in the pathophysiological mechanisms and cell survival. Biochem
Biophys Res Commun, 2006. 343(4): p. 1216-23.
182 Ana Marisa Chudzinski-Tavassi and Miryam Paola Alvarez-Flores

[44] Chudzinski-Tavassi, A. M. and Alvarez Flores M. P., Exploring new molecules and
activities from Lonomia obliqua caterpillars. Pathophysiol Haemost Thromb, 2005.
34(4-5): p. 228-33.
[45] Reis, C. V., et al., Lopap, a prothrombin activator from Lonomia obliqua belonging to
the lipocalin family: recombinant production, biochemical characterization and
structure-function insights. Biochem J, 2006. 398(2): p. 295-302.
[46] Reis, C. V., et al., In vivo characterization of Lopap, a prothrombin activator serine
protease from the Lonomia obliqua caterpillar venom. Thromb Res, 2001. 102(5): p.
437-43.
[47] Reis, C. V., et al., A Ca++ activated serine protease (LOPAP) could be responsible for
the haemorrhagic syndrome caused by the caterpillar Lonomia obliqua. L obliqua
Prothrombin Activator Protease. Lancet, 1999. 353(9168): p. 1942.
[48] Reis, C. V., et al., A prothrombin activator could be the major responsible for the
hemorrhagic syndrome accidents caused by the caterpillar Lonomia obliqua in Brazil.
Thrombosis and Haemostasis, 1999: p. 801-801.
[49] Reis, C. V., et al., A prothrombin activator serine protease from the Lonomia obliqua
caterpillar venom (Lopap) biochemical characterization. Thromb Res, 2001. 102(5): p.
427-36.
[50] Pinto, A. F., et al., Novel perspectives on the pathogenesis of Lonomia obliqua
caterpillar envenomation based on assessment of host response by gene expression
analysis. Toxicon, 2008. 51(6): p. 1119-28.
[51] Veiga, A. B., et al., A catalog for the transcripts from the venomous structures of the
caterpillar Lonomia obliqua: identification of the proteins potentially involved in the
coagulation disorder and hemorrhagic syndrome. Gene, 2005. 355: p. 11-27.
[52] Von Ihering, R., Estudo biológico das lagartas urticantes ou taturanas. Ann. Paulistas
Med. Cirurgia, 1914. 3: p. 129-139.
[53] Arocha-Pinango, C. L. and Layrisse M., Fibrinolysis produced by contact with a
caterpillar. Lancet, 1969. 1(7599): p. 810-2.
[54] Arocha-Pinango, C. L., [Fibrinolysis caused by contact with caterpillars: preliminary
communication]. Acta Cientifica Venezolana, 1967. 18(5): p. 136-9.
[55] Arocha-Pinango, C. L., et al., Six new cases of a caterpillar-induced bleeding
syndrome. Thromb Haemost, 1992. 67(4): p. 402-7.
[56] Couppie, P., et al., Venomous caterpillars in French Guyana: 5 cases. Annales de
Dermatologie et de Venereologie, 1998. 125(8): p. 489-491.
[57] Arocha-Pinango, C. L. and Guerrero B., Lonomia genus caterpillar envenomation:
clinical and biological aspects. Haemostasis, 2001. 31(3-6): p. 288-93.
[58] Chan, K., et al., Caterpillar-induced bleeding syndrome in a returning traveller. CMAJ,
2008. 179(2): p. 158-61.
[59] de Roodt, A. R., Salomon O. D., and Orduna T. A., [Accidents due to lepidoptera with
special reference to Lonomia sp]. Medicina (B Aires), 2000. 60(6): p. 964-72.
[60] Rubio, G. B., [Epidemiological surveillance of distribution of the caterpillar Lonomia
obliqua Walker, 1855, in the State of Parana, Brazil]. Cad Saude Publica, 2001. 17(4):
p. 1036.
[61] Lemaire, C., Revision of Genus Lonomia Walker [Lep-Attacidae]. Annales de la
Societe Entomologique de France, 1972. 8(4): p. 767-861.
 South American Lonomia Obliqua Caterpillars 183

[62] Pineda, D., et al., Síndrome hemorrágico por contacto con orugas del género Lonomia
(Saturniidae) en Casanare, Colombia: informe de 2 casos. Biomédica (Bogotá), 2001.
21: p. 328-332.
[63] Garcia, C. M. and Danni-Oliveira I. M., [Occurrence of accidents caused by Lonomia
obliqua Walker, in the State of Parana between 1989 and 2001]. Rev Soc Bras Med
Trop, 2007. 40(2): p. 242-6.
[64] Arocha-Pinango, C. L., Marval E., and Guerrero B., Lonomia genus caterpillar toxins:
biochemical aspects. Biochimie, 2000. 82(9-10): p. 937-42.
[65] Fraiha-Neto, H., et al., Síndrome hemorrágica por contato com larvas de mariposa
(Leptidoptera, Saturniidae). In: Instituto Evandro Chagas: 50 anos de contribuição às
ciências biológicas e à medicina tropical, 1986, Fundação Serviços de Saúde Pública
2: Belém. p. 811-820.
[66] Duarte, A. C., et al., Intracerebral haemorrhage after contact with Lonomia caterpillars.
Lancet, 1996. 348(9033): p. 1033.
[67] Arocha-Piñango, C. L., et al., Fibrinolytic and procoagulant agents from a saturnidae
moth caterpillar, in Hemostasis and animal venoms, P. H. and Markland F. S., Editors.
1988, Marcel Dekker Inc.: New York. p. 223-240.
[68] Guerrero, B. A., Arocha-Pinango C. L., and Gil San Juan A., Lonomia achelous
caterpillar venom (LACV) selectively inactivates blood clotting factor XIII. Thromb
Res, 1997. 87(1): p. 83-93.
[69] Abella, H. B., et al., Manual de diagnóstico e tratamento de acidentes por Lonomia.
Secretaria da Saúde e do Meio Ambiente, 1998: Porto Alegre. p. 19.
[70] Abella, H. B., et al., Acidentes com lagartas do gênero Lonomia registrados no Centro
de Informação Toxicológica do Rio Grande do Sul no período de 1997 a 2005, in
Toxico vigilância – toxicologia clínica: dados e indicadores selecionados, Secretaria
da Saúde, A. Nicolella, Editor 2006, Fundação Estadual de Produção e Pesquisa em
Saúde & Centro de Informação Toxicológica: Porto Alegre. p. 29–34.
[71] Burdmann, E. A., et al., Severe acute renal failure induced by the venom of Lonomia
caterpillars. Clin Nephrol, 1996. 46(5): p. 337-9.
[72] Rocha-Campos, A. C., et al., Specific heterologous F(ab')2 antibodies revert blood
incoagulability resulting from envenoming by Lonomia obliqua caterpillars. Am J Trop
Med Hyg, 2001. 64(5-6): p. 283-9.
[73] Caovilla, J. J. and Barros E. J., Efficacy of two different doses of antilonomic serum in
the resolution of hemorrhagic syndrome resulting from envenoming by Lonomia
obliqua caterpillars: a randomized controlled trial. Toxicon, 2004. 43(7): p. 811-8.
[74] Lorini, I., Taturana: a lagarta que mata. EMBRAPA-CNPT. 6pp, 1993.
[75] Correa, M. S., et al., [Lonomia erucism in Teresopolis, Rio de Janeiro State, Brazil:
report of a probable case and review]. Rev Soc Bras Med Trop, 2004. 37(5): p. 418-21.
[76] Oliveira Junior, J. B., Silva C. G., and Marchiori C. H., Himenópteros parasitóides
capturados em imaturos de Lonomia sp. (Lepidoptera: Saturniidae) oriundas de plantas
de cafeeiro. Revista Trópica – Ciências Agrárias e Biológicas, 2007. 1(1): p. 4.
[77] Moraes, R. H., Identificação dos inimigos naturais de Lonomia obliqua Walker, 1855
(Lepidoptera, Saturniidae) e possíveis fatores determinantes do aumento da sua
população, in Entomologia 2002, Escola Superior de Agricultura "Luiz de Queiroz" da
Universidade de São Paulo: Piracicaba. p. 58.
184 Ana Marisa Chudzinski-Tavassi and Miryam Paola Alvarez-Flores

[78] Gamborgi, G. P., Metcalf E. B., and Barros E. J., Acute renal failure provoked by toxin
from caterpillars of the species Lonomia obliqua. Toxicon, 2006. 47(1): p. 68-74.
[79] Lorini, L. M. and Corseuil E., Aspectos morfológicos de Lonomia obliqua Walker
(Lepidóptera: Saturniidae). Neotropical Entomology, 2001. 30(3): p. 373-378.
[80] Lorini, L. M., Zarbin P. H. G., and Tedesco C. D., Biology of laboratory-reared
Lonomia obliqua (Lepidoptera: Saturniidae). Florida Entomologist, 2007. 90(4): p.
770-771.
[81] Lorini, L. M., Rebelato G. S., and Bonatti J., Reproductive parameters of Lonomia
obliqua Walker, 1855 (Lepidoptera: Saturniidae) in laboratory. Brazilian Archives of
Biology and Technology, 2004. 47(4): p. 575-577.
[82] Fritzen, M., et al., Lonomia obliqua venom action on fibrinolytic system. Thromb Res,
2003. 112(1-2): p. 105-10.
[83] Lorand, L., Factor XIII: structure, activation, and interactions with fibrinogen and
fibrin. Ann N Y Acad Sci, 2001. 936: p. 291-311.
[84] Veiga, A. B., Pinto A. F., and Guimaraes J. A., Fibrinogenolytic and procoagulant
activities in the hemorrhagic syndrome caused by Lonomia obliqua caterpillars.
Thromb Res, 2003. 111(1-2): p. 95-101.
[85] Pinto, A. F., et al., Lonofibrase, a novel alpha-fibrinogenase from Lonomia obliqua
caterpillars. Thromb Res, 2004. 113(2): p. 147-54.
[86] Waismam, K., et al., Lopap: a non-inflammatory and cytoprotective molecule in
neutrophils and endothelial cells. Toxicon, 2009. 53(6): p. 652-9.
[87] Nascimento-Silva, V., et al., A pro-inflammatory profile of endothelial cell in Lonomia
obliqua envenomation. Toxicon, 2012. 60(1): p. 50-60.
[88] Berger, M., et al., Lonomia obliqua venomous secretion induces human platelet
adhesion and aggregation. J Thromb Thrombolysis, 2010. 30(3): p. 300-10.
[89] Berger, M., et al., Lonomia obliqua caterpillar envenomation causes platelet
hypoaggregation and blood incoagulability in rats. Toxicon, 2010. 55(1): p. 33-44.
[90] Reis, C. V., et al., In vivo characterization of Lopap, a prothrombin activator serine
protease from the Lonomia obliqua caterpillar venom. Thrombosis Research, 2001.
102(5): p. 437-443.
[91] Reis, C. V., et al., Lopap, a prothrombin activator from Lonomia obliqua belonging to
the lipocalin family: recombinant production, biochemical characterization and
structure-function insights. Biochemical Journal, 2006. 398: p. 295-302.
[92] Chudzinski-Tavassi, A. M., et al., A lipocalin sequence signature modulates cell
survival. FEBS Lett, 2010. 584(13): p. 2896-900.
[93] Fritzen, M., et al., A prothrombin activator (Lopap) modulating inflammation,
coagulation and cell survival mechanisms. Biochem Biophys Res Commun, 2005.
333(2): p. 517-23.
[94] Carrijo-Carvalho, L. C., et al., A lipocalin-derived Peptide modulating fibroblasts and
extracellular matrix proteins. J Toxicol, 2012. 2012: p. 325250.
[95] Chudzinski-Tavassi, A. M., et al., Effects of lopap on human endothelial cells and
platelets. Haemostasis, 2001. 31(3-6): p. 257-65.
[96] Carrijo-Carvalho, L. C., et al., A lipocalin-derived Peptide modulating fibroblasts and
extracellular matrix proteins. Journal of Toxicology doi:10.1155/2012/325250, 2012.
 South American Lonomia Obliqua Caterpillars 185

[97] Alvarez-Flores, M. P., et al., Losac, the first hemolin that exhibits procogulant activity
through selective factor X proteolytic activation. J Biol Chem, 2011. 286(9): p. 6918-
28.
[98] Hoffman, M. and Monroe D. M., Coagulation 2006: A modern view of hemostasis.
Hematology-Oncology Clinics of North America, 2007. 21(1): p. 1-+.
[99] Kisiel, W., Hermodson M. A., and Davie E. W., Factor-X activating enzyme from
Russells viper venom - Isolation and characterization. Biochemistry, 1976. 15(22): p.
4901-4905.
[100] Morita, T., Proteases which activate factor X, in Enzymes from snake venoms, Bailey
G. S., Editor 1998, Alaken Inc.: Fort Collins, Colorado. p. 179-208.
[101] Tans, G. and Rosing J., Snake venom activators of factor X: an overview. Haemostasis,
2001. 31(3-6): p. 225-33.
[102] Dimmeler, S., et al., Upregulation of superoxide dismutase and nitric oxide synthase
mediates the apoptosis-suppressive effects of shear stress on endothelial cells.
Arterioscler Thromb Vasc Biol, 1999. 19(3): p. 656-64.
[103] Rossig, L., et al., Nitric oxide inhibits caspase-3 by S-nitrosation in vivo. J Biol Chem,
1999. 274(11): p. 6823-6.
[104] Rhee, W. J., Kim E. J., and Park T. H., Kinetic effect of silkworm hemolymph on the
delayed host cell death in an insect cell-baculovirus system. Biotechnol Prog, 1999.
15(6): p. 1028-32.
[105] Kim, E. J., Park H. J., and Park T. H., Inhibition of apoptosis by recombinant 30K
protein originating from silkworm hemolymph. Biochem Biophys Res Commun, 2003.
308(3): p. 523-8.
[106] Maranga, L., et al., Enhancement of Sf-9 cell growth and longevity through
supplementation of culture medium with hemolymph. Biotechnol Prog, 2003. 19(1): p.
58-63.
[107] Souza, A. P. B., et al., Purification and characterization of an anti-apoptotic protein
isolated from Lonomia obliqua hemolymph. Biotechnology Progress, 2005. 21(1): p.
99-105.
[108] Greco, K. N., et al., Antiviral activity of the hemolymph of Lonomia obliqua
(Lepidoptera: Saturniidae). Antiviral Research, 2009. 84(1): p. 84-90.
[109] Carmo, A. C., et al., Expression of an antiviral protein from Lonomia obliqua
hemolymph in baculovirus/insect cell system. Antiviral Res, 2012. 94(2): p. 126-30.
In: Lepidoptera ISBN: 978-1-62417-248-9
Editors: Elia Guerritore and Johannes DeSare © 2013 Nova Science Publishers, Inc.

Chapter 7

ECOLOGY OF THE BLUE-STRIPED GRUB

MOTH PARASA LEPIDA (LEPIDOPTERA:
LIMACODIDAE) IN JAPAN

Kazuo Yamazaki1,, Kosuke Nakanishi2

and Hiroichi Sawada2,†
1
Osaka City Institute of Public Health and
Environmental Sciences, Tennoji, Osaka, Japan
2
School of Environmental Science,
University of Shiga Prefecture, Hikone, Shiga, Japan

ABSTRACT
The blue-striped grub moth Parasa lepida (Cramer) is an invasive alien species in
Japan; serious infestations occur in various street and fruit trees. To better understand its
present ecological impacts and to devise efficient control methods for such alien pests,
studies that compare their ecology among districts are useful. In this chapter, we
summarize the bionomics of P. lepida in Japan for comparison with their native ranges.
This species was first found in southern Japan in 1921; its distribution spread northeast
into the eastern mainland, where it is generally restricted to urban and suburban areas. It
emerges twice a year, but a portion of the population has a univoltine life cycle and
infests dozens of broad-leaved tree species, but not palms. The larvae have venomous
spines and modified setae that cause dermatitis in humans. Various natural enemies such
as pathogenic viruses, fungi, invertebrate predators, and birds attack immature stages of
this moth. Parasitoids play only a minor role in the regulation of these moth populations.
The cocoons are so durable that we can use them to examine population dynamics.
Intensive studies using life table analyses suggested that nuclear polyhedrosis virus
(NPV) might kill considerable numbers of second-generation larvae, reducing the number
of adults emerging from overwintering cocoons. Populations of this moth are controlled
by spraying insecticides and removing overwintering cocoons and aggregations of early-


Corresponding author: E-mail: kazuo-yamazaki@[Link].
†
Corresponding author: E-mail: sawada@[Link].
188 Kazuo Yamazaki, Kosuke Nakanishi and Hiroichi Sawada

instar larvae. Future perspectives for ecological studies of and control methods for this
moth are discussed.

INTRODUCTION
In this era of globalization, alien insects cause extensive damage to agricultural crops,
forestry products, and ornamental plants, harm humans, and have negative impacts on native
biota worldwide (Sharov et al. 2002, Cappaert et al. 2005, Benedict et al. 2007, Kenis et al.
2009). Some invasive alien insects have colonized and become problematic in multiple
regions of the world (Dafni 1998, Suarez et al. 2001, Matsumura et al. 2004, Hurley et al.
2007). Comparing life history, biotic interactions, and control methods of alien insects
between their native and introduced ranges or between several introduced ranges is a
promising method to clarify factors affecting invasion success and subsequent population
dynamics and to devise effective control strategies (e.g., Hurley et al. 2007).
The blue-striped grub moth Parasa lepida (Cramer) (Lepidoptera: Limacodidae)
originated in the tropical and subtropical oriental region. It is an invasive alien species in
Japan (Murakami and Washitani 2002) and is a major pest of various street and fruit trees and
useful plants. In this chapter, we review the ecological aspects of P. lepida in Japan, including
its life cycle, distributional spread, habitats, host plants, natural enemies, population
dynamics, and control methods. Similarities and differences in the ecology of P. lepida
between Japan and other countries are discussed, and perspectives on future ecological
studies and improvements to control strategies are described.

LIFE CYCLE
Parasa lepida adults are medium-sized limacodid moths. The length of the forewing
ranges from 15 to 22 mm (Oda and Hattori 1981, Umeya and Okada 2003, Figure 1a). Most
of the forewing is light green, but a dark brown half-moon patch and a wide brown band are
present at the base and along the outer edge of the forewing, respectively. The hind wing is
pale brown. Inoue (1982) treated the Japanese population as the original subspecies P. lepida
lepida, which is distributed from China to India. However, Howard et al. (2001) noted that P.
lepida could be classified into three subspecies: lepida in India and its adjacent areas, lepidula
in China and Japan, and media in Southeast Asia as far east as Bali. Adults usually emerge
twice a year, from early to late June and from early August to mid-September, in central
Japan (latitude, 31–36N; mean annual temperature, 13.6–18.6C) (Oda and Hattori 1981,
Natsuaki 1998), and from early April to early May and from mid-July to mid-August in
Okinawa in southern Japan (26N; 23C) (Higa and Kishimoto 1984). However, a small
proportion of the moths have a univoltine life cycle in western Japan (Sawada et al. 2008a).
Adult eclosion occurs mostly around sunset (Oda and Hattori 1981). Adults do not feed and
are attracted to light at night (Oda and Hattori1981, Natsuaki 1998). Copulation takes place
during dawn, before sunrise (4:00–6:00 AM) (Oda and Hattori 1981). Virgin females
first exhibit a calling behavior, males are attracted to them, and pairs end in copulation
(Figure 1a).
 Ecology of the Blue-Striped Grub Moth Parasa Lepida … 189

Figure 1. Life stages of Parasa lepida. (a) Adults during copulation, (b) egg batch, (c) gregarious
immature larvae, (d) mature larva, (e) cocoon from which an adult eclosed, (f) cocoon preyed on by a
bird. Scale lines = 10 mm.

Copulation lasts for several hours (Oda and Hattori 1981). (Z)-7,9-decadien-1-ol (Z7,9-
10:OH) has been identified as a sex pheromone component in this species (Wakamura et al.
2007, Islam et al. 2009). The adult life span is ca. 5–10 days in the laboratory (Oda and
Hattori 1981, Higa and Kishimoto 1984).
Eggs are laid on the underside of leaves in batches of 10–300 eggs (average: 20–45 eggs)
that are covered with a jelly-like substance (Oda and Hattori 1981, Higa and Kishimoto 1984,
Umeya and Okada 2003, Sawada et al. 2008e, Figure 1b). Hatching occurs 5–6 days after egg
deposition (Oda and Hattori 1981, Umeya and Okada 2003). Hatchlings molt into second-
instar larvae without feeding (Oda and Hattori 1981). Young larvae feed on leaf tissues in
190 Kazuo Yamazaki, Kosuke Nakanishi and Hiroichi Sawada

clusters on the underside of leaves (Figure 1c). As the young larvae feed, the epidermis of the
leaf surface is left behind, producing noticeable feeding traces (Sawada et al. 2008d). The
larvae gradually disperse as they grow, and solitary final-instar larvae feed on leaves from the
edge (Oda and Hattori 1981, Umeya and Okada 2003). Final-instar larvae are 22–25 mm
long, yellowish green, and have blue-stripes on their dorsal parts (Oda and Hattori 1981, Higa
and Kishimoto 1984, Figure 1d). Numerous venomous spines and modified setae are present
on their dorsal parts (Natsuaki 1998, see also Battisti et al. 2011 for the classification of
urticating hairs, Figure 1d). Among the spines, dozens of larger spines protrude from scoli
and are connected to poison glands (Oda and Hattori 1981, Natsuaki 1998). When humans
touch the larvae, the contact causes dermatitis (Otaki and Shinonaga 1995, Higa and
Kishimoto 1995, Natsuaki 1998). The causative venom of the dermatitis likely includes
histamine and unknown substances (Kawamoto 1978, Natsuaki 1998). The venomous spines
and modified setae of slug caterpillars are commonly effective defenses against vertebrate
(Foot 1922) and arthropod predators such as assassin bugs and paper wasps (Murphy et al.
2010, Greeney et al. 2012).
The larval period is ca. 38–46 days (Umeya and Okada 2003). Larvae pass up to nine
instars (Oda and Hattori 1981) and spin pressed spheroid cocoons (ca. 15 mm in length and
10 mm in width) on tree trunks, basal trunks, the underside of branches, and wood and stones
near host trees (Oda and Hattori 1981, Yamazaki et al. 1994a, b, 2007, Nakatomi 1987,
Natsuaki 1998, Umeya and Okada 2003, Sawada et al. 2008a–e, Nakano and Takimoto 2011,
Teramoto 2011, Figure 1e). The cocoons are chocolate brown and are covered with a dark-
gray silk sheet that contains bark tissue and thus resembles tree bark. Accordingly, new
cocoons are difficult to find with the human eye, compared with old cocoons that have lost
their sheet and have turned whitish brown. Many short poisonous setae are intermingled with
the silk when the cocoon walls are created (Oda and Hattori 1981, Natsuaki 1998). Thus,
contact with cocoons can also cause dermatitis in humans. The cocoons are so hard and stout
that they remain on tree trunks for 5–6 years after they are spun (Yamazaki et al. 2007). First-
generation prepupae pupate in a few weeks in the summer in cocoons, but second-generation
prepupae overwinter and pupate during the next spring (Oda and Hattori 1981, Higa and
Kishimoto 1984, Umeya and Okada 2003). Cocoons have emergence holes on the ceiling,
which are pushed up for the eclosion (Figure 1e).

DISTRIBUTION: EXPANSION AND ITS MECHANISM

This species was originally distributed in tropical and subtropical districts of oriental
regions such as southern China, Southeast Asia (Malaysia, Indonesia), and southern Asia
(India, Sri Lanka) (Oda and Hattori 1981, Inoue 1982); it is an invasive species in Japan. The
first collection record of P. lepida from Japan dates back to 1921 in Kagoshima Prefecture,
southern Japan (Kawada 1930, Figure 2). Afterward, there were no collection reports until
1960, when the species was rediscovered in Kagoshima (Takemura 1960). The reason that P.
lepida had almost disappeared from entomological literature for nearly 40 years is unclear.
However, P. lepida might not have been recorded because it was misidentified as other
congeneric species in Japan, namely P. consocia (Walker) and P. sinica (Moore).
Alternatively, this species may have remained latent, surviving in low densities and then
 Ecology of the Blue-Striped Grub Moth Parasa Lepida … 191

increasing for unknown reasons, or the moth may have been extirpated and then invaded
again. To unravel this issue, examinations of museum or private specimen collections could
be effective.
Many individuals were observed in the late 1970s in northern Kyushu, Osaka, Nara, and
Hyogo Prefectures (western Japan) (Oda and Hattori 1981, Inoue 1982, Miyata 1984,
Nakatomi 1987, Figure 2). In the 1980s, the population of P. lepida increased so rapidly that
most trees in urban parks in western Japan had cocoons on their trunks (Yamazaki 2001).
Additionally, P. lepida invaded Okinawa, the southern island of Japan, in the early 1980s
(Higa and Kishimoto 1984, Figure 2). In the late 1980s, the distribution of this limacodid had
spread to eastern Japan, such as Chiba, Tokyo, Saitama, Ibaraki, and Kanagawa Prefectures
(Nakano 2003, Kanai 2009, Figure 2). Occurrences of the moth have been increasing in
eastern Japan (Nakano 2003, Kanai 2009). The expansion of this species is thought to be due
to 1) the transportation of street and garden trees that harbor larvae or cocoons, 2) rapid
expansion of residential areas (i.e., suitable habitats) in suburban districts of western Japan,
and 3) global warming, which might facilitate northward distributional expansion (Miyata
1984, Higa and Kishimoto 1984, Natsuaki 1998, Okada and Umeya 2003). However,
empirical data supporting these hypotheses are lacking.

Figure 2. Distributional spread of Parasa lepida in Japan. Parasa lepida was first found and then
rediscovered in Kagoshima; its distribution subsequently spread to the northeast. However, there are no
records of this species in inland areas or on many islands.
192 Kazuo Yamazaki, Kosuke Nakanishi and Hiroichi Sawada

HABITAT AND HOST PLANTS

Parasa lepida is rarely found in primary and secondary forests, but it is very abundant in
urban and newly developed residential areas of western Japan. Trees that are planted along
streets or in parks, gardens, schools, and orchards are sometimes severely damaged by the
larvae (Oda and Hattori 1981, Nakatomi 1987, Yamazaki et al. 1994a, b, 2007, Umeya and
Okada 2003, Nishida et al. 2006, Sawada et al. 2008a–e).
Larvae feed on the leaves of various trees in Japan. Yamazaki et al. (1994a) examined
host plant use in this species based on cocoon inspection in Osaka, Kyoto, and Shiga
Prefectures of western Japan because the larvae usually spin their cocoons on the trunks of
their host trees. In total, 53 tree species from 26 families bore cocoons. The tree species that
suffered from heavy infestations were Morella rubra (Myriaceae), Quercus myrsinaefolia
(Fagaceae), Zelkova serrata (Ulmaceae), Magnolia kobus (Magnoliaceae), Liquidambar
formosana, L. styraciflua (Altingiaceae), Prunus  yedoensis (Rosaceae), Robinia pseudo-
acacia (Fabaceae), Triadica sebifera (Euphorbiaceae), Acer palmatum, A. buergerianum
(Sapindaceae), Elaeocarpus sylvestris (Elaeocarpaceae), Diospyros kaki (Ebenaceae), Styrax
japonica (Styracaceae), and Fraxinus japonica (Oleaceae). Eighty percent or more of the
individual trees of these species bore cocoons. The kinkgo tree Ginkgo biloba
(Gymnospermae, Ginkgoaceae) was also damaged, but the infestation rate was not high (35.5
%). The conifers, M. grandiflora, Liriodendron tulipifera (Magnoliaceae), and Nerium
indicum (Apocynaceae) were not used as hosts (Yamazaki et al. 1994a). Eucalyptus
(Myrtaceae) trees also were not used (Nasu et al. 2004). Thus, extreme toughness (M.
grandiflora), dense trichomes (M. grandiflora, L. tulipifera), and particular secondary
substances (conifers, L. tulipifera, N. indicum, Eucalyptus spp.) in leaves may hinder the
feeding activity or development of P. lepida larvae. Oda and Hattori (1981) also reported that
D. kaki was an important host of this moth. Nishida et al. (2006) investigated cocoon
distributions on 51 tree species on the campus of the University of Shiga Prefecture (western
Japan) and found cocoons on 26 species. The most heavily infested species were Q.
myrsinaefolia, T. sebifera, A. pycnanthum, A. palmatum matsumurae, P. lannesiana, and P. 
yedoensis. Umeya and Okada (2003) listed P. lepida as a pest of D. kaki, M. rubra, Acer spp.,
Platanus acerifolia (Platanaceae), Camellia spp. (Theaceae), Prunus spp., and Oleaceae.
The literature illustrates that P. lepida uses both deciduous and evergreen broad-leaved
trees. Yamazaki et al. (1994), however, reported that deciduous trees were preferred over
evergreens. Nishida et al. (2006) and Sawada et al. (2008b, d) documented that cocoon
density was much higher on deciduous trees than on evergreens in the first generation, but
cocoon density on the two tree groups was similar in the second generation. This pattern may
be attributable to the difference in leaf quality between deciduous and evergreens, as young
leaves are more abundant on deciduous trees in the first generation but not in the second
generation (Nishida et al. 2006, Sawada et al. 2008c). Limacodid slug caterpillars generally
avoid pubescent leaves but feed on leaves of a variety of trees irrespective of plant chemistry
and phylogeny (Lill et al. 2006).
In India and Southeast Asia, 78 plant species from 35 families, including Arecaceae,
Fabaceae, Moraceae, and Euphorbiaceae, have been recorded as host plants of this moth
(Robinson et al. 2001). Moreover, P. lepida is known as a notorious pest of fruit trees and
other useful plants, damaging coconuts, coffee, mango, cacao, and tea in India, Sri Lanka, and
 Ecology of the Blue-Striped Grub Moth Parasa Lepida … 193

Indonesia (Cranham 1966, Kalshoven 1981, Kapoor et al. 1985, Jeyabalan and Murugan
1996). Although most host plants are trees and shrubs, herbaceous crops such as Brassica
oleracea (Brassicaceae) and Oryza sativa (Poaceae) are also listed as its host plants
(Robinson et al. 2001). To our knowledge, P. lepida does not use palms as regular host plants
in Japan, although many palms are planted in streets and parks of coastal areas in southern
and western Japan. The larvae rarely feed on leaves of herbaceous plants in western Japan
(Yamazaki, personal observations).

NATURAL ENEMIES
A wide array of natural enemies attack P. lepida during all of its developmental stages,
from eggs to adults. The egg parasitoid Trichogramma dendrolimi Matsumura (Hymenoptera:
Trichogrammatidae) attacks the eggs, with the parasitism rate ranging from 2.3 to 36.6 %
(Sawada et al. 2008d). Parasitized eggs are easily identified due to their blackish color (Oda
and Hattori 1981, Sawada et al. 2008b, d). The ladybird beetle Harmonia axyridis (Pallas)
occasionally preys on eggs (Sawada et al. 2008d).
Entomopathogenic viruses (possibly nuclear polyhedrosis virus (NPV)) is one of the most
important mortality agents for larvae. Sawada et al. (2008a, d) reported that infections of
NPV could affect populations of second-generation larvae in a density-dependent fashion.
The possible importance of NPV for second-generation mortality was also suggested by Oda
and Hattori (1981). Larvae killed by NPV infections become swollen and yellowish in color
and are found in a typical wilted state, with their prolegs clinging to leaf petioles (Sawada et
al. 2008d, e). Infections by etomopathogenic fungi have been reported as an important cause
of mortality in first-generation larvae (Sawada et al. 2008e). The dead larvae are covered with
whitish fungal hyphae. Infection rates by entomopathogenic viruses and fungi sometimes
differ among host tree species (Sawada et al. 2008e). NPV is a known mortality agent of P.
lepida in India (Babu et al. 1988) and of another limacodid Scopelodes contracta Walker in
Japan (Aratake and Watanabe 1973). These entomopathogenic viruses and fungi also impose
considerable negative impacts on many other lepidopteran caterpillars, including the gypsy
moth Lymantria dispar L. (Elkinton and Liebhold 1990).
Various arthropod predators prey on larvae, including spiders, ladybird beetles H.
axyridis, the praying mantis Hierodula patellifera (Serville) (Sawada et al. 2008d), assassin
bugs Agriosphodrus dohrni (Signoret) (Nakano and Takimoto 2011), Isyndus obscurus
(Dallas) (Yano 1990), the carabid beetle Parena cavipennis (Bates) (Oda and Hattori 1981),
Polistes paper wasps (Sawada, unpublished data), and ants Formica japonica Motschulsky
(Yamazaki, unpublished data). Among them, A. dohrni can significantly reduce the number of
second-generation larvae on old cherry trees (Nakano and Takimoto 2011). Oda and Hattori
(1981) suggested that P. cavipennis adults and larvae are potential major natural enemies of
P. lepida larvae. Paper wasps attack young larvae on host leaves (Sawada, unpublished data).
Ants often attack and transport mature larvae to their nests while the larvae are searching for
suitable sites to construct cocoons around basal trunks (Yamazaki, unpublished data).
Although several parasitoids of Ichneumonidae and Tachinidae attack larvae and prepupae in
western Japan, the parasitism rate is usually less than 10 % (Sawada et al. 2008a, Yamazaki,
unpublished data).
194 Kazuo Yamazaki, Kosuke Nakanishi and Hiroichi Sawada

Several bird species, including the great tit Parus major, attack cocoons (prepupae or
pupae) of both the first and second generations (Yamazaki 2001, Nishida et al. 2006,
Yamazaki et al. 2007, Sawada et al. 2008a). Given that the period of overwintering cocoons
lasts for about 6 months and birds may experience food shortages during winter, the cocoons
are heavily preyed on by birds (Nishida et al. 2006, Sawada et al. 2008a). Parus major
usually preys on prepupae and leaves beak marks on cocoons (Figure 1f), but it sometimes
deftly opens the emergence hole and extracts the internal prepupae without leaving beak
marks (Sawada et al. 2008a). Therefore, the predation rate on cocoons can be underestimated
if only beak marks are used when assessing the impacts of avian predators on P. lepida
populations. When the moths eclose, they usually leave the pupal exuviae in the cocoon
(Figure 1e). These exuviae provide more reliable evidence of adult eclosion (Sawada et al.
2008a). The avian predation rate appears to be higher in suburban than in urban areas (e.g.,
Nishida et al. 2006, Yamazaki et al. 2007, Sawada et al. 2008a). Yamazaki et al. (2007)
reported that the avian predation rate was higher on cocoons that were attached to tree trunks
at heights between 0.5 and 1.5 m above the ground, where twigs had been cleared and prop
wood was affixed. This may reflect the improved foraging efficiency of birds in these
habitats. However, avian predation may be less intense on P. lepida cocoons that have
poisonous setae in the cocoon wall and on the integument of the prepupae than on cocoons of
other spineless lepidopteran species. For example, less than 20 % of P. lepida cocoons were
preyed on in an urban area in western Japan (Yamazaki et al. 2007), whereas more than 85 %
of palatable cocoons of Furcula furcula (Clerck) (Lepidoptera: Notodontidae) were attacked
(Yamazaki and Sugiura 2005). Similar relatively low predation rates (20–60 %) were reported
in two limacodid species that make cocoons in litter in the USA (Murphy and Lill 2010).
Prepupae frequently blacken to death within their cocoons, and this diagnosis may be
indicative of an infectious disease (Sawada et al. 2008a) or physiological death (e.g., winter
cold) (Oda and Hattori 1981).

POPULATION DYNAMICS
Despite its importance as a pest, P. lepida has rarely been the object of studies to clarify
the factors that regulate its population dynamics, except for a series of studies in Japan by
Sawada et al. (2008a–e). Sawada et al. (2008b, d, e) conducted a life table study of P. lepida
on the deciduous tallow tree T. sebifera and on an evergreen oak, Q. myrsinaefolia, on the
campus of the University of Shiga Prefecture (western Japan) over 3 years. In the first
generation, egg density was higher on T. sebifera than on Q. myrsinaefolia, and larval
mortality was lower on T. sebifera compared with Q. myrsinaefolia, resulting in higher
cocoon density on T. sebifera. In contrast, in the second generation, egg density was higher
on T. sebifera than on Q. myrsinaefolia, but larval mortality, possibly due to NPV, was much
higher on T. sebifera, leading to higher cocoon density on Q. myrsinaefolia (Sawada et al.
2008d). Because of the higher water content, lower carbon, and higher larval performance on
T. sebifera, the leaf quality of T. sebifera was superior to that of Q. myrsinaefolia for P.
lepida larvae (Sawada et al. 2008e). However, the higher larval density on T. sebifera due to
the superior leaf quality might facilitate NPV transmission among second-generation larvae
 Ecology of the Blue-Striped Grub Moth Parasa Lepida … 195

(Sawada et al. 2008e). Therefore, the interrelationships among leaf quality, larval density, and
infectious diseases may be important for population dynamics in this moth.
Nishida et al. (2006) and Sawada et al. (2008a) examined cocoon density and adult
emergence success from cocoons on 404 individual trees from 51 species over 5 years on the
same university campus. First-generation cocoon density fluctuated considerably year by
year, whereas second-generation cocoon density was stable. This population trend was caused
by inverse density-dependent population growth in the first generation and by density-
dependent and stabilizing population growth in the second generation (Sawada et al. 2008a).
As was exemplified on T. sebifera, in the first generation, eggs are likely to be laid on
superior-quality plants, and the larvae therefore suffer less mortality (Sawada et al. 2008d, e).
In contrast, in the second generation, NPV infection functions as an important factor,
imposing density-dependent mortality (Sawada et al. 2008a). These findings suggest that in
Japan, NPV infection in the second generation is a primary regulating factor of these moth
populations, although other mortality agents, such as avian and invertebrate predators, cannot
be ignored.

CONTROL METHODS
No quantitative studies have addressed the efficiency of various control methods for P.
lepida in Japan. In general, removing egg batches, young gregarious larvae, and
overwintering cocoons is a promising approach when the occurrence remains spatially
restricted (Oda and Hattori 1981, Higa and Kishimoto 1995). For example, in some personal
and rooftop gardens of urban Osaka, western Japan, the removal of overwintering cocoons
can often almost extirpate the local population (Yamazaki, personal observations). Because P.
lepida larvae and cocoons are easily transported with host trees, trees should be carefully
checked, and larvae and cocoons should be removed before planting. Spraying with organic
phosphorous pesticides, such as fenitrothion and nereistoxin analog (Cartap), is recommended
to kill larvae (Oda and Hattori 1981, Uezumi 1986, Higa and Kishimoto 1995). In Indonesia,
synthetic pyrethrinoids, carbaryl, and Bt are used to control P. lepida on coconuts (Desmier
de Chenon 1982).

CHARACTERISTICS OF JAPANESE POPULATIONS

Several unique ecological aspects have been observed in P. lepida of Japan compared
with its bionomics in other regions. First, in Japan, the moth experiences two generations per
year, with a portion of the populations having only one generation, whereas in India, five
generations take place using mango as a host plant (Kapoor et al. 1985). In Indonesia, only 68
days are required for individuals to grow from eggs to pupae during the rainy season
(Desmier de Chenon 1982), suggesting that several generations occur per year. Although in
southern Japan, including Okinawa (latitude, 26N; mean annual temperature, 23C), the
moth may be able to have one additional generation because of the warm climate, autumn
prepupae enter diapause in their cocoons to overwinter.
196 Kazuo Yamazaki, Kosuke Nakanishi and Hiroichi Sawada

Second, despite the availability of various palm species that are planted in urban areas of
Japan, the larvae rarely use them as host plants. However, many palms, such as Borassus
flabellifer, Cocos nucifera, and Nypa fructicans, are heavily infested by P. lepida larvae in
Southeast Asia (Desmier de Chenon 1982, Robinson et al. 2001). These two characteristics of
the Japanese populations may be attributable to the life history traits of the original
population, which were determined by the climate and vegetation of the original area.
Finally, larval mortality factors in Japan are different from those in Indonesia. Although
in Japan, the larvae are attacked by some ichneumonid and tachinid parasitoids, the parasitism
rate is usually low (less than 10 %) (Sawada et al. 2008A, Yamazaki, unpublished data).
However, in Indonesia, various parasitoids, including Braconidae, Ichneumonidae,
Tachinidae, and Sarcophagidae, parasitize the larvae and prepupae, and the pyralid caterpillar
Phycita dentilinella Hampson feeds on the cocoons, prepupae, and pupae, although
quantitative data of these parasitoids and predators have not been presented (Desmier de
Chenon 1982). Of these parasitoids, Apanteles parasae Rohwer (Hymenoptera: Braconidae)
appears to be the most important mortality agent (Desmier de Chenon 1982). Viral disease is
a very important cause of mortality in Japanese, Indian, and Indonesian populations, but the
viruses seem to differ among regions (Desmier de Chenon 1982, Babu et al. 1988, Sawada et
al. 2008a, d). Desmier de Chenon (1982) noted that the viral disease in Indonesia is
transmitted by Forcipomyia midges (Diptera: Cerapogonidae). Similarly, in a congeneric
species P. viridissima Holland, which defoliates the coconut palm and oil palm in Nigeria,
various natural enemies, including parasitoids, predators, and entomopathogenic microbes,
act in concert to regulate the moth populations (Igbinosa 1985a, b).
In Japan, occurrences of P. lepida are concentrated in urban and suburban areas, and
various street and fruit trees are sometimes severely defoliated (Oda and Hattori 1981,
Nakatomi 1987, Yamazaki et al. 1994a, b, 2007, Umeya and Okada 2003, Nishida et al. 2006,
Sawada et al. 2008a–e). In India, 56.2 % of mango leaves were damaged by the larvae
(Kapoor et al. 1985). In coconut plantations in Indonesia, coconut palms are heavily attacked,
especially when young plants are planted (Desmier de Chenon 1982). Severely defoliated
coconut palms produce fewer nuts for 40 months compared with non-damaged plants
(Desmier de Chenon 1982). In both Japan and its native regions, P. lepida may be mainly
restricted to anthropogenically transformed areas, and occasional outbreaks and subsequent
rapid declines due to infectious diseases and predation may recur.

FUTURE RESEARCH DIRECTIONS

Although in Japan, as stated above, various aspects of the ecology of P. lepida have been
studied for ca. 30 years, several issues remain to be addressed. First, the reason for the
distributional restriction to man-made habitats should be determined. Studies of moth
population dynamics have suggested that NPV infections could play an important role in
population regulation (Nishida et al. 2006, Sawada et al. 2008a–e). However, whether
infections by NPV and other microbes affect the partial occurrence of this moth in man-made
habitats is unclear. As cocoon predation by birds contributes to the considerable mortality in
P. lepida in Japan (Nishida et al. 2006, Yamazaki et al. 2007, Sawada et al. 2008a), it may
also be an important determinant of habitat preference. Pupal predators prevent outbreaks
 Ecology of the Blue-Striped Grub Moth Parasa Lepida … 197

from occurring in the winter moth Operophtera brumata (L.) (Lepidoptera: Geometridae),
and predator-rich forests rarely suffer from winter-moth outbreaks (Raymond et al. 2002).
Thus, predators and microbial infections are candidate primary factors driving habitat
preferences. The introduction of egg batches into natural habitats and subsequent population
censuses may provide a promising method to clarify this issue.
Second, the effects of P. lepida occurrences on other organisms also need to be examined
because several exotic insects have affected native flora, fauna, and ecosystem processes
(Kenis et al. 2009). For example, Kawamoto et al. (1984) reported that following an outbreak
of P. lepida, the population of a congeneric species, P. consocia, declined rapidly. Such
relationships can be the result of interspecific resource competition, interactions between two
species that are mediated by shared natural enemies, reproductive interference, accidents, and
other unknown mechanisms.
Finally, these studies should be integrated to devise efficient pest-control schemes to
maintain biodiversity and useful biotic interactions. Recently, such studies have gained
momentum in agroforestry systems (Sperber et al. 2004, Altieri and Nicholls 2008) and urban
environments (Ellis et al. 2005). To achieve this goal, additional comparative studies of P.
lepida ecology between Japan and its native regions are indispensable.

ACKNOWLEDGMENTS
We thank Takayoshi Nishida, Toshio Kitamoto, and Koh-ichi Takakura for their valuable
advice during our surveys and Mariko Furukawa for allowing us to use her photographs of P.
lepida eggs and adults.

REFERENCES
Altieri, M. A. and Nicholls, C. I. (2008) Ecologically based pest management in agroforestry
systems. In: Batish, D. R., Kohli, R. K., Jose, S. and Singh, H. P. (Eds.) Ecological Basis
of Agroforestry, 95-107. CRC Press, Boca Raton.
Aratake, Y. and Watanabe, H. (1973) A newly discovered nuclear polyhedrosis of the small
blackish cochlid, Scopelodes contracta Walker. Japanese Journal of Applied Entomology
and Zoology, 17, 132-136 (in Japanese with English summary).
Babu, M. P., Sheela, P. T. and Joseph, D. (1988) A multiple embedding virus of Parasa
lepida (Cramer) (Limacodidae: Lepidoptera) in India. Tropical Pest Management, 34,
107-108.
Battisti, A., Holm, G., Fagrell, B. and Larsson, S. (2011) Urticating hairs in arthropods: their
nature and medical significance. Annual Review of Entomology, 56, 203-220.
Benedict, M. Q., Levine, R. S., Hawley, W. A. and Lounibos, L. P. (2007) Spread of the tiger:
global risk of invasion by the mosquito Aedes albopictus. Vector-Borne Zoonotic
Diseases, 7, 76-85.
Cappaert, D., McCullough, D. G., Poland, T. M. and Siegert, N. W. (2005) Emerald ash borer
in North America: a research and regulatory challenge. American Entomologist, 51,
152-165.
198 Kazuo Yamazaki, Kosuke Nakanishi and Hiroichi Sawada

Cranham, J. E. (1966) Tea pests and their control. Annual Review of Entomology, 11,
491-514.
Dafni, A. (1998) The threat of Bombus terrestris spread. Bee World, 79, 113-114.
Desmier de Chenon, R. (1982) Latoia (Parasa) lepida (Cramer) Lepidoptera Limacodidae, a
coconut pest in Indonesia. Oléagineux, 37, 181-183.
Elkinton, J. S. and Liebhold, A. M. (1990) Population dynamics of gypsy moth in North
America. Annual Review of Entomology, 35, 571-596.
Ellis, J. A., Walter, A. D., Tooker, J. F., Ginzel, M. D., Reagel, P. F., Lacey, E. S., Bennett,
A. B., Grossman, E. M. and Hanks, L. M. (2005) Conservation biological control in
urban landscapes: manipulating parasitoids of bagworm (Lepidoptera: Psychidae) with
flowering forbs. Biological Control, 34, 99-107.
Foot, N. C. (1922) Pathology of the dermatitis cause by Megalopyge opercularis, a Texan
caterpillar. Journal of Experimental Medicine, 35, 737-753.
Greeney, H. F., Dyer, L. A. and Smilanich, A. M. (2012) Feeding by lepidopteran larvae is
dangerous: a review of caterpillars' chemical, physiological, morphological, and
behavioral defenses against natural enemies. Invertebrate Survival Journal, 9, 7-34.
Higa, Y. and Kishimoto, T. (1984) Background of importation and life cycle of Latoia lepida
(Cramer). Annual Report of Okinawa Prefectural Institute of Public Health, 18, 57-61 (in
Japanese).
Higa, Y. and Kishimoto, T. (1995) Background of caterpillar dermatitis caused by Latoia
lepida (Cramer) (Limacodidae) in Okinawa Prefecture, Japan. Annual Report of Okinawa
Prefectural Institute of Health and Environment, 29, 57-64 (in Japanese).
Howard, F. W., Moore, D., Giblin-Davis, R. M. and Abad, R. G. (2001) Insects on Palms.
CABI International, Wallingford.
Hurley, B. P., Slippers, B. and Wingfield, M. J. (2007) A comparison of control results for the
alien invasive wood wasp, Sirex noctilio, in the southern hemisphere. Agricultural and
Forest Entomology, 9, 159-171.
Igbinosa, I. B. (1985a) Life-table studies for the nettle caterpillar, Latoia viridissima Holland,
on the oil palm, Elaeis guineensis Jacq., and the coconut palm, Cocos nucifera L.
Agriculture, Ecosystems and Environment, 14, 77-93.
Igbinosa, I. B. (1985b) On the ecology of the nettle caterpillar, Latoia (=Parasa) viridissima
Holland (Limacodidae: Lepidoptera). Insect Science and Its Application, 6, 605-608.
Inoue, H. (1982) Latoia lepida (Cramer). In: Inoue, H. Sugi, S., Kuroko, H., Moriuti, S. and
Kawabe, A. (Eds.) Moths of Japan I, 300. Kodansha, Tokyo (in Japanese).
Islam, M. D. A., Yamakawa, R., Do, N. D., Numakura, N., Suzuki, T. and Ando, T. (2009)
Instrumental analysis of terminal-conjugated dienes for reexamination of the sex
pheromone secreted by a nettle moth, Parasa lepida lepida. Bioscience, Biotechnology,
and Biochemistry, 73, 1156-1162.
Jeyabalan, D. and Murugan, K. (1996) Impact of variation in foliar constituents of Mangifera
indica Linn. on consumption and digestion efficiency of Latoia lepida Cramer. Indian
Journal of Experimental Biology, 34, 472-474.
Kalshoven, L. G. E. (1981) Pests of Crops in Indonesia. (Revised and translated by van der
Laan, P. A.) PT. Ichtiar Baru, Jakaruta.
Kanai, N. (2009) First records of Parasa lepida (Limacodidae) in the western part of Ibaraki
Prefecture. Bulletin of Ibaraki Nature Museum, 12, 21-22 (in Japanese).
 Ecology of the Blue-Striped Grub Moth Parasa Lepida … 199

Kapoor, K. N., Deobhakta, S. R. and Dhamdhere, S. V. (1985) Bionomics of the slug

caterpillar, Latoia lepida (Cramer) (Lepidoptera: Limacodidae) on mango. Journal of
Entomological Research, 9, 235-236.
Kawada, A. (1930) A list of cochlidionid moths in Japan, with descriptions of two new
genera and six new species. Journal of Imperial Agricultural Experiment Station, 1, 231-
262, pl. 26.
Kawamoto, F. (1978) Studies on the venomous spicules and spines of moth caterpillars III.
Scanning electron microscopic examination of spines and spicules of the slug moth
caterpillar, Parasa consocia, and some properties of pain-producing substances in their
venoms. Medical Entomology and Zoology, 29, 185-196 (in Japanese with English
summary).
Kawamoto, F., Fujioka, H. and Kumata, N. (1984) Urticating hairs and venom of Parasa
lepida. Medical Entomology and Zoology, 35, 210 (In Japanese).
Kenis, M., Auger-Rozenberg, M. - A., Roques, A., Timms, L., Péré, C., Cock, M. J. W.,
Settele, J., Augustin, S. and Lopez-Vaamonde, C. (2009) Ecological effects of invasive
alien insects. Biological Invasions, 11, 21-45.
Lill, J.T., Marquis, R. J., Forkner, R. E., Le Corf, J., Holmberg, N. and Barber, N. A. (2006)
Leaf pubescence affects distribution and abundance of generalist slug caterpillars
(Lepidoptera: Limacodidae). Environmental Entomology, 35, 797-806.
Matsumura, C., Yokoyama, J. and Washitani, I. (2004) Invasion status and potential
ecological impacts of an invasive alien bumblebee, Bombus terrestris L. (Hymenoptera:
Apidae) naturalized in southern Hokkaido, Japan. Global Environmental Research, 8,
51-66.
Murakami, O. and Washitani, I. 100 of the Worst Invasive Alien Species in Japan.
In: The Ecological Society of Japan (Ed.) Handbook of Alien Species in Japan, 362-363.
Chinjin-shokan, Tokyo (in Japanese).
Murphy, S. M., Leahy, S. M., Williams, L. S. and Lill, J. T. (2010) Stinging spines protect
slug caterpillars (Limacodidae) from multiple generalist predators. Behavioral Ecology,
21, 153-160.
Murphy, S. M. and Lill, J. T. (2010) Winter predation of diapausing cocoons of slug
caterpillars (Lepidoptera: Limacodidae). Environmental Entomology, 39, 1893-1902.
Nakano, K. (2001) Distribution of blue striped nettle grub, Parasa lepida (Cramer)
(Lepidoptera: Limacodidae), in Kanto District. House and Household Insect Pests, 24,
61-62 (in Japanese).
Nakano, T. and Takimoto, G. (2011) Non-native species interactions between blue-striped
nettle grub moths and assassin bugs in Japan. Japanese Journal of Entomology (New
Series), 14, 11-20 (in Japanese with English summary).
Nakatomi, K. (1987) Latoia lepida (Cramer). In: Sugi, S. (Ed.) Larvae of Larger Moths in
Japan, 19. Kodansha, Tokyo (in Japanese).
Nasu, Y., Arita, Y., Kimura, M. and Ogata, A. (2004) Some lepidopterous pests of
Eucalyptus trees from Japan. Japanese Journal of Applied Entomology and Zoology, 48,
123-133 (in Japanese with English summary).
Natsuaki, Y. (1998) Parasa lepida causing dermatitis. In: Yasuda, T., Hirowatari, T. and
Ishii, M. (Ed.) Biology of the Microlepidoptera, 54-57. Bunkyo-shuppan, Osaka
(in Japanese).
200 Kazuo Yamazaki, Kosuke Nakanishi and Hiroichi Sawada

Nishida, S., Araki, Y. and Sawada, H. (2006) Seasonal population changes of the greenstriped
cochlid, Parasa lepida Cramer. Annual Report of the Kansai Plant Protection Society,
48, 115-117 (in Japanese).
Oda, M. and Hattori, I. (1981) The bluestriped (greenstriped) nettle grub, Latoia lepida
(Cramer): a new pest of Japanese persimmon. Plant Protection, 35, 401-405
(in Japanese).
Ohtaki, N. and Shinonaga, S. (1995) Dermatitis due to caterpillar of Latoia lepida in Saitama
Prefecture, Japan. Medical Entomology and Zoology, 46, 81-82 (in Japanese).
Raymond, B., Vanbergen, A., Watt, A., Hartley, S. E., Cory, J. S. and Hails, R. S. (2002)
Escape from pupal predation as a potential cause of outbreaks of the winter moth,
Operophtera brumata. Oikos, 98, 219-228.
Robinson, G. S., Ackery, P. R., Kitching, I. J., Beccaloni, G. W. and Hernández, L. M. (2001)
Hostplants of the Moth and Butterfly Caterpillars of the Oriental Region. The Natural
History Museum, London.
Sawada, H., Hori, Y., Nisida, S., Matsumoto , T. and Nishida, T. (2008a) Population
dynamics of an invasive grub moth Parasa lepida (Cramer) that damages ornamental
trees: the seasonal and annual fluctuations of the cocoon density. Japanese Journal of
Environmental Entomology and Zoology, 19, 115-124.
Sawada, H., Masumoto, Y. and Nishida, S. (2008b) Comparative life table study of the blue-
striped nettle grub moth Parasa lepida on deciduous and evergreen trees. Annual Report
of the Kansai Plant Protection Society, 50, 151-153 (in Japanese).
Sawada, H., Masumoto, Y. and Nishida, S. (2008c) Life table and survivorship curves of the
blue-striped nettle grub moth Parasa lepida. Annual Report of the Kansai Plant
Protection Society, 50, 155-157 (in Japanese).
Sawada, H., Masumoto, Y., Matsumoto, T. and Nishida, T. (2008d) Comparisons of cocoon
density and survival processes of the blue-striped nettle grub moth Parasa lepida
(Cramer) between deciduous and evergreen trees. Japanese Journal of Environmental
Entomology and Zoology, 19, 59-67.
Sawada, H., Shimomura, S., Nishida, T. and Matsumoto, T. (2008e) Causes of larval
mortality in relation to host plant quality in the invasive grub moth, Parasa lepida
(Cramer). Japanese Journal of Environmental Entomology and Zoology, 19, 69-78.
Sharov, A. A., Leonard, D., Liebhold, A. M., Roberts, E. A. and Dickerson, W. (2002) “Slow
the spread”: a national program to contain the gypsy moth. Journal of Forestry, 100,
30-35.
Sperber, C. F., Nakayama, K., Valverde, M. J. and Neves, F. de S. (2004) Tree species
richness and density affect parasitoid diversity in cacao agroforestry. Basic and Applied
Ecology, 5, 241-251.
Suarez, A. V., Holway, D. A. and Case, T. J. (2001) Patterns of spread in biological invasions
dominated by long-distance jump dispersal: insights from Argentine ants. Proceedings of
the National Academy of Sciences USA, 98, 1095-1100.
Takemura, Y. (1960) Local insect records with illustrations (IV). Satsuma, 26(35), 1-2
(in Japanese).
Teramoto, N. (2011) Parasa lepida (Cramer). In: Komai, F., Yoshiyasu, Y., Nasu, Y. and
Saito, T. (Eds.) A Guide to the Lepidoptera of Japan, 655. Tokai University Press,
Kanagawa (in Japanese).
 Ecology of the Blue-Striped Grub Moth Parasa Lepida … 201

Uezumi, Y. (1986) Slug caterpillars. In: Yamaguchi, A. and Ôtake, A. (Eds.) Diseases and
Invertebrate Pests of Fruit Trees. Zenkoku-noson-kyoiku-kyokai, Tokyo (in Japanese).
Umeya, K. and Okada T. (2003) Agricultural Pests in Japan. Zenkoku-noson-kyoiku-kyokai,
Tokyo (in Japanese).
Wakamura, S., Tanaka, H., Masumoto, Y., Sawada, H. and Toyohara, N. (2007) Sex
pheromone of the blue-striped nettle grub moth Parasa lepida (Cramer) (Lepidoptera:
Limacodidae): identification and field attraction. Applied Entomology and Zoology, 42,
347-352.
Yamazaki, K. (2001) Dynamic aspects of insect assemblages in urban setting. Nature and
Insects, 36(8), 8-12 (in Japanese).
Yamazaki, K., Kitamoto, T. and Nakatani, K. (1994a) Host plant selection of the blue-striped
nettle grub moth Parasa lepida I. Nature Study, 40, 20-23 (in Japanese).
Yamazaki, K., Kitamoto, T. and Nakatani, K. (1994b) Host plant selection of the blue-striped
nettle grub moth Parasa lepida II. Nature Study, 40, 43-46 (in Japanese).
Yamazaki, K., Kitamoto, T, Yariyama, Y. and Sugiura, S. (2007) An analysis of spatial
distribution in the exotic slug caterpillar Parasa lepida (Cramer) (Lepidoptera:
Limacodidae) at an urban coastal site in central Japan. Pan-Pacific Entomologist, 83,
193-199.
Yamazaki, K. and Sugiura, S. (2006) Possible avian predation on Furcula furcula cocoons
(Lepidoptera, Notodontidae) at an urban park. Transactions of the Lepidopterological
Society of Japan, 56, 165-170.
Yano, I. (1990) A host record of Isyndus obscurus (Dallas) (Hemiptera, Reduviidae).
Japanese Journal of Entomology, 58, 204.
In: Lepidoptera ISBN: 978-1-62417-248-9
Editors: Elia Guerritore and Johannes DeSare © 2013 Nova Science Publishers, Inc.

Chapter 8

BIOTRANSFORMATION OF SECONDARY PLANT

METABOLITES BY LEPIDOPTERA

Clécio Souza Ramos*

Department of Molecular Sciences, Rural Federal University of Pernambuco,
Recife-Pe, Brazil

ABSTRACT
It is presumed that the first insects in the fossil record from the middle of the
Carboniferous period were not herbivores, but rather saprophages, and that the habit of
feeding on green plants arose during the co-evolution of insects and plants. In response to
the change in the saprophagous habit of insects, plants developed chemical defenses
against them by accumulating toxic compounds known as secondary metabolites. In
response, insects developed strategies to overcome the chemical defense arsenal of
plants. There is still much discussion over exactly how this occurred, but there is
evidence that insects developed the ability to use the biotransformation mechanism to
detoxify these plant defense substances. The successful exploitation of plants as food by
the second largest order of insects, Lepidoptera, with over 160,000 species, can be
associated with the capacity of herbivorous species to minimize the harmful effects of
toxic plant substances through biotransformation as the main detoxification mechanism.
Since Lepidoptera species choose the host plant during the adult phase, their larvae must
adapt to the diet imposed on them by overcoming the possible chemical and physical
barriers. Despite a dearth of reports, studies of the metabolism of plant compounds by
Lepidoptera show that biotransformation of compounds to their less toxic and more polar
forms is a strategy employed by these insects. For example, larvae of the butterfly
Heraclides brasiliensis biotransform the compound 4-nerolidylcatechol, a natural
antioxidant and insecticide, the main constituent of Piper umbellata leaves, to its
respective acid, which is less toxic and more polar than 4-nerolidylcatechol The study of
the metabolism of plant compounds by Lepidoptera, besides generating information that
can help understand the feeding habits of insects and how they overcome plants’
chemical defense mechanisms, can also help in the search for new secondary metabolites
with potential as drugs or agricultural chemicals.

* Department of Molecular Sciences, Rural Federal University of Pernambuco, Recife-Pe, Brazil, CP 52.171-030,
email: clecio@[Link]
204 Clécio Souza Ramos

Of the extraordinary diversity found in the world’s biota, 60% corresponds to the
Insecta, of which 46% are phytophagous (Cronin and Abrahamson, 2001). Lepidoptera is
the second largest order of insects, with more than 160,000 identified species (Herrera
and Pellmyr, 2002) and its caterpillars are strongly attracted to plants. Initially, it was
presumed that the first insects, including Lepidoptera, in the fossil record, dating from the
middle of the Carboniferous period, were not herbivores but rather saprophages, and that
the habit of feeding on green plants arose during the co-evolution of insects and plants. In
response to the change in the saprophagous habit of insects, plants developed chemical
defenses against them by accumulating toxic compounds known as secondary
metabolites, causing insects to develop strategies to overcome the chemical defense
arsenal of plants. There is still much discussion over exactly how this occurred, but there
is evidence that insects developed the ability to use the biotransformation mechanism to
detoxify these plant defense substances
The chemical interaction between Lepidoptera and plants is a well-studied subject in
chemical ecology, with several papers available (Trigo, 2000; Brower and Brower, 1964;
Nishida, 2002; Schulz, 1998; Boppré, 1989). It is well known from early studies that
several deterrent chemicals from plants, such as cardenolides, pyrrolizidine alkaloids,
iridoid glycosides and aristolochic acids, are sequestrated by Lepidoptera, which use
these substances for their own purposes (Klitzke and Brown, 2000; Abe et al., 2001;
Nishida, 1995). In danaid butterflies, cardenolides have often been implicated in chemical
defense (Brower et al., 1982), and pyrrolizidine alkaloids in both chemical defense and
pheromone production (Schneider et al., 1975). However, the metabolic pathway of toxic
compounds from plants in Lepidoptera and other insect orders is underexplored, mainly
involving the natural diet of herbivorous insects, and is almost nonexistent when
involving the volatile chemical constituents of the host plants, although this study is a
crucial step to understanding how they overcome plant chemical defenses and adapt to
host plants (Schoonhoven et al, 2005).
The successful exploitation of plants as food by the second largest order of insects
can be associated with the capacity of these herbivorous to minimize the harmful effects
of toxic plant substances through biotransformation as the main detoxification
mechanism. Since Lepidoptera species choose the host plant during the adult phase, their
larvae must adapt to the diet imposed on them by overcoming the possible chemical and
physical barriers. Despite the dearth of reports, studies of the metabolism of plant
compounds by Lepidoptera show that biotransformation of compounds to their less toxic
and more polar forms is a strategy employed by these insects. This chapter describes the
metabolism of several plant secondary metabolites and their possible biotransformation
during the digestive process in Lepidoptera larvae.

SECONDARY METABOLITES ASSOCIATED WITH PLANT DEFENSES

AGAINST HERBIVOROUS INSECTS
Plants have evolved a wide array of defenses to reduce the injuries caused by herbivores.
Some of these defenses are physical, but the majority is chemical. The chemical compounds
associated with plant defenses are known as secondary metabolites. They not essential for life
and are found in only specific organisms or groups of organisms. They can be considered an
expression of the individuality of species. Secondary metabolites are not biosynthesized under
all conditions; in most cases these compounds’ function and benefit to the organism are not
yet known, though they usually contribute to survival. The list of chemicals that have
defensive value due to their ability to interfere with insect physiology or behavior is
 Biotransformation of Secondary Plant Metabolites by Lepidoptera 205

extensive. It is believed that most of the 200,000 known secondary metabolites are involved
in plant chemical defense systems and haven been formed over the millions of years plants
have coexisted with their attackers (Mazid et al., 2011).

Dhurrin Nicotine Psoralen

HO H
HO
O
O N
OH O
HO OH O O
N
Azadirachtin
O O OCH3 Limonene
Dillapiole HO
O OH O O
OCH3 O
O
O O

O OCH3 O OH
O
O
OCH3

Figure 1. Chemical structures of plant secondary metabolites with roles in chemical defense against
natural enemies, mainly insects.

Basically there are three overall groups of secondary metabolites: phenolic compounds,
alkaloids and terpenes, the last group including monoterpenes, sesquiterpenes and
triterpenoids as well as steroids (Figure 1). Phenolic compounds are derived from shikimic
acid or mevalonic acid. Alkaloids are derived from aromatic amino acids (phenylalanine,
tyrosine and tryptophan), which are in turn derived from shikimic acid and also from aliphatic
amino acids (ornithine and lysine). Finally, terpenes are biosynthesized starting from
mevalonic acid (in the cytoplasm) or pyruvate and 3-phosphoglycerate (in the chloroplast)

BIOTRANSFORMATION REACTIONS INVOLVED IN THE

DETOXIFICATION MECHANISM OF PLANT TOXIC
COMPOUNDS BY LEPIDOPTERA
The biotransformation of toxic compounds by insects in general involves the conversion
of these compounds to a less toxic or more polar form that facilitates their elimination. These
reactions entail diverse enzymes that can be produced by insects themselves or by microbial
symbionts (Herrera and Pellmyr, 2004). One of the most studied groups of enzymes that
metabolize a wide variety of toxicants is the cytochrome P450 mono-oxygenases, also known
as polysubstrate mono-oxygenases (PSMOs) or mixed-function oxydases (MFOs)
(Schoonhoven et al., 2005). These enzymes are called “P450” because there are so many of
them and they do so many things, a fact particularly relevant in the case of insect P450s
(Feyereisen, 1999). In insects, P450 cytochromes are involved in biosynthesis of ecdysteroids
and juvenile hormones as well as in metabolism and detoxification of insecticides. P450
206 Clécio Souza Ramos

cytochromes play crucial roles in defense against the natural products that insects have to
fend off in order to feed on otherwise toxic plants. The ability of an insect P540 cytochrome
to metabolize a specific natural product is often the key to the adaptation of herbivorous
insects to their host plants (Zagrobelny et al., 2004). Here we report some reactions of the
metabolism of toxic secondary plant metabolites in lepidopteras.
Alkaloids together with cyanogenic glycosides are among the best studied plant chemical
defense compounds against natural enemies, mainly against insects including Lepidoptera.
Exemples are pyrrolizidine alkaloids, which are a diverse class of natural compounds based
on a [3.3.0] azabicyclo ring, generally occurring as esters of a “necine base” with “necic
acids” as mono- or diesters. They are strong deterrents to insects and vertebrates (Trigo,
2000). Pyrrolizidine alkaloids can exist in two molecular forms, the pro-toxic free base and
the non-toxic N-oxide forms (Figure 2) (Hartmann, 2004).

HO
O
O
O H HO H
O O H HO O
OH OH
N N
HO
N O
Senecionine Retronecine Indicine-N-oxide

Figure 2. Example of natural pyrrolizidine alkaloids.

Various plant families, particularly the Asteraceae, Boraginaceae, Fabaceae,

Apocynaceae and Orchidaceae, produce and store pyrrolizidine alkaloids as N-oxides
(Hartmann, 1991; 1995; Mattocks, 1986; Borba et al., 2001). Pyrrolizidine alkaloids such as
N-oxides are quickly reduced to toxic form during digestion of herbivores, including
lepidopteras, after which they are absorbed through diffusion in the form of lipophilic free
base into the blood and the hemolymph, (Figure 3). Nevertheless, some herbivores, such as
the cinnabar moth Tyria jacobeae (Lepdoptera: Arctiidae), have developed mechanisms to
reconvert pyrrolizidine alkaloids in the toxic form to the nontoxic N-oxide form immediately
after their absorption from the digestive tract. These herbivores can then store the N-oxide in
their tissues for chemical defense against their own predators.

HO HO
O O
Reduced form
O O O O O O

Oxidized form

N N

O
Non-toxic N-oxide Toxic free base

Figure 3. The two forms (non-toxic and toxic) of pyrrolizidine alkaloids found in nature.
 Biotransformation of Secondary Plant Metabolites by Lepidoptera 207

Another group of secondary metabolites found in various plant tissues with chemical
defense roles against herbivores are cyanogenic glycosides (Figure 4). They are widely
distributed in more than 1000 species of food plants, such as in many species of the Rosaceae
family, such as the almond tree (Prunus dulcis), plum tree (Prunus domestica), cherry tree
(Prunus avium), peach tree (Prunus persica) as well as in the giant taro (Araceae: Alocasia
macrorrhizos), sorghum (Poaceae: Sorghum vulgare) and flax (Linaceae: Linum
usitatissimum) (Cade and Rubira, 1982 and Eisler, 1991). Cyanogenic glycosides are
secondary metabolites that are found in various plant tissues and produce HCN upon
hydrolysis.

OH
OH OH
HO O
HO O HO O
HO
OH H HO HO
OH OH OH
C
N C
C
N N
Prunasin Lotaustralin Epivolkenin

Figure 4. Chemical structure of cyanogenic glycosides found in plants.

Cyanogenic glycosides are not in themselves toxic, but are readily broken down through
enzymatic hydrolysis to give off the volatile poisons (Figure 5) cyanohydric acid or gas
hydrogen cyanide (HCN) when the plant tissues undergo injuries.

R OGly Glycosidase R OH R
C C C O HC N
R C N R C N R
Cyanogenic glycoside Cyanohydrin Ketone Cyanohydric acid

Figure 5. Enzymatic hydrolysis reaction of cyanogenic glycosides to give off cyanohydric acid. R
represents several alkyl or aryl substituents.

In intact plants, the enzyme and cyanogenic glycoside remain separated, but if the plant
tissue is damaged, for example by herbivorous insects, the two compounds come into contact
and cyanohydric acid is released. Cyanohydric acid is extremely toxic to a broad variety of
organisms due to its ability to link with metals (Fe2+, Mn2+and Cu2+) that are functional
groups of many enzymes, inhibiting processes like the reduction of oxygen in the cytochrome
respiratory chain, electron transport in photosynthesis, and the activity of enzymes like
catalase and oxidase (Cheeke, 1995; McMahon et al, 1995; Francisco and Pinotti, 2000).
The Neotropical butterfly Heliconius sara (Papilionoidea), whose larvae feed exclusively
on leaves of Passiflora auriculata (Passifloraceae) and accumulate cyanogenic glycosides,
can avoid the harmful cyanogenic effects. The monoglycoside cyclopentenyl cyanogens
(epivolkenin), obtained and sequestered from P. auriculata, are detoxified by larvae of H.
sara using a distinctive enzymatic mechanism not found in the host plants. This mechanism
involves substitution of the nitrile group by the thiol group, a metabolic reaction that pre-
empts the enzymatic release of cyanide (Engler et al., 2004) (Figure 6).
208 Clécio Souza Ramos

HO HO
O O
O OH H. sara O OH
HO HO
OH NC OH HS
HO H HO H

Epivolkenin Epivolkenin thiol

Figure 6. Biotransformation of epivolkenin into epivolkenin thiol by larvae of H. sara.

The reaction to cyanogenic glycoside observed in larvae of H. sara is new and it has not
been found in other organisms, including insects, where the HCN released is detoxified by
two main reactions. One route involves the formation of β-cyanoalanine from cysteine, which
is catalyzed by the enzyme β-cyanoalanine-synthase, after which β-cyanoalanine is converted
into asparagine. The other route occurs via conversion of HCN into thiocyanate, which is
catalyzed by rhodanese (Bordo and Bork, 2002; Miller and Conn, 1980) (Figure 7)

NH2  -Cyanoalanine NH2

synthase HO
HO SH
C N
O O
Cysteine  -Cyanoalanine
H C N
Hydrogen
Cyanide Rhodanese
S2O3- SCN- SO3-
Thiosulfate Thiocyanate Sulfite

Figure 7. The main routes for detoxification of cyanogenic glycoside in animals and plants.

Besides cyanogenic glycosides, probably the best-known conjugated defense compounds

are the glucosinolates (Figure 8). Glucosinolates are sulfur- and nitrogen-containing
compounds biosynthesized through amino acid metabolism and are found mainly in the
Capparales order and the Cruciferae and Capparidaceae families. Glucosinolates are known
for their deterrent activity in plants against generalist herbivores and other natural enemies,
including insects.

OH
HO O OH OH
OSO 3- OSO 3-
HO HO O HO O OH
OH OSO 3- N N
C HO HO
OH S OH S
N
Etraphyllin B sulfate Sinigrin Sinalbin

Figure 8. Chemical structure of glucosinolates found in plants.

The glucosinolate-derived isothiocyanates are metabolized into glutathione conjugates in

generalist lepidopteran herbivores, such as larvae of Spodoptera littoralis (Noctuidae),
Helicoverpa armigera, Trichoplusia ni (Plusiinae), Mamestra brassicae (Noctuidae) and
Plutella xylostella (Plutellidae) (Schramm et al., 2012). S. littoralis larvae fed with a diet of
 Biotransformation of Secondary Plant Metabolites by Lepidoptera 209

leaves of Arabidopsis thaliana (Brassicales) partially metabolize the compound 4-

methylsulfinylbutyl glucosinolate, a major constituent of the plant, turning it into
cysteinylglycine dipeptide, cysteinyl-conjugate and glycine.
Another plant family well known for the potent biological activity of its secondary
metabolites, including insecticidal activity, is the Piperaceae family, mainly leaves of species
accumulating lignans and neolignans (Vanin et al., 2008). Many larvae of Lepidoptera species
have a close relationship with its leaves. This family, together with the family Chlorantaceae,
has been considered as a basal Angiospermae (Jaramillo et al., 2004). The Piper genus is the
largest one, comprising more than 1200 species. Approximately 240 species of Piper L., 25
species of Ottonia Sprengel and two species of Pothomorphe Miquel have been described in
Brazil (Yuncker, 1973, 1975). The phytochemical investigations of Piperaceae species have
shown the accumulation of several insecticidal compounds in Piper nigrum L., P. betle L., P.
spirei C. DC., P. decurrens C. DC. and P. longun L. (Park et al., 2002; Navickiene et al.,
2000; Paula et al., 2000; Parmar et al., 1997; Chauret et al., 1996). Considering the ecological
importance of species of Piperaceae in the coevolution with insects, the discovery of the
metabolic pathway of their secondary metabolites in Lepidoptera is a relevant step for
elucidation of mechanisms used by this order of insects to overcome the insecticidal
compounds of Piperaceae (Dyer et al., 2004; Figueiredo and Sazima, 2000; Ramos and Kato,
2009; Ramos et al., 2009).

Figure 9. Chemical structures of compounds 1 and 2, larvae and pupae of H. brasiliensis in P.

umbellata (Ramos et al., 2012A).

Larvae of the butterfly Heraclides brasiliensis (Papilionidae) specifically feed on leaves

of some species of Piperaceae, such as Piper solmsianum, P. divaricatum, P. regnellii and P.
umbellata. All these species accumulate lignans and neolignans in their leaves.
The larvae of H. brasiliensis biotransform the compound 4-nerolidylcatechol (1), a
natural antioxidant and insecticide and the main constituent of Piper umbellata leaves, into its
respective acid, E-2,3-dihydro-3-(3,4-dihydroxyphenyl)farnesoic (2), which is less toxic and
more polar than 4-nerolidylcatechol (Figures 9 and 10) (Ramos et al., 2012).
The biotransformed product E-2,3-dihydro-3-(3,4-dihydroxyphenyl) farnesoic was also
found in larval and pupal tissues of the butterfly H. brasiliensis, but was not detected in the
tissues of the adult insect.
210 Clécio Souza Ramos

Figure 10. Chemical profiles (HPLC) of dichloromethane:methanol extracts from P. umbellata leaves
(a) and H. brasiliensis; larval feces (b); larvae (c); pupae (d); adults (e) (Ramos et al., 2012).

The evaluation of the toxicity of crude extracts from fecal material and leaves and of pure
compounds, 4-nerolidylcatechol and E-2,3-dihydro-3-(3,4-dihydroxyphenyl)farnesoic, by the
brine shrimp lethality bioassay (Artemia nauplii) indicated the biotransformed compound is
not toxic (LC50 of 18,617 µg/mL) while 4-nerolidylcatechol showed high toxicity (LC50 of
12.8 µg/mL). The extract of P. umbellate leaves showed moderate activity in the brine shrimp
test (LC50 = 523.3 µg/mL), while the fecal extract was shown not to be cytotoxic against A.
salina, probably in part due to the presence the metabolite E-2,3-dihydro-3-(3,4-
dihydroxyphenyl)farnesoic, displaying an LC50 value of 3,461 µg/mL (Ramos et al., 2012).
Other changes were observed in the biologic proprieties of P. umbellate. The evaluation
of the biological activity of both extracts showed that the antioxidant activity of the
metabolized P. umbellata leaves (fecal extract), as determined by the total phenol and
ABTS●+ methods, was higher than that of the leaves (Ramos et al., 2012A). The results
showed that the leaf and fecal extracts of P. umbellata both possess potent antioxidant
activity. However, the fecal extract was twice as active as the leaf extract. A direct correlation
was observed between the phenolic content and the antioxidant activity of the fecal and leaf
extracts, because phenolic compounds contribute directly to antioxidant activity (Re et al.,
1999). The higher total phenolic content observed for the fecal extract was associated with the
 Biotransformation of Secondary Plant Metabolites by Lepidoptera 211

release of phenolic compounds linked to the leaf cell walls during the insect’s digestive
process. In summary, the potentialization of the antioxidant activity of leaves of P. umbellata
after metabolism in H. brasiliensis can be associated with the biotransformation of the
compound 4-nerolidylcatechol into E-2,3-dihydro-3-(3,4-dihydroxyphenyl)farnesoic as well
as the increase in the total phenolic content of the fecal extract.
The type of reaction observed for 4-nerolidylcatechol in the digestive system corresponds
to the transformation of a vinyl group to a carboxylic acid. Similar reactions have been
described in Papilio polyxenes and Spodoptera frugiperda larvae (Ivie et al. 1983), which
were capable of biotransforming xanthotoxin into the two major metabolites 3 and 4 (Figure
11) as a result of the cleavage of the furan ring, possibly involving a transitory 20,30-epoxide
intermediate.

OCH3 OCH3 OCH3

O O O P. polyenes HO O O HO O O
O O
+
S. frugiperda
H² HO
Xanthotoxin 3 4
OH

Figure 11. Chemical structures of xanthotoxin and its major metabolites 3 and 4 biotransformed by P.
polyxenes and S. frugiperda larvae (Ivie et al., 1983).

Quadrus u-lucida (Hesperudae) (Figure 12) and Heraclides hectorides (Papilionidae)

(Figure 13) were capable of biotransforming tetrahydrofuran (-)-grandisin into the
corresponding mono-O-demethylated and di-O-demethylated phenolic derivatives when P.
solmsianum leaves were provided as their sole diet (Figure 14).

Figure 12. Quadrus u-lucida in leaves of P. solmsianum: Larvae, pupae, adult insect ad egg (Ramos
2006).
212 Clécio Souza Ramos

Figure 13. Butterfly Heraclides hectorides and their larvae in leaves of Piper solmsianum (Ramos
2006).

MeO OMe MeO OMe MeO OMe

O O Q. u lucida O
H. hectorides
MeO OMe HO OMe HO OH
Q. u lucida
OMe OMe
1 OMe OMe
6 OMe OMe

(-)-grandisin (-)-Grandisin (-)-Grandisin

mono-O-demethylated di-O-demethylated

Figure 14. Reaction of the metabolism of (-)-grandisin by larvae of H. hectorides and Q-u-lucida
butterflies.

Despite the lack of data on the toxicity of grandisin on such insects, some preliminary
hypotheses can be envisaged. The antifeeding activity of lignans and phenylpropanoids is
often reduced by an increase in their polarity, especially through hydroxylation and/or
glycosylation. On the other hand, non-polar substituents, such as methoxyl or methylene-
dioxyphenyl groups, could enhance toxicity (Harmatha and Nawrot, 2002). Nevertheless,
arguments for such processes as mechanisms to render compounds less harmful within the
insect digestive system are speculative and controversial. For instance, the phenolic
compounds produced by demethylation of aromatic methoxyl groups could be expected to be
unpalatable or at least to act as deterrents of insects. On the other hand, the defensive and
antioxidant properties of the phenolic compounds could represent a benefit for herbivorous
insects against their predators or to their overall health.
The demethylation reactions of lignan (-)-grandisin by the caterpillars Q. u-lucida and H.
hectorides are apparently regiospecific at para position, similar to the demethylation of the
lignan furofuran (+)-magnolin by the caterpillar of Spodoptera litura (Noctuidae) (Miyazawa
et al., 1995).
Known variously as the Oriental leafworm moth, cluster caterpillar, cotton leafworm,
tobacco cutworm, tropical armyworm, tobacco budworm and rice cutworm, S. litura is
considered a major polyphagous pest of various economically important crops such as cotton,
groundnuts, chilis, tobacco, castor beans, pulses and other crops in India, China and Japan
(Packiam and Ignacimuthu, 2012). It causes losses estimated at between 10 and 30% to these
crops (Ferry et al., 2004). Due to its economic importance, various diverse studies have
investigated the insecticidal potential of natural compounds as alternatives to control S. litura
caterpillars. The metabolism of some natural compounds known to have biological activities,
mainly the terpenoids, has been investigated in S. litura caterpillars. For example, the
terpenoids (+)-(1R) and (−)-(1S)-fenchone were biotransformed by S. litura yielding ten
 Biotransformation of Secondary Plant Metabolites by Lepidoptera 213

terpenoids by means of a region and stereoselective hydroxylation. (Miyazawa and

Miyamoto, 2005). (+)-Fenchone is found in fennel oil (Foeniculum vulgare), and (−)-
fenchone is obtained from Thuja oil (Thuja occidentalis). (+)-Fenchone was transformed into
five terpenoids, (+)-(1S,4R)-3-oxo-2,2,4-trimethyl-cyclopentylacetic acid, (+)-(1S,6S)-6-exo-
hydroxyfenchone, (+)-(1S,6R)-6-endo-hydroxyfenchone, (+)-(1R)-10-hydroxyfenchone and
(+)-(1S,5R)-5-exo-hydroxyfenchone (Figure 15). (-)-fenchone was biostransformed into five
terpenoid enantiomers from products of the biotransformation of (+)-fenchone.

HO H HO
S. litura HO 2C
H
O H O OH O OH O O O
(+)-Fenchone

Figure 15. Biotransformation of (+)fenchone in others five terpenoids by larvae of S. litura.

In another case, the monoterpene thymol and the phenylpropanoid trans-anethole were
metabolized into more polar compounds by S. litura larvae and Trichoplusia ni larvae
(Lepidoptera: Noctuidae) (Passreiter et al., 2005).
Thymol is the main constituent of the essential oil of thyme, Thymus Vulgaris
(Lamiaceae). It showed toxicity against late instar S. litura larvae. It has also been observed
that phenylpropanoid trans-anethole, obtained from the essential oil of anise, Pimpinella
anisum (Lamiaceae), has a synergistic effect on the toxicity of thymol to the larvae
(Hummelbrunner et al., 2001). Thymol is biotransformed into 3-O-β-glucoside while trans-
anethole undergoes hydroxylation of the side chain methyl group by both S. litura and T. ni
larvae (Figure 16).

trans-Anethole
OH

H3CO H3CO
Trichoplusia ni
S. litura
OH
OH OH
O O
HO OH
Thymol

Figure 16. Metabolism of trans-anethole and thymol by larvae of S. litura and Trichoplusia ni.

Another class of natural products active in the metabolic pathway in S. litura that has
been investigated is the isoflavones. These are natural compounds that form a quite distinct
subclass of flavonoids, known for their diverse antioxidant and antifungal activities. The
structural modifications of isoflavones by S. litura have been reported. The isoflavone 5,7,4’-
trimethoxyisoflavone undergoes trasformation to 5-hydroxy-7,4’-dimethoxyisoflavone, 7-
214 Clécio Souza Ramos

hydroxy-5,4’-dimethoxyisoflavone and 4’-hydroxy-5,7-dimethoxyisoflavone (Figure) after

metabolism by S. litura, while isoflavone 6,7,4’-trimethoxyisoflavone (2), and 7,4’-
dimethoxyisoflavone do not undergo transformations (Takahashi et al., 2006).

CH3O O
5-Hydroxy-7,4'-dimethoxyisoflavone

H3CO O
OH O
OCH3
S. litura HO O

OCH3 O
OCH3
5,7,4'-Trimethoxyisoflavone
OCH3 O
7-Hydroxy-5,4'-dimethoxyisoflavone OCH3

H3CO O

4'-Hydroxy-5,7-dimethoxyisoflavone OCH3 O
OH

Figure 17. Metabolism of isoflavone 5,7,4’-trimethoxyisoflavone by larvae of S. litura.

The study of the metabolism of plant compounds by Lepidoptera, besides generating

information that can help understand the feeding habits of insects and how they overcome
plants’ chemical defense mechanisms, can also help in the search for new secondary
metabolites with potential as drugs or agricultural chemicals. Thus, this study contributes to
the discovery of new natural bioactive compounds that can be used as alternative methods to
control insect pests.
In closing, during 400 million years of coevolution with plants, phytophagous insects
have developed diverse resistance mechanisms to cope with plant chemical defenses, and
even though many researchers have studied plant–insect interactions, our knowledge is still
limited. Elucidation of desintoxication mechanisms and reactions is important to understand
the adaptations of insects to certain plant species or groups, in particular the second largest
order of insects: the Lepidoptera.

REFERENCES
Borba, E. L., Trigo, J. R., Semir. 2001. Variation of diastereoisomeric pyrrolizidine alkaloids
in Pleurothallis (Orchidaceae). J. Biochem. Syst. Ecol., 29, 45.
Cade, J. W., Rubira, RJ. 1982 Cyanide poisoning of livestock by forage sorghums. Agnote,
Government of Victoria, Department of Agriculture, Australia.
Chauret, D C. C. B., Bernard, J. T. Arnason & T. Durst. 1996. Insecticidal neolignans from
Piper decurrens. Journal of Natural Products 59: 152–158.
Cheeke, P. R. 199), Endogenous toxins and mycotoxin in forage grasses and their effects on
livestock. J. Ani. Sci., 73:909-918.
 Biotransformation of Secondary Plant Metabolites by Lepidoptera 215

Passreiter, C. M., Wilson, J., Andersen, R., Isman, M. B. 2004. Metabolism of Thymol and
trans-Anethole in Larvae of Spodoptera litura and Trichoplusia ni (Lepidoptera:
Noctuidae) J. Agric. Food Chem., 52-2549-2551
Dyer, L. A., C. D., Dodson, J. Richards. 2004. Isolation, synthesis, and evolutionary ecology
of Piper amides. Pages 117–139. In: Dyer, L.A. & A.N. Palmer (eds.). Piper. A model
genus for studies of evolution, chemical ecology, and trophic interactions. Kluwer
Academic Publishers, Boston.
Eisler, R. 1991. Cyanide hazards to fish, wildlife, and invertebrates: A synoptic review.
Biological Report 85:1.23. Contaminant Hazard Reviews Report 23. U.S. Department of
the Interior, Fish and Wildlife Service, Washington, DC.
Engler, H.S., Spencer, K.C., Gilbert, L.E., 2000. Insect metabolism—preventing cyanide
release fromleaves. Nature 406:144–145.
Ferry, N., Edwards, M. G., Gatehouse JA, Gatehouse AMR. 2004. Plant–insect interaction:
molecular approaches to insect resistance. In: Sasaki T, Christou P, editors. Current
Opinion in Biotechnology. 15:155–161.
Feyereisen, R., 1999. Insect P450 enzymes. Ann. Rev. Entomology 44:507–533.
Figueiredo, R. A. & M. Sazima. 2000. Pollination biology of Piperaceae species in
southeastern Brazil. Annals of Botany 85:455–460.
Hartmann, T. In Herbivores. Their Interactions with Secondary Plant Metabolites. Vol. I.
Chemical Participants; Rosenthal, G. A.; Berenbaum, M. R., Eds.; Academic Press, San
Diego, CA, 1991, p. 79.
Hartmann, T., Witte, L. In Alkaloids: Chemical and Biological Perspectives; Vol. 9. Pelletier,
S.W. Ed.; Pergamon Press, Oxford, 1995, p. 155.
Hummelbrunner, L., Isman, M. B., 2001Acute, sublethal, antifeedant, and synergistic effects
of monoterpenoid essential oil compounds on the tobacco cutworm, Spodoptera litura
(Lep., Noctuidae). J. Agric. Food Chem. 49:715-720.
Francisco, I. A. 2000. Cyanogenic Glycosides in [Link], M. H. P., Brazilian Archives
of Biology and Technology. 43: 487-492
Jaramillo, M. A., Manos, P. S., Zimmer, E. A. 2004. Phylogenetic relationships of the
perianthless piperales: reconstructing the evolution of floral development. International
Journal of Plant Science 165:403–416.
Katharina Schramma, Daniel Giddings Vassão, Michael Reichelt, Jonathan Gershenzon, Ute
Wittstock. 2012. Metabolism of glucosinolate-derived isothiocyanates to glutathione
conjugates in generalist lepidopteran herbivores. Insect Biochemistry and Molecular
Biology 42:174-182
Takahashi, K., Araki H., Miyazawa M. 2006. Biotransformation of Isoflavones by the Larvae
of the Common Cutworm (Spodoptera litura). Chem. Pharm. Bull. 54:719—721.
Mattocks, A. R. Chemistry and Toxicology of Pyrrolizidine Alkaloids; Academic Press,
London, 1986.
Mazid, M., Khan, T. A., Mohammad, F. 2011. Role of secondary metabolites in defense
mechanisms of plants Biology and Medicine, 2: 232-249.
McMahon, J. M., White, W. L. B., Sayre, R. T. 1995. Cyanogenesis in cassava (Manihot
esculenta Crantz).J. Exp. Bot., 46:731-741
Zagrobelnya, M., Baka, S., Rasmussena, A. V., Jørgensenb, B. Naumannc, C. M., Møllera, B.
L. 2004. Cyanogenic glucosides and plant–insect interactions. Phytochemistry 65:293–
306
216 Clécio Souza Ramos

Mitsuo Miyazawa, Yohei Miyamoto; Biotransformation of (+)-(1R)- and (−)-(1S)-fenchone

by the larvae of common cutworm (Spodoptera litura). 2005. Journal of Molecular
Catalysis B: Enzymatic .32:123–130
Navickiene, H. M. D., Alécio A. C., Kato, M. J., Bolzani, V. S., Young, M. C. A. J.
Cavalheiro & M. Furlan. 2000. Antifungal mides from Piper hispidum and Piper
tuberculatum. Phytochemistry 6:621–626.
Park, I. K. Lee, G. S.. Shin, S. Park, J., Ahn, J. Y. 2002. Larvicidal activity of isobutylamides
identified in Piper nigum fruits gainst three mosquito species. Journal of Agricultural
and Food Chemistry 50:1866–1870.
Parmar, V. S., Jain, S. C. Bisht, K. S., Jain, R., Taneja, P., Jha, A., Tyagi, O. D., Prasad, A. K.
Wengel, J., Olsen C. E., Boll, P. M.. 1997. Phytochemistry of the genus Piper.
Phytochemistry 47: 597–673.
Paula, V. F., Barbosa, L. C. D., Demuner, A. J., Piló-Veloso D., Picanço, M. C. 2000.
Synthesis and activity of new amide derivative of piperine. Pest and Management
Science 56:168–174.
Ramos, C. S., Souza, L. J., Kato, M. J., Batista, R. 2012. Biotransformation of 4-
nerolidylcatechol by Heraclides brasiliensis (Lepidoptera: Papilionidae) reduces the
toxicity of Piper umbellata (Piperaceae). Chemoecology 19:73-80
Ramos, C. S., Kato, M. J., 2009. Hydrolysis of methyl benzoate from Piper arboreum by
Naupactus bipes beetle. J. Brazil. Chem. Soc. 20:560-563.
Ramos, C. S., Vanin, S. A., Kato, M. J. 2008. Metabolism of (-)-grandisin from Piper
solmsianum in Coleoptera and Lepidoptera species. Phytochemistry 69:2157–2161.
Ramos, C. S., Vanin, S. A., Kato, M. J. 2009. Sequestration of prenylated benzoic acid and
chromenes by Naupactus bipes (Coleoptera: Curculionidae) feeding on Piper
gaudichaudianum (Piperaceae). Chemoecology 19:73-80
Ramos CS, Ecologia química de insetos e espécies de Piperaceae. 2006. Thesis, São Paulo-
SP, Brasil.
Ramos CS, Paz AJLN, Silva TMG, Silva TMS. 2012A. Potentialization of antioxidant
activity of Piper umbellata (Piperaceae) leaves after their metabolism in Heraclides
brasiliensis larvae (Lepidoptera: Papilionidae). African Journal of Pharmacy and
Pharmacology. Accepted for publication.
Schoonhoven, L. M., van Loon, J. J. A., Dicke, M. Insect-Plant Biology, 2º edt, Oxford, New
York, 2005.
Schramma, K., Vassão, D. G, Reichelt, M., Gershenzon, J., Wittstock, U. 2012. Metabolism
of glucosinolate-derived isothiocyanates to glutathione conjugates in generalist
lepidopteran herbivores. Insect Biochemistry and Molecular Biology. 42: 174-182
Packiam, S. M., Ignacimuthu, S. 2012. Effect of PONNEEM on Spodoptera litura (Fab.) and
Its Compatibility with Trichogramma chilonis Ishii Brazilian Archives Of Biology And
Technology. 55: 291-298 .
Hartmann, T. 2004. Plant-derived secondary metabolites as defensive chemicals in
herbivorous insects: a case study in chemical ecology. Planta 219:1–4.
Yuncker, T. G. 1973. The Piperaceae of Brazil II: Piper – Group V; Ottonia; Pothomorphe;
Sarcorhachis. Hoehnea 3: 29–284.
Yuncker, T. G. 1975. The Piperaceae of Brazil V: Index. Hoehnea 5: 123–145.
 INDEX

allergic reaction, 170

# allergic rhinitis, 170
amino, 5, 10, 14, 15, 16, 17, 19, 25, 28, 43, 69, 101,
20th century, 50
124, 125, 127, 128, 205, 208
amino acid, 5, 10, 14, 15, 16, 17, 19, 25, 28, 43, 69,
A 124, 205, 208
amplitude, 59, 60
access, 57, 161 angiosperm, 118
accounting, 52 animal behavior, 105, 114
acetone, 18 ANOVA, 16
acetylcholine, 24, 101 antibody, 132, 135
acetylcholinesterase, 35 antigen, 42
acid, xi, 10, 14, 17, 25, 101, 125, 126, 127, 130, 139, antigenicity, 122
140, 141, 157, 177, 203, 205, 207, 209, 213, 216 antihistamines, x, 169, 170
acidity, 156 antioxidant, xi, 203, 209, 210, 212, 213, 216
action potential, 108 antivenom, 170, 180
active site, 25, 26, 28, 127 apoptosis, 5, 178, 185
acute renal failure, 171, 173, 181, 183 apples, 165
adaptation, 105, 206 Arabidopsis thaliana, 209
adaptations, 41, 214 Argentina, 171
adhesion, ix, 119, 120, 122, 123, 125, 136, 140, 177, arginine, 180
178, 184 Aristotle, 45
adhesion properties, 140 arrest, vii, 1, 2, 5, 9, 14, 42, 54
adults, xi, 9, 12, 15, 33, 36, 54, 137, 151, 152, 154, Artemia, 210
155, 157, 159, 161, 176, 187, 188, 193, 197, 210 arthritis, 171
adverse conditions, 2, 3, 4 arthropods, 17, 24, 71, 80, 84, 143, 155, 197
adverse effects, x, 68, 169, 171 articulation, 175
Africa, x, 3, 50, 55, 80, 83, 149 Asia, 50, 56, 190
age, 4, 172 asparagus, 55
agglutination, 126 asphyxia, 123
aggregation, 4, 37, 60, 83, 89, 90, 99, 100, 104, 108, assessment, 34, 71, 182
111, 113, 116, 123, 126, 129, 136, 140, 158, 184 assessment procedures, 71
agonist, 27, 30, 34, 75 asthma, 170
agriculture, viii, 45, 46, 48, 53, 76, 79, 80, 82, 86, atmosphere, 105
170 attachment, 135
alanine, 26 attractant, 112, 114, 116, 165
alfalfa, 85 Austria, 150
alien species, x, 187, 188 authorities, 78, 79
alkaloids, xii, 204, 205, 206, 214 avian, 194, 195, 201
218 Index

avoidance, 90, 118 brain tumor, 36

awareness, 78 Brazil, 169, 171, 172, 173, 174, 180, 182, 183, 203,
axons, 101, 102 209, 215, 216
breakdown, 104
breeding, 69, 83
B by-products, 53, 137

Bacillus subtilis, 123

background noise, 101, 103, 104 C
bacteria, 57, 121, 122, 123, 124, 125, 126, 127, 129,
130, 131, 132, 136, 139, 140, 141, 143, 144, 146, cabbage, 29, 104, 107, 114
147, 148, 156, 157, 178 cacao, 192, 200
bacterial infection, 123, 125, 127, 131, 137, 142, 148 cadmium, 36
bacteriophage, 124 calcium, 125, 178
bacterium, 121, 137 calyx, ix, 88
Bangladesh, 86 Canary Islands, 55
banks, 65 candidates, 116
barriers, ix, xi, xii, 64, 70, 119, 121, 203, 204 capsule, 4, 123, 136, 162, 164
base, 46, 54, 64, 80, 165, 175, 188, 206 carbohydrate, 125, 126, 141
beetles, 65, 70, 94, 96, 99, 100, 103, 104, 107, 111, carbon, 194
116, 118, 193 carboxyl, 28, 125
behaviors, ix, 88, 89, 93, 99, 105, 107, 109 carboxylic acid, 211
benefits, 66, 89 cardenolides, xii, 204
bias, 60 Caribbean, 79
bioassay, 99, 210 carnivores, 100
biochemistry, 34, 37, 43, 170 cascades, 124, 127, 129, 132, 138
biodiversity, 71, 197 case studies, 116
bioenergy, 83 case study, 216
biogas, 49 cash, 65
biological activities, 123, 212 cash crops, 65
biological activity, 209, 210 casting, 93, 110
biological control, viii, 45, 57, 68, 69, 85, 91, 113, catabolism, 30, 104
158, 198 cDNA, 5, 10, 11, 14, 17, 21, 23, 25, 26, 35, 36, 37,
biological media, 76 38, 41, 42, 43, 44, 138, 139, 145, 147, 171
biological processes, 5 cell culture, 178
biological sciences, 47 cell cycle, x, 9, 13, 42, 169, 177
biomolecules, 24 cell death, 32, 178, 185
biosensors, 97 cell differentiation, 5
biosynthesis, vii, 2, 16, 20, 22, 24, 41, 74, 121, 205 cell division, 2
biotechnology, 69, 83 cell line, 37, 135
biotic, 80, 154, 155, 156, 188, 197 cell membranes, 129
biotic factor, 154, 155, 156 cellular responses, ix, 119, 120, 121, 122, 127, 129
birds, xi, 155, 187, 194, 196 cellulose, 52
bisphenol, 27, 30, 34, 41 census, 47
bisphenol A (BPA), 28 central nervous system (CNS), 89, 97, 103, 105, 108,
bleeding, 122, 171, 172, 182 173
blends, 90, 96, 99, 103, 104, 105 challenges, 5
blood, 121, 135, 138, 171, 172, 173, 176, 177, 178, channel blocker, 73
179, 181, 183, 184, 206 chaperones, 4, 5, 13, 14, 32, 33, 35, 39
blood clot, 173, 177, 183 chemical, viii, x, xi, xii, 17, 30, 45, 48, 50, 53, 57,
bloodstream, x, 169 58, 64, 66, 67, 69, 71, 72, 75, 76, 77, 78, 85, 86,
body weight, 4, 172 88, 89, 93, 97, 101, 103, 105, 109, 110, 112, 114,
bonds, 130 115, 116, 149, 157, 158, 159, 198, 203, 204, 205,
brain, ix, 13, 36, 42, 88, 100, 101, 104, 113, 117 206, 207, 214, 215, 216
 Index 219

chemical characteristics, 17 complexity, 138

chemical industry, 48 complications, 173
chemical interaction, xii, 204 composition, viii, 15, 16, 45, 46, 84, 90, 91, 99, 107,
chemical properties, 71 143, 150
chemical structures, 67, 71 compounds, viii, xi, xii, 38, 45, 57, 66, 67, 71, 72,
chemicals, xi, xii, 68, 88, 89, 90, 127, 158, 203, 204, 75, 77, 89, 97, 99, 100, 101, 103, 104, 105, 108,
214, 216 112, 113, 143, 158, 164, 203, 204, 205, 206, 207,
chemotaxis, 108 208, 209, 210, 211, 212, 213, 214, 215
chicken, 129 conceptual model, 92
children, 120, 180 condensation, 155
Chile, 150 conduction, 157
China, 50, 53, 170, 180, 188, 190, 212 congress, 42, 81
chinese medicine, 170 conifer, 111, 118
chitin, 74, 121 conjugated dienes, 198
chloroplast, 205 conjunctivitis, 170
cholinesterase, 28 connectivity, 116
chorion, 152 consensus, 25, 26, 28, 33
chromatography, 89, 97, 112 conservation, 34
chromosome, 52 constituents, xii, 198, 204
chymotrypsin, 127 construction, 58
city, 119, 187 consumers, 79
classes, 4, 24, 26, 49, 71, 135, 136 consumption, 50, 76, 170, 171, 177, 178, 179, 180,
classification, vii, 57, 71, 72, 73, 74, 75, 122, 180, 198
190 consumption coagulopathy, 170, 171, 177, 178, 179
clean air, ix, 87, 92, 93 contaminant, 111
cleavage, 36, 127, 128, 178, 211 control measures, 56, 64, 65, 163
climate, 155, 156, 161, 195, 196 controversial, 212
climates, 49 convergence, 132
clinical symptoms, 176 cooperation, 130
cloning, 10, 25, 35, 36, 37, 38, 39, 41, 43, 138, 139, copper, 17, 49
140, 141, 143, 144, 145, 147 copulation, 188, 189
clusters, 54, 154, 156, 157, 159, 175, 190 correlation, 29, 47, 106, 210
coagulopathy, 172 correlations, 105
cocoon, 120, 153, 170, 171, 180, 189, 190, 192, 194, cosmetics, 52
195, 196, 200 cost, 62, 63, 64, 69, 157
coding, ix, 88, 109 cotton, viii, 42, 45, 48, 52, 54, 55, 59, 63, 64, 65, 69,
codon, 5, 10, 14, 17 76, 84, 85, 141, 212
coffee, 192 covering, 46, 120, 123
collagen, 126, 143, 178 crabs, 117
collateral, 157 cracks, 54
Colombia, 171, 183 critical density, 62
colonization, 111 crop, vii, viii, 45, 46, 47, 49, 50, 52, 53, 56, 57, 58,
color, 54, 153, 175, 176, 193 59, 60, 62, 63, 64, 65, 67, 68, 69, 72, 75, 76, 77,
commercial, 52, 67, 69, 170 78, 79, 80, 81, 83, 109, 158
commodity, 62, 63, 64 crop production, viii, 45, 46, 56, 57
common symptoms, 170 crop residue, 65
communication, 57, 67, 86, 90, 91, 111, 116, 117, crop rotations, 64
158, 182 crops, viii, 45, 46, 49, 53, 54, 55, 56, 57, 58, 62, 65,
communities, 65, 76 66, 67, 68, 69, 71, 76, 78, 83, 118, 120, 188, 193,
comparative analysis, 141, 145 212
compatibility, viii, 46, 57, 71, 72, 73, 74, 75 cues, vii, 1, 15, 93, 100, 108, 111, 112, 117
competition, 79, 85, 155, 197 cultivars, 50
complement, 141 cultivation, viii, 45, 50, 53, 57, 58, 64, 65, 68, 70, 80
220 Index

cultural practices, 64, 157 digestive enzymes, 121

culture, 27, 82, 159, 185 directives, viii, 46, 49
culture medium, 185 discrimination, viii, 57, 87, 90, 95, 96, 104, 107, 113
cumulative distribution function, 62 diseases, 50, 70, 156, 195, 196
cumulative percentage, 162 disorder, 182
cuticle, 16, 121, 124, 175 dispersion, ix, 4, 60, 87, 89, 91, 92, 93, 105, 112,
cyanide, 207, 215 116, 117, 158
cycles, 29, 31, 41 displacement, 89
cycling, 65 disseminated intravascular coagulation, 171, 172,
Cyprus, 55, 167 173, 177
cysteine, 25, 130, 147, 208 distribution, x, 46, 54, 56, 60, 67, 68, 83, 89, 92, 94,
cytochrome, 205, 207 105, 150, 162, 169, 171, 173, 179, 182, 187, 191,
cytokines, 123 199, 201
cytometry, 122, 133 diverse environmental cues, vii, 1
cytoplasm, 205 diversity, viii, xi, 24, 45, 46, 65, 98, 121, 145, 176,
200, 204
dizziness, 172, 173
D dopamine, 124, 127, 128
dosage, 116
damages, 154, 156, 158, 159, 179, 200
drawing, 102
danaid butterflies, xii, 204
drinking water, 78, 155
danger, viii, 87, 143, 164
drosophila, 5, 12, 14, 32, 33, 36, 37, 40, 42, 43, 100,
database, 10
102, 111, 113, 116, 123, 130, 131, 132, 135, 136,
deaths, 172, 173
137, 138, 143, 147, 148, 157
defence, 5, 135, 156
drugs, xi, 177, 203, 214
defense mechanisms, xi, 126, 203, 214, 215
dysphagia, 170
deficiency, 124, 130, 131
deforestation, 174
degradation, 13, 39, 131, 133, 176, 177 E
demographic change, 47
dendrites, 91, 101, 102 ecology, vii, x, xii, 56, 82, 83, 86, 115, 187, 188,
Department of Agriculture, 214 196, 197, 198, 204, 215, 216
deposition, 189 economic injury levels, viii, 45, 86
deposits, 55 economics, 62
deprivation, 4, 178 ecosystem, 46, 76, 162, 197
derivatives, 211 ecotoxicological, 55
dermatitis, xi, 170, 180, 187, 190, 198, 199 edema, 170, 173, 177
desiccation, 4, 5 editors, 112, 215
destruction, 156, 157, 173 egg, x, 32, 54, 149, 151, 152, 154, 155, 162, 169,
detectable, 3, 17, 97 175, 189, 193, 194, 195, 197, 211
detection, 89, 90, 91, 95, 101, 103, 104, 105, 108, Egypt, 50
138, 147, 153 elastomers, 49
detection system, 108 electron, 75, 199, 207
detergents, 52, 71 elucidation, 145, 209
detoxification, xi, xii, 126, 142, 203, 204, 205, 208 embryogenesis, 126
developed countries, 53, 76 emergency, 60, 62
developing countries, 48, 49, 50, 51, 53, 83 employment, 82
developmental process, vii, viii, 1, 2, 15 enantiomers, 213
dew, 155 encapsulation, ix, 119, 120, 121, 122, 123, 127, 136,
diet, xi, xii, 4, 203, 204, 208, 211 137, 142
dietary fiber, 49 encoding, vii, 2, 5, 6, 23, 27, 33, 35, 38, 39, 41, 42,
differential equations, 162 90, 137, 145, 146, 147
diffusion, ix, 87, 91, 93, 115, 206 endocrine, 13, 19, 22, 27, 28, 38
digestion, 198, 206 endothelial cells, 184, 185
 Index 221

endotoxins, 69 fat, 15, 16, 17, 18, 20, 21, 23, 25, 35, 39, 40, 123,
enemies, 67, 68, 69, 71, 75, 90, 159 124, 125, 127, 129, 130, 132, 147
energy, viii, 45, 46, 48, 65, 77 fauna, 157, 197
energy input, viii, 45, 48, 77 feces, 101, 210
engineering, 69 fertility, 76
England, 80, 163 fibers, 49
enlargement, 46 fibrin, 173, 177, 184
environment, 49, 56, 64, 67, 71, 76, 78, 88, 90, 91, fibrinogen, 173, 177, 179, 184
93, 97, 98, 100, 101, 105, 109, 115 fibrinolysis, 170, 179
environmental change, 85, 173 fibrinolytic, 173, 177, 180, 184
environmental characteristics, 48 fibroblasts, 184
environmental conditions, 2, 13, 67 field crops, 80
environmental degradation, 48 field tests, 84, 110
environmental impact, viii, 45, 85 filament, ix, 87, 93, 94
environmental stress, 36, 65 fine tuning, 110
environments, 76, 104, 109, 197 first generation, 154, 156, 157, 159, 192, 194, 195
enzyme, 26, 30, 31, 90, 124, 125, 128, 138, 139, 143, fish, 75, 215
177, 178, 185, 207, 208 Fish and Wildlife Service, 215
enzymes, 24, 38, 105, 108, 121, 205, 207, 215 flame, 97
epidemiology, 180 flavonoids, 213
epidermis, 132, 190 flight, x, 58, 60, 85, 86, 93, 95, 99, 105, 106, 110,
epithelium, 121, 175 111, 114, 117, 118, 126, 149, 155, 159, 161, 162,
equipment, 57, 65, 78 164
ester, 24, 30 flights, 54, 159
ester bonds, 24 flora, 197
ethology, 154 flour, 38
eukaryotic, 5 fluctuations, 60, 92, 93, 94, 96, 97, 105, 106, 112,
Europe, x, 50, 54, 56, 71, 80, 81, 83, 149, 150 113, 114, 200
European Commission, 78 fluid, 91, 92, 93, 109, 113
European Parliament, viii, 46 folding intermediates, 34
European Union (EU), 49, 50, 71, 78, 79, 82, 85 food, xi, xii, 46, 48, 49, 50, 53, 65, 76, 86, 90, 96,
evagination, 175 100, 120, 121, 134, 159, 194, 203, 204, 207
evidence, xi, 14, 36, 43, 76, 113, 158, 194, 203, 204 food additive, 49
evolution, xi, 14, 16, 24, 32, 33, 39, 40, 41, 42, 60, food production, 86
145, 176, 203, 204, 215 food web, 65
exaggeration, 48 forbs, 198
examinations, 191 forecasting, 78, 81, 162, 164
exploitation, xi, xii, 49, 203, 204 forecasting model, 78
exporter, 50 formation, ix, 3, 43, 119, 120, 122, 123, 126, 127,
exposure, 12, 13, 76, 95, 110, 121, 124, 133, 135, 129, 142, 161, 173, 177, 208
164, 180 formula, 157
expressed sequence tag (EST), 138, 139 fragments, 10, 126, 178
extracellular matrix, 178, 184 France, 35, 38, 50, 55, 79, 81, 150, 161, 162, 182
extraction, 19, 82, 157 frequency distribution, 164
extracts, 194, 210 fruits, 81, 216
FTICR, 143
functional analysis, 144, 148
F fungal infection, 147
fungi, xi, 36, 57, 69, 74, 91, 112, 115, 121, 126, 127,
factories, 50
129, 130, 139, 146, 154, 156, 157, 187, 193
families, 5, 9, 14, 15, 36, 40, 55, 104, 140, 151, 157,
fungus, 145, 165
170, 171, 179, 192, 206, 208
furan, 211
family members, 178
fusion, 108
farmers, 48, 56, 58, 64, 85
222 Index

harvesting, 57, 65
G hazards, 90, 215
headache, 172, 173
GABA, 72, 101
health, 64, 90, 172, 212
gel, 21
heat shock protein, vii, 1, 2, 3, 5, 9, 12, 13, 14, 32,
gene amplification, 42
36, 37, 38, 39, 40, 41, 43
gene expression, 2, 3, 17, 20, 21, 27, 29, 30, 31, 37,
height, ix, 87, 93, 98, 159, 175
42, 43, 125, 138, 140, 182
hematemesis, 173
gene regulation, 145
hematomas, 173
genes, vii, viii, 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13,
hematuria, 171, 173
14, 15, 18, 19, 20, 22, 23, 24, 27, 29, 31, 32, 33,
hemisphere, 71, 150, 154, 157, 198
34, 35, 36, 38, 40, 41, 69, 123, 126, 129, 132,
hemorrhage, 173
134, 138, 139, 140, 145, 146, 147, 148
hemostasis, 138, 173, 185
genetics, 36, 39, 134
hemostatic system, x, 169
genome, 86, 127, 129, 132, 134, 140, 145
histamine, 190
genomics, 39, 133, 134
history, 47, 188, 196
genre, 41
homogeneity, 65
genus, 52, 120, 170, 171, 179, 181, 182, 183, 209,
hormonal control, 33
215, 216
hormone, vii, 1, 2, 3, 13, 15, 16, 17, 18, 19, 22, 23,
Germany, 50, 102, 150
24, 25, 27, 28, 31, 32, 33, 34, 35, 36, 37, 38, 39,
gingival, 173
40, 41, 42, 43, 74
GIS, 82
hormones, 4, 13, 22, 27, 34, 35, 36, 205
gland, 41, 94, 158
host, ix, xi, xii, 55, 61, 62, 65, 68, 88, 89, 90, 91, 95,
global warming, 154, 191
96, 98, 99, 100, 103, 104, 108, 109, 110, 111,
globalization, 188
112, 113, 116, 117, 118, 126, 135, 136, 142, 150,
glucagon, 128
152, 153, 154, 175, 182, 185, 188, 190, 192, 193,
glucoside, 213
195, 196, 200, 201, 203, 204, 206, 207
glucosinolates, 104, 208
house, 199
glutathione, 208, 215, 216
human, x, 76, 80, 82, 144, 169, 170, 171, 173, 174,
glycine, 130, 209
176, 177, 178, 179, 181, 184, 190
glycoside, 207, 208
human capital, 82
glycosylation, 212
human health, 171
grants, 134, 179
human skin, x, 169, 176
graph, 155
humidity, x, 149, 151, 154, 155, 156, 157, 162
grasses, 52, 65, 214
humoral responses, ix, 119, 120, 122
Greece, 1, 3, 4, 45, 55, 83, 162, 164
Hunter, 85
green revolution, 46, 48, 82
hyaline, 122
greenhouse, 81
hybridization, 138
groundwater, 78
hydrocarbons, 67
growth, x, 2, 3, 24, 34, 36, 58, 61, 71, 75, 77, 130,
hydrogen, 207
136, 149, 151, 153, 154, 178, 185, 195
hydrogen cyanide, 207
growth rate, x, 149
hydrolysis, 207
Guatemala, 80
hypersensitivity, 170
guidance, 71, 118
hypertension, 170
guidelines, 64
hypothesis, 18, 104, 105
Guyana, 171, 180, 182

I
H
ICAM, 178
habitat, 60, 65, 82, 110, 116, 121, 154, 196
ice pack, 170
habitats, vii, 96, 98, 188, 191, 194, 196
ideal, 155, 173
hair, 90, 91
identification, 42, 46, 80, 82, 84, 97, 110, 117, 127,
harmful effects, xi, xii, 127, 203, 204
135, 136, 143, 182, 201
 Index 223

identity, 17, 25, 103, 109 96, 97, 98, 100, 102, 104, 105, 106, 107, 108,
immune function, x, 119, 120, 141 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
immune response, vii, 121, 122, 123, 124, 129, 130, 119, 120, 121, 122, 123, 124, 125, 126, 127, 129,
133, 134, 135, 136, 137, 138, 141, 142, 143, 144, 130, 133, 134, 135, 136, 137, 139, 140, 142, 143,
145, 147, 148 147, 148, 155, 156, 158, 160, 178, 188, 197, 199,
immune system, ix, 119, 120, 121, 126, 127, 130, 203, 204, 205, 206, 207, 208, 209, 212, 214, 216
135, 137, 142 insertion, 146
immunity, 120, 124, 130, 133, 134, 135, 137, 138, inspections, 159
139, 141, 143, 144, 145, 146, 147 institutions, 55
immunogenicity, 171 insulin, 3
immunoglobulin, 125, 140, 141 insulin signaling, 3
immunoglobulin superfamily, 140 integration, 48, 49, 68, 85, 86, 93
immunoprecipitation, 132 integrin, 137
impregnation, 158 integrins, 123, 126
improvements, 159, 188 integrity, 76, 114
in vitro, 39, 136, 143, 144, 145, 171, 177 integument, 24, 40, 121, 134, 194
in vivo, 40, 171, 177, 185 interface, 42
incidence, 60, 173, 179 interference, 135, 146, 197
income, 49, 82 interneurons, 110
incubation period, 175 intervention, 58, 59, 60, 165, 173
India, 188, 190, 192, 193, 195, 196, 197, 212 intracerebral bleed, 171
individuality, 204 intracerebral hemorrhage, 170, 173, 181
individuals, 3, 12, 17, 20, 60, 66, 67, 170, 191, 195 intron, 140
Indonesia, 190, 193, 195, 196, 198 invasions, 200
inducer, 27, 112, 133 invertebrates, 28, 121, 129, 143, 215
inducible protein, 139, 143 investment, 50
induction, x, 2, 7, 13, 21, 27, 34, 36, 37, 38, 41, 132, ionization, ix, 88, 90
138, 140, 142, 149, 151 ionized air, 97
industrialization, 76 Iran, 55
industry, 79, 170 Ireland, 35, 37
infection, ix, 65, 119, 120, 121, 122, 125, 126, 127, iron, 49
129, 131, 135, 145, 147, 148, 154, 156, 159, 195 irrigation, 46, 64, 157
infectious agents, ix, 119, 121 Islam, 86, 189, 198
infestations, x, 62, 65, 187, 192 islands, 191
inflammation, 124, 171, 177, 178, 184 isoflavone, 213, 214
infrastructure, 46 isolation, 17, 25, 100
ingest, 122 Israel, 55, 83, 112
ingestion, 39, 170, 180 issues, 3, 196
ingredients, 78 Italy, 50, 55, 120, 150, 162
inhibition, 24, 43, 126, 145, 154
inhibitor, 127, 131, 133, 143, 144
initiation, vii, 2, 5, 7, 8, 10, 16, 17, 18, 19, 22, 24, J
135, 139
Japan, v, x, 187, 188, 190, 191, 192, 193, 194, 195,
injuries, 170, 172, 204, 207
196, 197, 198, 199, 200, 201, 212
injury, viii, 45, 59, 60, 62, 63, 64, 83, 86, 176
joints, 171
innate immunity, 119, 121, 124, 133, 137, 138, 139,
140, 143
inoculation, 69 K
insecticide, xi, 40, 42, 48, 54, 55, 67, 82, 159, 203,
209 Kenya, 82
insects, vii, viii, ix, xi, xii, 1, 4, 5, 7, 8, 9, 13, 14, 15, kill, xi, 67, 187, 195
16, 17, 18, 22, 24, 26, 27, 28, 29, 33, 41, 42, 49, kinetics, 145
53, 65, 66, 69, 70, 75, 87, 89, 90, 91, 92, 94, 95, krypton, 97
224 Index

lysine, 205
L lysis, 177
lysozyme, 121, 129, 145
labeling, 158
landscape, viii, 46, 56, 87, 92, 96, 100, 109, 111, 162
landscapes, 65, 109, 114, 198 M
larva, x, 4, 5, 12, 31, 34, 41, 62, 101, 144, 152, 153,
154, 157, 169, 170, 176, 189 machinery, 78
larvae, vii, viii, ix, x, xi, xii, 1, 2, 3, 4, 6, 7, 8, 9, 10, macromolecules, 15, 16
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, magnitude, 95, 96
24, 25, 27, 29, 30, 31, 33, 34, 35, 37, 38, 39, 41, majority, x, 24, 50, 169, 204
43, 54, 59, 63, 70, 71, 80, 100, 119, 120, 121, Malaysia, 190
123, 124, 125, 126, 127, 132, 133, 135, 137, 142, mammals, 3, 76, 121, 124, 135, 142, 178
145, 147, 153, 154, 155, 157, 159, 161, 162, 164, man, 193, 196
165, 166, 169, 170, 171, 173, 174, 175, 179, 180, management, vii, viii, 13, 45, 48, 49, 55, 56, 57, 58,
187, 189, 191, 192, 193, 194, 195, 196, 198, 203, 59, 60, 62, 63, 64, 65, 67, 69, 71, 76, 77, 78, 79,
204, 207, 208, 209, 210, 211, 212, 213, 214, 216 80, 81, 82, 83, 84, 91, 112, 117, 118, 180, 197
larval development, 15, 25, 43, 122 mandible, 120
larval diapause, vii, 2, 3, 7, 9, 10, 12, 20, 24, 32, 33, manganese, 49
39, 42 manipulation, 69
larval stages, x, 4, 18, 23, 149, 151, 157, 161 masking, 104
Latin America, 50, 79, 80 mass, 4, 17, 49, 89, 90, 91, 92, 97, 100, 112, 158
lead, vii, ix, x, 2, 5, 20, 24, 50, 119, 120, 154, 157, mass spectrometry, 89, 92, 97
169, 170, 171, 172, 173, 177 matrix, 121, 134, 135
Leahy, 199 matter, 116
learning, 117 measurement, 97, 115, 116
legislation, 78, 79 measurements, 110, 112, 113, 115
legs, 176 media, 42, 54, 89, 93, 188
Lepidoptera, v, vii, viii, x, xi, xii, 1, 3, 5, 34, 35, 36, medical, 49, 170, 179, 180, 197
38, 45, 53, 54, 55, 56, 58, 60, 67, 74, 79, 80, 81, Mediterranean, 3, 34, 49, 54, 55, 82, 162
82, 83, 84, 86, 106, 109, 110, 112, 113, 114, 117, Mediterranean countries, 3
119, 120, 122, 124, 125, 126, 127, 128, 130, 132, melanin, 123
133, 134, 139, 145, 146, 150, 162, 163, 164, 165, melanization, ix, 119, 120, 126, 127, 128, 137, 138,
166, 167, 169, 170, 179, 180, 183, 184, 185, 187, 142, 143
188, 194, 197, 198, 199, 200, 201, 203, 204, 205, melon, 55
206, 209, 213, 214, 215, 216 membranes, 124
lesions, 170, 173, 174 messages, 116
leukocytes, 121, 124 metabolic, 32
life cycle, xi, 32, 49, 55, 56, 65, 70, 150, 153, 154, metabolism, xi, xii, 2, 24, 32, 203, 204, 205, 208,
187, 188, 198 211, 212, 214, 215, 216
ligand, 132 metabolites, xi, xii, 19, 23, 203, 204, 205, 206, 207,
light, 4, 15, 110, 153, 159, 175, 188 209, 211, 214, 215, 216
lignans, 209, 212 metabolized, 124, 208, 210, 213
linear model, 60 metalloproteinase, 127, 142, 143, 178
lipases, 34 metals, 207
lipids, 124, 126 metamorphosis, 15, 16, 19, 22, 23, 24, 29, 30, 37, 39,
liver, 124, 170 40, 42, 120, 129, 145, 176
livestock, 53, 214 methanol, 210
localization, ix, 2, 88, 101, 104, 144 methionine rich storage proteins family, viii, 2
longevity, 154, 178, 185 methodology, 159
low temperatures, 13 methyl group, 213
lumen, 101 Mexico, 119, 120, 134
Luo, 113 mice, 14
lymph, 91, 101, 121 microbial cells, 126
 Index 225

microorganism, 55, 126, 129, 130, 141 neuronal systems, 28

microorganisms, x, 119, 120, 121, 123, 124, 125, neurons, ix, 88, 90, 99, 101, 102, 103, 104, 108, 110,
128, 132, 133, 134, 139 112, 113, 114, 115
Middle East, 150 neuroscience, 113
migration, 123, 136 neutral, 112, 126
mildew, 50 neutrophils, 184
misuse, 76 Nigeria, 196
mixing, 89 nitric oxide, 137, 178, 185
model system, 13, 143 nitric oxide synthase, 185
modelling, 62, 114 nitrogen, 15, 208
models, vii, viii, x, 45, 46, 47, 49, 53, 61, 64, 65, 68, nodules, 123
75, 77, 79, 81, 97, 105, 120, 149, 159, 160, 162, nondiapausing larval instar, viii, 2
164, 165 non-polar, 212
moderate activity, 210 normal development, 4
modifications, 2, 4, 50, 150 normal distribution, 96
moisture, 79 North America, 56, 116, 185, 197, 198
molecular biology, 37 Norway, 115
molecular mass, 5, 14, 17 nucleotide sequence, 10, 32, 41
molecular weight, 10, 37, 67, 126 nucleotides, 5, 10, 14, 17, 25
molecules, viii, 67, 87, 88, 89, 91, 92, 93, 101, 108, nucleus, 131, 133
123, 124, 125, 127, 129, 130, 134, 147, 177, 178, nutrient, 16, 65, 178
182 nutrients, vii, 1, 3, 121, 126
momentum, 197 nutrition, 80, 90
Morocco, 55
morphogenesis, 2, 4
morphology, 99, 114, 122, 155, 171 O
mortality, 65, 69, 155, 171, 193, 194, 195, 196, 200
odor filaments, ix, 87, 93
mortality rate, 171
odor plumes, viii, 87, 90, 91, 92, 93, 96, 97, 105,
mosaic, 151
108, 111, 113, 114, 117
mosquitoes, 136
OECD, 83
motif, 27, 31, 38, 125, 126
oil, 52, 180, 196, 198, 213, 215
mRNA, viii, 2, 7, 9, 10, 11, 13, 14, 16, 17, 18, 20,
olfaction, 90, 93, 113, 116
21, 22, 23, 25, 26, 27, 28, 29, 31, 36, 44
oogenesis, 38
mRNAs, viii, 2, 6, 7, 10, 15, 18, 19, 21, 23, 25, 27,
opportunities, 109
29, 40
ordinary differential equations, 162
mucous membrane, 171, 173
organ, 59, 60
mutagenesis, 43
organic matter, 65
mutation, 130
organism, 5, 57, 61, 65, 71, 75, 88, 101, 123, 158,
204
N organs, 52, 62, 90, 93, 101, 136, 155, 157
ornamental plants, 55, 188
National Academy of Sciences, 85, 200 ornithine, 205
native species, 199 ovum, 154
natural compound, 30, 31, 206, 212, 213 oxidation, 127
natural enemies, xi, 48, 68, 69, 71, 75, 76, 77, 79, oxygen, 4, 17, 207
117, 150, 155, 157, 159, 187, 188, 193, 196, 197, oyster, 76
198, 205, 206, 208
natural habitats, 197
nausea, 172, 173 P
neolignans, 209, 214
Pacific, 201
nervous system, 89, 97, 103
pain, 170, 172, 177, 199
Netherlands, 42, 85
Pakistan, 81, 109
neuronal sensitivity, 110
226 Index

Paraguay, 171 physical mechanisms, 91

parallel, 3, 79 physical properties, 91
parasites, 57, 123, 165, 167, 173 physical structure, 97
partial thromboplastin time, 173 physics, 97, 105
pastures, 80 physiological, v, 33, 87, 166
pathogenesis, 143, 182 physiological mechanisms, viii, 2
pathogens, 49, 69, 100, 113, 115, 121, 123, 126, 129, physiology, 31, 34, 35, 37, 91, 204
135, 142 pigs, 53
pathology, 171, 173 plants, xi, xii, 24, 36, 52, 54, 55, 60, 66, 68, 69, 80,
pathophysiological, 171, 181 90, 96, 100, 103, 104, 106, 108, 112, 113, 117,
pathophysiology, x, 169, 177 188, 192, 195, 196, 203, 204, 205, 206, 207, 208,
pathways, ix, 2, 3, 119, 120, 124, 130, 131, 132, 136, 214, 215
144 plasma proteins, 124, 125
pattern recognition, ix, 119, 120, 126, 137, 138, 142, plasminogen, 177
147 platelet aggregation, 177
PCR, 6, 8, 10, 11, 12, 16, 18, 19, 20, 21, 22, 23, 25, platelets, 184
26, 29, 30, 31, 140 poison, 190
peptide, 41, 123, 126, 129, 134, 136, 146, 147, 148, Poland, 197
178 polar, xi, xii, 101, 203, 204, 205, 209, 213
peptides, ix, 119, 120, 121, 122, 124, 126, 129, 130, polarity, 212
131, 132, 133, 134, 135, 145, 146, 147 policy, 78, 81
periodicity, 84 pollen, 65, 104
permeability, 130 pollinators, 100
permission, 78 pollution, 90, 112
permit, 65, 79 polycarbonate, 28
Peru, 171 polymorphism, 139
pest populations, x, 56, 65, 69, 90, 149, 165 polypeptide, 24, 130, 131, 132, 133
pest regulation, 77 polypeptides, 5, 17, 38
pesticide, 48, 49, 63, 64, 77, 78, 79, 81 polystyrene, 4
pests, viii, ix, x, 45, 49, 50, 53, 55, 56, 58, 59, 62, 65, population, xi, 3, 35, 47, 58, 59, 60, 61, 62, 64, 65,
68, 69, 76, 77, 78, 81, 83, 85, 86, 90, 91, 99, 111, 69, 77, 80, 82, 84, 86, 91, 96, 102, 113, 115, 116,
119, 120, 149, 154, 159, 187, 198, 199, 214 139, 157, 158, 162, 163, 164, 173, 187, 188, 191,
phagocyte, 124 194, 195, 196, 197, 200
phagocytic cells, 121, 122 population density, 64, 96
phagocytosis, ix, 119, 120, 121, 122, 123, 125, 126, population growth, 69, 195
127, 128, 134, 135, 136, 143 Portugal, 55
pharmacology, 34 positive feedback, 128, 144
phenol, 134, 143, 210 positive interactions, 76
phenolic compounds, 205, 210, 212 potato, viii, 9, 12, 38, 42, 43, 45, 50, 51, 54, 69, 82,
phenotypes, 122 83, 84, 86, 117
phenylalanine, 17, 205 potential benefits, 58
pheromones, 89 poultry, 53
phospholipids, 178 praxis, 56, 60, 81
phosphorous, 195 predation, 65, 85, 96, 101, 194, 196, 199, 200, 201
phosphorus, 49, 97 predators, xi, 57, 65, 70, 71, 80, 91, 100, 155, 187,
phosphorylation, 131 190, 193, 194, 195, 196, 199, 206, 212
photographs, 197 prednisone, 170
photoperiodism, 32 preparation, 2, 109
photosynthesis, 207 prevention, 180
phylogenetic tree, 24, 26 primary olfactory centre, ix, 88, 90, 99, 101
physical characteristics, 92, 98 priming, 121
physical environment, 109 principles, viii, 46, 49, 76, 77, 113, 171
physical interaction, 91, 93 probability, 58, 59, 60, 96, 113
 Index 227

probability density function, 113 receptors, x, 15, 17, 33, 36, 73, 74, 90, 91, 102, 103,
probability distribution, 59 104, 105, 108, 109, 113, 116, 119, 120, 124, 128,
producers, 50, 52, 53 130, 132, 137, 140, 147, 178
programming, vii, 1, 3, 19 recognition, x, 48, 67, 99, 104, 118, 119, 120, 121,
pro-inflammatory, 184 123, 124, 125, 128, 130, 136, 138, 139, 140, 141,
project, 102 147, 148
proliferation, 173, 178 recommendations, 109
proline, 130 reconstruction, 16
propane, 30 recovery, 6, 7, 8, 10, 12, 14, 43, 173
protease inhibitors, 142, 144 redundancy, 110
protection, 3, 48, 50, 58, 60, 64, 71, 72, 78, 80, 81, regions of the world, 150, 188
86, 121, 127, 153, 158, 165, 178 regression, 60, 61, 62, 83
protective role, 129 regression line, 62
protein components, 175 regression model, 61
protein family, 10, 139 rehydration, 36
protein folding, 41, 42 rejection, 89
protein sequence, 14, 36 relative toxicity, 71, 75
protein synthesis, 3, 13, 17, 35, 145 relevance, 38
proteinase, ix, 119, 120, 124, 125, 127, 128, 129, renal failure, 170, 173, 184
131, 132, 133, 139, 142, 143, 144, 145 reproduction, 14, 15, 24, 39, 64, 65, 76, 90, 165, 176
proteins, vii, ix, 1, 2, 3, 4, 5, 9, 13, 15, 17, 18, 20, 22, reproductive organs, 28, 152
24, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, requirements, 62, 81
44, 49, 69, 74, 88, 91, 101, 119, 120, 121, 123, researchers, 3, 17, 19, 22, 161, 214
124, 125, 126, 129, 133, 135, 138, 139, 141, 145, reserves, 32
146, 170, 182, 184 residues, 25, 35, 49, 126, 127, 159
proteolysis, 127 resistance, viii, 3, 4, 40, 45, 48, 55, 57, 68, 69, 76,
proteomics, 133 77, 86, 90, 109, 113, 115, 131, 214, 215
prothrombin, x, 169, 173, 177, 181, 182, 184 resolution, 13, 99, 105, 106, 110, 111, 112, 114, 183
pruning, 157 resources, 58, 85, 180
pruritus, 170 respiration, 43
public health, 76, 172, 179 respiratory problems, 170
pulp, 49, 153, 157 response, vii, ix, xi, 1, 2, 3, 4, 6, 9, 13, 14, 23, 33, 35,
pupa, x, 5, 9, 18, 23, 65, 120, 149, 151, 153, 154, 38, 43, 48, 66, 76, 83, 88, 89, 95, 97, 99, 102,
158, 169, 170, 176, 180 106, 107, 108, 110, 112, 116, 119, 121, 123, 130,
purification, 138 132, 135, 137, 141, 148, 182, 203, 204
responsiveness, 7, 110
retardation, 5
Q Rhizopus, 156
rings, 111
query, 28
risk, 69, 71, 96, 101, 174, 197
quinones, 127, 128
risk assessment, 71
RNA, 6, 10, 18, 19, 20, 21, 23, 25, 86, 125, 135, 146
R RNAi, 125, 126, 133
root, 49, 50, 106
radius, 111, 116 roots, 55, 75
rainforest, vii rotations, 65
rape, 104 routes, 2, 157, 208
reactions, 136, 137, 142, 170, 205, 208, 211, 212, rubber, 171, 174
214 rules, 78
reactive oxygen, 121 rural areas, viii, 45, 176
reading, 5, 10, 14, 17 Russia, 50, 150
real time, 20
reception, 117
228 Index

society, 48, 164

S sodium, 73
solution, 69, 145, 163
safety, 48, 159
South America, v, vii, x, 169, 170, 171
saliva, 171
Southeast Asia, 170, 188, 190, 192, 196
scent, 91
Soviet Union, 50, 158
school, 78, 192
sowing, 56
science, 46, 48
Spain, 55, 149, 150, 153, 154, 162, 164
scope, 49
specialisation, 46
scripts, 75
species, vii, x, xi, xii, 1, 2, 3, 5, 9, 12, 13, 14, 15, 17,
second generation, 154, 157, 192, 194, 195
19, 20, 22, 25, 27, 28, 29, 36, 37, 52, 53, 54, 55,
secretion, 39, 180, 184
60, 61, 62, 64, 66, 67, 68, 69, 71, 75, 76, 78, 80,
seed, 50, 53, 54, 65, 75, 104, 106
85, 90, 91, 96, 100, 104, 111, 112, 120, 121, 123,
selectivity, 57
125, 126, 129, 130, 132, 133, 135, 149, 150, 151,
sensing, 126, 142, 147
155, 156, 158, 169, 170, 171, 172, 173, 176, 177,
sensitivity, 102, 114, 165
179, 184, 187, 189, 190, 191, 192, 193, 194, 195,
sensors, 138
196, 197, 199, 200, 203, 204, 207, 209, 214, 215,
sensory systems, 94, 109
216
serine, 25, 26, 28, 128, 142, 143, 144, 145, 177, 178,
species richness, 200
182, 184
spiders, 70, 155, 193
serum, x, 43, 144, 169, 170, 173, 178, 179, 183
spin, 55, 120, 190, 192
services, 86
Spring, 32, 76
sex, 41, 57, 58, 60, 80, 83, 84, 90, 91, 95, 99, 100,
Sri Lanka, 190, 192
105, 107, 108, 110, 112, 113, 114, 115, 158, 159,
stability, 22, 69, 89
167, 189, 198
standard error, 11, 21, 23, 25
sexual dimorphism, 153, 176
starch, 50, 52
shape, 58, 61, 62, 63, 96, 105, 109, 114, 151
state, 3, 15, 20, 23, 32, 47, 48, 58, 64, 126, 171, 172,
shear, 185
179, 193
shelter, 157
states, 49, 78, 126, 173
shock, 4, 6, 7, 8, 10, 12, 13, 14, 32, 33, 34, 35, 36,
statistics, 46, 78, 85, 116
37, 38, 39, 40, 41, 42, 43, 180
stem cells, 121
shortness of breath, 170
sterile, 57
showing, 43, 98, 107, 121, 173, 175
steroids, 205
shrimp, 210
stimulation, 117
shrubs, 65, 193
stimulus, 88, 89, 93, 95, 117
side chain, 213
stochastic model, 112, 162
side effects, viii, 45, 71, 78
stock, 53
signal peptide, 36
storage, vii, 1, 2, 3, 15, 16, 17, 18, 20, 24, 32, 33, 36,
signal transduction, 13, 124, 131, 133
37, 39, 40, 41, 42, 43, 44, 49, 55, 78
signaling pathway, 130, 131, 134, 147
strategy use, 68
signalling, 135, 136, 138, 147
stress, vii, 1, 2, 3, 4, 5, 6, 7, 9, 12, 13, 14, 32, 33, 34,
signals, viii, 67, 87, 93, 99, 100, 101, 103, 104, 105,
35, 38, 39, 43, 65, 185
107, 109, 115, 127, 143
stress response, 3, 35, 38
signs, 172
stressors, 5
silk, 55, 120, 125, 146, 153, 157, 170, 190
structural modifications, 213
silkworm, 5, 38, 39, 41, 42, 43, 120, 123, 129, 132,
structure, viii, ix, 16, 17, 25, 29, 30, 34, 35, 39, 40,
134, 138, 139, 140, 141, 142, 143, 144, 145, 146,
46, 65, 67, 74, 79, 87, 89, 91, 92, 94, 96, 97, 98,
148, 170, 180, 185
99, 101, 105, 106, 108, 110, 111, 112, 113, 114,
simple sampling, 58
130, 135, 136, 144, 145, 175, 178, 182, 184, 207,
simulation, 116, 164
208
sine wave, 79
subsistence, 53
skin, x, 169, 170, 173, 175
substitution, 30, 127, 207
smoke plume, viii, 87, 97, 113
substrate, 24, 30, 31
snake venom, 185
 Index 229

substrates, 128 tobacco, 41, 43, 54, 112, 120, 137, 141, 142, 148,
succession, 2 212, 215
sucrose, 49, 178 tonic, 108, 110
sugar beet, viii, 45, 49, 50, 54, 83 topology, 24, 99, 113
sugarcane, 50 total product, 53
sulfur, 208 toxicity, 68, 75, 76, 210, 212, 213, 216
sulphur, 97 toxin, 69, 177, 181, 184
Sun, 9, 140 trace elements, 49
supplementation, 178, 185 training, 78
suppression, 17, 20, 37, 69, 113 traits, 17, 69, 196
surface component, ix, 119, 120 transcription, 18, 19, 23, 27, 36, 39, 113, 131, 132,
surface layer, 115 133, 148
surveillance, 182 transcription factors, 39, 113, 131, 132, 133
survival, 4, 40, 64, 126, 155, 166, 178, 181, 184, transcripts, vii, 2, 5, 7, 9, 10, 11, 12, 13, 14, 15, 16,
200, 204 18, 20, 21, 22, 23, 25, 26, 27, 29, 37, 40, 43, 182
susceptibility, 50, 146 transduction, 113, 118, 140
sustainability, 46, 48, 83 transformation, 43, 83, 211
sustainable development, 79 transformations, 214
Sweden, 87, 110, 111 transgene, 133
swelling, 172 translation, 17
Switzerland, 162 translocation, 131, 133
symptomatic treatment, 170 transmission, 194
symptoms, 170, 171, 172, 173, 179 transport, 5, 33, 75, 89, 91, 121, 126, 142, 193, 207
syndrome, x, 169, 170, 171, 172, 173, 177, 179, 180, transportation, 191
181, 182, 183, 184 treatment, x, 4, 9, 12, 17, 19, 27, 28, 149, 150, 156,
synergistic effect, 213, 215 157, 169, 170, 171, 173, 177, 179
synthesis, vii, 2, 3, 13, 17, 20, 24, 27, 32, 38, 134, trial, 183
159, 215 triggers, ix, 119, 120
Syria, 55 trypsin, 127
tryptophan, 49, 205
turbulence, ix, 87, 93, 94, 96, 112
T Turkey, 55
turnover, 24
tactics, 49, 64, 76, 79, 134
tyrosine, 17, 124, 127, 128, 205
target, 56, 57, 68, 71, 75, 77, 80, 81, 123
taxa, 67, 109
techniques, 57, 58, 60, 70, 76, 94, 97, 133 U
technologies, ix, 48, 82, 88, 91, 105, 109, 158
technology, 46, 67, 69, 76, 77, 82 U.S. Department of the Interior, 85, 215
technology transfer, 46 U.S. Geological Survey, 85
temperature, vii, x, 1, 3, 4, 6, 9, 10, 12, 13, 34, 38, uniform, 46, 58, 60, 106
43, 60, 61, 62, 79, 80, 85, 93, 149, 151, 154, 155, unit cost, 158
156, 159, 161, 162, 167, 176, 188, 195 united, 47, 48, 50, 79, 82, 84, 85, 111
termination codon, 14 United Nations, 48, 79, 82, 84
terpenes, 205 United States, 47, 50, 82, 85, 111
territory, 89 univoltine life cycle, xi, 187, 188
testing, 71, 80 urban, xi, 173, 187, 191, 192, 194, 195, 196, 197,
tetrahydrofuran, 211 198, 201
threats, viii, 45, 49, 53, 55, 56, 58, 76 urban areas, 173, 194, 196
threshold level, 90 USA, 35, 37, 39, 43, 48, 69, 79, 82, 83, 110, 116,
thrombin, 173 117, 134, 143, 194, 200
thrombosis, 181 USDA, 48
time resolution, 97
tissue, 16, 37, 104, 127, 178, 190, 207
230 Index

Washington, 35, 215

V waste, 121
water, 4, 46, 50, 78, 89, 91, 93, 154, 155, 157, 194
variables, 62, 64, 155, 166
waterways, 65
variations, 48, 64, 133
weakness, 172
varieties, 46, 50, 52, 64, 65, 156
web, 70
vasculitis, 179
West Africa, 85, 150
vegetables, 54, 84
wildlife, 65, 215
vegetation, 54, 196
wind speed, 94, 106
velocity, 92, 93, 115, 118
windows, 42
Venezuela, 171, 180
wood, 116, 154, 190, 194, 198
vertebrates, 14, 28, 33, 49, 125, 129, 206
woodland, 117
victims, 174
workers, 170, 171, 174
viral infection, 125
World Wide Web, 81
viruses, xi, 57, 69, 74, 91, 121, 156, 187, 193, 196
worldwide, 50, 52, 53, 56, 69, 188
vitamin C, 49
worms, 65
volatile organic compounds, 97, 100, 116
wound healing, 123, 127
vomiting, 172, 173

Y
W
yeast, 14, 126, 130, 135
Wales, 80
yield, 47, 50, 52, 53, 55, 56, 62, 63, 156
walking, 94, 108, 116