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Library of Congress Cataloging-in-Publication Data

Insect reproduction / Simon R. Leather and Jim Hardie, editors.

p. cm.
Includes bibliographical references (p. ) and index.
ISBN 0-8493-6695-X (alk. paper)
1. Insects--Reproduction. I. Leather, S. R. (Simon R.)
II. Hardie, Jim.
QL495.I4985 1995
595.7’016--dc20 95- 16294

A Library of Congress record exists under LC control number: 95016294

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 INTRODUCTION
This book, consisting of ten review chapters contributed by leading workers in their
respective fields, from around the world, covers the whole subject of insect reproduction. It
begins with the basic physiological questions of insect reproduction, moves on to discuss the
new advances seen in the fields of behavioral and ecological mechanisms, and culminates by
examining the recent work on evolutionary biology and its application in the field.
Each chapter, although including a brief review of the basic seminal work, focuses mainly
on the advances made within the last ten years and highlights those areas in which the
respective authors see the greatest scope for further important advances. By allowing each
author full rein to explore their chapter subject using their particular "hobby horse," it has been
possible to make this not just a book of review chapters, but one in which exciting new ideas
have been raised.
This book should be of general interest to all entomologists, whether they are in pure or
applied fields, and should also be an important asset to any teaching program where entomol-
ogy is taught at the undergraduate and post-graduate level.

SRL, JH.
 THE EDITORS
Dr. Simon R. Leather is presently Lecturer in Applied Ecology and Pest Management at
Imperial College, London. He obtained his B.Sc. from the University of Leeds, England in
1977 with first class honors in Agricultural Zoology. After receiving his Ph.D. in 1980 from
the University of East Anglia in Norwich, he embarked on further research in aphid ecology.
Then followed a ten year spell with the British Forestry Commission where he worked in the
Research Division, primarily on the population biology of forest pests with particular refer-
ence to their reproductive behavior. He started his current position in 1992. Dr. Leather is a
Fellow of the Royal Entomological Society, a Member of the British Ecological Society, a
Member of the Flora and Fauna Preservation Society, and a Member of the Institute of
Biology. He sits on the Council of the Royal Entomological Society and edits their journal
Antenna.
Dr. Jim Hardie is presently a Principal Research Fellow at Imperial College, London. He
obtained a Ph.D. degree from the University of Birmingham, England in 1975 and a D.Sc.
from London University in 1989. He has worked in the field of aphid physiology for more than
twenty years and is regarded as one of the leading figures in this area. He is a Fellow of the
Institute of Biology, the Royal Entomological Society, and the Royal Microscopical Society.
 CONTRIBUTORS
Roger L. Blackman, B.Sc., Ph.D. Klaus H. Hoffmann, Prof. Dr.
Department of Entomology Department of Animal Ecology I
The Natural History Museum University of Bayreuth
London, England Bayreuth, Germany

Simon R. Leather, B.Sc., Ph.D.

Carol L. Boggs, Ph.D. Department of Biology
Center for Conservation Biology Imperial College of Science,
Department of Biological Sciences Technology, and Medicine
Stanford University University of London
Stanford, California Silwood Park
and Rocky Mountain Biological Ascot, England
Laboratory
Crested Butte, Colorado Athol McLachlan, Ph.D., D.Sc.
Department of Agriculture and
A. F. G. Dixon, B.Sc., D. Phil. Environmental Sciences
School of Biological Sciences University of Newcastle-upon-Tyne
University of East Anglia Newcastle-upon-Tyne, England
Norwich, England
Rachel Neems, B.Sc., Ph.D.
Department of Genetics
Cedric Gillott, B.Sc., Ph.D., D.Sc. University of Leeds
Department of Biology Leeds, England
University of Saskatchewan
Saskatoon, Canada Richard Wall, B.Sc., Ph.D.
School of Biological Sciences
Jim Hardie, B.Tech., Ph.D., D.Sc. University of Bristol
Department of Biology Bristol, England
Imperial College of Science,
Technology, and Medicine Christer Wiklund, Ph.D.
University of London Department of Zoology
Silwood Park University of Stockholm
Ascot, England Stockholm, Sweden
 TABLE OF CONTENTS
Chapter 1
Oogenesis and the Female Reproductive System .................................................................. 1
Klaus H. Hoffmann

Chapter 2
Insect Male Mating Systems .................................................................................................
33
Cedric Gillott

Chapter 3
Sex Determination in Insects ................................................................................................
57
Roger L. Blackman

Chapter 4
Hormones and Reproduction ................................................................................................95
Jim Hardie

Chapter 5
Fatal Attraction: The Disruption of Mating and Fertilization for Insect Control .............109
Richard Wall

Chapter 6
Parthenogenesis in Insects with Particular Reference to the
Ecological Aspects of Cyclical Parthenogenesis in Aphids ...............................................
131
A. F. G. Dixon

Chapter 7
Factors Affecting Fecundity, Fertility, Oviposition, and Larviposition in Insects ............ 143
Simon R. Leather

Chapter 8
Protandry and Mate Acquisition .........................................................................................
175
Christer Wiklund

Chapter 9
Swarm-Based Mating Systems ...........................................................................................199
Athol McLachlan and Rachel Neems

Chapter 10
Male Nuptial Gifts: Phenotypic Consequences and Evolutionary Implications................2 15
Carol L. Boggs

Index ....................................................................................................................................
243
 DEDICATION
This book is dedicated to our families in recognition of the support given during the
somewhat lengthy process that ensued once we had embarked upon this task. So, thank you
Gill, Fern, John, James, Thomas, and Matthew from Simon, and thank you Ros, Sally, Nicola,
and Robert from Jim.
 Chapter 1

OOGENESIS AND THE FEMALE REPRODUCTIVE SYSTEM

.
Klaus H Hoffmann

CONTENTS
I . Introduction ................................................................................................................... 1

I1. Morphology of the Female Reproductive System ........................................................2

A . External Genitalia ...................................................................................................
4
B . Structure of the Ovary ............................................................................................
4
C . Structure of the Female Accessory Reproductive System .....................................6
1. Spermatheca and Spermathecal Accessory Glands ........................................... 7
2 . Colleterial Glands and Mesodermal Accesso~yGlands .................................... 8
3 . Milk Glands ........................................................................................................
9
D . Endocrine Control of Differentiation of Accessory Glands and Ducts ............... 10

I11. Origin and Formation of the Germ Cells ...................................................................10

IV . Oogenesis .................................................................................................................... 11
A. Early Events in Oogenesis .................................................................................... 11
1. Oocyte Differentiation ..................................................................................... 14
2 . Endocrine Control of Early Oogenesis............................................................ 15
3 . Follicle Cell Differentiation ............................................................................. 15
4 . Trophic Function of Nurse Cells ..................................................................... 17
B . Previtellogenesis ................................................................................................... 19
C. Vitellogenesis ........................................................................................................19
1. Vitellogenin and Vitellin Chemistry ................................................................19
2. Vitellogenin Genes ...........................................................................................21
3 . Vitellogenin Synthesis ..................................................................................... 21
4 . Vitellogenin Secretion ...................................................................................... 22
5. Uptake of Vitellogenin by the Ovary ..............................................................23
D. Chorionization ....................................................................................................... 25
1. The Vitelline Membrane .................................................................................. 26
2. Chorion Formation ........................................................................................... 27

V. Ovulation and Oviposition ..........................................................................................

28

V1. Concluding Remarks ................................................................................................... 29

References ............................................................................................................................. 29

.
I INTRODUCTION
Insect reproduction results from a succession of interdependent steps which are often quite
different in nature and take place at various stages of the insect life cycle. The main reproduc-
tive events in females are sex determination. gonial mitoses and meioses. differentiation of the

.
0-8493-6695-X/95/$0.00+$.M
6 1995 by CRC PRSS Inc.
2 Insect Reproduction

reproductive organs, previtellogenesis and vitellogenesis, accessory gland functioning, sexual

behavior, mating, ovulation, and oviposition. It is the function of the female reproductive tract
to produce the eggs and to deposit them at an appropriate time and in an appropriate place.
In addition, the female reproductive tract must receive the spermatozoa from the male and
transport them to the spermatheca where they are stored until they are used to fertilize the eggs
as they are oviposited.
Insect reproduction strictly depends on environmental factors. Factors which may affect
reproduction include temperature, humidity, photoperiod, nutritive conditions, and a suitable
egg-laying substrate.
Regulation of insect reproduction involves numerous sensory receptors, nerve transmis-
sion, and integration in the brain, which regulates the synthesis of the two groups of insect
developmental hormones, the juvenile hormones and the ecdysteroids, and produces its own
neurohormones.' The mechanisms which regulate each reproductive step may vary with insect
species (see Chapter by Hardie).
Present knowledge of oogenesis has progressed variously, depending on the event consid-
ered. The best-known field is that of vitellogenesis and its endocrine regulation, while the
early events of ovarian development and its "fine-tuning" control by hormones are less well
understood.
Sexual reproduction is the general rule in insects, although many exceptions and modifi-
cations are observed (see Chapter by Dixon). In the more highly evolved social insects
(Hymenoptera), reproduction is limited to a small number of individuals, often one queen and
a small number of males (drones).

11. MORPHOLOGY OF THE FEMALE REPRODUCTIVE SYSTEM

The central site of egg production is the ovary. Ovaries are usually located dorsolateral to
the gut and each comprises a number of tubular ovarioles ensheathed by a network of
connective tissue (Figure 1). Each ovariole is composed of somatic and germ cell tissues. The
number of ovarioles per ovary varies from one (e.g., in some viviparous aphids and in dung
beetles) to about 3000 (in some higher termite queen^).^ Three basic types of ovary organi-
zation are found in insects (Figure 2). The panoistic ovarian, probably the most primitive one,
is found in the oldest families of insects, such as in Archaeognatha, Zygentoma, Odonata,
Plecoptera, and in most orthopteroid insects, but also in some Megaloptera (Corydalidae) and
most Siphonaptera. In the panoistic ovary, specialized nutritive cells are absent and most of
the informational resources of the oocyte are provided by the synthetic activity of the oocyte
nucleus itself. The polytrophic meroistic ovary is found in most endopterygotes, and in
Dermaptera, Psocoptera, and in Phthiraptera. In polytrophic ovaries, a number of nurse cells
are enclosed in each follicle along with an oocyte. In the Hemiptera, polyphage Coleoptera,
Megaloptera (Sialidae)/Raphidioptera, and also in the most "primitive" winged insects, the
Ephemer~ptera,~ telotrophic meroistic ovaries are found. In this ovarian type, a syncytium of
nutritive cells is connected with each oocyte by means of a trophic cord. In the vast majority
of insect species, the nutrive, or yolk, contribution is supplied largely by the fat body, but in
some cases the follicle cells can also serve as additional source of yolk. Accumulation of yolk
(vitellogenesis; Section 1V.C) normally occurs only in the terminal oocyte, that is, the oocyte
closest to the oviduct. Another follicle cell function is the formation of the protective layers
of the egg. These include the vitelline membrane and the chorion (Section 1V.D).
Besides the ovary, oviduct-associated secretory cells can have important contributions to
egg production (Section 1I.C). The most significant of such structures are the spermathecal
accessory glands and the female accessory glands, such as the colleterial glands. Last,
secretory functions of the vagina may play a role in egg production. Like other terrestrial
Oogenesis and the Female Reproductive System

$ p * r m o l h ~ c o l dond
common oviduct

FIGURE l. Female reproductive system: diagram of common type found in many insects. (From Gillott, C.,
Enromology, Plenum, New York, 1980, chap. 19. With permission.)

nutc

fc

PANOISTIC POLYTROPHIC TELOTROPHIC

FIGURE 2. Schematic diagrams showing the types of ovarioles. Meroistic ovaries may be organized in the
telotrophic and the polytrophic way, respe~tively.~.~~ch = chorion; fc = follicle cell; gm = germarium; nc =nurse cells;
nutc = nutritive cord; oo = oocyte; tf = terminal filament. (From Gillott,C., Enromology, Plenum, New York, 1980,
chap. 19. With permission.)
 Insect Reproduction

terao VIII. IX. and X

gonostyle

aonoooohvsis

FIGURE 3. The primitive structure of the pterygote ovipositor in the thysanuran Lepisma. (From Gillott, C . ,
Entomology, Plenum, New York, 1980, chap. 19. With permission.)

animals, insects have had to solve the problem of bringing together sperm and egg in the
absence of surrounding water (internal fertilization, see Chapter by Wall). Almost all insects
store the spermatozoa received from the male in a specialized organ, the spermatheca, until
they are used to fertilize the mature eggs.

A. EXTERNAL GENITALIA
The morphology of the organs specialized for copulation and oviposition is highly varied.
In the mayflies, the oviducts open directly to two genital pores behind the seventh abdominal
segment. In most insects, the appendages of the genital segments (eighth and ninth abdominal
segment) form an ovipositor. In the apterygotes and some of the winged insects, the ovipositor
is a simple opening both for copulation and for the deposition of eggs. The primitive structure
of the pterygote ovipositor can be seen already in the thysanuran Lepisma (Figure 3).4 Among
Pterygota, an ovipositor is found in Notoptera (Grylloblattodea),Dictyoptera, Ensifera, Caelifera,
and Hymenoptera, some Odonata and most Hemiptera, Thysanoptera, and Psocoptera. The
structure and elaborateness of the ovipositor is determined by the site of egg deposition. The
ovipositor of Hymenoptera may be considerably modified for boring, piercing, sawing, and
stinging. In the stinging Hymenoptera, such as bees, the eggs are released at the base of the
ovipositor, and the ovipositor is modified by the addition of poison glands and reservoirs that
evacuate the venom through the hollow sting. The ovipositor of Drosophila has sharpened
ends that penetrate the surface of fruit, while the ovipositors of some of the predatory wasps
are long (up to 15 cm in length) and sharp to penetrate the body of the insect prey.5

B. STRUCTURE OF THE OVARY

Insects have become particularly adept in manufacturing large numbers of oocytes within
the ovary. The fruit fly, Drosophila melanogaster, can produce, during a 10-week reproduc-
tion period, a quantity of eggs equivalent to 30 times her body eight.^ Her newly formed
oocytes can undergo a 100,000-fold increase in volume within 3 days. Such reproductive feats
are possible because of certain evolutionary adaptations: (1) Oocytes have developed methods
for incorporating massive quantities of female-specific proteins (vitellogenins) which are
transported in the hernolymph from the fat body to the ovary. (2) Mechanisms have evolved
for loading unfertilized eggs with the ribosomes, RNA, and long-lived mRNA that are
required in early embryogenesis. (3) The ovarioles are supplied by tracheae of the aeriferous
type, with a great diversity in the methods by which oxygen is delivered to the individual
Oogenesis and the Female Reproductive System

FIGURE 4. Panoistic ovariole of a cricket, Glyllus bimaculatus de Geer, with a mature egg in the terminal oocyte.
Photograph courtesy of K. H. Hoffmann, Bayreuth.

Panoistic ovarioles can be developed by blocking germ cell cluster divisions totally, as is
found in most "primitive" insects, or after germ cell cluster formation by final cleavage of
cystocytes, all of which develop as oocytes as found in stone flies or thripss In the panoistic
ovary, each of the ovarioles is composed of a terminal filament, the germarium, a series of
oocytes at the previtellogenic phase of development, one or more oocytes in the process of
vitellogenesis, and last, the mature egg (Figure 2A; Figure 4). The terminal filament is made
up of a group of flattened cells, surrounded by a basement lamina and an ovarian sheath, both
of the latter surrounding the entire ovariole. The oogonia are located in the most anterior
region of the germarium, followed by a zone of oocytes in the early stages of meiosis. At the
posterior end of the germarium, the oocytes are beginning to be surrounded by a monolayer
of follicle cells. The size increase of oocytes at the previtellogenic stage is accomplished by
an expansion of the cytoplasmic volume. In many cases, a multilayered pad of interfollicular
tissue is located between successive oocytes? In many insects with panoistic ovaries, vitello-
genesis commences in the penultimate oocyte only after ovulation at the terminal one (e.g.,
in Locusta migratoria and Schistocerca gregaria [Caelifera]),or in the case of ovoviviparous
cockroaches,after the loss of the egg case. The inhibitory effect is mediated by the interfollicular
cells which pass an inhibitory substance from the anterior to the more posterior oocyte. In
contrast, secretion from such cells located proximal to the oocyte stimulates vitellogenesis. In
other species (e.g., in Periplaneta americana [Blattodea] and Melanoplus sanguinipes
[Caelifera]), two or more oocytes may be vitellogenic at the same time, although at different
stages of the yolk deposition cycle. All the above-mentioned insects produce eggs in batches.
In the stick insect Clitumnus extadentatus, the different ovarioles can mature asynchronously,
and the female lays a few eggs per day for several weeks. The number of ovarioles in panoistic
ovaries can range from 4-3000 (5 in Acrididae, 15-30 in Tettigonioidea, 150-170 in Gryllidae,
about 3000 in Isoptera queens).
A terminal filament and a germarium are also found in polytrophic meroistic ovaries
(Figure 2B). In this case, the anterior region of the germarium contains one or more stem-line
oogonia and a number of daughter cells or cystoblasts. The cystoblasts divide to give a cluster
of cells remaining connected by structures called "ring canals" or "intercellular bridges." The
innovation of the polytrophic ovary is the differentiation of only one oocyte, which generates
from one, central cell of the cluster, whereas all other siblings are transformed into nurse cells.
6 Insect Reproduction

In many cases, clusters follow the 2"-rule ( l oocyte + 2"- 1 nurse cells), in which n represents
the number of cell cycles. Identical characters in polytrophic meroistic ovaries among hemi-
metabolous and holometabolous insects indicate a "basic type" of common rigi in.^ In Droso-
phila, where the number of cells in the cluster is 16, the clustering of the cells, as well as the
formation of the ring canals, is mediated by structures called fusomes. The fusomes contain
a random array of membranous vesicles and fibrils,'0." but no mitochondria and few ribo-
somes and microtubules. In the central region of the germarium, prefollicular cells grow
around the oocyte-nurse cell cluster, while in the posterior section of the germarium, typical
egg chambers are detectable; which means the oocyte-nurse cell complex is completely
surrounded by a unilayer of follicle cells. Previtellogenesis includes the enlargement of the
oocyte, an increase in the number of follicle cells, and the polyploidization of the follicle cells
and nurse cells. An epithelial sheath surrounds each ovariole and consists of a thin, acellular,
inner membrane; a median cellular network of muscle and tracheal cells; and an outer
epithelia1 membrane. The number of ovarioles in polytrophic ovaries can be highly variable
(usually 4 in the Lepidoptera, 10-30 in Drosophila, and 70-100 in Musca or Lucilia [Diptera]).
Commonly, a single oocyte per ovariole will become vitellogenic at one time.
Telotrophic ovaries differ from polytrophic ovaries by retention of all nurse cells in an
anterior trophic chamber and by changing oocyte-nurse cell determination. This type of ovary
developed independently three times (in Hemiptera, RaphidiopteraJMegaloptera [Sialidae],
and in polyphagous Coleoptera) from polytrophic ancestors and once directly from panoistic
ancestors in mayflie~.~ Despite fundamental differences between the subtypes of telotrophic
ovarioles, they share some common characters. As the oocytes move towards the region of
follicle formation, they become surrounded by prefollicular cells. The connection to the
tropharium is retained via a cytoplasmic strand, the nurse strand or trophic cord. The young
oocytes subsequently enlarge by incorporation of nurse cell material, transported through the
nutritive cords (previtellogenesis). The vitellogenic growth phase begins when yolk spheres
are observed to accumulate in the oocytes. The nutritive cords collapse during vitellogenesis.
The follicular cells surround each growing oocyte to form a monolayered epithelium, and this
tissue will secrete a vitellin membrane and the chorion. An interesting variant of the telotrophic
ovary is found in the polyphagous Coleoptera. The number of ovarioles remains more or less
constant in each species, but varies between species from 1 (some Scarabaeinae) to about 1000
in blister beetles (Meloe pro~carabaeus).~ In Creophilus maxillosus (Staphylinidae), the
differentiation of nurse cells and oocytes occurs within linear chains of sibling cells (linear
chain model). Only the most basal member of the sibling cluster develops into an oocyte; the
others differentiate into nurse cell^.^.^

C. STRUCTURE OF THE FEMALE ACCESSORY REPRODUCTIVE SYSTEM

The bottom of each ovariole forms a small duct or pedicel (Figure 1). The pedicels of each
group unite to form a calyx, and each calyx opens into a lateral oviduct. Usually the oviducts
of the two sides join to form a single median oviduct. The common oviduct is ectodermal in
origin and typically it is heavily invested with muscles. The lateral oviducts can be of
mesodermal or ectodermal origin. The presence or absence of a cuticular intima indicates the
origin. In Oncopeltusfasciatus (Hemiptera), the lateral oviducts undergo a drastic larval-adult
transformation during the last (fifth) larval stadium.12The long and thin larval oviducts shorten
and become very wide. This transformation is ecdysteroid dependent in a dose-related manner
and only takes place in the absence of juvenile hormone. The morphological transformation
is accompanied by dramatic cytological changes. Whereas the cells of the anterior part of the
oviduct commence with a strong secretory activity, the cells of the caudal part form a bizarre
pattern of cell projections which deposit the cuticle. The secretory material of the ductal
system may act as a lubricant for egg passage, as protective oothecal coverings, or as glues
to attach eggs to various substrates or to hold batches of eggs t ~ g e t h e rThe
. ~ distal end of the
common oviduct is called the gonopore, which serves for the discharge of eggs. In the
Oogenesis and the Female Reproductive System 7

Dermaptera, the gonopore is the external opening, located on the posterior part of the seventh
abdominal segment. In most insects, however, the gonopore opens into a genital chamber. The
opening to the outside is the vulva. The genital chamber can be of variable complexity and
is often associated with an ovipositor. Within some orders, an invagination of the primitive
genital chamber forms a distinct intermediate structure, the vagina, between the external vulva
and the gonopore. Generally, the vagina is not secretory and consists of a single layer of
epithelia1 cells, covered by a cuticular intima and surrounded by muscle. In many species, the
genital chamber has become modified to form a bursa copulatrix. An important function for
this organ is to receive spermatophores or seminal fluid. In Lepidoptera, the bursa remains in
the eighth abdominal segment, whereas the eggs are deposited through a separate opening, the
oviporus, on segment IX. In this case, the bursa is connected with the vagina by the seminal
duct. In other insects, however, there is no separation between the copulatory and egg-laying
apertures, and both of them open as the vulva on segment IX. In the bursa of some butterflies
(e.g., genus Danaus), tooth-like dentata are present and may function in tearing open the
spermatophore. Some secretory activity may also be associated with the bursa copulatrix,
since empty spermatophores are digested within the bursa of some insects. In ovoviviparous
and viviparous species, a brood pouch or uterus forms as an expansion of the vagina wall.
Two ectodermal glands (ectademia) are connected with the genital chamber or vagina. One
is the spermatheca in which spermatophoresare stored and that has a gland attached to its duct.
The other is a paired structure, the accessory glands or colleterial glands, with functions
associated with egg deposition.

1. Spermatheca and Spermathecal Accessory Glands

The morphology and arrangement, as well as the number of spermathecae, vary from
species to species. In most insects, the spermatheca is a single organ, spherical or ovoid in
form. In some insects, such as cockroaches and mosquitoes, secretory cells are associated with
the walls of the storage portion of the spermatheca, whereas in others, such as honeybees and
many beetles, the distal end of the spermatheca is specialized for secretion and is referred to
as the spermathecal accessory gland. In the genus Rhodnius (Heteroptera), the spermathecae
are a pair of blind tubules which open into the common oviduct near its junction with the
lateral oviducts.
In spite of the morphological diversity, the cellular elements of the secretory part of the
spermatheca are rather constant. A generalized diagram of a spermathecal secretory cell is
given in Figure 5. The cells form a cup-shaped cavity at their apical surface, and the membrane
in this region is thrown into numerous microvilli. The central cavity communicates with the
spermathecal lumen via a cuticle-lined ductule. In other forms, however, several ductules may
open at a single pore. The cuticle lining the ductule and the spermathecal lumen is produced
by separate duct cells which are interposed between the secretory cells and the lumen of the
spermatheca. Ultrastructural studies showed that the secretory cells have a phenotype associ-
ated with cells which are specialized for export protein synthesis?
The secretion produced by the gland cells is a mucoprotein or mucopolysaccharideand may
be used by the spermatozoa for an energy source.13 Removal of the spermathecal accessory
gland from females of Anthonomus grandis (Coleoptera) results in a gradual loss of motility
among spermatozoa in the spermatheca. The epithelium of the spermatheca, particularly in the
storage part, exhibits characteristics of ion-transporting epithelia and thus may be responsible
for providing an ionic milieu within the lumen of the spermatheca different from that in the
hemolymph. In some insects, the spermathecae may be sufficiently permeable to allow the
passage of various male secretory products.
Information on the control of spermatheca differentiation is rather limited. In the beetle
Tenebrio molitor, the differentiation of the spermatheca occurs in the pupal stage prior to the
eclosion to the adult. The differentiation process can be divided into three phases: ( l ) cellular
proliferation, (2) cellular morphogenesis, and (3) cuticulogenesis. From in vitro culture
 Insect Reproduction

FIGURE 5. Diagrammatic representation of a spermathecal secretory cell and its spatial relationship to the duct cell
and basement membrane. CC, central cavity; CU, cuticle; DC, duct or canal cell; DU, ductule; ER, rough endoplasmic
reticulum; LU, lumen; MV, microvilli; N. nucleus; SC, secretory cell. (From Kaulenas, M. S., Insect Accessory
Reproductive Structures. Function, Structure, and Development. Springer-Verlag, Berlin, 1992. With permission.)

experiments, it was concluded that cuticulogenesis is under control of 20-hydroxyecdysone;

the hormone is necessary to initiate cuticle deposition.14 With regard to the control of the
acquisition of definitive levels of differentiated functions by the spermatheca, some informa-
tion is available for a number of Orthopterans. In Chorthippus curtipennis (Caelifera,Acridinae),
allatectomy leads to degeneration of the spermatheca, while in Gomphocerus rufus (Caelifera,
Acridinae), allatectomy resulted in the inability of females to dissolve transferred spermato-
phores, suggesting a failure in the production of spermathecal proteolytic enzymes? The
results suggest that juvenile hormone is necessary to initiate and possibly maintain differentiative
secretory function in these grasshoppers. On the other hand, allatectomy in L. migratoria
(Caelifera, Locustinae) does not alter the histology of the spermatheca. In Rhodnius prolixus
(Heteroptera), removal of the neurosecretory cells in the pars intercerebralis results in a failure
of the secretion to appear in the lumen of the gland as a result of the absence of a myotropin
from the neurosecretory cells. The myotropin acts on the muscles of the spermatheca to
squeeze the secretion from the reservoir beneath the intima into the lumen.13

2. Colleterial Glands and Mesodermal Accessory Glands

Many insects produce protective coatings for the newly laid eggs. The Lepidoptera coat
individual eggs with a proteinaceous glue that hardens on contact with air and attaches the
eggs onto appropriate substrates. Accessory glands in the genus Musca (Diptera) contribute
in aiding in the fertilization process by providing secretions which assist in liberating sperm
acrosomal contents.15 Aquatic insects often produce egg cases of a gelled substance, but the
most complex of these structures may be the tough ootheca of cockroaches and mantids. In
mantids, the glands produce a polymerized protein foam. In cockroaches, several layers of
tanned protein form a complicated egg chamber with elaborate respiratory apparatus and
release valves. The sources of these protective devices are the colleterial glands which branch
off the vagina or genital chamber. In most cases, the colleterial glands are a pair of organs
composed of a number of multibranched tubules and are formed from invagination of the
epidermis (Figure 1). The morphology of the colleterial glands has been well documented.
Five cell types compose the left colleterial gland; four types of glandular cells are homologous
Oogenesis and the Female Reproductive System 9

with the dermal glands of the integument, and a chitinogenic cell type is homologous with the
epidermal cell of the integument. The latter cell type is found interspaced between the
glandular cells and secretes the protective intima which lines the lumen of the tubule. Each
of the gland cell types is distinct from the other, and is located in separate regions of the tubule.
Type 1 cells are found in the most posterior region of the tubule and are in a presecretory stage.
Type 2 gland cells are found anterior to type 1 cells and lack a well-defined rough endoplasmic
reticulum (ER), but are packed with mitochondria. The morphology of the type 3 cells
resembles that of both type 2 and type 4, and appears to be a transient intermediate form. The
type 4 cells dominate the anterior ends of the tubules and are packed with rough ER and with
mitochondria.16
The colleterial gland of P. americana has been studied most extensively?J3 The left side
tubules (type 4 cells) secrete the proteins (oothecins) which make up the structure of the
ootheca, together with a polyphenol oxidase (type 2 cells) as well as the precursor of the
tanning agent, the 4-0-P-glucoside of protocatechine acid. The right colleterial gland secretes
the enzyme P-glucosidase. The opening for the duct from the right gland is more anterior than
the opening for the duct from the left gland. At the time of ovulation (see Section VI), the
chorionated oocytes migrate down the oviduct to the genital atrium for fertilization. After
fertilization, the eggs are transported more posteriorly and pass the duct of the right gland
where the contents of the gland (P-glucosidase) are secreted onto the fertilized egg. Thereafter,
as the egg moves more posteriorly, it encounters the secretions of the left colleterial gland. The
subsequent mixing of the secretions from both glands results in the hardened ootheca.I6
Apparently, juvenile hormone affects the synthesis of oothecins in left colleterial
gland^?.'^.'^.^^ Analyses of the Periplaneta oothecin cDNA sequences and of the resultant
predicted amino acid sequences have confirmed the existence of I I major glycine-rich
oothecins which represent six size classes with molecular weights of 14.5, 15.5, 17-18.5,
23.5-26, 28.0, and 37-39 kDa, respectively.18The oothecin sequences have numerous simi-
larities to silkrnoth chorion proteins. In mantids, the chemistry of the ootheca proteins shows
some unique features. The glucosides identified in five mantid species are 3-0-P-glucosides
of N-P-alanyldopamine and N-(N-malonyl-P-alany1)dopamine.The light color of the ootheca
and the occurrence of phenolic compounds modified at the P position of the parent compounds
suggest that P-sclerotization occurs in mantid oothecae.
In S. gregaria and other Orthopterans, the foamy ootheca is produced, at least in part, by
mesodermal accessory glands (mesadenes) which consist of convoluted blind tubules opening
into the proximal end of the lateral oviducts. In spite of the great structural differences between
glands responsible for oothecal formation in cockroaches and locusts, similar mechanisms
may operate to harden the ootheca.

3. Milk Glands
In tsetse flies, members of the genus Glossina, the accessory glands of the female are
transformed into milk glands and supply a secretion upon which the developing larva feeds
(adenotrophic vivipary). In these flies, the female ovulates a single egg into a uterus, where
it hatches. The growth of the larva is rapid under such circumstances. In Glossina austeni for
example, development from an egg to a fully grown larva weighing some 30 mg requires only
9-10 days. The fully mature larva is then "larviposited." The milk gland in Glossina is a
branched tubular structure ramifying throughout the abdomen and emptying into the uterus via
a single muscular collecting duct which contains two channels.I3 The tubules consist of a
single layer of secretory cells similar to those in other accessory glands. The tubules undergo
cyclic changes in diameter, largely as a result of changes in the volume of secretory reservoirs.
The secretory reservoir is an extracellular structure, formed by a cup-shaped invagination of
the apical membrane of each of the secretory cells. In G. austeni, the tubular diameter reaches
a peak of about 100 pm 3 days before each larviposition, and a diameter of about 30 pm at
10 Insect Reproduction

each larviposition, followed by a resynthesis of the "synthetic machinery." Removal of the

corpora allata greatly reduces the production of milk in tsetse flies.
Milk glands also occur in some cockroaches. In the viviparous cockroach Diploptera
punctata, nutrient is supplied to the developing embryos, which increase in weight by a factor
of about 50 before they hatch. The developing embryos receive this nutrition by ingesting a
fluid secretion ("milk") in the brood pouch. The brood pouch is a part of the genital chamber.
The intima of this cuticle-lined chamber is penetrated by pores, each of which is the opening
of a ductule leading from a secretory cell. In Diploptera, juvenile hormone is necessary to
allow the decline of milk gland synthesis at the termination of pregnancy.lg

D. ENDOCRINE CONTROL OF DIFFERENTIATION

OF ACCESSORY GLANDS AND DUCTS
The dependency on ecdysone and 20-hydroxyecdysone of the organogenesis of accessory
glands and genital ducts has been demonstrated in several insect species.'-20The development
of the colleterial glands of P. americana, which occurs at the end of the last larval instar,
requires ecdysone. In young females of L. migratoria and S. gregaria, implantation of
additional corpora allata accelerated the development of both the oviducts and colleterial
glands. In T.molitor, Ephestia kiihniella and Samia cynthia, L. migratoria, and 0.fasciatus,
duct differentiation only occurs in the presence of ecdysone in vivo and in vitro. However,
other tissues of epidermic origin, such as the spermatheca, escape mitotic stimulation by
ecdysone.
Besides the effects exerted by hormones upon the reproductive organs, humoral relation-
ships between gonads and ducts have been demonstrated. In Drosophila, female ducts were
sometimes found to be attached to the gonads, thus causing degeneration. In the bugs
Dysdercus fasciatus and Triatoma infestans, atrophy of one ovary was observed together with
the regression of the upper part of the corresponding lateral oviduct, and the experimental
section of the oviduct had the same effect in both species.'

111. ORIGIN AND FORMATION OF THE GERM CELLS

Both sperm and eggs are derived from primordial germ cells set aside very early in the
development of the embryo. Among the orthopteroid insects, in species where the germ cells
appear early in development, such as in the house cricket Acheta domesticus and the grass-
hopper Melanoplus differentialis, the germ cells appear to be of ectodermal origin, forming
at the posterior pole of the egg at the time of mesoderm ~egregation.~ Later in development
in A. domesticus, they become associated with the mesoderm of the second and third abdomi-
nal segments; in M. differentialis they migrate into the coelomic cavities of the first to eighth
abdominal segments, where they associate with the splanchnic wall and form a genital strand,
from which the gonad differentiates later in embryogenesis. In species where the germ cells
are first recognizable slightly later in embryonic development, they are associated with the
median walls of the dorsal cavities in the abdominal segments (e.g., L. migratoria, Blattella
germanica, P. americana). In each of these species, a genital ridge containing the germ cells
is formed on each side of the embryo. The typical genital rudiment in these insects, during or
just after involution of the germ band (anatrepsis), consists of a terminal filament membrane;
a mesodermal dorsal cell mass, ventral to the filament membrane; a central cell mass,
composed of primordial germ cells and mesodermal cells; a ventral cell strand of mesodermal
cells which are the primordia of the gonadal portion of the genital ducts; and a surrounding
epithelia1 membrane which envelops all of the above (Figure 6).
Among the endopterygotes, germ cell formation is well understood in the Diptera. The
germ cells are formed from pole cells, which are established very early in development at the
posterior pole of the embryo. In Drosophila, about 18 energids (cleavage nuclei) enter the
posterior pole plasma and are pinched off as pole cells. The pole cells continue to divide, to
Oogenesis and the Female Reproductive System

\ TERMINAL FILIMENTS

FIGURE 6. Diagrammatic representation of the early development (late embryonic stage) of the female reproduc-
tive system in an orthopteroid insect.(From Kaulenas, M. S., Insect Accessory Reproductive Structures. Function,
Structure, and Development, Springer-Verlag. Berlin, 1992. With permission.)

produce eventually between 37 and 71 cells? Only some of the pole cells migrate to the
presumptive gonads, which lie on either side of the gut and are mesodermally derived. The
final number of pole cells in the gonad has an upper limit of about 13 pole cells per gonad at
about stage 16 of ernbryogenesi~.~~ The pole cells which fail to reach the gonad probably
degenerate later on. The determination of the pole cells as presumptive germ cells depends
upon the interaction of the entering energids with the cytoplasm of the posterior pole cells
(polar plasm). Likely candidates for cytoplasmic elements important for germ cell determina-
tion are polar granules, which are concentrated at the posterior pole of late stage oocytes and
early embryos.22Functionally similar posterior pole plasms, which determine germ cell
differentiation, occur in the Coleoptera and the Hymenoptera. In the Coleoptera, germ cells
become distinguishable at the time of blastoderm formation, at the posterior end of the egg.
In most Hymenoptera, germ cells first become recognizable during gastrulation or later,
forming from the mesodermal tube. In many Lepidoptera, germ cells appear at the posterior
pole just after blastoderm formation.
Germ cells (oogonia) are the only cells that normally exhibit genetic programs that lead to
the construction of eggs.

IV. OOGENESIS
The first events in oogenesis include mitosis, the onset of meiosis, and ovariole differen-
tiation. In the panoistic ovary, all oogonia (except stem line oognoia) are transformed to
oocytes, whereas in the meroistic type oogonia generate both oocytes and nurse cells.

A. EARLY EVENTS IN OOGENESIS

Since more information is available concerning oogenesis in D. melanogaster than for any
other insect, this fruit fly will be used to illustrate the early events of ovarian development that
are shared by many evolutionary advanced insects with a polytrophic meroistic ovary.6Each
Drosophila ovariole contains a collection of egg chambers in which each oocyte is one
member of a clone of interconnected cells. In brief, four consecutive mitoses of the cystoblast
and its daughter cells in region 1 of each germarium give rise to the 16 germ-line cells
(cystocytes), which remain connected via intercellular bridges due to incomplete cytokinesis
(Figure 7). King et al." suggest that the interactions of the centrioles and fusomes during the
cystocyte divisions are responsible for the multiple-branched canal system that results. In
region 2, the individual 16-cell clusters become separated by invading somatic follicle cells.
One cell with four intercellular bridges (ring canals), the prospective oocyte, moves from a
central position in the cell cluster to a posterior location between regions 2 and 3 of the
germarium. This spatial reorganization of the 16 gem-line cells results in a polarization of the
cluster: the prospective oocyte becomes positioned posterior to the remaining 15 cells, which
differentiate into polyploid nurse cells. The forming follicle has established an anteroposterior
 Insect Reproduction

FIGURE 7. Germarium and young follicles in Drosophila oogenesis. The insert shows a diagram of the steps in
the production of a clone of 16 cystocytes. By a series of four mitoses, each followed by incomplete cytokinesis, a
branching chain of 16 interconnected cells is produced. Cell 1 represents the later oocyte which moves from a central
position in the cell cluster to a posterior location. FC, follicle cell; NN, nurse cell nucleus; O N oocyte nucleus.
Photograph courtesy of H. 0 . Gutzeit, Dresden.

axis. The nurse cells grow and simultaneously transfer cytoplasmic macromolecules to the
oocyte (see Section 1V.C). The follicle cells begin to form a monolayered epithelium around
the germ-line cells in region 3 of the germarium. This process starts at the posterior end of the
follicle. At the anterior end, a special group of follicle cells forms a stalk ("stalk cells"), thus
separating the follicle from the germarium and releasing it into the ~ i t e l l a r i u m . ~ ~
In the silkrnoth Bombyx mori, there are only three cystocytes divisions resulting into eight
germ-line cells (n = 23). In the braconid wasp Habrobracon juglandis, the final number of
cystocytes per cluster is 32; in other wasps, the number is not fixed but varies from 20-80.24
Fleas with polytrophic meroistic ovarioles (some species of the Hystrichopsylloidea) have
germ cell clusters consisting of 32 cells ( F ) which are generated by five mitotic cycles during
the pupal stage. One of the cells containing five intercellular bridges becomes the oocyte; the
others serve as nurse cells. However, nurse cells remain small and show the same ultrastruc-
. ~ ~ species of lacewings do not obey the N = 2" rule.6 For
tural characters as the o o ~ y t eCertain
example, in Chrysopa perla, egg chambers contain 12-14 cystocytes. In this case, first- and
second-generation cystocytes divide in synchrony, whereas at M, (see Figure 7), cells 3,4,7,
and 8 divide; the rest do not. In the earwigs (Dermaptera), each follicle in the vitellarium
Oogenesis and the Female Reproductive System

FIGURE 8. Hypothetical diagrammatic representation of germ cell cluster formation in subgroups of Hemiptera:
(1) development begins in one persisting germ cell; (2) the germ cell divides by mitosis, followed by incomplete
cytokinesis. One of the daughter cells is determined as a presumptive nurse cell (black nucleus), the other will be a
presumptive oocyte (white nucleus); (2 a-c) germ cell division in scale insects (Coccina); (3) in other groups, the
presumptive nurse cell and the presumptive oocyte divide, giving rise to a cluster of four germ cells arranged in a
rosette configuration; (3a-c) germ cell division in aphids (Aphidina); (4) further divisions of presumptive nurse cells
and oocytes; (4 a-b) germ cell division in bugs (Heterotera), where the oocyte subclone has its divisions limited.
Asterisk = region of microtubule matter. For further details see text. (Reprinted from King, R. C. and Biining J.,
Comprehensive Insect Physiology, Biochemistry, and Pharmacology, Vol. 1, Kerkut, G. A. and Gilbert, L. L, Eds.,
Copyright 1985, ch. 3. With permission from Pergarnon Press Ltd., Headington Hill Hall, Oxford 0 x 3 . OBW, UK.)

consists of one oocyte and one nurse cell, surrounded by a single layer of follicle cells.
Formerly, the polytrophic meroistic ovary of earwigs has been looked at as a parallel devel-
opment, but new findings indicate only one origin of the polytrophic meroistic ovary (see
Section II.B).8
In all hemipteran species with telotrophic ovarioles, the germ cells are also clearly sepa-
rated into nurse cells and oocytes. The oocytes are found at the base of the tropharium (see
Figure 2C), whereas the nurse cells occupy its upper parts. Two models of germ cell cluster
formation have been proposed. Both assume that independent stem cells in the apical region
of the tropharium fuse to form germ cell clusters in which basally located germ cells are
subsequently determined as oocytes. Based on data derived from several groups of Hemiptera,
a new model has been advanced which assumes that cluster formation begins with a single
cell. The first division is a differential mitosis, leading to a presumptive oocyte and a
presumptive nurse cell. In scale insects (Coccinea), the presumptive nurse cell divides and its
apical descendant will divide again. The final configuration is a rosette of four cells in which
the intercellular bridges stay close together. During rosette formation, the intercellular bridges
then vanish, and the trophic core forms (Figure 8). The last step in cluster formation is the
polyploidization of nurse cells. In other groups, the presumptive nurse cell and the presump-
tive oocyte divide, giving rise to a cluster of four germ cells arranged in a rosette configura-
tion. In aphids, germ cells continue to divide, and subclones of 2"oocytes and 2" nurse cells
14 Insect Reproduction

arise. Rosette formation, formation of the trophic core, and polyploidization of nurse cell
nuclei are the same as proposed for scale insects. Further divisions of presumptive nurse cells
and oocytes lead to the situation in bugs (Heteroptera) with a constant number of oocytes, but
an increasing number of nurse cells.
In snakeflies (Raphidioptera) and alderflies (Megaloptera, Sialidae), the same type of
telotrophic ovary occurs. Cluster formation starts with germ cell migration into the ovariole
anlage. The number of germ cells increases as more germ cells enter the anlage and as those
already there divide. Dividing stem cells undergo complete cytokinesis next to the terminal
filament (apical region), whereas germ cell clusters arise by incomplete cytokinesis more
basally. The clusters are disc-shaped and oriented at right angles to the long axis of the
ovariole. Each cluster presumably contains 2" cells, with five as the maximum number of
division^.^ Prospective oocytes do not differ from nurse cells in their ultrastructure, except for
one fact: nurse cells lose their cell membranes totally to form a syncytium, whereas prospec-
tive oocytes remain in their original status, which they acquired at the end of cluster divi~ions.~
In polyphage Coleoptera, germ cells migrate in the ovariole anlagen, and cluster formation
is started by mitosis of each germ cell. Each germ cell can undergo only a limited number of
mitoses, each followed by incomplete cytokinesis. Later mitoses are highly synchronized
within each cluster, but not between different clusters. In most species, the clusters are
oriented parallel to the long axis of the tropharium. A three-dimensional network of interstitial
cells keeps the nurse cell nuclei in place, when nurse cell-nurse cell membranes are r e d ~ c e d . ~
Oocytes develop at the base of the tropharium, primarily connected to nurse cells by an
intercellular bridge. However, recent investigations have shown that cluster formation is more
complex than has been assumed before, and that ramifications of clusters occur, even between
oocytes.

1. Oocyte Differentiation
Immediately after the cluster of 16 cystocytes is found in a Drosophila germarium, both
four-canal cells (cells 1 and 2) form synaptonemal complexes in their nuclei during the time
they pass through germarial region 2. The synaptonemal complexes form during zygonema,
are completed during pachynema, and are responsible for the synapsis of homologous chro-
mosomes during meiotic pr~phase.~ Since cells 1 and 2 start meiosis, they are called pro-
oocytes. Sometimes cells 3 and 4 also form synaptonemal complexes and enter meiosis,
whereas cells 5-16 fail to enter meiotic prophase. In the posterior region 2, one of the two four-
canal cells loses its synaptonemal complexes and enters the cycle of endomitosis characteristic
of nurse cells. The other cell continues to develop as an oocyte and retains its synaptonemal
complexes during the previtellogenic stage of oogenesis in the vitellarium (see Section IV.B).
The divergence of the two pro-oocytes takes place in the region where follicle cells first
surround the 16-cell clone. It is suggested that the first pro-oocyte to come into contact with
a follicle cell is the one that receives the critical stimulus that causes it to continue on the
oocyte developmental pathway. Such cellular interactions between the germ-line cells and the
somatic follicle cells have been studied for a long time.26.27More recent data show that the
correct cellular organization and determination of cells in the germarium seem to depend on
the activity of several genes. For example, in Drosphila, genes like egaliterian and Bicaudal-
D are apparently involved in cystocyte diversification, since in mutant follicles a nurse cell
differentiates instead of the oocyte. The genes dicephalic and spindle-C are required for the
correct spatial arrangement of the cystocytes. Moreover, the mutant dicephalic illustrates the
importance of early cellular interactions between somatic follicle cells and germ-line cells.28
However, it remains to be analyzed which kind of specific signals the pro-oocytes receive in
the germarium.
In some carabid beetles, all cystocytes of a clone form synaptonemal complexes, and
consequently all nurse cells enter meiotic prophase together with the presumptive oocyte. In
some telotrophic ovaries, the cell which differentiates as an oocyte also seems to depend upon
Oogenesis and the Female Reproductive System 15

its position relative to certain somatic tissues. These facts again are best explained by
involving signals passed to the cystocyte by adjacent somatic cells.
In all insects with panoistic ovaries, fully activated oocyte chromosomes are required. In
these cases, the oocyte chromosomes enter a lampbrush state in the diplotene stage of meiotic
prophase. Lampbrush chromosomes also occur in many insects possessing telotrophic meroistic
ovaries.

2. Endocrine Control of Early Oogenesis

In the blood-sucking bug Panstrongylus megistus, determination of mitosis, the onset of
meiosis, and ovariole differentiation take place during the last larval instar and begin 24-48
h after a blood meal.' Observation of the neurosecretory (ns) cells of the pars intercerebralis
during the last larval instar from the time of the blood meal until imaginal moult showed the
presence of four distinct ns ceIl types with different patterns of activity. In type A cells, the
ns material is released before the mitotic crisis, whereas in type A' cells it is discharged before
the onset of meiosis. Hence the ns cells appear to trigger both mitosis and meiosis.
Electrocoagulation of pars intercerebralis prevented ovarian development and caused its
degeneration. Destroying only type A ns cells resulted in an inhibition of the mitotic crisis, but
the onset of meiosis was unaffected. Removal of the prothoracic glands showed that ecdysteroids
do not trigger the onset of mitosis, but are necessary for that of meiosis. Hemolymph
ecdysteroid determinations demonstrated two peak values; the first coincided with the onset
of meiosis, the second preceded moulting. Juvenile hormone (JH) does not seem to intervene,
either in early gonial mitosis or in the initiation of meiosis. In L. migratoria, P. americana,
and Gryllus bimaculatus (Ensifera, Gryllidae), it was shown that the meiosis reinitiation in the
vitellogenic oocyte (see Section 1V.C) is preceded by an increase of free ecdysone of the
oocytes. In vitro, the reinitiation of meiosis can be determined by the addition of e c d y ~ o n e . ~ ~
Not only the germ cells but also the mesodermal elements surrounding them have to go
through certain steps in order to develop adult structures, and these events are controlled by
hormones. It was shown both in vivo and in vitro that growth and differentiation of nurse cells
and follicle cells require the presence of 20-hydroxyecdysone, whereas JH may suppress the
effects of 20-hydroxyecdysone. In conclusion, 20-hydroxyecdysone and JH are both neces-
sary for ovariole differentiation and function, but the two hormones must act separately and
successively.

3. Follicle Cell Differentiation

During oogenesis, the follicle cells follow a characteristic differentiation program. Three
processes have been studied particularly thoroughly:

1. The production of localized developmental signals which bind to their respective

receptors in the oocyte membrane. These signals play a role in the axial determination
of the embryo.30
2. The contribution of the follicle cells to the synthesis and uptake of yolk proteins (see
Section IV.C.3).
3. The formation of the egg shell and its genetic control (see Section 1V.D).

General follicle cell morphology has been well described for the cockroach P. americana.
The earliest follicle cells are of the squamous type, with the apical ends of the cells applied
closely to the oolemma. As the oocyte grows, the follicle cells rapidly increase in number and
gradually become cuboidal. The follicle cells send out processes which interdigitate with the
microvilli of the oocyte. The cuboidal shape is maintained until prior to vitellogenesis when
the follicular epithelium becomes columnar. Similar arrangements have been found also in
other species (e.g., L. migratoria, Galloisiana nipponensis [Grylloblattodea], and ants of the
genus Formica [Hymenoptera]).At the start of the vitellogenic phase, the cell shape changes
 Insect Reproduction

FIGURE 9. Diagrammatic representation of the follicular epithelium from a Drosophila ovary (vitellogenic
follicle). The basement membrane (bm) with laminin (lam) in circular orientation is partly removed to expose the
basal face of the follicle cells. Int, high concentrations of PSP integrin at the contact site of the cells; mf, parallel
. microfilament bundles below the cell membrane which extend in the same circular direction as laminin; ics,
intercellular space. Figure courtesy of H. 0.Gutzeit, Dresden.

again to assume a somewhat spherical (P. americana) or flattened character (L. migratoria)?
Ultrastructural studies showed that the cytoplasm of the follicle cells at the late previtellogenic
and vitellogenic stages contain large numbers of mitochondria, multivesiculated bodies, and
Golgi complexes, which are characteristics of a highly active tissue. The columnar cells are
well supplied with rough ER and large amounts of ribonucleoproteins. In some dipteran and
hymenopteran insects, in addition to septate desmosomes and adhesion plaques, intercellular
bridges interconnect adjacent follicle cells. The bridges appear to result from incomplete
cytokinesis and may serve to synchronize differentiation and function of the follicular epithe-
lium. Several authors have described the occurrence of gap junctions between adjacent follicle
cells (L. migratoria), as well as between follicle cells and oocyte (R. prolixus, Tribolium
destructor [Coleoptera, Tenebrionidae], D. melanogaster)? The junctions disappear during
the chorion formation phase and during atresia.
The typical transition of follicle cell morphology from cuboidal to columnar and flattened,
with large intercellular spaces, suggests that cytoskeletal changes (microtubuli and microfila-
ments) are responsible for the cell shape transformations. The maintenance of the columnar
shape is associated with a well-organized, cylindrical orientation of the microtubular
cytoskeleton (P. americana). A random distribution of the microtubules might facilitate the
transition to a more flattened morphology. Alterations in microtubular association seem to be
juvenile hormone dependent. In Drosophila, parallel microfilament bundles were shown to be
present at the basal side of the vitellogenic follicle cells facing the basement membrane
(Figure 9). The density of the microfilament bundles increases during the course of oogenesis.
Indirect evidence from a variety of experiments using proteolytic digestion of collagen and
inhibitor studies suggests that the microfilaments are required for the adhesion of the follicle
cells to the basement membrane. In the absence of parallel microfilaments, the cells lose their
epithelia1 character and round Components of the extracellular matrix may affect the
organization of the cytoskeleton. Consequently, the parallel microfilaments, together with the
extracellular matrix glycoprotein laminin (and possibly additional components of the base-
ment membrane), act in concert in shaping the follicle.
The total number of follicle cells associated with a single oocyte varies with the develop-
mental stage and with the size of the oocyte. A vitellogenic oocyte of Leucophaea has
approximately 27,000 investing follicle cells. Drosophila follicles when first formed have 80
cells, increasing to about 1200 in mature follicles?
In Drosophila, a rather interesting additional feature is found. Once the maximum follicle
cell number is reached, some of the follicle cells undergo a series of migrations (Figure 10).
Oogenesis and the Female Reproductive System

FIGURE 10. Diagrammatic representation of Drosophila midvitellogenic follicle, indicating follicle cell migratory
pathways (1-5). FC, follicle cell; NC, nurse cell; 0, oocyte; ON, oocyte nucleus. (From Kaulenas, M. S., Insect
Accessory Reproductive Structures. Function, Structure, and Development, Springer-Verlag, Berlin, 1992. With
permission.)

The various cell migrations are microtubule de~endent.~' In addition to the involvement of
microtubules, the large, steady electric currents which have been shown to traverse the
follicles are proposed to direct the follicle cell migration.32
In biological systems, electric current is carried by ions, not by electrons. Ion asymmetries
within the follicle, which may be associated with the electrical phenomena, have been
confirmed, e.g., in Drosophila and Hyalophora cecropia (Lepidoptera) meroistic ovaries. In
the Cecropia moth, a potential difference of about 6 mV is observed over the 30-pm-wide open
cytoplasmic bridge which interconnects the oocyte with a trophocyte. The oocyte is at positive
potential as related to the tro~harium.~~Most studies of currents associated with the vitellogenic
phase of an oocyte have reported an anterior inward current and a posterior outward directed
current, rotationally symmetric about the oocyte's long axis. In polytrophic ovaries, the
anterior to posterior currents are suggested to provide an electrophoretic force for distributing
negatively charged nurse cell products to the vitellogenic oocyte. In panoistic ovaries, currents
in this type of oocyte might play a role in the intracellular distribution of cell organelles or
products throughout the oocyte (see Section 1V.C).
Before or during the vitellogenic stage, the follicular epithelia] cells become polyploid. In
panoistic ovaries, an exact doubling of DNA during polyploidization is typical, whereas in
meroistic ovaries, polyploidization is generally not exact. For example, in Drosophila hydei,
DNA sequences for the ribosomal RNAs are severely under-replicated, and it has been
suggested that polyploidization increases the concentration of those genes which play impor-
tant roles in follicle cell function^.^ All the major functions of the follicle cells seem to be
juvenile hormone dependent.

4. Trophic Function of Nurse Cells

In both the polytrophic and the telotrophic ovaries, the nurse cell-oocyte syncytium is a
polarized structure, with the site of "nutrient" formation distinct and separate from the
18 Insect Reproduction

recipient cell, the oocyte. The most significant difference between the two ovary types is the
greater separation between donor and recipient poles in the telotrophic ovary, making it more
difficult to use diffusion as a transport mechanism for nutrients.
The trophic function of the nurse cell is enhanced by endoreplication of the DNA. In
Drosophila, the nurse cells begin their cycle of endomitotic DNA replication in region 3 of
the germarium. In the vitellarium, each nurse cell undergoes another seven replications. The
maximum level of polyploidy reached by Drosophila nurse cells is 2'O. In the giant moth
Antheraea polyphemus, each of the seven nurse cells reaches ploidy levels approaching 216.34
In most polytrophic meroistic ovaries, even in Drosophila, an apical-basal gradient in
polyploidization exists among nurse cells. Highest ploidy levels are found in basal nurse
cells.35Whereas in panoistic ovaries an exact doubling of DNA content during polyploidization
seems to be typical, in meroistic ovaries, polyploidization is generally not exact, but some
sequences (e.g., ribosomal RNA genes, histone genes, telomere sequences, satellite se-
quences) can be under-re~licated.~~ Very young nurse cell nuclei have distinct polytenic
(giant) chromosomes, but chromosomes cannot be distinguished in large nuclei, as different
sections are replicated to varying degrees. In the last endoreplication cycle, all sequences,
including the previously under-replicated, replicate fully. In telotrophic ovaries, trophocyte
nuclear DNA also undergoes multiple rounds of duplication - a total of seven in Dysdercus
intennedius (Heter~ptera).~'
In the case of polytrophic ovaries, all or most of the nurse cell cytoplasm is transferred to
the oocyte towards the end of oogenesis, whereas for telotrophic ovaries the process could be
more selective. Among the major products accumulated by oocytes are large quantities of
mitochondria. Also produced are massive stores of ribosomes, which are used by embryonic
cells during the early periods of development, when little or no rRNA synthesis takes place.
In most meroistic ovaries, the expanded ribosomal gene numbers in the polyploid nurse cells
provide sufficient templates for the massive rRNA synthesis. In a few cases, however,
additional extrachromosomal rDNA amplification is encountered, as in water beetles.38 A
large variety of nonribosomal transcripts is also synthesized in nurse cells and transferred to
the oocyte. Among the most interesting and important gene transcripts synthesized in nurse
cells and stored in oocytes are those which specify embryo p ~ l a r i t y . ~Other
~ . " ~mRNAs which
are transcribed in the nurse cells and later transferred to the oocyte include those for heat-
shock protein^.^'
Despite a substantial amount of work, the mechanisms of transport of macromolecules
from the trophic cells to the oocyte remain to be totally defined. In polytrophic ovaries, the
total nurse cell cytoplasm flows through the ring canals into the oocyte during the final phase
of vitellogenesis. Electrophysiological studies have shown that electrophoresis may regulate
the distribution of charged molecules between the nurse cells and oocyte. The electrophoretic
current from nurse cells to oocyte is driven by the voltage gradient produced by an egg
chamber (see Section VI.A.3). Since the equilibrium potential of the nurse cells is several mV
more negative than that of the oocyte, macromolecules carrying a net negative charge may be
carried by electrophoresis to the oocyte. However, some authors have failed to demonstrate
intercellular electrophoresis in movement of materials in polytrophic ovaries. The potential
difference between nurse cells and oocyte, therefore, may serve primarily as a regulatory gate
effect rather than providing the principal force for macromolecule transport? In a variety of
polytrophic ovaries, the nurse cell cytoplasmic streaming can be reversibly inhibited by
cytochalasins, so it is likely that microfilament contraction plays some role in the cytoplasmic
streaming phenomenon, possibly by squeezing the nurse cell contents into the ~ o c y t e . ~ ~
However, mechanisms for capturing specific regionalized compounds in the oocyte must also
exist.
In telotrophic ovarioles, the distance between the place of synthesis of macromolecules (the
trophic cells in the tropharium) and the place of deposition (the growing oocytes in the
vitellarium) can be enormous (see Figure 2). Therefore, most workers have assumed that
Oogenesis and the Female Reproductive System 19

molecules are actively transported to the oocyte. After the discovery of a system of parallel
microtubules in the nutritive cords of heteropterans, suggestions were advanced that these
organelles may play a role in the active transp~rt.~ However, various species of polyphage
Coleoptera have nutritive cords that lack microtubules, and in other insect species, the
microtubules in the nutritive cords are randomly oriented. Another favored mechanism for the
transport of molecules involves electrophoresis and a flow of material assisted by differences
in hydrostatic pressure between the trophic area and the oocyte, which may be created by ionic
current asymmetries around the o ~ a r i o l e sBesides
.~~ active transport, peristaltic movement of
the musculature of the epithelia1 sheath which surrounds the ovariole may cause some
cytoplasm to flow from the tropharium to the oocytes. Additional work is required to resolve
all the components acting during macromolecule transport in polytrophic as well as in
telotrophic ovaries.

B. PREVITELLOGENESIS
The period when young oocytes enlarge by incorporation of nurse cell material (see Section
IV.A.4) is called the previtellogenic growth phase, or just previtellogenesis. Previtellogenesis
is difficult to investigate because it takes place chiefly in the penultimate oocyte during
vitellogenesis of the terminal oocyte and is thus simultaneously subject to its own control and
the control exerted on the terminal oocyte. Generally, previtellogenesis begins in young
adults, late in pupal development, in nymph, or last instar larvae. However, in insects with
panoistic ovarioles, previtellogenic growth begins during earlier nymphal stages. In those
insect species which hibernate as adults, growth of the oocytes may be stopped at the
beginning of previtellogenesis. The arrest and the onset of previtellogenesis are part of the
adult diapause and may be under hormonal control1 (see Chapter by Hardie).

C. VITELLOGENESIS
Vitellogenesis is the most important metabolic event in the adult life of the female insect.
The vitellogenic growth phase begins when yolk spheres are first observed to accumulate in
the oocytes. Vitellogenesis often occurs in the adult insect but also may take place earlier. For
example, in Sialisflavilatera (Megaloptera), vitellogenesis begins early in pupal life. Such a
shift into preadult stages will become necessary when the adult lives only a few days, as in
species with polytrophic meroistic ovaries that do not feed as adults. In many insects, however,
vitellogenesis and egg production is dependent on food availability. An extreme example of
a cyclic yolk production with feeding as an initial trigger for vitellogenesis is found, e.g., in
Aedes, Phormia, or Rhodnius (anautogenous insects).
Vitellogenesis involves the production of female-specific proteins termed vitellogenins
(vg) and their entry into the oocyte. When vitellogenin is taken into the oocyte, it is processed
to vitellin (vn). The vitellogenins are mostly produced in the fat body but may be also
produced in the ovary. They are transported by the hemolymph, in which their titer is high
during vitellogenesis, and accumulate in the oocyte against a concentration gradient 20-100
times their concentration in the hemolymph. In most species, vitellins comprise 60-90% of the
total soluble egg yolk protein. As noted above, vitellogenesis occurs in the terminal oocyte
within an ovariole, yet in many species the process is highly synchronized among ovarioles
and between ovaries. The synchronization results in a production of egg batches. In some
females, vitellogenesis in the penultimate oocyte appears to be inhibited even after the
terminal oocyte has completed its yolk deposition and has become chorinated, provided that
the mature egg is not laid.

1. Vitellogenin and Vitellin Chemistry

Data on yolk protein chemistry have expanded since new techniques of DNA analysis are
providing cloned cDNA and genomic-DNA with which the relationships between vitellogenin
genes and the final secretion products can be studied (see Section IV.C.2).
20 Insect Reproduction

In all insect species investigated, vgs and vns are irnrnunochemically identical. They are
glycolipophosphoproteins of native molecular masses ranging between 190 and 650 kDa and
often are composed of several polypeptides of variable sizes. In some species, endogenous
proteolytic cleavage changes the pattern of vn peptides compared to vgs. In Leucophaea
maderae, B. germanica, and L. migratoria, there seem to be large precursor molecules that are
proteolytically cleaved either in the fat body, hemolymph, or oocyte it~elf.4~ Also, subtle
differences in vg and vn lipid and carbohydrate moieties may exist. However, the differences
between vg and vn are small and, therefore, the chemistry of both polypeptides will be
discussed together.
Cloning the vitellogenin genes enables the putative amino acid sequences of the primary
products to be determined; but only complex protein chemistry will unravel the processing and
modification of these molecules during their secretion, transport, uptake, and depo~ition.4~
Harnish and White46have characterized the vitellins of a number of insect species and report
the existence of three definable groups. The largest group (group I) comprises insects from
several orders, including Ephemeroptera, Orthoptera, Dictyoptera, Hemiptera, Demaptera,
Coleoptera, and Lepidoptera. The native proteins are between 380 and 470 kDa, and upon
denaturation two distinctly different size classes of subunits are released. The high molecular
mass group ranges from 100-180 kDa, the low mass group from 43-86 kDa. The simplest
patterns exhibit one polypeptide in each size class, but several in each class is very common.
Group I1 vitellins occur in the orders Hymenoptera (e.g., Apis mellifera) and from the more
ancient dipterans, the suborder Tipolomorpha (Aedes aegypti). In denaturing SDS gels, group
I1 vitellins release only high molecular weight polypeptides. The third type (group 111) is found
only in higher Diptera (e.g., Drosophila, Calliphora, Lucilia). These proteins appear to have
molecular masses of about 200 kDa and are composed entirely of small polypeptides of about
50 kDa. The evidence for this schema, however, is not entirely convincing.
Carbohydrate has been found covalently linked to purified vgs and vns in every instance
it has been sought. The average carbohydrate content is 1-1 1%. In many cases, mannose and
glucosamine were the only identified sugars involved. In group I proteins, the oligosaccha-
rides are attached only to the heavy subunit. Lipids are also integral to all vgs and vns
characterized (7-15%). Phospholipids, diacylglycerides, and cholesterol comprise the bulk
lipid components. Vns appear to contain less lipids than vgs. In locust eggs, conjugated
ecdysteroids have been found noncovalently bound to vitellin.
Esterified phosphate is another integral part of some vgs and vns. In L. maderae vitellogenin,
the covalently attached phosphorus is distributed in an uneven fashion among the five
subunits. Phosphorylation of vitellogenin occurs posttranslationally in the fat body endoplas-
mic reticulum.
The fat body of vitellogenic mosquitoes was found to synthesize and secrete another
protein, in addition to vitellogenins, that accumulated in developing oocytes. This 53-kDa
protein is glycosylated, and immunoblot analysis demonstrated the immunological identity of
the 53-kDa polypeptides from the fat body and the ovary.47In eggs of some lepidopteran
insects, vitellin comprises only half of the total yolk proteins, and the yolk contains significant
amounts of other kinds of proteins. Silkworm (B. mori) eggs contain a vitellin (M, 420 kDa)
belonging to group I which consists of two heavy subunits (178 kDa) and two light subunits
(42 kDa). The second major yolk protein group is composed of non-sex-linked serum proteins
with a molecular mass of 30 kDa. They are also produced in the fat body, released into the
hemolymph, and finally sequestered into developing oocytes. Bombyx 30-kDa proteins are a
mixture of three monomers (29.5 to 32 kDa) and contain various lipids and carbohydrates. The
third main protein of silkworm eggs is the so-called egg-specific protein, which is produced
by the ovary itself and accumulates especially in developing oocytes. The egg-specific protein
is a trimer (225 kDa) of two heavy subunits (72 kDa) and one light subunit (64 kDa).48
Oogenesis and the Female Reproductive System 21

2. Vitellogenin Genes
A large amount of information has been developed in the last decade on vitellogenin gene
sequences. Most of the information is on the genes of Drosophila. In Drosophila, three distinct
genes coding for yolk proteins are present, namely, YP1, YP2, and YP3. The genes were
shown to be single copy, with the YPl and YP 2 closely spaced and YP3 approximately 1000
kilobases (kb) distant on the X chromosome. YP1 and YP3 each have a single species of
transcript, of about 1.6 and 1.54 kb, respectively. YP 2 produces transcripts of two sizes, 1.59
and 1.67 kb. All three genes have been ~ e q u e n c e dWith
. ~ ~ the availability of base sequences
for the yolk protein genes, more recent work has been concentrating on their regulation40(see
also Section IV.C.3).
A. aegypti and L. migratoria are two other insects in which the yolk protein genes have been
cloned. In both, the primary transcripts are very large, over 6000 nucleotides in length. The
results on A. aegypti suggest that there may be a total of five different vitellogenin genes.50
Sequence information and analysis of any gene control region are not yet available. In L.
migratoria, two genes, VgA and VgB, coding for vitellogenins have been identified, with only
little homology between them (at the 5' ends)." Both are located in the X chromosome. The
homologous regions in their 5' flanking sequences may be important for their control by
juvenile hormone.

3. Vitellogenin Synthesis
The fat body is the major, and in many cases, the only site of vitellogenin synthesis. Among
some of the Holometabola (Diptera, Lepidoptera, Coleoptera), however, the ovarian follicle
cells are also involved in yolk protein production. In the Diptera, the same structural genes,
synthesizing identical proteins, are active in both the fat body and the follicle cells.
Three cell types are commonly found in the insect fat body - trophocytes, urocytes, and
mycetocytes. The trophocyte is the principal cell type and it functions in a metabolic and
storage capacity. The cells are characterized by the presence of lipid droplets, protein spheres,
and glycogen granules in a metabolically active cytoplasm.16 The majority of the trophocytes
are found at the periphery of the fat body lobe. This distribution allows the trophocytes to
absorb or release products efficiently into the hernolymph.
In the majority of insects, juvenile hormone appears to be the key element in the control
of yolk protein production (see Chapter by H ~ d i e )The . ~ ~primary mode of action for this
hormone is at the fat body by initiating vitellogenin synthesis, with a secondary function in
the regulation of yolk uptake by the ovary (Section IV.C.5). The induction of vg synthesis by
JH in the fat body provides a system of hormonal control of gene expression. In response to
JH stimulation, the nucleus of the fat body trophocytes enlarges, while the cytoplasm develops
extensive rough ER and Golgi complexes. At the macromolecular level, the cells undergo
rapid synthesis of DNA, RNA, and protein in response to JH. In L. migratoria, a primary
stimulation by JH or JH mimics results in a rapid synthesis of rRNA, while the accumulation
of vitellogenin mRNA can be detected only after a lag phase. A second dose of JH leads to
a more rapid accumulation and translation of vg mRNA, but lowers the production of rRNA.
The picture of JH action obtained in insect fat body parallels the finding for steroid hormone-
stimulated vitellogenin synthesis in vertebrate systems.52
Since JH is a terpenoid and therefore different in chemical structure from that of a steroid
hormone, it is of interest to know whether a specific JH receptor is present in the cytosol and
nuclei of the target tissues. Cytosolic receptors may function in the translocation of the
hormone to nuclear acceptor molecules, the latter being essential for the initiation of gene
tran~cription.~~ Cytosolic fat body preparations of L. maderae adults contained a population
of JH binding compounds with a high affinity (K, ca. 1 nM),which could not be found in
nymphal tissues. A JH binding compound with similar affinity was extracted from nuclei of
22 Insect Reproduction

vitellogenic fat body cells of L. maderae. Putative juvenile hormone receptors have also been
identified in locust (L. migratoria) fat bodies. In the absence of JH, the adipokinetic hormone
(AKH-I), which is involved in mobilizing diglycerides, may inhibit vitellogenin gene expres-
sion in the locust fat body.54Signals from the ovary are supposed to terminate vitellogenin
synthesis in the fat body since ovariectomized females continue to produce vitellogenin,
which accumulates in the hemolymph. These signals may operate via modulating the activity
of the corpora allata.5s
The majority of insects conform more or less to the regulating scheme described above, but
some display significant variations. For example, among Coleoptera, while JH is necessary to
set off the initial vitellogenic response, continued yolk production then becomes autonomous.
In many Lepidoptera, vitellogenesis appears to be a part of a programmed developmental
response to metamorphosis. Among the Hymenoptera, honeybee queens show no dependence
on JH or ecdysone for the production of vitellogenins. In some Hemiptera, ecdysone seems
to be responsible for triggering elevated levels of yolk protein production.
The induction of vitellogenin synthesis is normally limited to adult females. It is possible,
however, to induce vitellogenin synthesis in adult males and in nymphs with large doses of
juvenile hormone or JH analogues. This has been demonstrated for L. migratoria and several
species of Di~tyoptera.~~ In male fat bodies of some Diptera, vitellogenin synthesis could be
induced by 20-hydroxyecdysone, and not by JH.57Female- and male-produced vitellogenins
may be different in their polypeptide compositions.
Details of ovarian yolk protein production are best understood for Drosophila. Using a
radioactive labeled probe containing the coding regions of yolk protein genes and in situ
hybridization techniques, it has been shown that the follicular epithelium is the specific site
of vitellogenin synthesis. The maximum level of yolk protein synthesis by the follicle cells
occurs in early vitellogenic stages. The follicular epithelium contributes ca. 35% of the yolk
proteins 1 and 2 to the total oocyte content, but only about 10% of the yolk protein 3
polypeptide. Since all three yolk proteins are transcribed at similar rates, yolk protein 3 mRNA
seems to be destabilized in the ovarian follicle cells, accounting for the reduction in its steady
state level. In the housefly Musca domestica, at the start of vitellogenesis the fat body appears
to be the main site of vitellogenin synthesis; later, the dominant role is taken over by the
ovaries. Overall, the follicle cell contribution of yolk proteins to the oocyte exceeds that of the
fat body.
As in the Diptera, the yolk proteins of the Lepidoptera are synthesized in the fat body and
the ovarian follicle cells. In Lepidoptera, however, the follicle cell vitellogenins are the
product of genes different from those responsible for vg synthesis in the fat body. In a moth,
Plodia interpunctella, fat body and follicle cell yolk proteins show no immunological cross-
reactivity, either as native proteins or as individual subunits.
Diptera also handle the hormonal regulation of vg synthesis somewhat differently from
most other insects (see above). 20-Hydroxyecdysone appears to be the main hormonal trigger
in the activation of the vitellogenin genes, but JH is involved in facilitating 20-hydroxyecdysone
action in the fat body and in the regulation of yolk protein uptake by the ovary58(see Chapter
by Hardie). In mosquitoes, an oostatic hormone may act to inhibit vitellogenin p r o d u c t i ~ n . ~ ~

4. Vitellogenin Secretion
The mechanism for the export of the yolk proteins both from the fat body and the follicle
cells involves the usual route through the Golgi and exocytosis at the plasma membrane. The
carbohydrate moieties may confer a certain degree of stability to the protein subunits, ensuring
proper assembly or preventing aggregation prior to secretion.60In a cockroach, B. gennanica,
the vg precursor accumulates but is not secreted when the animal is treated with tunicamycin.
Similar observations were done on the export of fat body proteins in Galleria mellonella
(Lepidoptera). In dipteran follicle cells, the export mechanism can be disrupted by colchicine
and other microtubule inhibitors, suggesting an important role of these cytoskeletal compounds.
Oogenesis and the Female Reproductive System 23

Yolk proteins excreted from the follicle cells are not normally liberated into the hemolymph
at large, but possibly are presented directly to the oocyte surface.

5. Uptake of Vitellogenin by the Ovary

Because of the heterosynthetic nature of vitellogenins in most insects, the oocytes are
highly specialized for the specific accumulation of the fat body vitellogenins. The pathway for
internalization of vitellogenins and other yolk protein precursors is similar in all types of
ovarioles. The onset of vitellogenic uptake is characterized by the formation of gaps and
spaces between the follicle cells (Figure 11). Many researchers have utilized various dyes and
tracers to demonstrate that this interfollicular route of uptake is universal among insects.
Vitellogenins appear to concentrate in the perioocytic space and from there are later taken up
by micropinocytosis of the oocyte membrane. The enlargement of the interfollicular channels
occurs at the onset of vitellogenesis and has been termed patency. In R. prolixus, this condition
has been shown to be a development in response to juvenile hormone. In other insects, JH also
seems to be involved in yolk protein uptake, but in these cases the precise role of the hormone
is not entirely clear (see Chapter by Hardie). The follicle cells lose patency at the time of
cessation of vitellogenesis and the deposition of the chorion.
Current evidence clearly points to yolk protein uptake being a receptor-mediated process.
The uptake is highly selective for vgs, saturable, species specific, energy dependent, and
sensitive to conditions of pH, temperature, and divalent cation concentrations (Ca2+).Studies
on binding of vg to membranes isolated from follicles have been made on only a few insects.
Binding under equilibrium conditions demonstrated the existence of a saturable, single class
of binding sites of follicle or ovary membranes (except for the cockroach Nauphoeta cinerea,
where two separate binding sites for vg have been suggested). The dissociation constant (K,)
varied between 100 nM (for L. migratoria) to 13 nM (for Manduca sexta). In L. migratoria,
the vitellogenin receptor protein has been isolated and purified by immunaffinity chroma-
t~graphy.~' It is an acidic, negatively charged (p1 = 3.5) 180 kDa glycoprotein (nonreducing
conditions) with large amounts of N- and 0-linked oligosaccharides,among them neuraminic
acid, which has been found to be essential for receptor function. The vg receptor was localized
in oocyte membranes and in endocytotic vesicles. So far, vg receptors of two other insects
have been visualized using the ligand blotting technique, from N. cinerea (M, = 200 kDa) and
A. aegypti (M, = 205 kDa).62The fact that the receptors bind vg only when separated under
nonreducing conditions suggests that disulfide bonds within the receptor molecule are neces-
sary for its biological activity.
Receptor-bound vitellogenins accumulate in specialized regions of the surface membrane,
the coated pits, and are then internalized by the formation of coated vesicles (Figure 12).
Coated vesicles are suggested to be universal organelles for a specific macromolecular
transport in eukaryotic cells. The characteristic feature of the coated vesicles is an outer
proteinaceous polyhedral cage enclosing its membrane. The major protein of this cage (50%
of the total protein) is "clathrin." Three molecules of clathrin heavy chain (M, 180 kDa)
together with three molecules of clathrin light chain (33 and 36 kDa) form a subunit of the coat
called triskelion. The triskelions are self-assembledinto the polyhedral cage on the membrane
of the coated pits. Once the coated vesicle is formed and pinched off the plasma membrane,
rapid uncoating follows, releasing the vesicle and allowing the coat components to recycle to
the plasma membrane.62 The released vesicles fuse with endosomes. Within the endosomal
compartment, the adsorbed yolk precursors dissociate from the membrane to become homo-
geneously distributed in the lumen. As a final transformation, the transitional yolk body
changes into a mature yolk body with the crystallization of the vitellins. Mature yolk bodies
will store the yolk proteins until initiation of embryonic development, during which they are
utilized.
The developmental processes of vitellogenin synthesis and vg uptake by the maturing
ovaries occur independently of the presence of conspecific males. However, in Drosophila,
24 Insect Reproduction

FIGURE 11A

FIGURE 11B

FIGURES 11A and B. Follicular epithelium in Blatfella germanica oocytes. Sections at the equatorial zone in
oocytes from 3-day-old (A) and 5-day-old (B) females with large intercellular spaces (arrows) in (B). Photographs
courtesy of X. Belles, Barcelona.
Oogenesis and the Female Reproductive System

FIGURE 12. Schematic representation of the vitellogenin internalization pathway in mosquito oocyte: cl, clathrin;
cp. coated pit; CV,coated vesicle; end, vesicular endosome; FC, follicle cell; Itc, tubular endosome labeled positively
with anti-vg antibodies; mv, microvilli; myb, mature yolk body; rc, receptor; tyb, transitional yolk body; utc, tubula~
compartment labeled negatively with anti-vg antibodies; vg, vitellogenin. (Reproduced, with permission, from the
Annual Review of Entomology, Vol. 37, p. 217, 01992 by Annual Reviews Inc.)

it has recently been shown that mature males significantly accelerate the onset of vitellogen-
esis, and thus ovarian maturation overall, by about 4 days. Although the proximate stimulus
is not known, it is conceivable that social signals from the males (during courtship attempts)
elicit changes in the hormonal levels of females, thereby initiating the events leading to
vitellogenesis. Again, this effect of male behavior on female reproductive biology is similar
to that reported in vertebrate^.^^

D. CHORIONIZATION
When vitellogenesis is completed, the vitelline membrane and, later, the chorion (eggshell)
are formed (Figure 13).
26 Insect Reproduction

FIGURE 13. Follicle cells from a basal oocyte of Blattella germanica at late chorion formation. The perioocyte
the innerchorion layer(1CL) and the complex outer chorion layer (OCL)
space contains the vitelline membrane (VM),
showing columnar projections (P). IS, intercellular space; L, lipid droplet. Photograph courtesy of X. Belles,
Barcelona.

1. The Vitelline Membrane

Early observations suggested that the vitelline membrane was produced by the oocyte
itself. This may be the case in some species, for example, in grasshoppers and Lepid~ptera.~
However, more recent studies have demonstrated that the follicle cells secrete material which
forms the vitelline membrane in a large number of insects. Although differences in details of
this process have been encountered in all of the systems, vitelline membrane production is
preceded by an extensive hypertrophy of rough ER and formation of Golgi complexes,
followed by an accumulation of secretory granules (vitelline membrane body precursors) in
the apical zones of the follicle cells. In Simulium vittatum and other Diptera, the build-up of
the ER and Golgi vesicles occurs at midvitellogenesis.By the time that vitellogenesisis almost
complete, the Golgi complexes, containing both dense and fibrous materials, become even
more prominent, and the previtelline membrane secretory substance begins to be deposited
between the follicle cells and the oocyte. The secreted material then begins to coalesce and
gradually forms the vitelline membrane (see Figure 13). Most of the ultrastructural observa-
tions of vitelline membrane formation in exopterygotes support the above outline for the
process.
Electron microscopical studies on vitelline membrane formation in L. migratoria showed
that this structure is composed of two ultrastructurally distinguishable components, the
vitelline membrane bodies (VMB) and a fine granular material which eventually cements the
VMBs together to give the complete vitelline membrane. The observation that, in L. migratoria,
the first condensation of VMBs occurs in the vicinity of the oocyte membrane may indicate
that the oocyte is the source of VMB secretion, whereas the second vitelline membrane
component is clearly the product of the follicle cells. Other support for a dual origin of the
vitelline membrane has come from studies on Lepidoptera. In the butterfly Calpodes ethlius,
a distinct vitelline membrane is detectable at the end of the yolk uptake phase. It has an
electron-dense layer away from the oocyte and an electron-lucent layer apposing the oocyte.
Both the oocyte and the follicle cells contain coated vesicles, which appear to be in the process
of exocytosis. The electron-dense material seems to be exocytosed from the follicle cells,
whereas coated vesicles at the oocyte surface exocytose the electron-lucent granular material.
Oogenesis and the Female Reproductive System

micropylar
openings

inner opening

C vitelline membrane

---...-. .-..
endochorion

~ m i c r o n v l a r canal

FIGURE 14. Egg of Locusfa migraforia with a section through the chorion along the micropylar axis. (From
Gillott, C., Entomology, Plenum, New York, 1980, chap 19. With permission.)

Once fully formed, the vitelline membrane is completely electron dense.64 A definitive
decision on the source of the various contributors to the vitelline membrane cannot be made
until it can be demonstrated, with molecular biological techniques now becoming available,
that definitive vitelline membrane proteins are synthesized by the oocyte.

2. Chorion Formation
The chorion is usually secreted entirely by the follicle cells and can be seen to comprise
two main layers, an endochorion adjacent to the vitelline membrane and an exochorion
(Figures 13 and 14). In some insects, e.g., Acrididae, the shell takes on a third layer, the
extrachorion, as an oocyte moves through the common oviduct. Although the follicle cells are
mesodermal derivatives, the chorion is cuticle-like in nature and contains layers of proteins
and lipoproteins, some of which are tanned by polyphenolic substances released by the cells.
With chorion secretion, the follicle cells complete their duties and then die.
At the physiological level the chorion functions in protecting the oocyte from mechanical
stresses, such as from predators, as well as environmental stresses, such as dessication and
drowning, while at the same time permitting gas exchange and sperm ~enetration.~~ In some
species, a wax layer is formed immediately above the vitelline membrane by the coalescence
of oil droplets secreted by the follicle cells which renders the chorion waterproof. Viewed
from the perspective of the oocyte, the next structure is the basal or inner chorion layer
(crystalline chorion layer). The crystalline chorion layer, although flexible, puts a limit to the
volume that the oocyte can achieve. Speculations on other functions include a role in the
confinement of the wax layer, as well as allowing for gas exchange, through plastron
28 Insect Reproduction

respiration or directly. Distal to the basal chorion is the trabeculate layer which is considered
to be a part of the endochorion. The trabeculate layer is characterized by the presence of
cavities and pores. The small pores may be formed by the withdrawal of follicle cell processes
during andlor after the deposition of this layer. The cavities may interconnect and form
extensive channels. The channels may serve as air spaces and open to the exterior via
aeropyles (gas exchange). In other cases, the cavities are filled with a mucus-like substance
which serves as a reserve to surface-localized adhesive material used to attach eggs to the
substrate. The outermost layer of the endochorion, if present, is characterized by the presence
of lamellae, based on the helicoidal arrangement of stacks or fibrils. The lamellar layer may
be traversed by pores to the exterior. In many Lepidoptera, the lamella layer serves as the outer
portion of the shell. In most other insects, however, an exochorion is present. The exochorion
consists largely of mucoprotein and contains polysaccharides. In some insects, a ring of
follicle cells near the anterior end of the oocyte secretes no exochorion, so that a line of
weakness is created at this point, which facilitates hatching. Also, certain follicle cells appear
to have larger than normal microvilli which, when withdrawn after chorion formation, leave
channels to permit entry of sperm (micropyles; see Figure 14).
The molecular analysis of chorion formation has advanced rapidly. Recent reviews on the
biochemistry of chorion proteins, chorion gene structure, and chorion gene expression were
presented by Regier and K a f a t o ~and~ ~by Kaulenas9
In general, it is assumed that choriogenesis is independent of hormonal control and is
initiated at an appropriate late stage of vitellogenesis in response to local signals. However,
some information is available that shell formation might depend on brain neurohormones,
juvenile hormone, and 20-hydro~yecdysone.'.~~,~~ In L. migratoria, ecdysteroids are synthe-
sized by the epithelium of the follicle cells at around the time of chorion formation during a
short period of 8-12 h. However, ecdysteroid synthesis in follicle cells can also occur earlier,
particularly in ovoviviparous or viviparous species, such as in cockroaches and in Glossina.
Besides helping to regulate chorionization, at least part of these ecdysteroids enter the oocyte,
where they are mostly present as polar or apolar conjugates and seem to control the first events
in embryogenesis. In a cricket, G. bimaculatus, coincident changes in ecdysteroid production
for both the ovaries and the abdominal integument were observed.68The role of epidermal
ecdysteroids during oogenesis is not yet clear.

V. OVULATION AND OVIPOSITION

Ovulation includes the opening of both the oocyte follicle and interfollicular tissue, and
involves contractions of ovarioles, pedicels, and oviducts resulting in the expulsion of the egg
from the ovary into the oviducts. The follicle then collapses and the follicular cells undergo
autolysis. The degenerating follicular cells are generally referred to as a "corpus luteum", and
structures of this type have been described in several insect species.69Ovulation may occur
just before oviposition, or a larger period of time may elapse between the two processes.
Several species, including cockroaches and flies, are ovoviviparous and retain their eggs in the
genital pouch without supplying them with anything but water. Viviparous insects, on the
other hand, supply food to their progeny, either from modified follicle cells or from the
accessory glands.
Although in many insects ovulation is rapidly followed by oviposition, there are indications
that the two processes are separately'controlled. In R. prolixus, ovulation is a result of ovarian
motility induced by a neurosecretory myotropin released from the corpus cardiacum. The
hormone is released in mated females when mature eggs are present in the ovary, as signaled
by an increase in ecdysteroid titer in the hemolymph. In other species, ovulation seems to be
controlled in a similar way.
Oogenesis and the Female Reproductive System 29

In some insects, oocyte resorption, termed oosorption, may take place under various
unfavorable environmental conditions. Starvation or the lack of food, mating, or a suitable
place to oviposit are frequent causes, but factors such as temperature and change in photope-
riod, social life, or maternal care also induce oosorption. In general, oosorption occurs when
the external factors do not allow either the survival of eggs and larvae or egg deposition.*O
Oosorption may occur either in young previtellogenic oocytes or in vitellogenic oocytes, and
even in chorionated eggs. Oocytes may grow to a certain size and then stop, while the follicle
cells begin to change from cuboidal to irregular shapes. The transport of material from the
interfollicular spaces to the oocyte ceases, owing to the breakdown of the microvilli of the
oocytes and follicle cells. Hydrolytic enzymes produced in the follicle cells cause oocyte lysis,
breaking down first the protein and then the lipid yolk globules, penetrating the oocyte and
finally themselves degenerating. Often it can be observed that only certain follicles degener-
ate, while others continue to develop. The appearance of oosorption processes is apparently
caused by a decrease in the activity of the corpora allata, but the brain certainly intervenes too,
by regulating corpora allata functioning and/or acting humorally on the ovary.

VI. CONCLUDING REMARKS

The development of the female reproductive system is reasonably well understood at the
structural or morphological level across a wide range of insect species. Details of the molecu-
lar mechanisms involved in the development processes, and especially of the genetic control
of gene expression, have been explored in a much lower number of insects. Insect oogenesis
comprises many stages which are regulated by certain humoral factors, including neurohor-
mones, juvenile hormone(s), and ecdysteroids, whose importance and modes of intervention
vary depending on the species. Detailed knowledge on female reproductive biochemistry,
physiology, and endocrinology in a great many insects will be necessary, e.g., to provide a
basis for using insect hormones, hormone agonists, or hormone antagonists as "third genera-
tion pesticides" in insect pest control. Beyond that, ovarian development and oogenesis are
affected by environmental factors, including temperature, humidity, photoperiod, or the
finding of food. Therefore, it seems impossible to study the physiology and endocrinology of
insect reproduction without taking into account its ecological conditions.

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30 Insect Reproduction

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38. Dittmann, F., Steinbriick, G., and Miinz, A., Amplification of tropharium rDNA in the telotrophic ovariole
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43. Diehl-Jones, W. and Huebner, E., Pattern and composition of ionic currents around ovarioles of the
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44. Della-Cioppa, G. and Engelmann, F., The vitellogenin of Leucophaea maderae. Synthesis as a large
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49. Yan, Y. L., Kunert, L. J., and Postlethwait, J. H., Sequencehomologies among the three yolk polypeptides
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Wolfner, M. F., and Hagedorn, H. H., Isolation of mosquito vitellogenin genes and induction of expression
by 20-hydroxyecdysones, Insect Biochem., 16, 761, 1986.
51. Locke, J., White, B. N., and Wyatt, G. R., Cloning and 5' end nucleotide sequences of two juvenile
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52. Hagedorn, H. H. and Kunkel, J. G., Vitellogenin and vitellin in insects. Annu. Rev. Entomol.. 24,475, 1979.
53. Engelmann, F., Regulation of vitellogenesis in cockroaches, in Cockroaches as Models for Neurobiology:
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58. Kelly, T. J., Adams, T. S., Schwartz, M. B., Birnbaum, M. J., Rubenstein, E. C., and Imberski, R. B.,
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61. Ferenz, H.-J., The locust oocyte vitellogenin receptor-function and characteristics, in Advances in Inverte-
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37, 217, 1992.
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32 Insect Reproduction

66. Pascual, N., Cerdh, X., Benito, B., T o m h , J., Piulachs, M. D., and BeliCs, X., Ovarian ecdysteroid levels
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 Chapter 2

INSECT MALE MATING SYSTEMS

Cedric Gillott

CONTENTS
I . Introduction .................................................................................................................
33

I1. Structure ......................................................................................................................

34
A. Internal Organs ..................................................................................................... 34
B . External Genitalia ................................................................................................. 36
C. Specialized Mating Systems ................................................................................. 37

I11. Development ................................................................................................................ 37

A. Embryonic Development ....................................................................................... 37
B . Postembryonic Development ................................................................................38
1. General ............................................................................................................. 38
2. Endocrine Regulation ....................................................................................... 38

IV. Functional Aspects ......................................................................................................39

A. Spermatogenesis and Sperm Storage ................................................................... 39
1. Spermatogenesis............................................................................................... 39
2. Sperm Release and Storage ............................................................................. 40
B . Accessory Gland Activity ..................................................................................... 40
1. Nature and Formation of Secretion ................................................................. 41
2. Endocrine Regulation and Effect of Mating ................................................... 41
C. Spermatophore Formation .................................................................................... 42
D. Seminal Fluid ........................................................................................................44
E. Fecundity-Enhancing and Receptivity-Inhibiting Chemicals .............................. 44
1. Source and Nature ............................................................................................ 45
2. Site and Mode of Action .................................................................................46
F. Other Functions .....................................................................................................47

Acknowledgments .................................................................................................................
49

References .............................................................................................................................
49

.
I INTRODUCTION
Among the problems that required solution in the evolution of the Insecta as a predominantly
terrestrial group was the bringing together of sperm and egg in the absence of an aquatic
environment. The solution came through the formation. in most insects. of a spermatophore
within which the sperm could be safely transferred to the female reproductive tract. avoiding
the risk of both desiccation and predation . In relatively few species. spermatophores are not
found and sperm transfer is achieved by means of an intromittent organ . Though sperm
production and transfer are the primary functions of the male reproductive system. a number

.
0-8493-6695-X/95/S0.00+$.50
O 1995 by CRC Press Inc.
34 Insect Reproduction

of secondary functions have evolved, including sperm storage, the generation of "signals,"
either physical or chemical, that induce significant changes in the female's fecundity andlor
receptivity, and the transfer of nutrients to the female.
This chapter will provide the structural and functional background for ensuing chapters that
deal with more specific aspects of male insect reproductive biology. In addition, it will focus
on selected aspects of the functions of the male reproductive system that are not covered
elsewhere but are of particular interest to the author. In keeping with the extreme morphologi-
cal diversity of the Insecta, the structural, physiological, and biochemical nature of the male
system is widely varied. It is not the purpose of this chapter to encyclopedically describe this
plethora of detail, but rather to note the generalities that have been established and to point
out areas where understanding is still weak.

11. STRUCTURE
A. INTERNAL ORGANS
The male internal organs comprise paired testes (fused to form a single median structure
in Lepidoptera), paired vasa deferentia and seminal vesicles, a median ejaculatory duct, and
in most species, accessory glands of varied origin and complexity (Figure 1).
Within each testis is a varied number of tubular follicles bound together by a connective
tissue sheath. Each follicle connects with a short vas efferens, the vasa efferentia from each
gonad opening either confluently or in a linear sequence into the vas deferens. Within each
follicle, groups of germ cells in various stages of spermatogenesis (see Section 1V.A) may be
seen in final instar larvae or adults. As well, glandular cells may occur which, on the basis of
their staining properties, have been proposed to produce the nourishment necessary for both
maturation of the sperm and their maintenance (storage) within the seminal vesicle. The
glandular cells commonly take the form of an ensheathing epithelium around the developing
germ cells, the whole structure being known as a "cyst." Though the cyst wall usually breaks
open in the final stages of spermatogenesis, the sperm within may remain as a bundle even
after insemination. In Acrididae (Orthoptera), one cyst cell differentiates as a "nurse cell"
during the spermatid stage.' The spermatids in each bundle then become oriented and embed
their heads into the cell, which produces a large quantity of mucoprotein, the sperm and
mucoprotein cap constituting a spermatodesm. Cantacuzbnel speculates that the cap does not
serve directly as a source of energy for the sperm while they are stored in the seminal vesicle;
rather, the enzymes which, she suggests, reside within it degrade the nutrients released by the
epithelium which lines the seminal vesicle. The literature also contains a number of reports
that implicate the testes as the site of production of chemicals that modify female fecundity
andlor receptivity, but these are more appropriately dealt with in Section 1V.E.
The vasa deferentia are typically assumed to be merely tubes for conducting the sperm from
the testes to the seminal vesicles; indeed, the latter are frequently dilations of the vasa
deferentia. However, light micro~copical~-~ and a few electron m i c r o s ~ o p i c a lstudies
~ - ~ ~ sug-
gest that these tubes have important glandular and phagocytic functions in some species.
Rojas-Rousse2and Gerber et aL5propose that the secretion is used to nourish the sperm within
the male reproductive tract, while the presence of numerous lysosomes in the vas deferens
cells of Drosophila melanogaster may indicate a role in digestion of aged or degenerate
sperm.ll This function is also proposed for the phagocytic cells in the upper vas deferens of
the lepidopterans Anagasta kiihniella8and Calpodes ethlius.12Other proposed functions of the
vas deferens secretion include involvement in spermatophore production in Trichoptera13and
the blister beetle, Lytta nuttalli,14 and production of the spermatodesm in Tettigoniidae
(Orth~ptera).~
In the great majority of insects, the ejaculatory duct is of ectodermal origin and, as such,
has a cuticular intima. However, in all Lepidoptera, Diadromus pulchellus (Hymenoptera),
Plecia nearctica (Diptera), and Nezara viridula (Herniptera) an anterior mesodermal component
Insect Male Mating Systems

FIGURE 1. Representative male reproductive systems (not to scale). A. Melanoplus sanguinipes (Orthoptera);
B . Tenebrio molitor (Coleoptera); C. Musca domestica (Diptera); D. Anagasta kiihniella (Lepidoptera). Abbrevia-
tions: AG, accessory glands; BAG, bean-shaped accessory gland; CS, cuticular simplex; D, duplex; ED, ejaculatory
duct; ES, ejaculatory sac; LHT, long hyaline tubule; LVD, lower vas deferens; SHT, short hyaline tubules; SV, seminal
vesicle; T, testis; TF, testis follicles; TAG, tubular accessory gland; UVD, upper vas deferens; VD, vas deferens;
WT. white tubules; 1-8, Eight regions of the noncuticular simplex. (A, original; B, redrawn from Dailey, P.J. et al.,
Journal ofMorphology, Vol. 166. Copyright O 1980John Wiley & Sons, Inc. Reprinted by permission of John Wiley
& Sons, Inc. C, redrawn from Luther S. West: The Housefly: Its Natural History. Medical Importance, and Control.
Copyright O 1950 Comstock Publishing Co., Inc. Used by permission of the publisher, Cornell University Press; D,
redrawn from diagram supplied by Dr. J.G. Riemann.)

occurs.15The term "simplex" is traditionally used for the ejaculatory duct of Lepidoptera, with
the corresponding sections being referred to as the anterior "noncuticular" and posterior
"cuticular" simplex regions; further, the noncuticular simpiex is subdivisible into as many as
seven or eight distinct segments (Figure ID),each with its own tinctorial and ultrastructural
characteristics (see, for example, References 12 and 16).
36 Insect Reproduction

Secretory cells of both ectodermal and mesodermal origin have been described from the
ejaculatory duct, and a variety of functions have been proposed for their products. In Lepi-
doptera and Coleoptera, components of the spermatophore are derived from the secretions.
The noncuticular simplex of some Lepidoptera also produces a sperm activator (see Section
IV.F).I7-l9In the midge Chironomusplumosus, which lacks accessory glands, seminal fluid is
produced by the ejaculatory duct cells.20An enzyme, esterase 6, produced in the anterior
ejaculatory duct of D. melanogastefll may have diverse roles in the species' reproductive
biologyz2(see Section 1V.F). Receptivity-inhibiting substances (see Section 1V.E) have also
been reported from the ejaculatory duct of Musca d o m e ~ t i c aand ~ ~Stomoxys ~ a l c i t r a n s . ~ ~
The forms and functions of the accessory glands (= collateral glands), which parallel the wide
diversity of external form and habits of the Insecta, have been considered in detail by Gillott.15 In
the great majority of insects, the glands are mesodermal and are named "mesadenia". This is clearly
the primitive arrangement, though, in a few groups, substitution of ectodermal for mesodermal
components in the reproductive system generally results in the formation of ectodermal glands
(ectadenia) (see Section III.A).Accessory glands are primitively absent in Thysanura, Ephemeroptera,
Plecoptera, Dermaptera, and most Odonata, and have been secondarily lost in many Diptera.
The accessory glands occur in most species as a single pair of tubular structures, though
in Coleoptera there are commonly two or three pairs, and in Thysanoptera and Acrididae
(Orthoptera) multiple pairs of tubules occur. In the latter arrangement, the cytology of a tubule
is generally uniform throughout its length, whereas in species with a single pair of tubules
there are often regional or intercellular differences. The secretion of the accessory glands,
indeed of individual tubules, is a complex mixture, and this is reflected in the variety of
functions that have been ascribed to it, including spermatophore, mating plug and seminal
fluid formation; fecundity enhancement and receptivity inhibition; sperm activation; and
supply of nutrients to the female. In view of the central importance of the accessory gland
secretion to the reproductive biology of the male, its nature and some of its major functions
are discussed as distinct topics in Section IV.

B. EXTERNAL GENITALIA
The male external genitalia include two components, the basic structures common to all
species and derived from the primary phallic lobes of the embryonic tenth (larval ninth)
abdominal segment25and secondary structures unique to groups or species formed on adjacent
segments. Because of the enormous diversity in form of the external genitalia among different
insect groups, only the basic plan will be considered here. For information on specific groups,
the reader is referred to the work of M a t ~ u d a . ~ ~
The phallic lobes arise as paired ectodermal outgrowths of the ventral surface of the
segment, though only in Ephemeroptera do they remain separate to form the paired penes seen
in the adult. In Thysanura the lobes meet in the midline to form a short tubular "penis," a
misnomer because the structure is not an intromittent organ. In Odonata, the genitalia on the
tenth segment are greatly reduced and, instead, secondary structures develop on the second
and third abdominal segments (see Section 1I.C). In most other insects, each phallic lobe is
divided to form a median mesomere and a lateral paramere. Between the fused mesomeres is
the gonopore from which the ejaculatory duct arises. Elongation of the mesomeres produces
an intromittent organ, the aedeagus, whose opening is the phallotreme and inner channel the
endophallus. In some insects, the endophallus is eversible through the phallotreme so that, in
effect, the gonopore becomes the external opening. Normally, the parameres differentiate into
elongate clasping organs. However, in some cases they fuse with the mesomeres to form the
phallobase. This can become an elongate structure, the phallotheca, that encloses the now
eversible aedeagus. This telescopic arrangement (i.e., phallotheca, aedeagus, and endophallus),
when everted by hemolymph pressure during copulation, may form a very elongate penis that
deposits sperm deep within the female system.
Insect Male Mating Systems 37

C. SPECIALIZED MATING SYSTEMS

According to M a t ~ u d ain, ~Ephemeroptera
~ the primary phallic lobes of the juvenile stage
are replaced by adult structures that develop within or in association with those of the nymph;
that is, phallic lobe development is biphasic in this order, in contrast to that in all other orders
in which continuous development towards the adult form occurs. Further, in this order each
imaginal phallic lobe remains separate and does not subdivide into mesomere and paramere.
Rather, each lobe develops as a penis, and from each gonopore an ejaculatory duct grows
anteriorly to fuse with the vas deferens on each side.
In Odonata, the phallic lobes do not develop to any significant degree so that males have
no aedeagus and only an inconspicuous ejaculatory duct. Thus, a unique method of sperm
transfer has evolved in this order.27Prior to mating, a male coils his abdomen forwards so that
the gonopore comes into contact with the intromittent organ on the third abdominal sternum,
and a spermatophore is transferred. During copulation, the male grasps the female's head
(Anisoptera) or prothorax (Zygoptera) by means of his terminal appendages (modified cerci).
The interlocking of the male and female parts may be extremely precise, thus playing an
important role in species isolation. The female then rotates her abdomen under that of the male
until her genital opening contacts the intromittentorgan when the spermatophoreis transferred.
Sperm transfer in Strepsiptera, many Cimicoidea, and at least one species of Anthocoridae
(Hemiptera) is achieved by hemocoelic Though the details vary, in this most
unusual form of insemination the penis penetrates the integument or vagina wall so that sperm
is deposited in the hemocoel, sometimes into a special structure, the spermalege. Eventually,
some of the sperm makes its way to the conceptacula seminales for storage, the rest being
phagocytosed either by hemocytes or by cells of the spermalege. HintonZ8suggested that
hemocoelic insemination may serve to provide females with nutrients to enable them to
survive for longer periods in the absence of suitable food.

111. DEVELOPMENT

A. EMBRYONIC DEVELOPMENT
The origin and development of the germ cells and gonads is varied, though two distinct
trends can be noted, namely, earlier segregation of the primordial germ cells and the restriction
of these cells to fewer abdominal segment^.^'.^^ In some Thysanura and Orthoptera, the germ
cells do not become distinguishable until they appear in the splanchnic walls of several
abdominal segments. In Locusta migratoria, for example, they arise in the second through
tenth pairs of abdominal somites although they persist only in pairs three to These
segmental groups of cells proliferate and fuse longitudinally to form the gonad on each side.
In contrast, in Dermaptera, Psocoptera, Homoptera, and many endopterygotes the germ cells
become obvious during blastoderm formation as roundish cells at the posterior end of the egg.
In due course, in exopterygotes the cells move anteriorly through the yolk to become enclosed
within the splanchnic mesoderm of the thirdfourth abdominal segments. The germ cells
subsequently separate into left and right halves, from which the testes develop. In endopterygotes,
the picture is similar except that division of the germ cells into two groups occurs prior to
migration through the y01k.~'.~*
In exopterygotes, the vasa deferentia develop from one or more posterior pairs of abdomi-
nal somites (e.g., those of segments nine and ten in p h a ~ m i d s while
~ ~ ) the ejaculatory duct is
an ectodermal invagination, usually arising behind the ninth sternum. In lower endopterygotes,
for example, Tenebrio molitor,6 embryonic development of the gonoducts is similar to that of
exopterygotes, and all the elements of the reproductive system can be identified at hatching.
In Diptera and Lepidoptera, the ductal components develop postembronically from a single
midventral or a bilateral pair, respectively, of genital imaginal discs that arise late in embryo-
g e n e ~ i s . ~In
~ -all
~ ' insects except, apparently, Acrididae (Orthoptera) the formation of the
38 Insect Reproduction

primary phallic lobes and subsequent development of the external genitalia occur during the
larval stage.26

B. POSTEMBRYONIC DEVELOPMENT
1. General
In exopterygotes, the reproductive system, like other organ systems, grows steadily during
the larval period; differentiation of the various components does not occur, however, until the
final juvenile stadium. In contrast, in endopterygotes very little growth occurs through the
larval period; rather, growth and differentiation occur simultaneously and are compressed into
the pupal stadium. "Differentiation," including in its broadest sense both spermatogenesis and
the organogenesis of the tubular components, is regulated hormonally as well as being affected
by various exogenous factors. The endocrine control of spermatogenesis is discussed by
Hardie in Chapter 5.

2. Endocrine Regulation
As with the differentiation of other tissues, it is changes in the hormone balance within the
insect that induce development of the reproductive tract. Though I am not aware of studies on
the phenomenon, it seems reasonable to propose, in light of what is known regarding hormonal
events during metamorphosis, that changes in hormone titers in earlier instars permit (and
possibly promote) the mitotic division of the undifferentiated reproductive tract cells and the
slight growth of the system seen especially in exopterygotes.
In the final juvenile instar, there occurs a marked decline in the level of circulating juvenile
hormone (JH), as well as one or more surges in the level of circulating e c d y ~ t e r o i d .This ~~.~~
change in the ratio of the two principal "players" permits the expression of adult characters,
though the specific roles of the two hormones are only slowly becoming clear. JH appears to
influence metamorphosis in two ways: first, by inhibiting the secretion of prothoracicotropic
hormone, ecdysteroid production and release is prevented; and second, though this is more
speculative, it may directly prevent the action of ecdysteroid at the organltissue l e ~ e l .
Our rather limited understanding of the involvement of hormones in preimaginal growth
and differentiation of the male reproductive system has recently been reviewed by H a ~ p . ~ '
Almost nothing is known about the site and mode of action of JH. In the locusts L. migratoria
and Schistocerca gregaria, implantation of two corpora allata into the abdomen of male fourth
(penultimate) instar nymphs leads to sterility.' However, this treatment or injection of syn-
thetic JH does not exert its effect on spermatogenesis, which proceeds normally, but prevents
differentiation of the vas deferens, which remains a thin, solid cord of cells.42These treatments
also prevent differentiation of the accessory whereas the reverse (i.e., allatectomy)
results in precocious metamorphosis of these structure^.^^ Perhaps more important,
Cantacuzi?ne'sl data show that the timing of the treatment is critical, the greatest effect being
achieved when insects are treated midway through the stadium. The accessory gland trans-
plantation experiments of G a l l o i ~ support ~ ~ . ~ ~this point. Implantation of these glands from
final instar nymphs of varied ages into adult hosts shows that only those glands from nymphs
2, 3, or more days into the instar are competent to differentiate. This critical period (days 2
and 3) coincides with a marked decrease in JH titer (and a small increase in the level of
ecdy~teroid)."~ As all these investigations utilized whole insects, it is unclear whether JH is
acting directly or indirectly on the tissues whose development it is influencing.
Mere absence of JH is insufficient for development to proceed; ecdysteroid (probably in
the form of ecdysterone) must be present both for growth and for differentiation of the
reproductive tract. The involvement of ecdysteroid has been investigated in several
endopterygotes, most notably Ephestia kiihniell~,~' Samia cynthia,1° Heliothis v i r e ~ c e n s ,
Bombyx m~ri,~O and T. m o l i t ~ r . ~In
' " ~Lepidoptera, both the sperm ducts and the genital discs
(which form the seminal vesicles [= upper vasa deferentia of some authors] and the remaining
Insect Male Mating Systems 39

components of the tract, respectively) grow and differentiateunder the influence of ecdysteroid.
Apparently, ecdysteroid exerts both direct and indirect effects. In S. cynthia1° and B. m~ri,~O
ecdysteroid is able to directly promote development of the tract in vitro, whereas in H.
virescens, ecdysterone alone has no e f f e ~ t . ~Development
~ , ~ ~ . ~ ~of the sperm ducts or genital
discs does occur when fat body or testis sheath is present in the culture medium in addition
to the hormone, or when the tissues are cultured with aqueous extract of fat body previously
exposed to ecdysteroid for 24 hours.
Only in T. molitor and H. virescens does the site and mode of action of the ecdysteroid
appear to have been s t ~ d i e d . ~In~ the
+ ~ bean-shaped
'-~~ glands (BAGs) of Tenebrio (Figure 1B)
there are two bouts of mitosis as growth occurs, the second of which coincides with the peak
of ecdysteroid in the pupal stage. In vitro studies showed that the second burst of mitosis
requires ecdysteroid for its oc~urrence?~ the hormone promoting the flow of cells from the G,
into the G, and S phases.52Though Grimnes and H a ~ showed p ~ ~ that the same concentration
of ecdysterone which produced maximum rates of mitosis also promotes formation of char-
acteristic secretions and adult-specific antigens in the BAGs, the latter activity in vivo is not
immediately induced by the ecdysterone but begins at the end of pupation when levels of the
hormone are In H. virescens genital discs, ecdysterone in combination with fat body or
testis sheath stimulates tritiated thymidine incorporation into DNA, suggestive of mitotic
divi~ion."~

IV. FUNCTIONAL ASPECTS

A. SPERMATOGENESIS AND SPERM STORAGE
1. Sperrnatogenesis
In insects, as in other animals, spermatogenesis comprises three phases: (1) the multipli-
cation phase (spermatocytogenesis); (2) the meiotic phase; and (3) the maturation phase
(spermiogene~is).~~ In many insects, this temporal sequence of events is "fixed" spatially; that
is, the phases occur in the distal, middle, and proximal regions, respectively, of each testis
follicle. In the multiplication phase, the spermatogonia (cells derived from germ cells)
undergo a species-specificnumber (usually, five to eight) of mitoses. For most insects (but not,
apparently, Diptera), these divisions (and hence the daughter spermatogonia) occur within a
"cyst," that is, a capsule of somatic cells formed initially around either a single spermatogo-
nium or a clone of daughter spermatogonia after one or two divisions. When mitosis is
complete, the spermatocytes, as the cells are now known, undergo two meiotic (maturation)
divisions. Generally, four spermatids result from each spermatocyte, each having a haploid
chromosome complement. However, especially among Diptera, variants of the meiotic pro-
cess occur, so that only one or two spermatids are formed from each spermatocyte. During
spermiogenesis, the spermatids differentiate into flagellated spermatozoa. At this time, the
enveloping cyst celis are rich in glycogen and may be supplying nutrients; in addition, they
phagocytose cytoplasmic remnants sloughed by the spermatids as they transform into sperma-
tozoa. In Acrididae (Orth~ptera)'.~~ and Cole~ptera,~'a mucopolysaccharide "cap," the
spermatodesm, comes to envelop the sperm heads near the end of spermiogenesis, though it
is by no means clear that this is a product of the cyst cells. According to Cantacuzbne,' the
cap is not a direct source of nutrients; rather, it contains enzymes that degrade nutritive
molecules secreted by the seminal vesicles into which the mature sperm are now transferred
and stored. In Tettigoniidae (Orthoptera), the spermatodesm is formed from secretion of the
intratesticular region of the vas deferens.'
In Lepidoptera, two types of sperm are produced, eupyrene (nucleate), which fertilize the
eggs, and apyrene (anucleate), whose largely speculative functions may include facilitating
eupyrene sperm movement from the testes to the vas deferens, assisting the eupyrene sperm
in their migration within the female reproductive tract, providing nourishment for the eupyrene
sperm, the female herself, or the zygote, and playing a role in sperm competition either by
40 Insect Reproduction

eliminating sperm deposited in previous matings or by preventing further matingsS8(and see

also Section 1V.F). The two types can be distinguished as early as the secondary spermatocyte
stage, following which their changing morphology follows very different paths.59 Eupyrene
sperm formation begins somewhat earlier than that of apyrene sperm, the former being
initiated in the last or penultimate larval instar, the latter in the last larval or pupal instar,
depending on the species. The eupyrene sperm, like the sperm of other types of insect, remain
in bundles until they have been transferred to the female tract; however, the apyrene sperm
bundles dissociate, releasing individual sperm, as they move out of the testis.
For most insects, formation of spermatogonia and spermatocytes is reported to occur in the
final nymphal or pupal instar, so that the testes of the adult contain only spermatids and
spermatozoa or only spermatozoa in those forms with a very short adult life. These observa-
tions led, more than 40 years ago, to the proposal that spermatogenesis was regulated by the
morphogenetic hormones, ecdysone and JH, the former promoting and the latter inhibiting the
process. However, in representatives from diverse orders, meiotic and even premeiotic phases
of spermatogenesis can be found in the adult t e ~ t i s .Dumser
~ ~ , ~ and
~ Da~ey@-~~%uggested that
the hormones affected only the rate of spermatogonial mitosis, not differentiation per se.
However, the subsequent discovery of ecdysteroids in adult males of several species and of
other effects of both e c d y s t e r o i d ~and
~ ~ ~JH" on germ cell maturation and viability has
reopened the question of the endocrine control of sperm production. See Chapter 5 for a full
discussion of this topic.

2. Sperm Release and Storage

In the majority of insects, the bundles of mature sperm are moved by peristalsis to the
seminal vesicle where they are stored until insemination. However, except in Lepidoptera,
information on this process is lacking. For several species of Lepidoptera, it has been observed
that release of sperm (both types) from the testis and its subsequent movement through the
upper vas deferens and seminal vesicle to the ductus ejaculatorius duplex (Figure 1D) shows
a daily rhythm (see Reference 64). This rhythm has since been shown to be circadian in
with the clock and its associated photoreceptor located in the testis-vas deferens
complex.66Correlated with the rhythm of sperm movement is cyclical secretory activity by the
epithelium of the upper vas deferens, though the purpose of this carbohydrate-rich secretion
remains unclear.@ However, in their passage toward the ductus ejaculatorius duplex, the
sperm undergo complex morphological changes, especially with respect to the fibrous sheath
that surrounds individual eupyrene sperm.67The significance of these changes remains un-
clear, but it may be speculated that they relate somehow to the ability of the sperm to fertilize.
The overall significance of this periodic sperm release and movement is related to the fact that
males normally mate but once a day, and at this time they ejaculate all the sperm stored in the
duplex. The timing of sperm release and movement (which is species specific) is such that the
sperm produced each day move into the duplex shortly after the end of the male's daily period
of receptivity to female sex pheromone. Thus, even if a male does mate, a substantial amount
of new sperm will be available for insemination the next day.65Though the usefulness of this
type of a system is readily apparent for species that have diurnal mating rhythms, it remains
to be determined how sperm release and storage is regulated in species whose males are more
opportunistic with respect to mating.

B. ACCESSORY GLAND ACTIVITY

As noted earlier, the accessory glands take on a vast array of forms, and their secretion a
spectrum of functions. Both these aspects have been extensively studied over the past two
decades, and the major conclusions are presented in reviews by H i n t ~ nLe0pold,6~
,~~ Chen,7O
Happ," and Gillott.15Also, the endocrine control of accessory gland activity has recently been
revie~ed.'~
Insect Male Mating Systems 41

1. Nature and Formation of Secretion

Numerous histochemical studies have shown the secretion to be a mixture or chemical
complex of protein, carbohydrate, and iipid, and electron microscopy has confirmed that the
glandular epithelium is designed for production of these materials. Also noteworthy is that
some of these early studies have demonstrated the regional variation that occurs both within
and between accessory gland tubules with respect to both amount and nature of secretory
components. Electrophoretic studies, in some cases combined with histochemistry, have
revealed that the secretion contains many distinct proteins (e.g., >40in the BAGSof T e n e b r i ~ ~ ~ ) ,
including glycoproteins and lipoproteins. Some of these proteins are enzymic in nature while
others, which may be quantitatively dominant, play a structural role (see below for details).
Free amino acids and small peptides have also been identified. Among the carbohydrates not
complexed with protein are glycogen, mucopolysaccharides (acidic, basic and neutral), glu-
cose, and inositol. Neutral lipids and phospholipids have been reported for some species.
Guanosine 3', 5'-cyclic monophosphate occurs in large amounts in the glands of Acheta
domesticus and several other Gryllinae (Orthoptera), where it may have a role in sperm
metabolism and a ~ t i v i t y .Uniquely,
~ ~ . ~ ~ the glands of male Hyalophora cecropia store massive
quantities of JH-I and JH-I1 which are transferred to the female during c ~ p u l a t i o n .Also,
~~-~~
the accessory glands of male Blaberoidea (Dictyoptera) accumulate uric acid (up to about 90%
of the dry weight in Blattella g e n n a n i ~ a ~which
~ ) , is then secreted as the outermost layer of
the spermatophore, possibly to dissuade the female from eating the spermatophore before
sperm evacuation has o c ~ u r r e d . However,
~ ~ . ~ the observation by Mullins and KeilSobthat
radiolabeled urate produced by males and ingested by females (as part of the spermatophore)
appears in the ootheca suggests that it may represent an important nitrogen resource for
reproductive success (and see Chapter 10).

2. Endocrine Regulation and Effect of Mating

Almost a11 investigations of the hormonal regulation of accessory gland activity have
focused on the role of JH. This is quite understandable in view of the demonstration,more than
50 years ago?' of the importance of the corpora allata in male reproductive activity and the
long-standing presumption that ecdysteroids would not occur in adults following the degen-
eration of the moult glands at metamorphosis. However, several demonstrations of ecdysteroids
in adult male^,^*-^^ combined with the realization that, in insects which are sexually mature at
eclosion, the accessory glands acquire their secretion during the pupal instar when the corpora
allata are inactive, raise the possibility that in some species ecdysteroids may be involved.
In males of most species studied, allatectomy prevents or greatly retards the accumulation of
secretion by the accessory glands, an effect that can be reversed by application of JH. Without
exception, the effect of JH is at the level of protein synthesis, and in the relatively few investigations
where it has been examined, JH has been shown to regulate the synthesis of specific proteins. For
example, in allatectomized Melanoplus sanguinipes the reduced ability of the long hyaline tubule
(Figure 1A) to accumulate secretion is due to the failure of the tubule to synthesize a glycoprotein,
LHPI, which comprises 51% of the total protein in the tubule of mature virgin males.85-86 Parallel
ultrastructural studies show that allatectomy causes changes in the protein-synthesizing machinery
of the gland cells and, in some cases, in the appearance of the s e c r e t i ~ n . ~ ~ - ~ ~
It must be emphasized that the majority of studies, in which whole insects have been used,
do not clarify whether the action of JH on the accessory glands is direct or indirect. A few
authors have used decapitation,%or have removed both the corpora allata and the cerebral
neurosecretory ~ystem,9'-~~ followed by JH application, or have used an in vitro system72.94-96
to confirm that JH acts directly on the glands. How and where JH acts in the accessory glands
is, at this time, a matter of conjecture, though Yamamoto et al?5 have obtained evidence
suggesting the existence of a membrane receptor protein for JH in D. melanogaster.
42 Insect Reproduction

Early reports that ecdysterone promoted protein-synthetic activity in accessory glands must
be treated cautiously because pharmacological, rather than physiological, doses of hormone
were e m p l ~ y e d .The
~ ~ .stimulation
~~ of RNA and protein synthesis by ecdysterone has been
observed under both in vivo and in vitro conditions in pharate and newly emerged adults of
Chilo partellus and Spodoptera l i t ~ r a . ~ ~These- ' O ~ are interesting observations, because in
other Lepidoptera ecdysteroid titers are known to be very low just prior to eclosion. Further,
as noted earlier (Section 1II.B.l), though ecdysteroid is required in T. molitor and B. mori to
render the accessory glands competent to produce secretion, when production begins at the
end of pupation, ecdysteroid levels are low; in other words, in these species protein synthesis
is not directly regulated by e c d y ~ t e r o i d s . ~ ~ . ~ ~
Only a handful of reports indicate that neurosecretory factors directly affect accessory
gland protein synthesis. For example, in Rhodnius prolixus, removal of the median neuro-
secretory cells reduces protein accumulation by the transparent accessory glands, an effect
which can be only partially overcome even by multiple doses of JH-I.93 In vitro studies
subsequently confirmed that a polypeptide from the brain stimulated protein synthesis in the
transparent accessory glands.Io2
An aspect of endocrine control that merits further examination i's the movement of proteins
between the accessory glands and the hemolymph. For example, in M. sanguinipes there are
immunologically similar proteins in the accessory glands, fat body, and hemolymph.lo3The
accessory glands can accumulate these proteins from the hemolymph in normal, but not in
allatectomized,males. In C. partellus, in vitro CO-cultureof accessory glands from adult males
with fat body and hemolymph from larvae injected several hours previously with [35S]-
methionine has demonstrated that the larval proteins are synthesized in the fat body, released
into the hemolymph, and are then accumulated by the accessory glands. Accumulation is
enhanced by ecdysterone or its agonist RH 5849 but inhibited by JH-I.Io4"Conversely, the
transparent accessory gland of Rhodnius synthesizes and releases into the hemolymph a 170-
kDa polypeptide,IWband Sevala and DaveylWbsuggest that the apparently redundant control
of protein accumulation in the transparent accessory gland by both JH%and neurose~retion'~~
may ultimately be explained by assigning a specific function, synthesis, or release, to each of
the hormonal factors.
That mating leads to enhanced synthesis of accessory gland secretion has been shown for
various However, to date there is no clear understanding of the control
pathway by which the mating effect is exerted. Baumann's study'0Ssuggested that in Droso-
phila funebris mechanical emptying of the gland was not the stimulus for renewed secretory
activity, and this author speculated that there might be "a neurohormonal influence." For D.
melanogaster, also, copulation-enhanced protein synthesis has been suggested to involve
neurohormonal factors based on the parallel effects of mating and JH a p p l i c a t i ~ n . In '~~
contrast, though JH is essential for the normal expression of protein synthesis in the accessory
glands of M. sanguinipes,lo7it does not mediate the copulation effect. This is readily seen in
mated but allatectomized M. sanguinipes which still exhibit a three-fold increase in accessory
gland protein synthetic activity compared with unmated allatectomized controls.lo7

C. SPERMATOPHORE FORMATION
Spermatophores are usually presumed to have evolved in association with the taking up of
terrestrial life by insect ancestors and have as their primary function the delivery of sperm to
the female reproductive tract. However, as Daveylo8pointed out, some marine Crustacea and
many aquatic Annelida produce a spermatophore, so that, for Insecta, this should not be
thought of as a new structure but rather as an existing struciure that has taken on a new
function. Though detailed descriptions of the mechanical aspects of spermatophore formation
are available for many species, aspects of the process such as the nature of the stimuli that
initiate it, its control and coordination, and its biochemical nature are relatively unexplored.
This is unfortunate, given the centrality of this method of sperm transfer in the evolutionary
Insect Male Mating Systems 43

success of insects and, from the pest management point of view, its importance in the life
history of species.
Generally, spermatophores have their most complex form in primitive groups; in advanced
endopterygotes they may be relatively simple or have been secondarily lost. Paralleling this
trend is a change in the site of formation of the spermatophore. GerberIogrecognized four
categories of spermatophore formation based on spermatophore complexity and site of
formation. In the most primitive, the first male-determined method, the complex spermato-
phore is formed in the ejaculatory duct or copulatory organ of the male. Such a method is
typical of orthopteroid insects. In the second male-determined method, which characterizes
some Hemiptera, Coleoptera, and a few Diptera, the spermatophore is again formed in the
copulatory sac, but the latter is everted into the bursa copulatrix of the female. After copula-
tion, the sac is withdrawn, leaving the usually less complex spermatophore in the bursa. In
Trichoptera, Lepidoptera, some Coleoptera, and a few Diptera, the spermatophore forms
directly in the female tract which thus determines its shape (first female-determined method);
the spermatophore is relatively simple but nonetheless, like those formed by the male-
determined methods, still encloses the sperm. In the second female-determined method, the
accessory gland secretions do not enclose the sperm; rather, they follow them into the female
genital tract where they may temporarily harden to form, for example, the mating plug of
mosquitoes and the honeybee and the sphragis of some Lepidoptera. It is speculated that these
"barriers" may prevent loss of semen or further transfer of sperm in a subsequent mating.
In most early analyses of spermatophore formation, only histological methods were em-
ployed, so that the origin and nature of spermatophore components remained unclear. A few
authors have used histochemistry or surgical removal of certain accessory gland structures to
determine the source and broad chemical nature of particular components (see Gillott15 for
references). However, it is only relatively recently that information on the biochemical nature
of individual spermatophore components and their mode of formation has become available.
For example, in T. molitor the use of monoclonal antibody techniques has permitted Happ's
group to trace three structural proteins (spermatophorins)from their site of production, distinct
cell types in the BAGS,to specific layers within the ~permatophore.~l@~~~ Amino acid analysis
of one of these spermatophorins showed that >25% of its residues were proline, which is in
keeping with the large amounts of this amino acid reported for other insect structural proteins
(in cuticle, egg shell, and ootheca) and for ~ollagen."~ Though having immunological
identicality, it is clear that the nature of the precursor accessory gland secretion may differ
from that of the spermatophorin. This was first noted by Grimnes and HappuOas a change in
solubility and electrophoretic mobility, leading these authors to speculate that proteolytic
cleavage of the precursor may have occurred. A proteolytic enzyme probably involved in the
formation of a spermatophorin has been identified in accessory gland secretions of M.
~ a n g u i n i p e sA
. ~precursor
~~ protein of m01 wt 85 kDa, which appears to be produced primarily
in short hyaline tubule 3 (Figure lA), is cleaved by a trypsin-like enzyme to form the
spermatophorin SP62 (m01 wt 62 kDa), a major component of the outer layer of the spermato-
phore. The major products of the white gland tubules of Melanoplus (Figure 1A) are also
spermatophorins, but it is not known whether they, too, are derived by proteolysis of higher
molecular weight precursors. The function of an aminopeptidasealso identified in Melanoplus
accessory gland secretion remains unknown.'13
Except in GryIlidae (Orthoptera), where a spermatophore is preformed by the male and
carried until copulation takes place, spermatophore formation begins only after copulation is
initiated, that is, the male has achieved the correct mating position. Sensory input, especially
tactile but for some species chemical and visual, triggers the process, which is under motor
neuronal control. For most insects studied, the central nervous system must be intact in order
for spermatophore formation to start. For example, in Locusta the head ganglia are necessary
at this stage to interpret input from the cerci, though decapitation 15 min or more after
initiation does not disrupt spermatophore formation.'14 Control of the subsequent stages of
44 Insect Reproduction

spermatophore formation resides in the terminal abdominal ganglion, removal of which causes
abrupt termination of the process. The presence of the female is necessary only at the start,
presumably related to the need for correct sensory input via the cerci. Separation of copulating
pairs beyond 15 min does not disrupt spermatophore formation though the structures are
malformed because, Gregoryii4suggested, the spermatophore desiccates and the spermathecal
duct serves as a mold for the spermatophore tube in this species.
In Teleogryllus commodus and perhaps other species of crickets where the spermatophore
is produced prior to mating, formation of the spermatophore is under circadian control; that
is, a new spermatophore is produced during each 24-h period.ii5According to Loher,li5who
employed a variety of surgical procedures, the pars intercerebralis region of the brain may
serve a dual function in regulating spermatophore formation. First, it may hormonally regulate
the synthesis of the raw materials in the accessory glands and, second, it may be the
coordinating center for release of the materials from the glands as the spermatophore is being
formed. However, Loher's workii5does not clarify whether the pars intercerebralis serves
only as a trigger, the rest of the process being regulated within the terminal abdominal
ganglion as in other insects, or whether it controls the entire event. The site of the circadian
control center is also undetermined but may be within the optic lobes, as their destruction leads
to random generation of spermatophores throughout the 24-h period.

D. SEMINAL FLUID
At the outset, it is important to clarify the term "insect seminal fluid" because, in contrast
to the mammalian situation in which seminal fluid is a secretion of the seminal vesicles, in
many insects the seminal vesicles are not glandular in nature. Potentially, the fluid that comes
to bathe the sperm may be derived from any or all glandular parts of the male system, and its
nature may change as secretions are added to or removed from it during insemination and
when it reaches the female system.
The following discussion will deal only with our somewhat limited knowledge of the
general composition and functions of insect seminal fluid. The nature and functions of specific
components will be examined in parts E and F of this Section. The obviously minute quantities
of semen together with the high density of sperm make the obtaining of seminal fluid samples
for analysis difficult. Further, depending on its source (e.g., ejaculate, spermatophore, or
spermatheca), its composition will vary. Perhaps not surprisingly, a variety of potential energy
sources for the sperm have been identified. For example, Apis mellifera seminal fluid contains
trehalose, glucose, and fructose though the latter becomes virtually undetectable 40 min after
ejaculation, perhaps because of its use by the sperm.ii6The three sugars occur throughout the
reproductive tract with the greatest concentration in the testes and penis bulb. Histochemical
studies have demonstrated the existence of glycogen in the seminal fluid of Periplaneta
americanaii7and S. g r e g ~ r i aThe
. ~ ~lipid
~ in the semen of A. mellifera is almost entirely of
spermatozoan origin rather than in the seminal fluid.Ii9The amino acids (free and bound) in
A. mellifera semen apparently resemble those of mammalian semen though their specific
origin (sperm or seminal fluid) and functions are undetermined.I2OWhile Blum et a1.Ii6report
that the semen of A. mellifera contains calcium, sodium, manganese, magnesium, copper, and
iron (of which only the last three are detectable in sperm), there appear to have been no
quantitative studies on the inorganic constituents of seminal fluid despite several reports on
the activation of sperm by buffers at various pH values.

E. FECUNDITY-ENHANCING AND RECEPTIVITY-INHIBITING CHEMICALS

In addition to its obvious function of sperm transfer, for many species mating has other
important effects, including the stimulation of egg production and the rendering of the female
unwilling to remate. In some species (for example, cockroachesi21)the stimulus given by
mating is physical in nature; that is, the insertion of the spermatophore stretches the wall of
the bursa copulatrix, which causes the female to become unreceptive. For others, chemicals
Insect Male Mating Systems 45

in the seminal fluid stimulate egg production andlor render females unreceptive. These
pheromones have been named fecundity-enhancing (FES) and receptivity-inhibiting sub-
stances (RIS), re~pectively.'~~
Both FES and RIS "inform" the female that she has been inseminated. For the FES, the
significance lies in the fact that in most insects unfertilized eggs are inviable; thus, it is critical
that oviposition not occur until a supply of sperm is available. For the RIS, the rendering of
the female unreceptive "guarantees" that only the first (fittest) male's sperm will be used to
fertilize the eggs. It may also provoke males to actively seek virgin females in the population
and, for females of some species, to switch their behavior from mate-seeking to food- and/or
oviposition-site seeking. Although the FES and RIS play distinct roles, it is appropriate to deal
with both concurrently in view of their similar sites of production and chemical nature. Indeed,
in some species it seems likely that the same substance may serve as both a FES and a RIS.
Gillott and FriedelIz2and GillottI5have provided detailed reviews of FES and RIS.

1. Source and Nature

In males of most species where FES or RIS have been identified, the accessory glands are
their source. However, in Musca, males of which lack such glands (Figure lC), the FESIRIS
is produced in the upper third of the ejaculatory duct.Iz3The testes apparently produce these
pheromones in H. c e c r ~ p i a ,~' ~r i~c k e t s , ' ~and R.~proli~us,'~'
~.'~ (though in the latter species,
an effect of the seminal fluid merely stretching the spermathecal wall cannot be ruled out). It
should also be noted that for none of these species is it clear whether glandular cells in the
gonads or the sperm are the source of the pheromone. In some Drosophila species, there is
evidence for the existence of both short- and long-term MS, the former being produced by the
accessory glands, the latter by the
With one exception, all FES and RIS characterized to date are peptides or proteins. For
example, the monocoitic substances reported from M. domestica, Cochliomyia hominivorux
and Phormia regina are peptides with molecular weights estimated at 750 to 3000 Da.I3O
Likewise, the RIS (PS-1) of D. funebris has a molecular weight of approximately 2700 Da and
comprises 27 amino acid r e s i d u e ~ .PS-1' ~ ~ exists in two forms, differing in a single amino acid
(valine or leucine) at position 2.l3l The sex peptides of D. melanogaster and D. sechellia,
which serve as both FES and RIS, each comprise 36 amino acids and differ from each other
at only three positions. Perhaps not surprisingly, the D. sechellia sex peptide stimulates
oviposition and inhibits receptivity in D. melanogaster, as well as in D. simulans and D.
mauritania, all four species belonging to the melanogaster species subgroup. However, the D.
melanogaster peptide has no effect on virgins of D. funebris and vice versa.132
On the other hand, the FES of M. ~anguinipes,'~~ L. r n i g r a t ~ r i a ,and
' ~ ~Aedes a e g ~ p t iare
l~~
proteins of molecular weight 30, 13, and 60 kDa, respectively. The FES of the crickets A.
d o r n e s t i ~ u sand
~~~T. . cornrnod~s'~~
~~~ is a prostaglandin synthetase enzyme complex which,
after transfer during mating, promotes prostaglandin production in the female reproductive
system.
Three D. melanogaster accessory gland proteins and the genes which code for them have
been studied by Wolfner's Of these, perhaps the most interesting is msP 355a, a
basic protein with many features typical of peptide pheromone and hormone precursors;
indeed, part of its amino acid sequence is similar to that of the egg-laying hormone (ELH) of
the mollusc Aplysia ca1iforni~a.l~~ In the accessory glands, msP 355a exists mainly in a form
of molecular weight 37 kDa. During mating, it is transferred to the female genital tract; some
of the 37-kDa fraction passes unchanged into the female's hemolymph, but the rest undergoes
rapid proteolysis, first to a 29-kDa, then to a 22-kDa fragment. However, the ELH-like
segment is retained by both these smaller m0lecu1es.l~~ The second protein in the trio is msP
355b, an acidic protein 90 amino acids in length and having a molecular weight of 11-14
kDa.l4I It, too, is transferred during mating, and some enters the female hemolymph. However,
that which remains in the genital tract is not proteolysed and, along with the msP 355a
46 Insect Reproduction

fragments, unstored sperm, and other secretions, is expelled from the genital tract some 2-3
h after copulation terminates. DiBenedetto et al.,I4Oon the basis of their analysis of the gene
that codes for it, have characterized msP 316, a small basic protein made up of 52 amino acids.
Prostaglandins have been identified from the male reproductive organs of B. r n ~ r iand ,~~~
greater levels of these compounds have been noted in mated females compared with virgins.143
However, it is not clear whether these increases are the result of direct transfer from the male
or are an effect of mating.

2. Site and Mode of Action

The most common effect of FES is to stimulate oviposition, though the manner in which
this is achieved is not well understood. Extracts of Locusta male accessory glands stimulate
contractions of the lateral oviduct, an effect that can be partially mimicked by octopamine and
forskolin, suggesting the involvement of octopaminergic re~ept0rs.l~~ And recently, specific
peptides with myotropic activity on the oviduct have been isolated from male accessory
glands, spermatophores and spermathecae of mated fern ale^.'^^.^^^ Obviously, oviposition is a
complex process, both mechanically speaking and in terms of when it occurs. Not surprisingly,
it is controlled both hormonally and neurally (for review, see Lange14'). Perhaps the role of
these male-derived substances is to prime the oviductal musculature so that, at the appropriate
time and place for egg laying, the nervous system can provide the fine control.
In contrast, in RhodniusI2' and T e l e ~ g r y l l u the
s ~ ~FES
~ acts on the spermatheca, which is
stimulated to produce a hormone of unknown nature (the "spermathecal factor") and prosta-
glandins, respectively. Similarly, in Hyalophora the FES is released into the bursa copulatrix
where it triggers production of a hormone.124For other species, it seems likely that the FES
passes through the wall of the female reproductive system to some other site of action. Thus,
in D. funebris, FES can be detected in all parts of the body 2 h after mating,148and in both M.
~anguinipesl~~ and M. d o m e s t i ~ asome
' ~ ~ male accessory gland proteins pass unchanged into
the hemolymph of the female (though in neither species is it clear whether the FES is one of
these).
The remaining links in the pathway culminating in egg laying are not well known. For a
number of species (references in References 15,147), the cerebral neurosecretory cells are
known to produce an ovulation- or oviposition-inducinghormone (a myotropin) whose release
may be facilitated by the FES. In Rhodnius, for example, ecdysteroids released from the ovary
as eggs mature can initiate electrical activity in, and release of myotropin from, specific
median neurosecretory cells. However, this occurs only in mated females, that is, in the
presence of the spermathecal factor which, it has been p r ~ p o s e d , ~may ~ ~ unmask
"~ aminergic
receptors within the brain allowing the ecdysteroids to act. In contrast, in B. mori the FES may
act on the terminal abdominal ganglion, whose spontaneous activity is increased after mating
or application of extracts of the male reproductive system.152a Further work showed that the
sensitivity of the ganglion to Ringer's solutions containing varied amounts of NaCl and KC1
is changed after mating, prompting the suggestion that the FES might act by altering the
permeability of the neural sheath surrounding the gang1i0n.l~~~
A second potential role for an FES is to enhance egg development. This has been proposed
by several authors, though only in a few species has a direct link been demonstrated.
B a ~ m a n n , using
l ~ ~ an in vitro system, showed that uptake of I4C-labeled amino acids into
proteins was increased in the ovaries of virgin D. funebris injected with FES. Decapitation
immediately after mating prevents this increase, leading Baumanr~l~~ to suggest that the FES
acts via the neuroendocrine system.
In mosquitoes, also, the FES promotes vitellogenesis, possibly by inducing the ovaries to
produce a hormone whose function is to stimulate release of egg development neurosecretory
hormone from the corpora ~ a r d i a c a .For ' ~ ~anautogenous mosquitoes, an additional means of
promoting egg development has been suggested,154namely, that the FES causes acceleration
Insect Male Mating Systems 47

of blood-meal digestion. Again, the evidence suggests that this effect is exerted via an
endocrine pathway. Even in nutritionally stressed female mosquitoes, accessory gland im-
plants trigger egg development, leading Klowden and Chambers155to suggest that the FES is
acting as a primer pheromone that switches the female's metabolic priorities from self-
sustenance to egg production.
In the bean weevil Acanthoscelides obtectus, the sole demonstrated role for the FES is
stimulation of egg development; that is, the pheromone does not promote egg laying.lS6
The demonstration that mated females carrying an hsP70-msP 355a fusion gene lay 20%
more eggs than controls has led Monsma et al.I4l to speculate that msP 355a may have some
role in egg production. The function of msP 355b is unknown. Like msP 355a, msP 316 has
features common to precursors of peptide hormones though its role remains unknown.I4O
Much less information is available on the site and mode of action of RIS, though, in all
instances, the pheromone exerts its influence via the neuroendocrine system. In H. cecropia,
the RIS, like the FES (with which it may be chemically identical), may stimulate the wall of
the bursa copulatrix to release a hormone.124Originally, it was proposed that this hormone
acted on the corpora cardiaca to stop release of the calling hormone; however, more recent
has ruled out any involvement of the corpora cardiaca in calling behavior, and the
remaining steps in the pathway remain unknown. The brain appears to be the site of action of
the RIS in Musca, as decapitated, decerebrated, or cervically ligated virgins will mate as many
as five times in an 8-h period.158Further, radiolabeled male material transferred during mating
accumulates in the head r e g i ~ n , ' ~ Oleading
J ~ ~ Leopold et a1.Is9to propose that the RIS binds
to receptor sites in the head (brain?) to induce refractoriness. Interestingly, in view of the
apparently identical nature of the RIS and the FES in Musca, refractoriness (which in this
insect is marked by withdrawal of the ovipositor) is induced by high concentrations, whereas
oviposition (requiring extension of the ovipositor) is triggered by low concentrations of the
pheromone.
In contrast, Gwadz's work160 suggests that in Aedes aegypti the RIS acts on the terminal
abdominal ganglion, with the brain, the suboesophageal ganglion, and the thoracic ganglionic
mass having no direct involvement in the control of sexual behavior.
An aspect worthy of study is the interaction between RIS and JH, the latter enhancing
receptivity as females become sexually mature. It is unlikely that RIS directly affects corpus
allatum activity as refractoriness is life-long, though a female goes through several cycles of
egg production, the latter correlated in many species with changes in corpus allatum activity.
As proposed earlier,15it may be that the RIS and JH compete for the same receptor sites within
the central nervous system, with the RIS being successful in normally monogamous species.
In this context, it is noteworthy that when less than normal amounts of RIS are transferred
during mating (e.g., by forcibly separating mating pairs or mating females to already multiply-
mated malesl6I), receptivity returns after a variable period of time.

F. OTHER FUNCTIONS
Several other functions have been ascribed to components of the accessory gland secretion
or to secretory products of other parts of the male tract. Most of these relate to maturation,
activation (motility), or energy metabolism of the sperm; in addition, for some species, the
spermatophore represents a source of nourishment for the female.
How sperm move from the spermatophore to the spermatheca is largely unknown though
it presumably involves either active movement on the part of the gametes or peristalsis of the
wall of the female tract so as to "squeeze" sperm out of the spermatophore andlor to draw
sperm up the spermathecal duct. In Rhodnius, there is good evidence for production by the
opaque accessory glands of a peristalsis-inducing s e ~ r e t i 0 n . Normally,
l~~ the spermathecae
begin to fill with sperm within 5 to 10 min of the end of mating. However, in females mated
to males whose opaque glands have been removed, the spermathecae are still empty even 5
h later. This is not due to a malformed spermatophore (which is a product of the transparent
48 Insect Reproduction

accessory glands) or to an action on the sperm (which even if killed can still be moved into
the spermathecae). In isolated preparations of the reproductive tract, the opaque gland secre-
tion induces peristalsis, ruling out central nervous control; however, D a ~ e yalso l ~ ~showed that
the secretion acts on the peripheral nervous system rather than directly on the musculature of
the reproductive tract. A similar function for the accessory gland secretion has been suggested
for several other species; in all cases, however, an effect on the sperm per se rather than the
tract musculature cannot be ruled out.
In Saturniidae (Lepidoptera), the noncuticular simplex (see Figure ID) produces a sperm
activator (it is not specified whether apyrene, eupyrene, or both types of sperm are affected).I8
The molecule, which is reported to be a peptide of molecular weight about 3100 Da,163
apparently diffuses through the spermatophore wall (already formed from secretions emanat-
ing further up the simplex) and may work by disrupting the sperm plasma membrane.Ibl A
detailed study of sperm activation of Bombyx mori has been undertaken by Osanai and
colleagues, who have demonstrated a remarkable proteolytic cascade within the spermato-
phore that affects both apyrene and eupyrene perm.'^^-^^' During spermatophore and seminal
fluid formation, various components are added from different regions of the ejaculatory duct,
the final (but key) participant being i n i t i a t ~ r i nan
, ~ endopeptidase
~~ released by the proximal
segment of the duct.166Four distinct roles have been identified for this enzyme. It digests the
coating around the apyrene sperm whose resultant activity then serves to stir the seminal
it also digests the intercellular glue that binds together the eupyrene sperm, leading
to their release and a~tivati0n.l~~ Its third role is to split proteins (produced elsewhere in the
simplex) on the C side of arginine r e ~ i d u e s and , ~ ~its
~ fourth role is to activate an arginine
carboxypeptidase produced in the ampulla.168The arginine then released by the action of this
e~opeptidasel~~ is hydrolyzed to ornithine and urea under the catalytic action of arginase from
the seminal vesicles.170From the ornithine is derived glutamate, then 2-oxoglutarate, which
serves both as a substrate for sperm respiration and as a promoter of pyruvate 0xidati0n.l~~
Trehalases have been detected in the accessory glands of male P. a m e r i ~ a n a and l ~ ~in the
BAGS and spermatophore of T.m01itor.I~~ Characterization of the enzyme174shows that it has
high specificity towards trehalose. However, the few published analyses do not suggest that
this sugar normally occurs in very significant amounts in insect seminal fluid. Thus, a role for
this enzyme, for example, in sperm metabolism, remains unclear.
The anterior ejaculatory duct of D. melanogaster produces an enzyme, esterase 6, that is
transferred to the female in the seminal fluid.21Esterase 6 appears to have two purposes: it may
be involved in lipid catabolism in the ejaculate, thus affecting sperm motility;175it was also
proposed22that the enzyme catalyzed the conversion of cis-vaccenyl acetate (produced in the
male's ejaculatory bulb and also transferred during mating) to cis-vaccenol, the latter then
being released by the female as an antiaphrodisiac. However, attempts to confirm this have
been u n s u c ~ e s s f u l . ~ ~ ~
In addition to the RIS described earlier, a second sex peptide has recently been identified
and characterized in the accessory glands of D. f ~ n e b r i s . lThis ~ ~ 63-amino acid-containing
molecule has several similar properties and sequence homologies with known protease
inhibitors, including the ability to inhibit acrosin, a trypsin-like endopeptidase associated with
the acrosome of mammalian sperm. Thus, Schmidt et speculate that this peptide tempo-
rarily inactivates the acrosomal proteases until the appropriate moment for egg fertilization.
Males of some species produce a very large spermatophore (e.g., in some Gryllidae and
Tettigoniidae [Orthoptera] it may be 40% of the male's body weight178),part or all of which
is eventually eaten by the female; in others, for example M. ~anguinipes,'~~ the male transfers
several spermatophores in sequence during a single mating. Though there have been claims
that in some species the spermatophore is digested within the female reproductive tract,
demonstrations of the existence of hydrolytic enzymes in this region are virtually nonexistent.
It has been shown, however, that material from the spermatophore does enter the female's
hemolymph and, in some species, the ovaries, leading to the proposal that the male may
Insect Male Mating Systems 49

thereby make a nutritional contribution to the female or, more specifically, to egg develop-
The subject of male nutrient investment is discussed by Boggs (Chapter 10).
ment.149-180-183

ACKNOWLEDGMENTS
Original work of the author cited in this review is supported by the Natural Sciences and
Engineering Research Council of Canada. Thanks are extended to Dr. J.G. Riemann for
provision of a diagram of the male flour moth reproductive system and to Mr. D. Dyck for
assistance in the preparation of the figure.

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50 Insect Reproduction

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158. Leopold, R.A., Terranova, A.C., and Swilley, E.M., Mating refusal in Musca domestica: effects of repeated
mating and decerebration upon frequency and duration of copulation, J. Exp. Zool., 176, 353, 1971.
159. Leopold, R.A., Terranova, A.C., Thorson, BJ., and Degrugillier, M.E., The biosynthesis of the male
housefly accessory secretion and its fate in the mated female, J. Insect Physiol., 17, 987, 1971.
160. Gwadz, R.W., Neuro-hormonal regulation of sexual receptivity in female Aedes aegypti, J. Insect Physiol.,
18, 259, 1972.
161. Smith, P.H., Gillott, C., Barton Browne, L., and van Gerwen, A.C.M., The mating-induced refractoriness
of Lucilia cuprina females: manipulating the male contribution, Physiol. Entomol., 15, 469, 1990.
162. Davey, K.G., The migration of spermatozoa in the female of Rhodnius prolixus Stil, J. Exp. Biol., 35,694,
1958.
163. Shepherd, J.G., A polypeptide sperm activator from male saturniid moths, J. Insect Physiol., 21, 9, 1975.
164. Shepherd, J.G., Sperm activation in saturniid moths: some aspects of the mechanism of activation, J. Insect
Physiol., 20, 232 1, 1974.
Insect Male Mating Systems 55

165. Osanai, M., Aigaki, T., and Kasuga, H., Arginine degradation cascade as an energy-yielding system for
sperm maturation in the spermatophore of the silkworm, Bombyx mori, in New Horizons in Sperm Cell
Research, Mohri, H., Ed., Japan Scientific Society Press, Tokyo, 1987, 185.
166. Aigaki, T., Kasuga, H., and Osanai, M., A specific endopeptidase,BAEE esterase, in the glandula prostatica
of the male reproductive system of the silkworm, Bombyx mori, Insect Biochem., 17, 323, 1987.
167. Osanai, M., Kasuga, H., and Aigaki, T., Induction of motility of apyrene spermatozoa and dissociation of
eupyrene sperm bundles of the silkworm, Bombyx mori by initiatorin and trypsin, Invertebr. Reprod. Dev., 15,
97, 1989.
168. Aigaki, T., Osanai, M., and Kasuga, H., Arginine carboxypeptidase activity in the male reproductive glands
of the silkworm, Bombyx mori, Insect Biochem., 18, 295, 1988.
169. Kasuga, H., Aigaki, T., and Osanai, M., System for supply of free arginine in the spermatophore of Bombyx
mori. Arginine-liberating activities of contents of male reproductive glands, Insect Biochem., 17, 3 17, 1987.
170. Osanai, M., Aigaki, T., Kasuga, H., and Yonezawa, Y., Role of arginase transferred from the vesicula
seminalis during mating and changes in amino acid pools of the spermatophore after ejaculation in the
silkworm, Bombyx mori, Insect Biochem., 16, 879, 1986.
17 1. Osanai, M., Aigaki, T., and Kasuga, H., Energy metabolism in the spermatophore of the silkmoth, Bombyx
mori, associated with accumulation of alanine derived from arginine, Insect Biochem., 17, 71, 1987.
172. Takahashi, S.Y., Higashi, S., Minoshima, S., Ogiso, M., and Hanaoka, K., Trehalases from the American
cockroach, Periplaneta americana: multiple occurrence of the enzymes and partial purification of enzymes
from male accessory glands, In!. J. Invertebr. Reprod., 2, 373, 1980.
173. Yaginuma, T. and Happ, G.M., Trehalase from the bean-shaped accessory glands and the spermatophore
of the male mealworm beetle, Tenebrio molitor, J. Comp. Physiol. B, 157, 765, 1988.
174. Ogiso, M., Shinohara, Y., Hanaoka, K., Kageyama, T., and Takahashi, S.Y., Further purification and
characterization of trehalases from the American cockroach, Periplaneta americana, J. Comp. Physiol. B, 155,
553, 1985.
175. Gilbert, D.G., Ejaculate esterase 6 and initial sperm use by female Drosophila melanogaster, J. Insect
Physiol., 27, 641, 1981.
176. Vander Meer, R.K., Obin, M.S., Zawistowski, S., Sheehan, K.B., and Richmond, R.C., A reevaluation
of the role of cis-vaccenyl acetate, cis-vaccenol and esterase 6 in the regulation of mated female sexual
attractiveness in Drosophila melanogaster, J. Insect Physiol., 32, 681, 1986.
177. Schmidt, T., Stumm-Zollinger, E., Chen, PS., Biihlen, P., and Stone S.R., A male accessory gland peptide
with protease inhibitory activity in Drosophila funebris, J. Biol. Chem., 264, 9745, 1989.
178. Gwynne, D.T., Male nutritional investment and the evolution of sexual differences in Tettigoniidae and other
Orthoptera, in Orthopteran Mating Systems, Gwynne, D.T. and Morris, G.K., Eds., Westview Press, Boulder,
CO, 1983, 337.
179. Pickford, R. and Gillott, C., Insemination in the migratory grasshopper, Melanoplus sanguinipes (Fab.),
Can. J. Zool., 49, 1583, 1971.
180. Boggs, C.L. and Gilbert, L.E., Male contribution to egg production in butterflies: Evidence for transfer of
nutrients at mating, Science, 206, 83, 1979.
181. Huignard, J., Transfer and fate of male secretions deposited in the spermatophoreof females ofAcanthoscelides
obtectus Say (Coleoptera Bmchidae), J. lnsect Physiol., 29, 55, 1983.
182. Markow, T.A. and Ankney, P.F., Drosophila males contribute to oogenesis in a multiple mating species,
Science, 224, 302, 1984.
183. Boucher, L. and Huignard, J., Transfer of male secretions from the spermatophore to the female insect in
Caryedon serratus (01.): analysis of the possible trophic role of these secretions, J. lnsect Physiol., 33, 949,
1987.
 Chapter 3

SEX DETERMINATION IN INSECTS

Roger L. Blackman

CONTENTS
I. Introduction .................................................................................................................
57

11. General Aspects of Sex Determination in Insects ....................................................58

A. XX/XY Systems ..................................................................................................
58
B. XX/XO Systems ...................................................................................................
59
C. Multiple Sex Chromosome Systems .................................................................... 61
D. "Multiple Factor" Systems ..................................................................................
63
E. Haplodiploid Sex Determination ..........................................................................63
F. The Molecular Basis of Sex Determination ......................................................... 64
G. Dosage Compensation .........................................................................................
67

111. Sex Determination in Different Groups of Insects ..................................................... 67

A. Apterygota .............................................................................................................
67
B. Primitive Exopterygota ........................................................................................ 67
C. The Orthopteroid Orders ..................................................................................... 68
D. The Hemipteroid Orders ....................................................................................... 71
E. Neuropteroidea and Coleoptera ...........................................................................75
F. Hymenoptera .......................................................................................................
77
G. The Panorpoid Orders ........................................................................................... 78

IV. Evolution of Sex Chromosomes and Sex Determination in Insects ..........................85

References ............................................................................................................................
86

I. INTRODUCTION
Sex determination is the process by which the gender of a bisexual organism becomes
fixed, so that the individual progeny develops either as a son or a daughter. As is the case with
other fundamental biological processes, evolution has in the course of time produced a
seemingly infinite variety of ways of achieving this one essentially simple objective, and
classical genetic and cytogenetic observations have, over the years, combined to display a
bewildering diversity of sex-determiningmechanisms. Much of this work has been on insects,
from the first recognition of sex chromosomes in the heteropteran Pyrrhocoris apterus,'
through the classic experiments of bridge^^-^ on Drosophila and Golds~hmidt~.~ on Porthetria
dispar, to the recent molecular work elucidating the hierarchy of regulatory genes responsible
for the sex of fruit f l i e ~ . ~ . ~
In the general literature on sex determination, two works stand out?,1° each with a radically
different approach to the subject. Both cover the full range of sex-determining systems, but
the cytogeneticist Whiteg gives pride of place to evolutionary changes in the sex chromo-
somes, whereas the evolutionary geneticist Bulllo pays more attention to the underlying

0-8493-6695-X/95/50.MkS.50
8 1995 by CRC Press. Inc.
58 Insect Reproduction

mechanisms. The problem with reviewing sex determination in insects at the present point in
time is that, for all orders except Diptera, the greater part of the evidence is cytological, and
even the most basic information about the genetic systems involved is not usually available.
It is nevertheless worthwhile to follow the lead of Nothiger and Steinmann-Zwicky,ll and look
for general principles of sex determination that could be applicable to all insects, and possibly
to all biparental organisms.
In this chapter I shall start by reviewing the main types of sex determination found in
insects, then outline what is known about sex chromosome systems in each insect order, and
end with some discussion of the evolutionary implications of what we now know about sex
determination.

11. GENERAL ASPECTS OF SEX DETERMINATION IN INSECTS

The sex of an insect is almost always determined genetically. Hermaphroditism, in the
sense of the same genotype producing functional male and female organs in the same
individual, seldom occurs in insects; it seems to have evolved only once, in one genus of scale
insects (Icerya). An oft-quoted second example of insect hermaphroditism,in the termiticolous
phorid group Termitoxeriinae, has now been refuted.I2 (True hermaphrodites should not be
confused with gynandromorphs and intersexes - genetically abnormal individuals - which
are of common occurrence in insects.) Environmental factors may sometimes influence the
genetic determination of sex (see BergerardI3for review), but the type of environmental sex
determination that occurs widely in reptiles, for example, where the sex of an individual is
decided by the environment of the egg after it has been fertilized, seems to be rare in insects.

A. XXIXY SYSTEMS
Most bisexual organisms produce a 1:1 sex ratio, and this can be achieved simply by having
one sex (the heterogametic sex) produce two genetically different types of gamete, and the
other sex (the homogametic sex) produce gametes of only one of these types. The two types
of gamete carry different sex factors (here given the notation S, and S,), which segregate from
one another in Mendelian fashion in the meiosis of the heterogametic sex:

s,s, X s,s, (parents) + 1:l s,s, and sls2(progeny)

The heterogametic sex is the male in most insects, but female in Lepidoptera, Trichoptera, and
some Diptera.
Although these sex factors are conventionally regarded as alternative alleles at the same
locus, it is usually the case that only one sex factor plays an active part in the determination
of sex. The other "sex factor" may merely be the corresponding site on a homologous
chromosome: e.g., a nonfunctioning (null) allele, or the location at which the sex factor is
inserted, in the case of a transposable element.
In some cases, sex factors may be inherited as single genes, recombining freely with other
genes on the same chromosome pair, although they are more often than not tightly linked to
other genes involved in sex differentiation. Very often, however, chromosomes carrying sex
factors are cytologically distinct (heteromorphic), so that the inheritance of sex can be
observed cytologically:

XX X XY (parents) + 1:1 XX and XY (progeny)

X and Y chromosomes usually pair at meiosis before segregating to opposite poles, but there
is normally little or no recombinational exchange between them. This is called an XXIXY, or
XY (male) sex determination system. When the female is the heterogametic sex, the sex
chromosomes are sometimes termed Z and W, and the system called ZWIZZ:
Sex Determination in Insects 59

ZW (female parent) X ZZ (male parent) 4 1:l ZZ and ZW (progeny)

but this terminology has now been largely abandoned in the literature on insect cytogenetics
as an unnecessary complication. The fact that the female is the heterogametic sex can be
indicated by putting the heterozygous genotype first; i.e., XY/XX or XY (female) sex
determination.
At this stage it is important to address two common misconceptions about sex chromo-
somes, which can easily hinder understanding of sex-determining mechanisms and their
evolution. The first point concerns the common notation of heteromorphic sex chromosomes
throughout both plant and animal kingdoms as X and Y, which might be thought to imply
some degree of homology, not just at the sex-determining loci but of the chromosome as a
whole, across major groups of organisms. On the contrary, there is no doubt that heteromor-
phic sex chromosomes have evolved many times independently in different taxa.I4 The Y
chromosome in particular can sometimes be an extremely labile structure, apparently under-
going cycles of degeneration and regeneration within taxa. These will be discussed further
later, and in order to emphasize this lability it is sufficient here to note that what seem to be
major changes in sex chromosome constitution, such as the formation of a Y chromosome de
novo (a "neo-Y"), can be found even within a single species.
The second common misconception is that the Y chromosome always has a dominant,
male-determining function. This is certainly the case in mammals and in some insects, but the
more general condition in insects with XXJXY systems is for the Y chromosome to have an
essentially passive role, influencing sex merely by segregating opposite the X at meiosis. The
sex of the zygote is then determined by the balance between the actions of regulatory genes
on the X chromosome and on the autosomes. This is roughly equivalent to the "genic balance"
model developed by Bridges3from his work on Drosophila. The zygote must be homozygous
for a sex factor in order to be female (XX), so this has also been termed a "recessive-X
~ystem".'~
Genetic studies are obviously needed to establish for certain whether the system operating
in any one species is based on a dominant Y or genic balance. For species with heteromorphic
sex chromosomes, deductions are possible by observing the sex of individuals with abnormal
sex chromosome constitutions. The two most informative abnormalities are XXY and XO. If
XXY individuals of a species which normally has an XXIXY system are male, then there is
obviously a dominant Y factor operating, but if XXY is female, then the Y chromosome is
likely to be sexually inert, and the X has an active, although recessive, role. Frequently,
aberrant individuals completely lacking a Y chromosome (i.e., with X 0 constitution) are at
least viable enough to observe their sex; such individuals will be female in dominant-Y
systems, but male in genic balance systems, as the latter require XX for female determination.
Evidence like this is available for only relatively few insects, mostly Diptera, in which occur
both dominant-Y (e.g., Phormia regina, Lucilia cuprina15) and recessive-X systems (e.g.,
Drosophila melanoga~ter,~ Glossina palpali~'~). However, there are good reasons for believ-
ing that genic balance is the most general condition in insects, stemming from the widespread
occurrence of XXIXO sex determination in many insect order^.^

B. XXJXO SYSTEMS
In organisms with heteromorphic sex chromosomes, X-Y recombination is usually sup-
pressed, and the Y chromosome tends to be more degenerate than the X, often having few or
no functional alleles. This degeneration of the Y is generally perceived as a progressive
evolutionary phenomenon.I0Various explanations for this have been offered.l4.I7For example,
because the Y is permanently heterozygous and nonrecombinant, selection must act at the
level of the entire chromosome, so it evolves as "an asexual component of an otherwise sexual
genome".18 Deleterious mutations (often nonfunctional alleles) will tend to accumulate in the
absence of recombination by the process known as "Muller's ratchet,"lg which may be
 Insect Reproduction

(a) "Muller's ratchet" (h) "Hitchhiking"

deleterious
mutation

beneficial
mutation

FIGURE 1. Degeneration of a Y chromosome by operation of (a) Muller's "ratchet," and (b) "hitchhiking." In a
population of Y chromosomes free from recombination (left), some Ys will have one or more mutations to
nonfunctional alleles (black segments), with different Ys mutated at different loci. If the selective disadvantage per
locus is small and population size is small enough relative to the mutation rate, the class of Y chromosomes with no
mutations may be lost by chance and, because there is no recombination, it cannot be restored. Next the class canying
just one mutation becomes vulnerable to chance loss, and so on, so that as time goes on the mean numbers of mutations
per Y chromosome gradually increases. If a favorable Y-linked mutation should occur, however (a),it could spread
rapidly through the population by selection, carrying with it any nonfunctional alleles that happen to be present on
the Y chromosome in which it originated ("hitchhiking"), leading to fixation of nonfunctional alleles and accelerating
the degenerative process.

accelerated by a "genetic hitchhiking" effect18 (Figure 1). However, there is still no entirely
satisfactory explanation.
The end point of such an evolutionary process may be the complete loss of the Y
chromosome, so that males are XO. An XX/XO sex determination system works in exactly
the same way as an XX/XY system, with the X moving to one pole in male meiosis, so that
sperm are either with or without an X:

XX (female parent) X X 0 (male parent) + 1:1 XX and X 0 progeny

Obviously, since there is no Y, such a system must be based on genic balance. XXIXO sex
determination occurs in almost all orders of insects, both primitive and advanced. It is almost
certainly the ancestral form of sex determination of orthopteroid insects and probably of other
major orders such as Hemiptera and Coleoptera. It is even possible that the ancestors of all
insects had X 0 males, and that all insect Y chromosomes have arisen de novo.
Y chromosomes in insects often seem to arise as a result of centric fusion between an X
chromosome and an autosome (e.g., Figure 2). A recently formed, autosomally derived Y
chromosome (a neo-Y) is often easily recognized because it is likely to be still homologous
with the autosomal part of the neo-X, and therefore synapses with it at meiosis. This homology
may be gradually lost in the course of evolution as X-Y recombination becomes suppressed,
and secondary structural and genetic changes occur independently on both the neo-X and the
neo-Y.
Clearly, a neo-Y chromosome cannot carry a dominant factor for male determination, and
sex determination in such cases must be based on genic balance, as in the ancestral XXIXO
system. Neo-XY systems occur most frequently in groups in which XXIXO systems are
common; these include the orthopteroid orders, Odonata, the hemipteroid orders, and Co-
leoptera. Each of these groups will be discussed later. In all of them, there are also species with
multiple sex chromosomes.
Sex Determination in Insects

< plus qy

Autosome

FIGURE 2. White'sg model of the origin of neo-XY sex determination from an X 0 condition in the heterogametic
sex. After breakage near the centromeres of an X and an autosome, centric fusion occurs, creating a neo-X
chromosome. When this fusion has reached fixation in the population (i.e., when the original unfused Xs no longer
occur), the original homologue of the autosome involved in the fusion will be confined to the male line and will act
as a "neo-Y", segregating opposite the neo-X at meiosis. (White9believed that centromeres were never situated at
the extreme ends (telomeres) of chromosomes, and therefore his models assume arm breaks and exchanges, followed
by loss of a minute chromosome. Alternative models of centric fusion involving breakage within centromeres are
discussed by John and H e ~ i t t . ' ~ ~ )

C. MULTIPLE SEX CHROMOSOME SYSTEMS

Chromosomal differences between male and female insects normally involve only one
chromosome pair (X and Y, or just the X in XX/XO systems), but there are numerous cases
in most insect orders where the difference between the sexes involves a larger number of
chromosomes. The most common "multiple" sex chromosome systems have just two Xs (the
notation used for the male condition X,X,Y, or X,X,O, and the notation for the sex determi-
nation system is X,X,X,X2/X,X2Y,or X,X,X2X2/X,X20),although species are known with
almost any number of Xs from 1 to 6 (and in one extreme case, 12), and any number of Ys
from 1 to 6.
The origins of X,X,X2X2/X,X2Ysex determination are usually fairly simply explained,
with good evidence provided by the way in which the X,, X,, and Y associate at metaphase
I of spermatogenesis. Figure 2 showed the origin of a neo-XY condition by centric fusion,
resulting in a metacentric X (i.e., with the centromere near the center of the chromosome) and
an acrocentric Y (with the centromere at one end). If a further centric fusion should occur, this
time between the Y chromosome and another acrocentric autosome, then an X,X,Y condition
will arise, with a metacentric Y (Figure 3).
Alternatively, an X,X2Y condition may be derived directly from an XO, by a reciprocal
translocation between the X and one member of an autosome pair, the other member of the
autosome pair becoming a neo-Y (Figure 4).
The other common derivation of a multiple sex chromosome system is that found in
organisms with holocentric chromosomes (i.e., chromosomes with diffuse centromeric activ-
ity), such as the Hemiptera and Dermaptera, where X,, X,, etc., have almost certainly arisen
simply by dissociation (fission) of the single original X chromosome into two or more parts,
which still segregate to the same pole at meiosis (Figure 5). Such X dissociations have
occurred in both XXIXY and XX/XO systems, a single X dissociation giving X,X,X,Xfl,X,Y
and X,X,X,X~,X,O, respectively. Multiple Ys have also arisen by dissociation in some
species. It is characteristic of multiple sex chromosome systems formed by dissociation that the
X,, X,, etc. are much smaller than the original X, and that there is no accompanying change in
the number of autosomes.
Multiple sex chromosomes are cytogenetically interesting, because they show very clearly
the fixation of different types of chromosomal rearrangement, but they have little or no
 Insect Reproduction

-
plus c@
Autosome (lost)

FIGURE 3. Origin of an X,X,Y condition in the heterogametic sex (leading at fixation to an X,X,X,XdX,X,Y sex
determination mechanism), by centric fusion between an acrocentric Y and an acrocentric autosome, to give a
metacentric "neo-Y" ("neo" because part of it is recently derived from an autosome), and a neo-X,. The arrangement
of sex chromosomes on the spindle of the first meiotic division, with homologous sections associated, is shown
diagrammatically. (Adapted from White, M. Animal Cytology and Evolution, 3rd ed., Cambridge University Press,
Cambridge, U.K., 1973.)

.....
......
......
......
.: :.....
......
:::.
......
......
......
......
....... ......
......
......
......
.......
.....
...... ......
......
......
......
......
......
.......
...... ......
......
....
......
......
..... X2

neo-y

Autosome
1
FIGURE 4. Origin of an X,X,Y condition in the male (leading at fixation to X,X,X,X~X,X,Y sex determination)
from an XXIXO system, by reciprocal translocation between a single metacentric X in the male and a metacentric
autosome. The arrangement on the spindle of the first meiotic division, with homologous sections associated
terminally, and X, and X, moving to the opposite pole from the Y chromosome, is shown diagrammatically. (Adapted
from White, M. Animal Cytology and Evolution, 3rd ed., Cambridge University Press, Cambridge, U.K., 1973.)

FIGURE 5. Multiple X chromosomes (in [b] and [c]) are derived from an XY system (a) by simple dissociation.
Segregation of Xs and Y at spermatogenesis is represented diagrammatically, with autosomes not shown. Usually in
such systems the Xs and Y do not associate, but show "distance" (or "touch-and-go") pairing.
Sex Determination in Insects 63

significance in relation to the molecular genetic basis of sex determination. Of more interest
in this respect are the so-called "multiple factor" systems of certain Diptera.

D. "MULTIPLE FACTOR" SYSTEMS

Species with multiple sex chromosomes are nevertheless still likely to have only a single
sex-determining locus - for example, on just one of the Xs in a multiple X system. But more
complex sex-determination systems are known where one species may have sex-determining
factors at several different loci, and sometimes even different individuals within the same
population have their sex factor on different chromosomes. Such complex systems cannot be
detected cytogenetically, and they have only been recognized in genetically well-studied
organisms with an abundance of genetic markers. In insects, this means certain species of
Diptera.
The type of multiple factor system most commonly observed in Diptera and found in
members of both the Nematocera and the Cyclorrhapha, can be symbolized in a general way
as follows

Females Males
SlSl S2S2 Slsl s2s2
SlSl S2s2

S, and S, behave as dominant male-determining factors, segregating, respectively, at the two

different loci 1 and 2, and always restricted to the male line. This 2-locus system, with S, and
S,, can be generalized to any number of loci:

Females Males
....
S I S l S2S2 S3S3 Slsl S2S2 S3S3....
sls, S2s2s,s ,....
s,s, s2s2S,s ,....

So for any species with a multiple factor system, there is always just one female genotype,
homozygous for all sex factor loci, and n distinct male genotypes, each heterozygous at just
one of the n loci. Sometimes a sex factor locus may be on a cytologically distinct sex
chromosome, so that S,, for example, is manifestly a Y chromosome; but sex factors may
equally occur on chromosomes that are in all other respects autosomal, in which case there is
no obvious cytological difference between the sexes. Dipteran geneticists have termed the
autosomal male-determining factors "M factors". Green20suggested that M factors at different
loci in any one species were perhaps all actually the same gene, transposed to several different
sites in the genome. The evidence now strongly favors this interpretation in several cases
(discussed later under Diptera). This explains the exclusive nature of their occurrence in
individual males, but makes the term "multiple sex factors" something of a misnomer.
This kind of sex determination may occur in other insects, especially where heteromorphic
sex chromosomes have not been detected or do not occur regularly, but the necessary genetic
evidence is lacking for other orders apart from Diptera.

E. HAPLODIPLOID SEX DETERMINATION

No general survey of sex determination in insects would be complete without mention of
haplodiploidy, of which Hymenoptera are, of course, the leading exponents. Haplodiploidy
may also be general to Thysanoptera, and is found in some species of Homoptera and
Coleoptera (as well as occurring widely in mites and ticks). The genetic mechanisms involved
have been the subject of much speculation (reviewed by C r o ~ i e r ~Indeed,
~ ~ ~ ~at) first
. sight, it
is difficult to see how any genetic mechanism at all can be operating, as haploid males simply
64 Insect Reproduction

have half the female dose of all genes. However, several hypotheses based on multiple sex
factors have been suggested, and one has some experimental foundation.
It has long been known that, in certain Hymenoptera, inbreeding results in diploid male^,^^-^^
indicating that not only haploids but diploid homozygotes are male. This can be explained if
there are multiple sex factors (S,S2S,...) - possibly alternative alleles at a single locus -
segregating in opposition:

Females Males
SlS2, SIS,, SzS,, ... S,, S,, S,, ... (if eggs unfertilized)
or S$,, S,S2, S$,, ... (in inbred populations)

The sex factors appear to complement one another, and this has therefore been termed a
complementary sex-determining mechanism.
While this explanation fits members of several groups of Hymenoptera very well (e.g.,
Habrobracon, Apis, Neodiprion, and Solenopsis), it cannot apply generally, because some
other Hymenoptera inbreed considerably, yet fail to produce diploid males. To accommodate
this problem, the hypothesis can be modified to involve multiple loci.21The theory is that
diploids would then have to be homozygous at all loci in order to be male, and this would only
be likely after long-term intensive inbreeding.

F. THE MOLECULAR BASIS OF SEX DETERMINATION

Thus there is a variety of ways in which sex can be determined in insects. Sex factors can
apparently determine either maleness or femaleness, can be dominant or recessive in their
action, can be single or multiple, and can occur on sex chromosomes or autosomes. How can
all this be explained in molecular terms?
Our knowledge of the molecular biology of sex determination is almost entirely restricted
to D. melanogaster, but at least in this one species some of the details of the mechanism are
now worked out. The key gene is Sexlethal (Sxl), which is located on the X chromosome. This
gene is essential for determination of females, but completely functionless in males, so that
it can be eliminated by mutation without affecting the male phenotype. There is a maternal
gene, daughterless (da), that has to be active for Sxl to function, because a mutation at the da
locus causes mothers to produce only sons; however, da is not normally involved in sex
determination. Activation of the female-determining function of Sxl in Drosophila is in fact
dependent in some way on the ratio between the number of X chromosomes and the number
of sets of autosomes (henceforth X:A). Thus, if X:A is 1.0 (as in diploid eggs with two X
chromosomes), Sxl produces an active product that causes the embryo to develop as female,
but if X:A is 0.5 (e.g., diploid eggs with XY or XO) then Sxl is silent and the embryo develops
as male.
The genes repressing Sxl in male eggs have not been identified, but in Drosophila they must
be located on the autosomes, because a genotype with one X chromosome and one set of
autosomes (X + A) is female, whereas the addition of another autosome set (X + AA) results
in a male. The molecular nature of the X:A signal is a source of continuing ~peculation.~~
ChandraZ7proposed that the normal, diploid forms of both sexes produce the same limited
number of repressor (R) molecules; two X chromosomes can bind all the R molecules so that
Sxl can be transcribed, but one X chromosome leaves sufficient R molecules to repress Sxl.
One currently favored form of this hypothesisz8has the X:A signal produced by two or more
X-linked genes (e.g., sis-a and sis-b in Figure 6) whose products act as "numerator elements,"
and are titrated by certain autosomal products ("denominator elements"), so that a sufficient
concentration of sis products to promote transcription of an active product by Sxl will only be
achieved in females (Figure 6).29.30
Sxl regulates the differentiation of the female tissues, in Drosophila acting entirely through
its control of a locus on chromosome 3, transformer (tra), which is also only functional in
Sex Determination in Insects

Female Determination Male Determination

Mother

product needed 0 .
to activate S?d

autosomal repressor
genes (postulated) m/ lm]
products bind
to repressor molecules

... chromosome
t
non-functional
product

lZq-1
- ........ . . ...
.-...
autosome 3

FIGURE 6. Simplified model of a possible mechanism for genetic control of sex determination in Drosophila
melanogaster. Female determinationand differentiation (left) depends on the product of the key gene Sex lethal (Sxl).
The product of the maternal gene daughterless (da) needs to be present for Sxl to be active, but this is normally
supplied to both male and female eggs. It is thought that unidentified autosomal genes produce a similar concentration
of repressor molecules ("denominator elements": R) in both sexes. Female eggs, with two X chromosomes, produce
twice as many "numerator elements" (i.e., products of X-linked loci such as sis-a and sis-b) as male eggs, so that there
is an excess of unbound molecules to promote the female-determining activity of Sxl. The active product of Sxl
influences the transcription of the product of the autosome 3 gene transformer (tra), which in turn acts on the
doublesex locus (dsx: see text and Figure 7).

females. The tra product collaborates with the product of another gene (tra-2) to control the
expression of another locus on chromosome 3, doublesex (dsx) (Figure 6). The dsx locus is
active in both sexes and provides the double switch mechanism necessary to ensure that
development proceeds only as either one sex or the other; it consists of two cistrons, d s p and
dsd, only one of which functions in each sex. In female eggs (i.e., when both tra and tra-2
are active), dsd is active and its products repress the male sex differentiation genes, whereas
in male eggs the products of dsx" repress female sex differentiation genes.
It is now known that regulation at all the three main stages occurs at the level of RNA
splicing$ that is to say, the primary gene products are the same in both sexes, but they are
"edited" by the splicing out of different sections (introns) of RNA to produce the male- and
female-specific messenger RNAs (Figure 7). The male-specific messenger RNAs of both Sxl
and tra include a stop codon which truncates the open reading frame so that the transcript is
nonfunctional. This explains why mutational loss of these genes has no effect in males.
66 Insect Reproduction

Primary gene products

Female-specific M
splicing 9 Default splicing
A
1121415161718
v
STOP

U
Female-specific
splicing 9 Default splicing

. . .:.
or STOP
tra-2

Female-specific &X
splicing 9 Default splicing

FIGURE 7. Production of sex-specificmessenger RNAs (mRNAs) from the primary gene products of the Sexlethal
(Sxl), rransformer (tra), and doublesex (dsx) genes of Drosophila melanogaster by differential splicing. Primary
transcripts of these genes are shown in the center; the boxes represent coding regions (exons), the horizontal lines
joining them represent introns (which do not form part of an active product), and the female-specificand male-specific
patterns of splicing are depicted, respectively, above and below the structures of the primary transcripts. The mRNAs
generated by this process are depicted to left (female) and right (male) of the primary transcripts. In males the mRNAs
result from the default pattern of splicing. which in the cases of Sxl and tra includes a stop codon rendering the mRNA
nonfunctional. The female-specific product of Sxl regulates its own activity by positive feedback, and regulates tra
activity by promoting the female-specific product of that gene, which in turn plays its part in directing the female-
specific pattern of splicing of the dsx gene. (Adapted from Baker, B. Annu. Rev. Gener.. 17, 345, 1983.)

Thus, sex determination in Drosophila depends on a hierarchical system of regulatory

genes. Can such a mechanism be generally applicable, given the apparently diverse systems
of sex determination found in other organisms? Nothiger and Steinmann-Zwicky " speculated
on various mutations in the regulatory system that could, together with changes in the sex
chromosomes, explain most of the variations observed in insects. They regarded the action of
the double switch gene, dsx, as likely to be basic to the sex determination of all insects, and
therefore not capable of functional mutation. They postulated that the primitive system in
insects was probably represented by species in which the male is heterozygous at a single sex-
determining locus. Using the notation introduced earlier in this chapter, in dominant-Y
systems the sex factor S, acts as the male determiner by repression of the key gene Sxl, whereas
the sex factor S, does not code for functional product, thus allowing Sxl to be active. If
heteromorphic sex chromosomes are involved, then the male sex factor S, (or strictly S,, since
its effect is dominant) would be located on the Y chromosome, but it could be transposed to
different locations in the genome, or copied to different locations to form a "multiple factor"
(M) system. Recessive-X systems, which occur commonly and widely in insects, are expli-
cable in terms of a genic balance as already discussed for Drosophila; the repressor gene is
on an autosome, and Sxl is only activated if two X chromosomes are present, i.e., in individuals
homozygous for S,.
It is possible to interpret other, less common, types of sex determination in terms of
mutations of the key gene Sxl, of its repressor, or of the maternal gene da. Some of these
special cases will be referred to later in this chapter.
Sex Determination in Insects 67

G. DOSAGE COMPENSATION
Organisms in which the Y has little or no homology with the X or does not exist at all (XO),
have a gene dosage problem. An XX female has two copies of every X-linked gene, while an
XY or X 0 male has only one copy. Mammals compensate for this by inactivation of one of
the two X chromosomes in female somatic tissues. However, in Drosophila, where polytene
chromosomes make it easy to study the level of transcriptional activity, dosage compensation
is achieved in a different way. Both X chromosomes are active in female tissues, but the
transcription rate is only half that of the single X chromosome in males, which produces just
as much RNA as the two Xs in females put t~gether.~"' The hyperactivity of X-linked genes
in male Drosophila appears to be due to a set of genes (msl) which are inhibited when the key
gene Sxl is active and are therefore only functional in males8
The only other information on dosage compensation in insects is in Orthoptera. Rao and
Ali32showed that both X chromosomes in hepatic cecal cells of female Acheta domesticus
were euchromatic (i.e., transcriptionally active), and provided some evidence - using an
indirect measure of transcriptional activity of unproven reliability - that the single X
chromosome in the male may be hyperactive, as in Drosophila. On the other hand, females
of the mole cricket Gryllotalpa fossor (=africana?) seem to have only one arm of one X
chromosome transcriptionally active in hepatic cecal cell~,3~,"which resembles the system in
mammals. However, there is evidence that activity or inactivity of the X chromosomes in
Orthoptera may differ among tissues.35
In Lepidoptera, the limited evidence available from the differential activity of sex-linked
loci suggests that members of this order may manage without a dosage compensation mecha-
nism. Indeed, Johnson and Turner36suggested that in mimetic butterflies the dosage differential
may be used to advantage, in order to limit expression of a polymorphism to the female sex.

111. SEX DETERMINATION IN DIFFERENT

GROUPS OF INSECTS
A. APTERYGOTA
Of the four most primitive extant orders of insects, only the Collembola have been studied
sufficiently to warrant generalization, and in these the male is the heterogametic sex and is
normally X0,37J8 but in Neanuridae, species with X 0 and others with XY are known.39
Presumably the XY species are neo-XY, but there is no cytological evidence to confirm this.
In Neanura monticola, with X 0 males, the X chromosome shows considerable polymorphism
with large amounts of heterochromatin (probably repetitive, noncoding DNA) in high altitude
population~.~~ In the Thysanura, Themobia domestica possibly has X,X20males.4O In Protura,
on the other hand, no instances of an XXKO system have been reported; very few of the
species examined had morphologically differentiated sex ~hromosomes.4'~~~ No representa-
tives of the Diplura seem to have been examined cytologically.

B. PRIMITIVE EXOPTERYGOTA
XX/XO sex determination predominates in the Odonata, possibly in the Ephemer~ptera~~
(although these are poorly studied), and certainly in the main Orthopteroid orders (Dictyoptera-
Phasmida-Orthoptera). Where an XXIXY system occurs in these groups, it is usually clear that
it is a neo-XY system, formed by fusion of an X with an autosome (Figure 2), so that the neo-
Y is homologous with a large part of the neo-X. In the anisopteran families of Odonata, for
example (reviewed by Kia~ta,"~")most species are XXIXO, but there are apparent neo-XY
systems in 15 species scattered through the families Gomphidae, Aeschnidae, Cordaliidae, and
Libellulidae, representing about 4% of the dragonflies then studied. Species with neo-XY
generally have, as might be expected, one less autosome pair than related species with an X 0
68 Insect Reproduction

system; e.g., Aeshna crenata has 2n = 28 and X 0 males, whereas A. grandis has 2n = 26 and
neo-XY males.46 Kia~ta,4~ followed by Tyagi,4' explained an evolutionary decrease in the
number of autosomes in the family Gomphidae as a succession of fusions and translocations
between the neo-Y, the neo-X, and autosomes, the outcome of each step being a secondarily
derived X 0 system with one fewer autosome pairs. However, there is no clear cytogenetic
evidence that the sex chromosomes are involved in these changes of karyotype.

C. THE ORTHOPTEROID ORDERS

In the Plecoptera, which are generally thought to be an orthopteroid order that retains
primitive features, several species in different genera have X 0 males, always with a very large
metacentric X chromosome that moves in a highly characteristic way in the first meiotic
division.48 XY males (presumably neo-XY) are only recorded for one species (Perla
(=Paragnetina) immarginata), but some species of Perla have a multiple X chromosome
system apparently derived from XX/XO, males being X,X20, and in Perlodes there are three
species known with X,X,X30 males. The two or three Xs in these species are much smaller
than the single X of X 0 and XY males in related species, and associate together in the first
meiotic division. Their mode of origin is a mystery, as there is no simple way in which a large
metacentric chromosome can give rise to several smaller elements.
The cytogenetics of the other orthopteroid orders has been comprehensively reviewed by
H e ~ i t (Orthoptera)
t~~ and WhiteS0 (Grylloblattodea, Dictyoptera, Isoptera, Phasmida,
Dermaptera, and Embioptera), so information about the sex chromosome systems of these
groups will only be summarized here and up-dated. Dictyoptera (Blattodea + Mantodea),
Phasmida, Orthoptera, and Embyoptera all seem to be primitively XXIXO, whereas in
Isoptera and Dermaptera XX/XY predominates and is possibly the primitive condition.
In Blattodea (cockroaches), males seem to be invariably X 0 where both sexes have been
k a r y ~ t y p e d Whiteso
.~~ suggested that this stability of the sex determination system could be
due to the fact that the X is almost always metacentric, and therefore not so readily available
for centric fusion with an autosome to generate a neo-XY system. Yet it is difficult to see why
this argument does not equally apply to Phasmida, which also have a metacentric X yet
frequently develop a neo-XY system.
Isoptera (termites) are generally thought to have arisen from primitive Blattodea but,
whereas the most primitive cockroach examined cytologically has XX/X0,52 the most primi-
tive extant termites seem to be mainly XX/XY.53Nevertheless, some species do have XX/
XO?4 and fusions and translocations between sex chromosomes and autosomes are so com-
mon in termite^^^"^ that XX/XO could still be the primitive c o n d i t i ~ n . ~ ~
Several species of Kalotermitidae in southern U.S. and the Caribbean form remarkable
chains or rings of up to 19 chromosomes in male meiotic n~clei,~'-~O often involving more than
half the total chromosome complement. The chains are thought to be due to a series of
reciprocal translocations involving both the sex chromosomes and the autosomes of one
chromosome set, these changes being restricted entirely to the male line, so that all the
chromosomes involved function together as a multiple Y chromosome complex (Figure 8).
Females are structurally homozygous and form normal bivalents at meiosis. The genetic
consequences of such an arrangement are quite profound; for example, they restrict many
alleles to males and increase the genetic similarity of offspring to the same-sex parent and to
same-sex siblings. The idea that this unusual system has played a significant part in the
development of eusociality in termites61is, however, somewhat undermined by the fact that
the most extreme rearrangements are found in only a few of the more primitive termites.
Mantodea also seem to have an XX/XO mechanism with a metacentric X chromosome as
the primitive condition, but members of the largest subfamily Mantinae consistently have an
X,X,X2X2:X,X2Ymechanism that has aroused considerable interest among cytogeneticists.
White62proposed that this was derived from XXIXO by translocation between the X and a
metacentric autosome (Figure 4), and all subsequent evidence has been consistent with this
Sex Determination in Insects 69

FIGURE 8. Diagram illustrating how a series of reciprocal translocations, involving one member of each of six
autosome pairs and the Y chromosome (a), could lead to a ring of linked chromosomes in male meiosis of the termite
Incisirermes schwarrzi. The reciprocal interchange set is stippled, and only occurs in males. Chromosome pairs not
involved in translocations (right) form normal bivalents. Sizes of chromosomes are arbitrary; the X and Y chromo-
somes have not actually been distinguished from the autosomes or from each other in this species. (Adapted from
Syren, R. and Luykx, P,,Nature (London), 266, 167, 1987.)

hypothesi~.~~ All Mantinae have a remarkably consistent chromosome complement, with 2n

(male) = 27 (only one exception is known - see below). Presumably the X,X,Y system had
a single origin in the evolution of this subfamily, and some of the species with X,X2Y males
currently placed in other subfamilies are perhaps wrongly classified. However, the African
mantid genus Compsothespis has an X,X2Ysystem with much smaller sex chromosomes, and
2n (male) = 23; at least in this case, an independent origin seems likely (see WhiteS0for further
details). It is not at all apparent why this system has proved so successful for mantids. The
mechanism itself does not seem very efficient; complications often seem to arise in correctly
forming an X,X2Y trivalent in the first division of spermatogenesis, and consequently in
correctly segregating the X, and X, into one daughter spermatocyte and the Y into the other.
~ ~ for Mantis religiosa that the first meiotic division of those
Callan and J a ~ o b sshowed
spermatocytes that fail to form the X,X,Y trivalent is inhibited, thus preventing the formation
of aneuploid sperm. Liebenberg et al.@ reported a single male of Polyspilota aeruginosa
(Mantinae) with 2n = 28 instead of the usual 2n = 27, and an X,X2Y,Y, mechanism. The origin
of the extra Y (neo-Y) in this one aberrant case is unclear.
Whitesolisted 57 species of Phasmida (stick insects) with identified sex chromosomes, of
which 49 are reported to have X 0 males - undoubtedly the primitive condition - and 7
species (in 6 separate genera) have a neo-XY system arising through fusion of an X with an
autosome (see Figure 2). The one other species studied, Didymuria violescens, occurs in
Southeast Australia, where it has at least 10 chromosomal races, occupying contiguous
distribution areas, and including both X 0 and neo-XY forms.6s Several independent origins
of a neo-XY system can be traced from X 0 ancestors.50
In the well-known parthenogenetic stick insect Carausius morosus, males and masculin-
ized females (intersexes or sex mosaics) appear occasionally in laboratory cultures, and their
numbers can be enhanced by various treatments, e.g., subjecting the eggs to high (30°C)
temperature,'j6centrifuging the eggs,'j7X-irradiating egg or o ~ c y t e sor, ~injecting
~ the mother
with pterine derivative^.^^ Females and masculinized females have three metacentric chromo-
somes that are regarded as sex chromosomes because of their behavior in meiosis. Males lack
one of these sex chromosomes or a segment of one of them.'O The method of sex determination
is difficult to work out because the female karyotype is highly aberrant due to its long history
of parthenogenesis. It has been suggested that C. morosus originated as a triploid or tetra-
p10id.~~ However, whatever their origins, both sex chromosome and autosome complements
70 Insect Reproduction

are now aneuploid, and cannot be regarded as comprising any particular number of chromo-
some sets. Male determination presumably occurs because of a change in the genic balance
between factors on the sex chromosomes and on the autosomes (so that, assuming that the
molecular model established for Drosophila applies, the key female-determining gene Sxl is
repressed). It is not clear why intersexes, which retain the female karyotype, arise under
certain conditions; one possibility is that high temperatures, etc. prevent splicing of female-
specific messenger RNAs. General inactivation of the sex chromosomes by
heterochromatinization has been suggested70 to function in sex determination, but such
heterochromatinization has only been observed in germ-line (spermatogonial) interphase
nuclei, and it is not known whether it occurs in embryonic somatic cells.
Only eight species of Embioptera have been studied cytologically (four in each of the
families Oligotomidae and Embiidae), and all have an odd number of chromosomes in male
somatic cells, indicating that sex determination is probably XX/XO, with the X chromosomes
large and m e t a c e n t r i ~Nothing
.~~ is known about sex determination in Zoraptera.
H e ~ i tcomprehensively
t~~ reviewed the extensive cytogenetic studies that have been car-
ried out on the Orthoptera proper (Saltatoria). Since Hewitt's review, there have been signifi-
cant contributions on the sex chromosome systems of neotropical Acridoidea (about 200
species72),the acridoid subfamilies Catant~pinae,'~and Pam~haginea,~~ Indian Orthoptera (30
species7s), and certain Tettigon~idea?~-~~ XX/XO sex determination is found in the great
majority of species in all subdivisions of the order, both primitive and advanced, and is
undoubtedly the primitive condition for the Orthoptera as a whole. The only exception is the
relic group Grylloblattodea, with XY males in the only two species studied,'O but in the face
of all the other evidence, this must be regarded as a derived state. About 8% of species have
X Z X Y or XlXlX2X,/XlX2Ysystems, which occur in every major subdivision of the group
and are usually clearly evolved secondarily from an XX/XO condition by centric fusion
(Figures 1,2). Two cases are known, one in Eumastacoidea ("Morabinae species P45b")79and
the other in Tettigonoidea (Callicrania ~ e o a n e i of
) ~ the
~ neo-X being formed by "tandem
fusion" of an autosome to the centromeric end of the original X. In both these cases, the neo-
Y forms a terminal connection with the neo-X at meiosis, and this neo-XY bivalent divides
equationally at first meiotic division, so that the X and Y do not segregate until the second
division ("postreductional meiosis"). The mantid type of origin of an XlX2Ysystem, directly
from XX/XO by translocation between an X and an autosome, is not known to occur in
Saltatoria, perhaps because autosomes in this order are predominantly acrocentric? making
centric fusions a more likely occurrence.
The neo-X produced by centric fusion between an acrocentric X and an acrocentric
autosome is likely to be large and metacentric, and the neo-Y (the original autosome) is
acrocentric (see Figure 1); the majority of cases of neo-XY systems in Saltatoria have sex
chromosomes of this form (see Table 8 in H e ~ i t t ~Likewise,
~). neo-X,X,Y males produced as
a result of a Y-autosome fusion have a metacentric X, and Y and an acrocentric X, (Figure
2); again, the majority of X,X,Y systems in Saltatoria conform to this pattern. This may reflect
the recent origin of many of these systems because, once a neo-Y is formed, it is subject to
very different evolutionary pressures from the original autosome. Several species have been
studied that have both X 0 and neo-XY p o p ~ l a t i o n s ; ~presumably
~ , ~ ~ , ~ ~ the
- ~ ~neo-XY system
is only very recently established in such populations, and in some cases the early stages of
differentiation of the neo-Y from its homologue, now part of the neo-X, can be observed. The
neo-Y may acquire heterochromatic segments, and pairing between the neo-X and neo-Y may
become restricted to terminal regions, so that crossing-over is limited, paving the way for
further differentiation of the genetic role of the neo-Y from that of its former homologue.
In time, as discussed earlier, the neo-Y is likely to degenerate; an example of this may be
the Gryllacridoid genus Dolichopoda, where the "neoW-XYsystem is possibly as old as the
genus itself, and all species studied have a large metacentric X and a small dot-like Y.83
However, no instance has yet been identified in Orthoptera of the complete loss of a neo-Y,
Sex Determination in Insects 71

to revert to an X 0 system, which suggests that the neo-Y may acquire and retain some
functional male-linked loci.49
The earwigs (Dermaptera) seem to stand somewhat apart from the other orthopteroid
orders, and this is reflected in their chromosomes, which have diffuse centromeric activity like
those of Hemiptera, and in their sex determination system, as the primitive condition for the
group seems to be XXIXY rather than XX/XO. Only two species with X 0 males are recorded,
belonging to different families.84Multiple sex chromosomes are very common, occurring in
about half the species that have been karyotyped, with similar frequency of incidence in all
families. Multiple Xs have probably arisen by simple dissociation of the existing X chromo-
somes, as in other insects with holocentric chromosomes (Figure 5). They form a close cluster
on the spindle at first meiotic division, and all move together to one pole, while the X moves
to the opposite pole. The ubiquitous earwig Foficula auricularia is unusual in having two
alternative Y chromosomes, one of which ("Y,") is mitotically unstable so that it tends to
accumulate in number, and individual males may have up to four copies (XY,Y2Y,Y2).
Mosaic males have been recorded with different numbers of Y chromosomes in the cells of
each testisg4

D. THE HEMIPTEROID ORDERS

The Psocoptera are generally regarded as close to the basal hemipteroid stock, and all the
32 species so far examined cytologicallysSseem to have XX/XO sex determination. Nothing
is known about the sex-determining mechanisms of biting and sucking lice (Mallophaga and
Siphunculata), as no sex chromosomes have been identified in any of them. When a female
human louse (Pediculus humanus) is mated with a single male, the sex ratio of the progeny
is strongly biased toward one or other sex, and unisexual broods are common.86Contrary to
White? no information is available about the progeny of females mated more than once, and
it seems likely on the available evidence that maternal factors are involved in the determina-
tion of sex in lice, as in certain Diptera (e.g., Chrysomya).
Most species of Heteroptera have XX/XY sex determinati~n,~' but there are some groups
- e.g., 124 species in the related families Coreidae and Alydidae - that are almost exclu-
sively XX/XO.ggX 0 males also predominate in the supposedly more primitive Heteroptera
(Gerromorpha; but see Calabrese and Tallericog9),and Ueshimas7 concluded that the XY
system in Heteroptera, despite its widespread occurrence, is derived from a primitive X 0
condition. Nokkala and N ~ k k a l aon , ~the other hand, argued that XY was ancestral. Clearly,
XY systems are ancient and well-established in terrestrial Heteroptera; the X and Y chromo-
somes generally show little or no evidence of the homology expected of a neo-XY system and
undergo a characteristic pattern of meiotic behavior in which they usually segregate at the
second division (for details, see Whiteg(pp. 620-62 1) or Ueshimag7).The scattered occurrence
of X 0 species within genera must surely be due to secondary loss of the Y chromosome, and
such loss may have occurred early in the evolution of many families of terrestrial Heteroptera.
However, this does not rule out the possibility that the common ancestor of all Heteroptera was
XO, as in Psocoptera, and that XX/XO sex determination may be the primitive condition in
some families of Gerromorpha. The problem can only be resolved when the cytology of
members of the most primitive groups, Enicocephalomorpha and Dipsocoromorpha, now
thought to have a sister-group relationship with all other Heter~ptera,~' as well as of the relic
family Peloridiidae (suborder Coleorrhyncha), have been examined. The only information for
these groups so far is for one species of Dipsocoromorpha, males of which were tentatively
recorded as X0.92Multiple sex chromosomes are common in Heteroptera, and may be derived
from either XY or X 0 systems. Apparently they are in most cases due to dissociation of the
X chromosome into two or more smaller parts, which group together on the spindle of the
second meiotic division and move en bloc to one pole (see Figure 5). In some species, the
number of X chromosomes varies; the best-known example is the bedbug Cimex lectularius,
where the number of separate X elements varies from 2 to 15.
72 Insect Reproduction

Messthaler and T r a ~showed

t ~ ~ that the Y chromosome was heterochromatic and therefore
transcriptionally inactive in all stages of spermatogenesis of the milkweed bug, Oncopeltus
~ can be little doubt that the Y chromo-
fasciatus. Despite the reservations of T h o m a ~ ?there
some in Heteroptera is genetically inert, and that sex determination is based, as in most insects,
on a "recessive-X" (i.e., genic balance) system. Otherwise it would be impossible to explain
how the secondary loss of the Y chromosome could occur in so many groups without
concomitant loss of genetic viability.
Sex determination in Homoptera-Auchenorrhyncha is predominantly XX/X0.95,96A few
species with XY males occur within genera and subfamilies that are otherwise exclusively
XO, and in such cases it is often clear that the Y is a neo-Y; i.e., the homologue of an autosome
that has recently fused with the X. Such a neo-Y pairs with the autosomal part of the neo-X
in meiosis and segregates from it at the first divi~ion.~' In several species of Oncopsis, both
X 0 and neo-XY males occur in the same or different populations; the XY state results from
fusion of the X chromosome with a different autosome in each species.98
The Homoptera-Sternorrhyncha include some of the most specialized hemipteroid fami-
lies, and the basic system of sex determination is often obscured, especially in groups with
well-developed parthenogenesis. The Psylloidea are the least reproductively specialized, and
here again sex determination is predominantly XXKO. Only three species with XY males
have been found in a total of 39 species examined, all apparently recently derived from X 0
by X chromosome-autosomefu~ion.~~-lO~ The Aleyrodoidea have received very little attention
from cytogeneticists. On the basis of early cytological work on three species,lo2J03and the
observation that males are only produced in laboratory populations by unmated females, the
general presumption is that all male aleyrodids are haploid? It would be preferable to have this
confirmed for more species before assuming that haplodiploidy is of general occurrence in this
group. The factors invoking male determination are unclear, but the cytological mechanism
in those species studied seems to be the same as in Hymenoptera, with meiosis replaced by
a single mitotic division, each primary spermatocyte giving rise to only two spermatids.
Populations of Trialeurodes vaporariorum seem to have an approximately 1:l sex ratio in
field populations in both Europe and North America,Io4which is unusual for a haplodiploid
system. However, thelytoky is a complicating factor in interpreting sex ratios in this species.
The populations originally introduced from North America to England consisted almost
exclusively of thelytokous females,Io3and it is not known what proportion (if any) of females
reproduce thelytokously in present-day populations. In Bemisia tabaci, which has not been
studied cytologically, thelytoky is unknown and the number of males produced seems to be
temperature dependent.los
It is impossible to do justice here to the remarkable sex determination systems of scale
insects (Coccoidea), and for details the reader is referred to the authoritative reviews by
NU^.^^^.'^^ All the different systems are believed to have evolved from an ancestral XXIXO
system which is still found in some members of the more primitive families (Ortheziidae,
Margarodidae, Phenaeolaechiidae). Some of the margarodids (Icerya and four closely related
genera) have evolved male haploidy, and in some species of Icerya there is the further
development of hermaphroditism, with morphologically female individuals maturing haploid
sperm and diploid ova in an ovotestis. In hermaphrodite Ice~ya,fertilization is usually between
eggs and sperm of the same individual; nevertheless, some eggs apparently remain unfertilized
and give rise to functional haploid males. In all the more advanced families of Coccoidea, the
paternal set of chromosomes is rendered inactive in most tissues by heterochromatinization
during the development of male embryos (the "lecanoid" and "Comstockiella" systems; see
NurIo6). It seems that sex in these families is determined maternally rather than by the
genotype of the zygote, because the sex ratio is greatly affected by the age of the female at
mating and by environmental conditions such as temperature.'07 It is not clear, however,
whether the inactivation occurs after, and as a consequence of, the embryo already having
Sex Determination in Insects 73

been determined as male, as in Sciaridae (see Diptera, below), or whether the inactivation
process itself provides the mechanism for male determination.Io7
Bulllo pointed out that the evolution of these advanced coccoid systems from XXIXO is
something of a mystery, because the heterochromatinized paternal chromosomes are elimi-
nated in spermatogenesis, so that all sperm carry only the maternal genome. There is thus no
genetic polymorphism among sperm to serve as a basis for sex determination, which effec-
tively means that the advanced coccoid systems can never have coexisted with a system such
as XXIXO, and must therefore have evolved through a form of sex determination without male
heterogamety. There are a few coccid species (e.g., Lachnodius eucalypti) without identifiable
sex chromosomes, and which do not undergo heterochromatinization of one chromosome set
in the male (2N-2N of NurIM).Nur thought that these were probably derivatives from forms
with heterochromatinization,but Bull's argument makes it more likely that they are represen-
tative of this intermediate stage, evolved from XXIXO prior to the origin of
heterochromatinization, which is in line with the original views of Brown.Io8 Haig has
developed a model for the evolution of the advanced coccoid systems based on sex ratio
theory .log
Aphids (Aphididae) all have XX/XO sex determination. An XXKY system would be an
impossibility for these cyclically parthenogenetic insects, because most species exist through
the summer as all-female, thelytokous populations, and during this period the Y chromosome
would have "nowhere to go". Aphids produce males parthenogenetically. To develop as X 0
males, oocytes have to lose half the sex chromatin of the parent female. This is achieved in
a single egg maturation division, as in the thelytokous production of females, but the X
chromosomes pair during prophase1'Oand then undergo a sort of "mini-meiosis" on their own,
first separating the products of pairing and then dividing equationally with the autosomes, all
on the spindle of the single maturation d i v i ~ i o n . ~ l This
l . ' ~ ~peculiar cytological mechanism for
male determination is of special interest because it is normally triggered by environmental
conditions and mediated by a low level of juvenile hormone in the haemolymph; males can
be induced by treatment with precocene, which destroys the corpus allatum, and inhibited by
the juvenile hormone analogue kin~prene."~ The environmental factors are normally photo-
period (actually the length of the dark phase) and temperature in Aphidinae, but may be
nutritional in other groups, and in some species, males appear spontaneously or after a
genetically programmed number of thelytokous generations.Il4
The spermatogenesis of aphids is also relevant to their sex determination, because the
fertilized eggs must all develop as thelytokous females, so all the sperm from X 0 males must
carry an X chromosome. This is achieved by a peculiar first meiotic division in which the X
is stretched on the spindle before passing into one of the daughter spermatocyte nuclei, after
which the daughter nucleus without an X degenerates.Il5
Multiple X chromosome systems occur in some aphids, apparently as a result of dissocia-
tion of the original X, and the separate elements all behave in the same way in m e i o ~ i s . l ' ~ * ' ~ ~
The greenideine species Schoutedenia ralumensis (=lutes) has what was presumably origi-
nally an X,X,X2X2/X,X,0 system, but it has become modified in a remarkable way by
consistent association or temporary fusion of one member of an autosome pair with X, and
the other member of the same pair with X,.lk8Male determination necessarily retains both
these autosomal homologues (AA), so males receive the two elements X, + A and X, +A. One
of the X chromosomes (it is not clear whether it is X, or X,) then has to lose its connection
with the autosome at anaphase I of spermatogenesis, so that males can transmit one X + A and
one X to the next generation (Figure 9). How this peculiar system evolved as a stable
mechanism for sex determination is something of a mystery.
Multiple X chromosome systems with X,X2 males also occur in the primitive aphidoid
families Phylloxeridae and Adelgidae (see BlackmanH9for review), and show some unusual
features in the few species studied. In particular, there seem to be species in each group which
have evolved a potential for male-linked inheritance "by proxy," which overcomes the total
 Insect Reproduction

1 Spermatocyte ll nucleus lacking

0 xl and xz degenerates

FIGURE 9. Sex chromosome-autosome associations in the aphid Schoutedenia ralumensis (=S.lurea). For simpiic-
ity only, the X chromosomes and the pair of autosomes (AA) associated with them are shown. Female somatic cells
(a) have four long chromosomes of unequal length, representing X,, (X, + A), X, and (X, + A). Male somatic cells
and spermatogonia (h) have the longest two chromosomes, which are (X, + A) and (X, + A). In spermatogenesis, at
prophase of the first meiotic division, the autosomes attached to X, and X,, being homologous, pair in parallel (c,d).
When the cell divides (anaphase), either X, or X, loses its connection with the autosome (e; shown here as X,, but
it is uncertain which). The lost autosome passes into one daughter cell which lacks both X chromosomes and
degenerates. The other daughter cell has both X chromosomes, with one autosome still attached to one of them (0;
it divides equationally to give spermatids with the same chromosome constitution. Presumably, for the system to be
stabilized, oogenesis must somehow result in oocytes with the complementary arrangement; i.e., if spenn have (X,+
A) and X,, oocytes will have X, and (X, + A). It is not known how this is achieved. (Based on Hales, D. Chromosoma
98, 295, 1989.)

absence of males during the parthenogenetic (thelytokous)part of the life cycle, by having two
cytologically distinct types of all-female line; one leading eventually to male production and
the other to sexual females. In Phylloxera caryaecaulis, studied by the pioneer cytogeneticist
T. H. Morgan,120one member of the smaller "pair" of X chromosomes seems to be limited to
the male-producing line, and behaves differently from the other in its pairing relationships
during sex determination and spermatogenesis (Figure 10). In the adelgid Gilletteella (=Adelges)
cooleyi, Steffan12' found one member of the longer pair of X chromosomes dissociated into
two parts in about 50% of thelytokous females, and in the somatic cells of males, but not in
sexual females. Further work is needed on these groups to confirm and extend these findings.
The last hemipteroid order to be considered is the Thysanoptera (thrips), both suborders of
which (Terebrantia, Tubulifera), on the basis of the few species that have been studied
cytologically, have haploid males.122.123
The cytological mechanism involved is not very clear,
but seems to differ from that of Aleyrodoidea and Hymenoptera, and must be independently
derived. Instead of meiosis being replaced by a single mitotic division, as in other insects with
haploid males, two meiotic divisions are retained; the first is apparently equational, giving rise
to two similar-sized spermatocytes, but the second produces one large functional spermatid
Sex Determination in Insects

Female-Producing Male-Producing
Line Line

Polar plate of

+ +
Stem mother's egg

#- Somatic metaphase of

R.#
parthenogenetic generation Ot3
+
Polar plate of Polar plate of
6 egg
J

Anaphase
9 egg + +
Somatic metaphase
of 6 e-
Somatic metaphase O O Q

of sexual 9

Anaphase I
sexual
egg

and oocyte 9 line 6 line

nucleus

FIGURE 10. Chromosome cycle of Phylloxera caryaecaulis, redrawn from M~rgan.~~O Autosomes are shown
black, X chromosomes white, except for one member of the smaller pair (X,) in the male line, which is stippled to
show its differential behavior and possible role in sex determination. In the line leading to production of sexual
females (left), small and large X chromosomes seem to be consistently associated, in the somatic cells of both
parthenogenetic and sexual females and throughout oogenesis. In the line leading to male production (right), the small
and large X chromosomes are likewise associated throughout the parthenogenetic phase, but during maturation of
eggs destined to become male, the Xs exchange partners, so that the two large X chromosomes form one pair, and
the small Xs another. Consequently, at maturation division of male eggs, the small and large X chromosomes
segregate from each other independently. Males have X,X,O; half of them apparently have X, and X, associated
together as in females, and half have them separate. Sperm with separate X, and X, are believed to give rise to the
parthenogenetic line that will produce the males of the next bisexual generation.

and one much smaller one that rapidly degenerates.lZ2As in the Aleyrodoidea, the factors
invoking male determination are unclear; sex ratios show considerable variation within and
between species,lZ4but the interpretation of these in genetic terms is complicated by the
occurrence of thelytokous parthenogenesis in many of the best-studied species.Iz5 In
Elaphrothrips tuberculatus, females have unisexual broods, the males being produced vivipa-
rously and the females oviparously; more males seem to be produced when the offspring are
larger and fitter in the favorable nutritional conditions of spring.lZ6

E. NEUROPTEROIDEA AND COLEOPTERA

The Neuropteroidea (Megaloptera, Raphidioptera, and Plannipennia) are generally re-
garded as an early branch in the phylogeny of the endopterygote insects, but no species have
 Insect Reproduction

FIGURE 11. Diagrammatic drawings of first meiotic metaphase of male of (a) the neuropteran Macroneurus
appendiculatus, showing "distance pairing" of X and Y chromosomes, and (b) the megalopteran Neohermes
filicornis, showing X and Y forming a bivalent like the "parachute bivalent" (Xy,) of Coleoptera. Structure of the
parachute bivalent is shown in (c). (Based on Hughes-Schrader. S. Chromosoma. 81, 307, 1980.)

been found with XXIXO. Almost all species studied seem to have XXIXY sex determination,
with a few showing multiple X systems. The X and Y chromosomes of Raphidioptera and
Plannipennia (=Newoptera sensu stricto) behave in a very consistent fashion during spermato-
genesis (Figure l la). They are both small chromosomes that apparently lack any homology,
because they never pair to form a bivalent in the first meiotic division, and regularly take up
positions in opposite halves of the spindle before segregating into the daughter spermato-
c y t e s . 9 ~In~ the
~ ~ two species of Megaloptera that have been studied, however, the X and Y
chromosomes form a bivalent that positions itself with the autosomes on the equator of the
spindle and segregates synchronously with them at the first meiotic division.128The Y
chromosome is much smaller than the X, and in one species the bivalent looks very like the
"parachute" bivalent (Xy,) found in Coleoptera (Figure l lb,c, and see below).
Thus, the sex chromosome systems of the Neuropteroidea seem to provide useful phylo-
genetic evidence pointing to a sister-group relationship between Raphidioidea and Plannipennia,
and also supporting the often-held view (e.g., Henning129)that the Megaloptera are the sister
group to the Coleoptera.
The Coleoptera show a great diversity of sex chromosome systems, although the underly-
ing genetics of sex determination may well be far less variable, and is likely to be based on
a recessive-X mechanism, except where male haploidy has evolved. Coleopteran cytogeneti-
cists have accumulated information about the sex chromosome systems of over 2500 species.
Fortunately, the comprehensive reviews by Smith and VirkkiI3O and Virkkil3I mean that only
a brief overview and some updating are necessary here.
The peculiar symbols used in the literature on beetle sex chromosomes are somewhat
daunting to the nonspecialist, but can be simply explained. They symbolize the appearance
and behavior of the sex chromosomes in the first meiotic division of the male beetle. Sex
chromosome symbols are written together if there is any sort of pairing between them to form
a bivalent (e.g., XY), but separated by a plus sign (e.g, X+Y) in the much rarer cases where
they do not pair. The Y chromosome is usually very small in Coleoptera, and this is indicated
by writing Xy instead of XY. In most Polyphaga with Xy, the minute Y is attached by both
its arms to the larger X, so that it resembles a parachutist suspended below the "canopy"
formed by the X (Figure 1lc). The formation and structure of the parachute has recently been
studied by silver staining;132it is believed to have a role in assisting the regular segregation
of the X and Y at first meiotic division. When the Xy bivalent takes this form, then a subscript
Sex Determination in Insects 77

"p" (for parachute) is added: Xy,. XX/XO systems in Coleoptera are represented by a single
X, rather than as XO. Systems with multiple small Y (=y) chromosomes involved in a single
parachute are written Xyy,, Xyyy,, etc.
Two main types of neo-XY system occur in beetles; those with a large Y, probably derived
from an X 0 system by X-autosome fusion (e.g., Figure 2), and those where an autosome has
apparently undergone a reciprocal translocation with either the X, or the y, of an Xy, system
to give an "X,neoX-neoY,", or some other complex system in which the original parachute
has elements (neo-X, neo-Y) associated with it in the first division of m e i o ~ i s . ' ~ ' . ' ~ ~
The more primitive beetles (Adphaga) differ from the Polyphaga in that Xy, systems are
virtually absent except in a few Dytiscidae (records of Carabidae with Xy, are apparently
q~estionablel~~). XXIXO is most frequent in Adephaga, occurring in about 53% of species,
with 29% having XX/XY (or XX/XY).'~~ XY systems predominate, however, in the carabid
genus Bembidion (176 out of the 205 species examined135).Tiger beetles (Cicindelidae)
mostly seem to have multiple X systems, with 2, 3, or 4 X chrom~somes.l~~
More than half of over 2000 species of Polyphaga examined cytologically have Xy, sex
chromosome systems, which are well represented in every major family, and are generally
thought to be the ancestral condition for the whole suborder. Whether Xy, is the primitive
condition for all beetles is not quite so clear, because of its rarity in Adephaga, although the
recent finding of a sex parachute in a megalopteran makes it more likely. Only single species
have been examined in each of the two primitive beetle suborders Archostemmata and
Myxophaga, and they may both be ~ntypical.'~~,'~'
Since Virkki's 1984 review,13' there have been significant studies on the sex chromosome
systems of C h r y s ~ m e l i d a e , ' ~
Histeridae,laIndian
~J~~ Staphylinidae,I4' Indian C u r c u l i ~ n i d a e , ~ ~ ~
32 other Indian beetle species,143Tenebri~nidae,'~~ ~ ~ 50 Russian beetle
B r ~ c h i d a e , 'and
species.146
Apparently the related order Strepsiptera is still cytologically unknown.

F. HYMENOPTERA
All the Hymenoptera except the few species that are thelytokous have haploid males,
produced from unfertilized eggs. The origin of haplodiploidy in this group presumably
therefore dates back to its inception in the early Mesozoic or late Palaeozoic. There seems little
doubt that this form of sex determination has been the key factor enabling the development
of eusociality in the higher groups of the order.I4'
Possible genetic mechanisms underlying haplodiploid sex determination have already been
outlined. At least two different models are necessary to fit the observed facts, one involving
multiple alleles at a single locus, and the other involving multiple l o ~ i . ~ The
~ . single-locus
~~~.'~~
mechanism (see p. 64) results in up to 50% of the fertilized eggs in inbred populations
developing as diploid males, which generally have low viability and fertility.21J50Diploid
males have been reported from several species of Tenthredinoidea, Ichneumonoidea, Apoidea,
and Formic~idea.'~~ Not all the reported instances can be attributed to inbreeding, but the
single-locus model seems to be established for one or more species in each of the above-cited
subfamilies, suggesting that it is ancestral to the Hymenoptera as a whole; e.g., the sawfly
(Neodiprion nigroscutum, the braconid Habrobracon hebetor, the ichneumonid Diadromus
p~lchellus,~5' the honeybee Apis mellifera, and the fire ant Solenopsis i n v i ~ t a . ' ~ ~
If single-locus sex determination occurs generally in the ichneumonid and braconid para-
sitoids reared and released as biological control agents, they may suffer from reduced viability
if inbred populations are used, because of diploid male produ~tion.'~~ In several species of
Chalcidoidea, however, in which sibmating is common in nature, inbreeding has not led to the
male-biased sex ratios that would be expected if diploid males were being produced, and a
multiple-locus model seems to be necessary. A single-locus scheme also does not seem to
explain sex determination in six species of meliponine bees, which did not produce diploid
males when ~ i b m a t e d , although
'~~ diploid males were later obtained in another meliponine
78 Insect Reproduction

species.ls4 Neither single-locus nor multiple-locus models seem applicable to the bethyloid
Goniozus nephantidis, which typically has within-brood mating and hence marked inbreed-
ing.155Generalizations would be unwise in the present state of knowledge, but it seems that
single-locus sex determination is likely to occur in Hymenoptera that generally practice
outbreeding, or in the higher social groups where the production of diploid males can be
controlled; for example, diploid male honeybee larvae are eaten by workers about 72 h after
eclosi~n.~~~
There have been estimates of the number of sex-determining alleles for several species,
either by crossing different lines (9 alleles, in H. hebetor), or by a statistical calculation based
on the incidence of diploid males in natural populations (99-19 in A. mellifera, depending on
population size;Is720 in Melipona compressipes f a ~ c i c u l a t a ;15
~ ~in~ S. invictalS2).
The pteromalid (chalcidoid) wasp Nasonia vitripennis has on occasions produced fully
fertile diploid males in laboratory cultures, but will not do so in response to intensive
inbreeding.158It is difficult to explain sex determination in this species, even as a multiple-
locus me~hanism.'~ Nasonia has been studied particularly with regard to the ability of the
female wasp to manipulate sex ratios by controlling sperm access to eggs, and in the course
of those studies several apparently extrachromosomal factors were discovered that influence
sex. One of these is of particular interest because it is transmitted paternally, but then
inactivates the paternal chromosome set by heterochromatinization in the fertilized egg, so
that genomically haploid, all-male broods are 0btai11ed.I~~ It thus mimics the normal mode of
sex determination of some scale insects and of sciarid flies. The transmitting agent has now
been identified as an accessory (B) chromosome, termed the paternal sex ratio or PSR
c h r o m o ~ o m e .In
~ ~effect,
~ . ~ ~the
~ PSR chromosome "jumps" from one haploid set to another
at the expense of the chromosomes with which it is associated and is thus an extreme example
of "selfish" DNA.
Most gall-forming Cynipoidea have two generations per year, one thelytokous and the
other bisexual. For several common species it has been shown that females of the unisexual,
thelytokous generations differ in the eggs that they lay, producing either only haploid (male)
eggs or only diploid (female) eggs.162The females of the bisexual generation are also of two
types, one giving rise only to the male producers of the next unisexual generation, the other
only to the female producers. Thus each female of the bisexual generation has grandchildren
of only one sex. Possible underlying genetic mechanisms were discussed by C r o ~ i e r . ~ ~

G. THE PANORPOID ORDERS

It is generally agreed that the remaining insect orders -including Lepidoptera and Diptera
- form a monophyletic group, with the Mecoptera close to its main stem. It is therefore of
interest that, whereas only a minority of species in the higher panorpoid orders have an
XX/XO system, X 0 males are found in all species of Mecoptera so far examined except one
(which has a clearly derived multiple sex chromosome system, with X,X,Y males)? It seems
likely, therefore, that the variety of sex chromosome systems found in the remaining orders
were all derived from an XXIXO system.
Very little is known about sex determination in the highly specialized order Siphonaptera
(fleas), which is placed somewhat uncertainly in the panorpoid complex. Of the four species
examined, two were probably XXIXY, and two apparently had multiple sex c h r o m o s ~ m e s ; ~ J ~ ~
males of one of these latter were X,X,Y, and of the other possibly XlYlX,Y2.
At some stage in one of the two main branches of panorpoid evolution - that leading to
the Trichoptera and Lepidoptera - the XX/XO system was replaced by a system involving
female heterogamety (XYIXX or ZWEZ). S ~ o m a l a i n e ndiscussed
'~~ the similarity between
trichopteran and lepidopteran sex chromosome systems. Both Trichoptera and Lepidoptera
have numerous small holocentric chromosomes, with sex chromosomes that are hardly
distinguishable in either mitotic or meiotic cell divisions, so much of the early work on sex
determination in Lymantria dispaP and Bombyx m ~ r i was l ~ ~done without any cytological
Sex Determination in Insects 79

information. R o b i n ~ o n ' s list

' ~ ~of chromosome numbers of over 1000 species of Lepidoptera
has no information on sex chromosomes. However, Smith's167discovery that all or part of the
unpaired Y (or W) chromosome in females is heterochromatic in interphase nuclei - and
hence that nuclei of female Lepidoptera with XY sex chromosome constitution contain a
dense "sex chromatin body" that is not found in male cells - provided a simple method for
determining the sex chromosome system. Traut and M o ~ b a c h e rand ' ~ ~E n n i ~between
l ~ ~ them
found a sex chromatin body in cell nuclei of females of 151 out of a total of 185 species
studied. Where females of a species had no sex chromatin body, female somatic or oogonial
cells of that species generally had an odd number of chromosomes, one less than in the male,
indicating an XO/XX (or ZO/ZZ) sex chromosome system. X 0 females are more common in
some families (e.g., Lasiocampidae, Yponomeutidae, Noctuidae) than others. Genera in
several families contain both XY and X 0 species.169A very few species have one or two
heterochromatic bodies in male as well as female nuclei, presumably due to presence of
constitutive heterochromatin on other chromosomes apart from the Y.
Multiple sex chromosomes (females with XY,Y,) have been found in two species of
Pyraloidea and two of Tortri~oidea.'~~ The two Y chromosomes, probably arising by simple
dissociation, both associate with the X in the first meiotic division of oocytes to form a
trivalent. In female somatic cell nuclei of three of the four species, it was possible to observe
two sex chromatin bodies, corresponding to Y, and Y,.
Some species of Solenobia (Psychidae) have thelytokous races, and a special mechanism
is necessary to ensure that progeny are all heterogametic like their mother. In S. triquetrella,
which has both diploid and tetrapioid thelytokous races, the nucleus that develops as an
embryo is derived by fusion of two of the four nuclei resulting from meiosis; as this fusion
is always between nonsister nuclei, the sex chromosome heterozygosity is preserved.171
Even though very few species have been studied in any detail, it is clear that the genetic
mechanisms involved in sex determination in Lepidoptera must be almost as variable as they
are in Diptera (see below). In the silkworm B. mori, it has long been known that the Y
chromosome of the female carries a dominant female-determining gene.16sDiploid, triploid
and tetraploid individuals are female as long as there is at least one Y chromosome, even when
the X:Y ratio is 3:l. Intersexes do not occur. Thus any male-determining factors on the X or
on autosomes must be very weak in their effect. On the other hand, there is abundant evidence
from Goldschmidt's work that sex determination in L. dispar depends on a delicate and
evolutionarily labile balance between the strengths of a male-determining factor or factors on
the X chromosomes and one or more female-determining factors, probably on the Y (reviewed
by White;9 but see also Clarke and Ford17,).
In several lepidopteran families, including some La~iocampidael~~ and S a t ~ r n i d a e ,in~ ~ ~ . ~ ~ ~
the same superfamily as Bombyx, the occurrence of XO/XX sex chromosome systems seems
to rule out any mechanism based on a dominant Y-borne sex factor and, where closely-related
X 0 and XY species occur, it seems likely that the Y has little or no role in sex determination.
In Ephestia, at least half the Y chromosome can be lost without any effect on sex determina-
tion174(although the female-determining factor could still be located on the remaining half).
It seems possible, therefore, that the vital and dominant role of the silkworm Y chromosome
is somewhat unusual.
The other main branch of the panorpoid complex - that leading to the higher Diptera -
also seems to have undergone major changes in the mechanism of sex determination early in
its evolution. The most primitive group of Diptera-Nematocera, the Tipuloidea, includes
species with XY males, such as Pales (=Nephrotoma) ferruginea, in which the X and Y
behave in an almost identical fashion to those of Neuroptera-Plannipennia; i.e., they do not
form a bivalent in the first division of meiosis, and take up positions in opposite halves of the
spindle ("distance pairing"), before segregating to the p01es.l~~ Even in Tipuloidea, however,
there is evidence of a change in the sex determination mechanism which forms the basis for
the variety of systems in the Diptera as a whole. In P.fermginea, XXY individuals are male,176
80 Insect Reproduction

indicating that sex determination is based on a dominant-Y mechanism, which is unlike all the
other insect groups covered so far.
The X and Y in most tipulids are very small, and in Tipula caesia and T. pruinosa they have
"disappeared; it seems probable that the sex chromosomes in these two species, or at least
the Y chromosome, have fused with members of a pair of autosomes, so that one of the three
pairs of autosomes now bears the sex-determining locus? A similar change seems to have
occurred in the two species of the tipulid subfamily Limnobidae (=Limoniinae) studied by
Wolf:177Dicranomiya (=Limonia)tnnotata and Thaumastoptera calceata. A species of Liriope
in the family Ptychopteridae, which is placed phylogenetically somewhere between Tipulidae
and Psychodidae, provides support for this idea;178it has heteromorphic sex chromosomes (X
and Y), but they have "acquired" homologous regions so that they pair to form a bivalent in
meiosis, suggesting that they are a neo-X and neo-Y formed by translocation or fusion with
a pair of autosomes. In this case, the size of the original Y, or the size differential between the
original X and Y, was presumably large enough to ensure that the neo-X and neo-Y are
recognizably heteromorphic.
In the Psychodoidea, which appear to be a branch of the dipteran phylogeny arising
between the Tipuloidea and Culicoidea, only a few species of sand flies (Psychodidae) have
been studied c y t ~ l o g i c a l l y , but
~ ~ ~these
- ~ ~provide
~ a similar picture to the Tipuloidea. Most
species have 2n = 6 or 2n = 8 without recognizable sex chromosomes, but one (Phlebotomus
pemiciosus) had 2n = 10 including a small heteromorphic pair of sex chromosomes~79-
presumably the more primitive condition.
In Culicoidea, where many more species have been studied cytogenetically, only the
Chaoboridae and the culicid subfamily Anophelinae have heteromorphic sex chromosome^.^^^.'^^
The Chaoboridae and one anopheline species (Chagasia bathana) have 2n = 8, with acrocen-
tric X and Y, whereas other Anophelinae and all other Culicidae studied have 2n = 6, perhaps
as a result of sex chromosome-autosome fusion. X and Y chromosomes in most mosquito
species can, however, be distinguished by their different patterns of staining with Giemsa or
quinacrine ("G, C, or Q bands"; e.g., Newton et al.Ix4),and slight intraspecific or interspecific
differences size can often be attributed to different-sized blocks of constitutive heterochroma-
tin (repetitive, noncoding DNA) (e.g., Mezzanotte et aI.lg5).Anopheles X and Y chromosomes
have extensive heterochromatic regions.lX3
The dominant, male-determining locus (M) of Aedes aegypti is on one member of the
shortest chromosome pair near the centromere,Ix6and has been similarly located in several
species of Culex,Ix3but in one strain of C. tritaeniorhynchus in Japan, "M" is on one of the
longest chromosome pair.Ig7Thus, the sex determinant can alter its position in the genome, a
phenomenon which comes into its own in the next superfamily, Chironomoidea.
Members of the other family usually included in Culicoidea, the Dixidae, lack heteromor-
phic sex chrorno~omes,~~~ as in the Chironomoidea.
Sex determination in Chironomoidea-or at least, in Simuliidae and Chironomidae, since
the Ceratopogonidae are little studied - is characterized by two features: (1) there are almost
always three chromosome pairs, none of which are heteromorphic; and (2) there is usually a
dominant male-determining factor that can apparently occur almost anywhere in the genome
and often differs in location between closely related species (Figure 12). In the Eusimulium
vemum complex alone, for example, five out of the six chromosome arms are involved in sex
determination in different species and sibling species.'8g In the E. aureum species group,
which is unusual in having 2n = 4, either of the two chromosome pairs may serve as the sex
chromosomes, and sex factors may occur in any of the four chromosome arms.Ig0
Sometimes the location of the sex factor varies within species, in which case sex determi-
nation operates as a multiple factor system. For example, the Australian Chironomus oppositus
species complex includes one form, whitei, which is apparently polymorphic for four different
sex factor locations, with up to three locations occurring in any one population.lgl Many other
examples are now available which support the idea that the sex-determining locus in
Sex Determination in Insects

FIGURE 12. Diagrammatic illustration of the mobility of the male-determining factor in blackflies (Simuliidae).
The diagram also shows the standard notation used by blackfly cytogeneticists for the six arms of the three
chromosome pairs (I, 11, and 111) of the normal blackfly chromosome complement. Any of these six arms can function
as the sex chromosomes, due to transposition of the male-determining factor (M) between chromosomes.The location
of M could also be switched from one arm to the other of the same chromosome by a pericentric inversion; for
example, an inversion of the section bracketed by a dotted line on chromosome I (although paracentric inversions -
those not involving the centromere - are much more common in blackflies).

Chironomoidea - and in many of the higher Diptera discussed below - acts as, or is
associated with, a transposable element, and can thus be excised and moved to multiple
locations in the gen~me.~O
The location of the male sex factor can sometimes be detected cytologically in polytene
chromosomes by minor differences in the banding pattern, or by sex-linked inversions.
Inversions arise when sections of the chromosome of varying length are excised and then
reinserted in the chromosome the "wrong way around". Individuals heterozygous for an
inversion can be detected by examining the sequence of bands in the polytene chromosomes,
which is inverted in a section of one chromosome in comparison with its homologue. If an
inversion is close to or encompasses the sex factor locus, as seems to happen very frequently
in blackflies, then it will be partially or completely sex-linked.192 The frequency of sex-linked
inversions also fits the idea of a transposable element being involved in sex determination,
because the breakpoints for inversions can also be the sites of excision or insertion of
transposable elements.193
R o t h f e l ~ considered
l~~ that the ancestral condition for Simuliidae and related groups was
a complete absence of differentiation of the chromosome carrying the male-determining factor
(the notation used for undifferentiated, homomorphic sex chromosomes by simuliid cytoge-
neticists is )dY,). While this may well be true, it is also possible for the )dYo condition to be
secondary; if, for example, a sex locus associated with an inversion is transposed out of the
inversion to a new genomic site, so that the inversion is no longer ~ e x - l i n k e d . ~ ~ ~ . ~ ~ ~
In Chironomus tentans, males are normally heterozygous for a dominant male sex factor,
but one population was found that seemed to have female heterogamety; this was interpreted,
on the basis of crosses between populations, as due to a dominant female sex f a ~ t o r . ' ~ . ' ~ ~
Nijthiger and Steinmann-Zwicky1Ipostulated that this situation might arise by a null mutation
of the key gene Sxl, accompanied by loss of the dominant male sex factor (M). It has also been
suggested, however, that a model involving a weakened male determiner could provide a
better explanation of the published result^.^^'.^^^
The remaining groups of Nematocera all have an achiasmate male meiosis, a feature that
links them cytogenetically with the higher Diptera. The sex chromosomes of Thaumaleidae
and Bibionoidea are usually small, do not form a bivalent, and show "distance pairing" in the
first meiotic division, as in tip~1ids.I~~ The Mycetophilidae also have XY males, but the related
82 Insect Reproduction

families Sciaridae and Cecidomyidae have developed remarkably aberrant chromosome sys-
tems, with more chromosomes in the germ line than in the soma (reviewed in detail by
White9). Neither of these families have Y chromosomes, so that the genic balance between X
chromosomes and autosomal factors must be the basis for sex determination.
In Sciara, male somatic cells are XO, but the germ line is XX and, after passing through
a highly peculiar spermatogenesis, the sperm are homogametic and all carry two X chromo-
somes. Oocytes are normal, with a single X, so all zygotes have three X chromosomes, with
the potential to develop as either sex. Either one or two of the three X chromosomes are
eliminated from presumptive somatic cells at the seventh or eighth cleavage division, to
determine the soma of the embryo as either female (XX) or male (XO), respectively. (Germ-
line cells later lose one X chromosome, irrespective of the sex of the embryo, so that they are
XX in both sexes.) The sex of the offspring - i.e., whether one or two X chromosomes are
eliminated from somatic cells - depends entirely on the genetic constitution of the mother.
Certain species of Sciara are monogenous, i.e., they invariably have unisexual progenies, so
that there are two kinds of mother, male producing and female producing. The latter are
thought to be heterozygous for a dominant factor (F), presumably acting through the cyto-
plasm of the egg to cause the soma of the embryo to develop as female. The ratio of male- to
female-producing mothers in such species, and hence the resulting sex ratio, is approximately
1:l. Thus, sex is inherited genetically, but the inheritance is displaced back to the maternal
generation. The genetic mechanism could be a null mutation of the daughterless gene or its
equivalent, as discussed for Chrysomya below. Other species of Sciara have females that
normally produce progeny of both sexes, however, and sex determination in other genera of
Sciaridae has still hardly been studied, so it would be unwise to generalize. Haig'99 reviewed
the chromosome system of Sciara coprophila and developed a model for its evolution based
on sex ratio theory.
Cecidomyidae have even more aberrant chromosome systems, with numerous extra ("E")
chromosomes in the germ line that are eliminated from somatic cells in early cleavage
division^.^ In most species studied, there are six chromosomes in male somatic cells and eight
in female somatic cells, so that the sex chromosome system is XlXlX2X2/XlX20. This is the
case in the hessian fly, Mayetiola destr~ctor,2~.20' despite early reports of eight chromosomes
in the somatic cells of both sexes. As in Sciara, sex is determined by a maternal factor rather
than by male heterogamety. Males although X,X,O are homogametic, producing only XlX2
sperm, so that zygotes are all XlX1X2X2.In male embryos, two X chromosomes (one X, and
one X,) are eliminated from presumptive somatic cells at a separate, later cleavage division
than that at which the E chromosomes are eliminated; e.g., in Wachtliella persicariae, E
chromosomes are eliminated at the fourth cleavage division and X chromosomes at the
seventh.202
As in Sciaridae, many cecidomyids have unisexual pro genie^,^^^^^ but others have the
same mothers producing both male and female progeny, and the system by which sex is
controlled is unclear. Heteropeza pygmaea is best studied in this respect, but Heteropezinae
differ from other cecidomyids in that the male somatic cells appear to be haploid, with five
chromosomes, whereas female somatic cells have ten chromosomes. However, this only
applies when the progeny are produced pedogenetically; in H. pygmaea, females reproducing
as adults lay only female-determined eggs, but these have five chromosomes as in
pedogenetically produced male Sex determination therefore cannot be based on
haplodiploidy, and does not seem to have a genetic basis at all. Went and Camenzind205
cultured larval ovaries of H. pygmaea in vitro, using as culture medium the hemolymph of
larvae that had been previously kept in different nutritional environments, and were able to
show that the sex of the progeny was dependent on the nutritional conditions experienced by
the mother during development.
The more primitive groups of Brachycera have received very little attention from cytoge-
neticists. In the Tabanoidea, Rhagionidae and Stratiomyidae have XY males where these have
Sex Determination in Insects 83

been studied,206whereas in Asiloidea, the asilid Dasyllis (=Laphria) grossa is reported to have
an XXIXO system.207In the more advanced groups of Brachycera (=Cyclorrhapha), there has
been detailed work on sex determination mechanisms of representatives of five families:
Phoridae (Megaselia scalaris), Muscidae (Musca domestica), Calliphoridae (Chrysomya
ru$facies Lucilia cuprina), Tephritidae (Ceratitis capitata), and, of course, Drosophilidae (D.
melanogaster, D. miranda). These five families span four superfamilies of Cyclorrhapha, so
may be fairly representative of the range of mechanisms in the higher Diptera as a whole.
In the phorid fly M. scalaris, X and Y chromosomes are not morphologically differentiated,
and the male-determining factor (M) is capable of being located on any of the three chromo-
some pairs,208much as in chironomids. In laboratory strains the chromosome (Y) carrying the
M factor could be distinguished from its homologue (X) using a combination of cytogenetic
and molecular technique^.^^ The segment of the Y chromosome carrying M was found to be
conserved in comparison with the homologous region of X, when two unrelated strains were
compared. Nevertheless, when the two strains were crossed, four cases were found where the
M factor had moved to a different chromosome. The frequency of this change was about
0.06%, which is comparable with known rates of movement of transposable elements in other
organisms.210The conservation of the M-containing chromosomal region observed in pure
strains perhaps indicates that a specific location is favored under certain circumstances, and
this could be the first step in the differentiation of new heteromorphic sex chromosomes.
The housefly M. domestica provides some particularly interesting examples of evolution
of sex-determining systems in progress. It was fully reviewed by Bull,lo but since then there
have been further interesting developments. Earlier European work established that sex
determination in houseflies was XXIXY, with heteromorphic sex chromosomes and a presum-
ably dominant male sex factor on the Y. Apart from the Y-borne sex factor, X and Y
chromosomes seem to have few or no functional coding regions and are heterochromatic. In
strains of non-European origin, however, various sex factors have been found on the auto-
somes, especially a male determiner (M) near the centromere on autosome 3, and a female
determiner (F) on autosome 4 which is epistatic to (i.e., ovemdes) any number of male-
determining factor~.~" In continental Europe, samples from Denmark to Sicily taken in 1975-
1981 showed a latitudinal cline: north European populations were all XXIXY, whereas in
south and central Italy all populations were XXIXX with sex determined autosomally, the X
being totally neutral with regard to sex determination. In southern France, Yugoslavia, and
northern Italy, intermediate, mixed populations occurred with all combinations of X and Y in
either A very similar north-south cline was found in Japan.213The changes in sex
determination mechanisms in both southern Europe and Japan are believed to be recent.
Various models (e.g., Jayakar2I4)have been advanced to explain this phenomenon; possibly
climatic influences are involved, or perhaps the driving force is selective insecticide pressure,
as there is now good evidence that pyrethroidJDDT resistance (the "knockdown factor," Kdr)
is genetically linked with the male-determining locus on autosome 3.215However, recent
changes have also occurred in the sex-determination system of housefly populations in
southeast England, involving an apparent increase in frequency of a male factor on the X
c h r o m ~ s o m e , ~and
~ ~there
. ~ ~ 'is no evidence that loci associated with insecticide resistance (or,
in fact, any other functional genes) occur on the housefly X chromosome. Or has the
resistance-conferring gene also been transposed to the X from autosome 3, along with the male
sex factor? This seems quite possible, since a laboratory housefly strain in Australia was
shown to have DDT resistance linked to a male sex factor, but in this case on autosome 2.218
Few other Muscidae have been studied cytogenetically. Recent work on Hydrotaea
meriodionalis indicates a similar story to M. domestica, with a dominant autosomal male
determiner in some populations and others with XY males.219In the closely related
Anthomyiidae, the cabbage root fly, Delia radica (=Hylemyia brassicae), is now known to
have a male-determining factor on an autosome (chromosome 6), whereas D. antiqua has a
small heteromorphic X and Y.220
84 Insect Reproduction

Sex determination in houseflies, with male determiners at various locations in the genome,
and the presence of a dominant female determiner in some populations, seems a very different
mechanism to the Drosophila system, based on an X:A ratio, but there are ways of deriving
one from the other fairly simply. Nothiger and Steinmann-Zwickyl' suggested, for example,
that the dominant female determiner (F) could be a mutation of the key gene Sxl to an
irrepressible condition, so that it cannot be turned off by M. A similar conclusion was reached
by Inoue and H i r o y i ~ h i ; ~their
~ ' model for housefly sex determination incorporates the
discovery of a mutation tra, closely linked with F on autosome 4; when this is present in the
mother, it causes progeny to develop as males even in the absence of any M factors.
In fact, a genic balancelrecessive-X system does operate in another muscid, the tsetse fly
G. palpalis, which also resembles Drosophila in that the Y chromosome carries some loci that
are necessary for sperm viability, but is not involved in sex determination.16
Blowflies (Calliphoridae) generally have small, heteromorphic, and mainly heterochro-
matic, sex chromosomes. The Y chromosome in L. cuprina carries a dominant male sex factor,
located near its c e n t r ~ m e r e .In ~ ~Calliphora
~ . ~ ~ ~ erythrocephala, however, the small hetero-
chromatic pair are no longer sex chromosomes, and the male-determining locus is on one of
the other chromosomes, where it is recognizable as a small heterozygosity of the chromomere
pattern of the polytene chromosome.224And in the monogenic blowfly C. rufifacies, sex is
controlled by a dominant female determining factor (F') in the mother, Flfmothers producing
only daughters andflmothers producing only sons, which are therefore also of ff genotype.
U l l e r i ~ h , 2in~some
~ elegant experimental work, transplanted pole cells (primordial germ cells)
between female embryos of Flf and ff genotypes. The resultant mothers were germ-line
mosaics for Flfand ff, and both the donor and recipient genotypes were expressed, resulting
in a mixture of male and female progeny. Thus, the F'gene product is synthesized by the germ-
line cells themselves, rather than by maternal somatic cells. Ullerich also did pole cell
transplantations between male and female embryos. These resulted in germ-line mosaics that
were completely fertile and heterosexual; the donor cells underwent sex reversal and devel-
oped as male or female according to their mother's genotype and irrespective of their own
genotype. Thus, a genotypically male germ cell can develop as a functional oocyte in a female
host, a genotypically female germ cell can develop as a functional sperm in a male host, and
sex is determined solely by regulatory factors provided by maternal somatic cells.
Nothiger and Steinmann-Zwickyll postulated that F'in Chrysomya is similar or identical
to the daughterless gene (da)of Drosophila, which is necessary in the mother in order for the
key gene Sxl to be active (Figure 6). If f is the null (mutant) allele da-, then in homozygous
condition it will render Sxl of embryonic germ cells inactive, so that all progeny will be sons.
DNA sequence homology has now been demonstrated between the da gene of Drosophila and
a polytene band on the Chrysomya chromosome that carries the F ' l o ~ u s strongly
, ~ ~ ~ support-
ing this hypothesis.
Tephritidae mostly have heteromorphic sex chromosomes (XXIXY), and in several cases
a dominant-Y system has been demonstrated, e.g., in the medfly C. ~ a p i t a t aX,X,Y . ~ ~ ~ males
occur in some s p e c i e ~ , 9 Indian
. ~ ~ ~ species in four genera of Trypetinae apparently have
homomorphic sex chromosomes,229and female heterogamety (XYKX) has been demon-
strated in some Australian species.230In C. capitata, the sex chromosomes are almost entirely
heterochromatic, and the Y chromosome can suffer large deletions without any obvious ill
effect; the male-determining factor is located on its long arm close to the c e n t r ~ m e r eSome
.~~~
repetitive DNA sequences that are specific to or concentrated in the Y chromosome of C.
capitata were recently is0lated.~3~
The sex determination mechanism of D. melanogaster was discussed earlier in this chapter,
and there are numerous recent r e v i e ~ s . ~ .AS . ~ ~ ~Drosophila
~ ~regards - ~ ~ ~ other than D.
melanogaster, the most interesting developments have been with D. miranda, a species in the
obscura group which has an XlX2Y system, the X, and the Y being recently derived (i.e., a
neo-X and neo-Y) by translocation to the original Y chromosome of one member of the third
Sex Determination in Insects 85

autosome pair found in the closest relatives (D. pseudobscura, D. persimilis), leaving its
homologue as a neo-X. Chromosome 3 of D. pseudobscura/persimilis is also homologous to
the right arm of chromosome 2 of D. melanogaster. Thus, a very comprehensively mapped
chromosome segment has quite recently become a neo-Y, providing considerable scope for
study of the degenerative changes that follow from its permanent heterozygosity, and the
consequent accumulation of nonfunctional alleles. Comparisons of the neo-Y and its recently
homologous neo-X have particularly shown that the neo-Y has acquired inserted DNA
sequences that are not present in the neo-X, and appear to represent a novel transposable
element that may be involved in the degenerative p r o ~ e s s . ~ ~ ' - ~ ~ ~

IV. EVOLUTION OF SEX CHROMOSOMES AND SEX

DETERMINATION IN INSECTS
It has taken quite a lot of words to provide an outline review of the many and various
methods by which sex determination is achieved in the different orders of insects. It is clearly
important to distinguish between the sex chromosome systems, which display to the cytoge-
neticist a remarkable diversity in their form, behavior, and extent of evolutionary change
within and between groups, and the underlying molecular mechanisms, which may perhaps
show less variation. However, for most insect groups, the molecular genetics of the sex-
determining process is still merely a matter of speculation or extrapolation from the paradigm
of Drosophila.
Much of the discussion on sex chromosome evolution has centered on the Y chromosome.
The ideas about the progressive evolutionary degeneration of the Y chromosome discussed
earlier in this chapter were developed primarily with regard to vertebrate systems, particularly
mammals where the Y chromosome bears a dominant male-determining l o ~ ~ sThe .
various models that have been proposed241all assume a primitive condition where the sex
chromosomes are undifferentiated, homologous, and in fact essentially autosomal except at
the sex-determining locus, but in time become progressively differentiated as the Y acquires
noncoding DNA and the X acquires a system of dosage compensation.14 Bullioreviewed the
evidence for progressive sex chromosome differentiation in other groups including insects,
and concluded that it could be applied more generally. Nothiger and Steinmann-Zwicky"
postulated that the various genetic mechanisms for sex determination in higher Diptera arose
by mutations from a primitive state with undifferentiated sex chromosomes and a dominant
male determiner, as in some mosquitoes.
However, when insects are looked at as a group, certain qualifications to the model of
progressive sex chromosome differentiation and Y degeneration are necessary. First, a model
that is applicable to the genic balance systems that seem to predominate in insects has not yet
been developed.I4 Second, the XXIXO system predominates in the lower insect orders, and
must be the ancestral condition for several major groups, if not for the class Insecta as a whole.
Thus, for many insects with XXIXY, if not the majority, the XY condition has arisen by a
major chromosomal rearrangement, rather than by a progressive, gradual change from a
primitively undifferentiated state. The subsequent evolution of the neo-Y and the homologous
region of the neo-X may be comparable in many ways to the process of mammalian sex
chromosome differentiation, but the presence from the start of a fully differentiated X
chromosome, coupled with the lack of sex determiners on the Y, must have consequences that
need to be fully addressed. It would be instructive to compare the molecular changes in the
recently acquired neo-XY system of Drosophila miranda with the changes following a recent
acquisition of "autosomal" sex determination due to the transposition of a male determiner
(M) to a new location, e.g., in certain mosquito species or, very recently, in certain populations
of M. domestica.
Third, although there are examples of loss of homology between a neo-Y and a neo-X, and
evidence of accumulation of nonfunctional alleles and repetitive DNA on the Y chromosome,
86 Insect Reproduction

there is a lack of information about the circumstances which determine whether a neo-XY
becomes an evolutionarily stable XY system, or proceeds inevitably towards complete degen-
eration and eventual loss of the Y chromosome. In the cytologically well-studied Orthoptera,
which show much evolutionary change in the sex chromosomes including numerous examples
of de novo acquisition of XY systems, there are no clear cases where the Y chromosome has
been secondarily lost. It seems that a stable condition may sometimes be reached, where it is
advantageous to have sex-linked genes retained on the Y chromosome. In Coleoptera, Xy,
systems with a "degenerate Y" are very ancient and show great evolutionary stability. In
Heteroptera, secondary loss of Y chromosomes seems to have occurred many times in the
course of evolution at the family level, but not between closely related species, indicating that
it does not happen fast or frequently.
This leads to the fifth and final point of qualification, which was discussed by Feraday et
al.'95 specifically with respect to the evolution of the sex chromosomes of Simuliidae. There
has been a tendency to regard sex chromosome differentiation as an inevitable sequence of
events, under the influence of mutation and random drift, rather than as an adaptive process.
In Simuliidae, any of the three chromosome pairs can be heterozygous for the male-determin-
ing sex factor. Usually the only cytological differentiation between the "X" and the "Y" is in
the form of inversions, which may be sex-linked but do not form part of any progressive
evolutionary sequence of sex chromosome differentiation.lg5Whitegpointed out that if certain
autosomal alleles are polymorphic and exert different selective pressures in the two sexes, then
it is advantageous to have them linked to the sex chromosomes. In Orthoptera this may be
accomplished by centric fusions between sex chromosomes and autosomes to give neo-XY
systems.242In those Diptera which have single locus, dominant male sex factors, the linkage
may be more easily obtained by transposing the sex locus to another position in the genome.
Selective advantage is thus important in establishing a new sex chromosome system, and
presumably continues to influence the nature and extent of any subsequent differentiation of
the X and Y chromosomes.

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191. Martin, J. and Lee, B. T. O., A phylogenetic study of sex determiner location in a group of Australasian
Chironomus species (Diptera, Chironomidae), Chromosoma, 90, 190, 1984.
192. Rothfels, K. H., Chromosomal variability and speciation in black flies, in Insect Cytogenetics, Blackman,
R. L., Hewitt, G. M,, and Ashburner, M., Eds., Blackwell, Oxford, 1980, 207.
193. Smith, P. a. and Corces, V. G., Drosophila transposable elements: mechanisms of mutagenesis and
interactions with the host genome, Adv. Genet., 29, 229, 1991.
194. Mason, G. F., Sex chromosome polymorphism in the Simulium tuberosum complex (Lundstrom) (Diptera:
Simulidae), Can. J. Zool.. 62, 647. 1984.
195. Feraday, R. M., Leonhardt, K. G., and Brockhouse, C. L., The role of sex chromosomes in black fly
evolution, Genome, 32, 538, 1989.
196. Thompson, P. E. and Bowen, J. S., Interactions of differential primary sex factors in Chironomus tentans,
Genetics, 70, 49 1, 1972.
197. Feraday, R. M., Weak male-determining genes and female heterogarnety in Chironomus tentans. Can. J.
Genet. Cytol., 26, 748, 1984.
198. Martin, J. and Lee, B. T. 0..Are there female heterogametic strains of Chironomus tenrans Fabricius?, Can.
J. Genet. Cytol., 26, 743, 1984.
199. Haig, D., The evolution of unusual chromosome systems in sciarid flies: intragenomic conflict and the sex
ratio, J. Evol. Biol., 6, 249, 1993.
200. Stuart, J. J. and Hatchett, J. H., Cytogenetics of the hessian fly. I. Mitotic karyotype analysis and polytene
chromosome correlations, J. Hered., 79, 184, 1988.
201. Stuart, J. J. and Hatchett, J. H., Cytogenetics of the hessian fly. 11. Inheritance and behaviour of somatic
and germ-line-limited chromosomes, J. Hered.. 79, 190, 1988.
202. Geyer-Duszyh, I., Experimental research and chromosome elimination in Cecidomyidae (Diptera).
Chromosoma, 11,499, 1959.
203. Kozlova, L. V., [On monogeny of gall midges, Aphidoletes aphidimyza Rond. (Diptera: Cecidomyidae)],
Nauchn. Tr. Leningr. Ord. Skh. Inst., No. 374, 11, 1979.
204. Went, D. F. and Camenzind, R., Sex determination in the dipteran insect Heteropeza pygmaea, Genetica,
52/53, 373, 1980.
Sex Determination in Insects 93

205. Went, D. F. and Camenzind, R., Haemolymph-dependent sex determination in a paedogenetic gall midge,
Natunvissenschafen, 64, 276, 1977.
206. Boyes, J. W., The chromosomes of Rhagionidae, Stratiomyidae and Xylomyidae (Diptera), Can. J. Genet.
Cytol., 15, 255, 1973.
207. Metz, C. W., Chromosome studies in the Diptera. IV. Incomplete synapsis in Dasyllis grossa. Biol. Bull.
(Wood's Hole, Mass.), 43, 253, 1922.
208. Mainz, F., The genetics of Megaselia scalaris Loew (Phoridae): a new type of sex determination in Diptera,
Am. Nut., 98, 415, 1964.
209. Willhoeft, U. and Traut, W., Molecular differentiation of the homomorphic sex chromosomes in Megaselia
scalaris (Diptera) detected by random DNA probes, Chromosoma. 99, 237, 1990.
210. Traut, W. and Willhaft, U., A jumping sex determining factor in the fly Megaselia scalaris, Chromosoma,
99,407, 1990.
21 1. Diibendorfer, A., Hiltiker-Kleiner, D., and Nothiger, R., Sex determination mechanisms in dipteran
insects: the case of Musca domestica. Semin. Dev. Biol.. 3, 349, 1992.
212. Franco, M. G., Rubini, P. G., and Vecchi, M., Sex-determinantsand their distributionin various populations
of Musca domestica L. of western Europe, Genet. Res., 40, 279, 1982.
213. Tomita, T. and Wada, Y., Multifactorial sex determination in natural populations of the housefly (Musca
domestica) in Japan, Jpn. J. Genet., 64, 373, 1989.
214. Jayakar, S. D., Some two-locus models for the evolution of sex-determining mechanisms, Theor. Popul.
Biol.. 32, 188, 1987.
215. Shono, T. and Scott, J. G., Autosomal sex-associated pyrethroid resistance in a strain of housefly (Diptera:
Muscidae) with a male-determining factor on chromosome three, J. Econ. Entomol., 83, 686, 1990.
216. Denholm, I., Franco, M. G., Rubini, P. G., and Vecchi, M., Identification of a male determinant on the X
chromosome of a housefly (Musca domestica L.) population in south-eastEngland, Genet. Res., 42.31 l, 1983.
217. Denholm, I., Franco, M. G., Rubini, P. G., and Vecchi, M., Geographical variation in house fly (Musca
domestica L.) sex determinants within the British Isles, Genet. Res., 47, 19, 1986.
218. Kerr, R. W., Inheritance of DDT resistance in a laboratory colony of the house fly, Musca domestica. Aust.
J. Biol. Sci., 23, 377, 1970.
219. Loeschke, V., Nielsen, B. 0..and Andersen, D., Chromosomal variation, segregation and sex determination
in Hydrotaea meridionalis (Diptera: Muscidae), Hereditas, 118, 229, 1993.
220. Samoylov, Yu. B., [Genetic control for the cabbage root fly 11. Localization of male determining factor in the
cabbage root fly Delia brassicae Bouche], Genetika, 21, 1810, 1985.
221. Inoue, H. and Hiroyishi, I., A maternal effect sex transformation mutant of the housefly Musca domestica,
Genetics, 12,469, 1986.
222. Maddern, R. H. and Bedo, D. G., Properties of the sex chromosomes of Lucilia cuprina deduced from
radiation studies, Genetica. 63, 203, 1984.
223. Bedo, D. G. and Foster, G. G., Cytogenetic mapping of the male-determining region of Lucilia cuprina
(Diptera: Calliphoridae), Chromosome, 92, 344, 1985.
224. Ribbert, D., Die Polyt;inchromosomender Borstenbildungszellen von Calliphoraeryrhrocephula, Chromosomu,
21, 296, 1967.
225. Ullerich, F.-H., Analysis of sex determination in the monogenic blowfly Chrysomya rujifacies by pole cell
transplantation, Mol. Gen. Genet., 193,479, 1984.
226. Clausen, S. and Ullerich, F.-H., Sequence homology between a polytene band in the genetic sex chromo-
somes of Chrysomya rufifacies and the daughterless gene of Drosophila melanogaster, Natunvissenschaften,
77, 137, 1990.
227. Lifschitz, E. and Cladera, J. L., Ceratitis capirata: cytogenetics and sex determination,in Fruit Flies. Their
Biology, Natural Enemiesand Control (World Crop Pests 38). Robinson, A. S. and Harper, G., Eds., Elsevier,
Amsterdam, 1989, 3.
228. Solferini, V. N. and Morgante, J. S., X,X,X,X,:X,X,Y mechanism of sex determination in Anastrepha
bistrigata and A. serpentina (Diptera: Tephritidae), Rev. Bras. Genet., 13, 201, 1990.
229. Grewal, J. S. and. Kapoor, V. C., Karyotypes of some fruitfly species (Tephritidae) of India, in Fruit Flies
of Economic Importance (CEC/IOBCInt. Symp.. Rome, 7-IOApril 1987), Cavallaro, R., Ed.,A. A. Balkema,
Rotterdam, 1989,237.
230. Bush, G. L., Female heterogamety in the family Tephritidae (Acalyptrata, Diptera),Am. Nar., 100, 119, 1966.
231. Anleitner, J. E. and Haymer, D. S., Y-Enriched and Y specific DNA sequences from the genome of the
Mediterranean fruit fly, Ceratitis capitata, Chromosomu, 101, 271, 1992.
232. Hodgkin, J., Drosophila sex determination: a cascade of regulated splicing, Cell, 56, 905, 1989.
233. Slee, R. and Bownes, M., Sex determination in Drosophila melanogaster, Q. Rev. Biol., 65, 175, 1990.
234. Torres, M. and Sanchez, L., The segmentation gene runt is needed to activate Sex-lethal, a gene that controls
sex determination and dosage compensation in Drosophila, Genet. Res.. 59, 189, 1992.
235. Steinmann-Zwicky, M., How do germ cells choose their sex? Drosophila as a paradigm, BioEssays. 14,513,
1992.
94 Insect Reproduction

236. Cline, T. W., The Drosophila sex determination signal: how do flies count to two?, Trends Genet., 9, 385,
1993.
237. Steinemann,M. and Steinemann,S., Evolutionary changes in the organization of the major LCP gene cluster
during sex chromosomal differentiation in the sibling species Drosophila persimilis, D. pseudobscura and D.
miranda, Chromosoma, 99, 424, 1990.
238. Ganguly, R., Swanson, K. D., Ray, K., and Krishnan, R., A BamHI repeat element is predominantly
associated with the degenerating NEO-Y chromosome of Drosophila miranda but absent in Drosophila
melanogaster genome, Proc. Natl. Acad. Sci. U.S.A., 89, 1340, 1992.
239. Steinemann, M., Steinemann, S., and Lottspeich, F., How Y chromosomes become genetically inert, Proc.
Natl. Acad. Sci. U.S.A., 90, 5737, 1993.
240. Ohno, S., Evolution of sex chromosomes in mammals, Annu. Rev. Genet., 3, 495, 1969.
241. Jablonka, E. and Lamb, M. J., The evolution of heteromorphic sex chromosomes, Biol. Rev. Camb. Philos.
Soc.. 65, 249, 1990.
242. Charlesworth,D. and Charlesworth, B., Sex differences in fitness and selection for certain fusions between
sex chromosomes and autosomes, Genet. Res., 35,205, 1980.
 Chapter 4

HORMONES AND REPRODUCTION

Jim Hardie

CONTENTS
I. Introduction .................................................................................................................
95

Hormones and Females ............................................................................................... 96

A. Oogenesis ..............................................................................................................96
1. Cockroaches ..................................................................................................... 96
2. Dipterans ......................................................................................................... 97
a. Mosquitoes .................................................................................................. 97
b. Flies .............................................................................................................
98
3. Hemipterans .....................................................................................................99
4. Homopterans ....................................................................................................99
B. Regulation of Sex Pheromone Release .............................................................. 101

111. Hormones and Males ................................................................................................ 101

A. Spermatogenesis.................................................................................................. 102
B. Sperm Release and Maturation ........................................................................... 103
C. Gonadal Development ........................................................................................ 103

IV. Summary ....................................................................................................................

103

Acknowledgments ...............................................................................................................
103

References ...........................................................................................................................
104

I. INTRODUCTION
This chapter concerns the endocrine control of reproduction in female and male insects.
The major gonadotropic hormones are juvenile hormones (JHs) and ecdysteroids, the same
hormones that control metamorphosis and moulting. This parsimony in hormone complement
occurs because the windows of sensitivity for development and reproduction are, to an extent,
isolated from each other and the temporal distribution of hormone receptors differs between
different tissues. Thus, during larval and pupal stages, JHs and ecdysteroids are responsible
for development, while in the adult (and in certain instances preadult) stages they take on
gonadotropic functions. Juvenile hormones are synthesized and released from the corpora
allata which are present in all insect stages. The corpora allata are possibly the only site of
synthesis, although recently male accessory glands have been reported to produce JH.' In
contrast, during larval and pupal stadia, ecdysteroids are synthesized and released, for the
main part, from the prothoracic glands, which, with certain exceptions (e.g., in Apterygota
which alternately moult and reproduce as adults and in solitary locusts), atrophy at the final
moult. In adult females, ecdysteroid synthesis occurs in the ovaries, or more specifically in the
follicle cells of the ovaries, and in males they are synthesized by the testes, while other

0-8493-6695-X/95/$0.MkSSM
O 1995 by CRC Press. Inc.
96 Insect Reproduction

possible sites for production have been ~uggested.~

With today's more sensitive techniques for
hormone isolation and identification together with molecular biological procedures, the con-
trol of JH and ecdysteroid titers and their actions at the gene level are becoming more
under~tood.~ Recent reviews of the hormonal control of insect reproduction include references
4 through 7.

11. HORMONES AND FEMALES

The endocrine control of reproduction in female insects varies with species; this is, perhaps,
not surprising as the reproductive strategies also differ (e.g., oviparous, ovoviparous, vivipa-
rous, sexual, parthenogenetic). The present chapter will concentrate on presenting some of the
different endocrine strategies with reference to recent investigations of some well-researched
insects. Chapter 1 describes the events of oogenesis with some details on endocrine effects on
female accessory glands.

A. OOGENESIS
1. Cockroaches
In all species of cockroaches investigated, including oviparous, ovoviparous, and vivipa-
rous species, JH has been shown to play a major gonadotropic role. Mated females of the
oviparous American cockroach, Periplaneta americana, undergo the cyclical production of
oothecae (egg cases containing up to 16 eggs) every 4 to 5 days. Removal of the corpora allata
from immature females prevents any previtellogenic ovarian development, while in mature
females it prevents further ootheca formation by inhibiting vitellogenin synthesis and ~ p t a k e . ~
Reimplantation of corpora allata or treatment with JH restores the reproductive c y ~ l e . ~The
.'~
precise corpora allata control of the cycle of ootheca production has been the subject of many
studies. In vitro culture of corpora allata under conditions that allow the synthesis of JH (JH
111 in this species) has shown a corresponding cycle of JH synthesis and release." More
recently, it has been shown that in vivo titers of JH I11 show concomitant cyclical rises, with
peak titers occurring during vitellogenesis and low titers at the point where oothecae are
deposited.I2Surgical techniques, more refined than complete allatectomy, have been imple-
mented and show that after unilateral allatectomy (removing one of the pair of corpora allata),
ootheca production continues, but at a slightly lower rate, while severing all the nervous
connections to the corpora allata had a similar effect.13 The corpora allata effects on ootheca
production could, therefore, be accommodated by the loss of one corpus allatum and, more
important, the cyclical activity appeared to be driven by hemolymph-borne factors. However,
the same study showed that the cyclical production of oothecae could be reinstated in
allatectomized females by treatment with a potent JH analogue which also speeded up the
ootheca cycle in intact insects. The study concluded that the ootheca production cycle was not
driven by the cycle of JH synthesis and release, but that JH sewed only to control the speed
of an endogenous reproductive cycle: lower JH levels slowed the cycle while higher levels
speeded it up; in the absence of JH the cycle stopped.13 Control of JH synthesis in vivo may
well be effected by peptide factors such as allatostatins (which inhibit JH synthesis by the
corpora allata), recently identified in the brain of the viviparous cockroach, Diploptera
punctata, and shown to be effective in P. americana.14
Ecdysteroids are produced by the ovaries of adult co~kroaches,~~ but their role has not been
fully elucidated. It has been proposed that the CO-occurrenceof ecdysteroid and choriogenesis
might indicate a role at this stage in egg development. They may have an inhibitory efect on
JH secretion, and their continued presence in the ootheca indicates that they supply the
requirements of the embryo^.'^.'^
Hormones and Reproduction

v+
Adult Eodysis Blood Meal

ma~ng--Err,l
brain
l
corpus allaturn
I -
behaviour
fat body O W
1
TMOF
i+
competent fat body resting W H
CCSF

t oocyts maturation
WTULOQENlN -----#-vit~ltOgOni~ OVW
l

FIGURE l. Factors regulating ovarian development in the mosquito, Aedes aegypti. CCSF = corpus cardiacum-
stimulating factor; MNC = medial neurosecretory cells: EDNH = egg development neurosecretory hormone;
TMOF = trypsin-modulating oostatic factor. (Modified from Hagedom, H. Comprehensive Insect Physiology, Bio-
chemistry and Pharmacology, Kerkut, G. and Gilbert, L., Eds. Pergamon, Oxford, 1985 page 165.)

2. Dipterans
In a number of Diptera, ecdysteroids replace JH as the major gonadotropic hormone
stimulating vitellogenin synthesistuptake.

a. Mosquitoes
The hormonal control of egg maturation in Aedes aegypti, an anautogenous mosquito
(requiring a blood meal to develop the first and subsequent egg batches) has been studied
inten~ively.~ A summary of the endocrine interactions is shown in Figure 1. Adult emergence
triggers the release of JH from the corpora allata.I8JH then acts on the ovary, which contains
the primary follicles, to stimulate growth to around twice the original size and form a "resting
stage" ovary which is complete within 3 days of emergence. This previtellogenic development
of the ovary also involves the production of endocytic organelles by the oocytes, which then
become competent for protein uptake.19The "resting stage" ovary now exerts an inhibition of
JH secretion,20but does not develop further until a blood meal has been taken. This initial rise
in JH titer also stimulates mating behaviorZ1and induces a competence in the fat body to
respond' to ecdysteroids. The fat body remains unresponsive if allatectomy is performed at
, ~ ~in vitro experiments confirm that JH promotes the competence required
adult e c l o s i ~ nand
for an ecdysteroid response.23
After a blood meal, egg development neurosecretory hormone (EDNH)" is released from
the corpora cardiaca, having been produced in the protocerebrum by the medial neurosecre-
tory cells, and acts on the "resting stage" ovary to induce the production of e ~ d y s o n eThis
.~~
EDNH release, also, involves a corpus cardiacum-stimulating factor (CCSF) which is pro-
duced by the resting o ~ a r y .Ecdysone
~ ~ . ~ ~is secreted into the hemolymph and converted to 20-
hydroxyecdysone, which then acts on the competent fat body to stimulate vitellogenin synthe-
sis and release. Additionally, the 20-hydroxyecdysoneinduces the separation of new follicles,
the secondary follicles, from the g e r m ~ i aControl
. ~ ~ of the endocytotic uptake of vitellogenin
by the oocytes has yet to be elucidated, but it is possible that both 20-hydroxyecdysone and
an unidentified brain hormone effect vitellogenin uptake.29After a short delay, blood meals
also induce a 24- to 36-h pulse of elevated JH titer which coincides with decreased levels of
98 Insect Reproduction

JH-esterase activity.I8 This meal-induced JH pulse stimulates the new follicles to develop to
the resting stage and a renewed competence of the fat body to respond to ecdysteroids. The
next blood meal reinitiates the egg maturation cycle. Further blood meals are usually taken
only after egg batches are laid. If taken before oviposition, the mature postvitellogenic oocytes
produce an oostatic hormone, providing an inhibitory feedback which prevents further ovarian
development and vitellogene~is.~~ This oostatic hormone has recently been characterized as a
decapeptide which inhibits synthesis of a trypsin-like enzyme in the midgut of female A.
aegypti; it thus prevents the blood-meal digestion and, indirectly, vitell~genesis.~~
Aedes atropalpus is an autogenous mosquito species which does not require a blood meal
to produce the first egg batch. Nevertheless, hormonal control of the ovarian cycle appears
similar to the anautogenous A. aegypti except that emergence is the stimulus for EDNH release
and development of the first egg batch.5 Indeed, peptides have now been isolated that induce
ecdysteroid release from ovaries (i.e., GDNH-like) of both species.32

b. Flies
Both JH and ecdysteroids are again implicated in the hormonal control of egg maturation
in the higher Diptera (Cyclorrhapha), and finer details are still being revealed. The major JH
produced by the corpus allatum of adult Cyclorrhaphan flies (Drosophila melanogaster,
Calliphora vomitoria) has recently been identified as the bisepoxide of JH III.33.34
In an anautogenous colony (requiring a protein meal before egg development) of the black
blowfly, Phormia regina, it appears that JH is required for the uptake of vitellogenin by the
~ o c y t e Treatment
.~~ of sugar-fed adult females with JH I11 or a JH analogue resulted in a
proportion of follicles with oocytes containing an opaque material. Immunological procedures
showed that this opaque material was not vitellogenin, and it was concluded that protein
uptake by the oocytes had been stimulated, but, in the absence of vitellogenin, the opaque
material comprised other hemolymph proteins. Sugar-fed flies also retained low levels of
ecdysteroids, but after a protein meal, ecdysteroids increased, the ovaries being the major
source, and vitellogenin uptake by the oocytes followed.36Previous experiments had shown
that treatment with precocene I1 (a pro-allatocidin compound which prevents JH synthesis by
the corpus allatum) inhibited oocyte development beyond the previtellogenic stage, but did not
prevent vitellogenin synthesis or release into the hemolymph, albeit levels were lower than
controls.37Recently it has been shown that a peptide factor from the midgut is released in liver-
fed females, which stimulates neurosecretory cells in the brain to initiate the neuroendocrine
cascade leading to o o g e n e ~ i sA. ~summary
~ of the proposed endocrine interaction for this fly
species is presented in Figure 2 and includes a possible factor released from the brain which
induces synthesis and release of ecdysteroids from the ovaries (modified from References 36
and 38).
It has been shown in other flies that both the fat body and the ovaries produce ~ i t e l l o g e n i n . ~ ~ . ~ ~
In Drosophila, genes for three vitellogenins (termed yolk proteins 1,2, and 3) are expressed
in the fat body and follicuiar cells of the ovary but the regulation of expression differs between
the tissues. Experiments with ligated abdomens showed that ecdysteroids and JHs could
induce vitellogenin formation but, in addition, JH promoted vitellogenin uptake by the
oocytes. Further studies on the relative amounts of the three vitellogenins indicated that JH
stimulated normal synthesis and uptake in the ovary but abnormal synthesis by the fat body,
while ecdysteroids had no effect on the ovary but induced normal synthesis by the fat body.5
This simplistic model may have to be modified in view of more recent work which indicates
the presence of other factors mediating nutritional effects on vitellogenin production and
~ p t a k e . ~By
~ .comparison,
~' in the housefly Musca domestica, ecdysteroids and a JH analogue
have been shown to induce vitellogenin mRNA in both the fat body and the ovaries. However,
the JH analogue was the least potent of the two and proved more effective on the ovary than
on the fat body.40
Hormones and Reproduction

Protein Meal

corpus wdiacum corpus allaturn

l

FIGURE 2. Factors regulating ovarian development in the black blowfly,Phormia regina. (Modifiedfrom Yin et a1.S3&)

3. Hernipterans
The blood-sucking bug Rhodnius prolixus was used by Wigglesworth to identify JH as a
hormone controlling both metamorphosis and reproducti~n.~~ He demonstrated that the re-
moval of the corpus allatum prevented vitellogenesis in the ovaries of adult females. This
insect has continued to be utilized for the examination of reproductive endocrinology, particu-
larly by Davey and colleagues, and our knowledge of humoral events controlling ovarian
development and oviposition has become increasingly detailed. JH probably stimulates
vitellogenin synthesis in the fat body, but the study of vitellogenin uptake by the oocytes has
taken research ~ r e c e d e n c eIn
. ~mated
~ females, the JH has a three-fold action in the stimulation
of vitellogenin uptake by the terminal oocytes. Vitellogenins are large protein molecules, and
their access to the oocyte cell membrane is facilitated by JH-induced changes in shape of the
surrounding follicle cells and the resulting enlargement in the intercellular spaces (increased
"patency"; see Chapter However, the presence of JH is also necessary during follicle cell
development in order that cells become competent to respond to later increases in JH t i t e ~ - . ~ ~
The uptake of vitellogenin by the oocyte is a calcium-dependent receptor-mediated response,
and the receptor binding of vitellogenin is also enhanced by JH.46Recent studies also provide
evidence of a neural inhibition of corpus allatum activity coming from the brain.47
In addition to the gonadotropic effects of JH, Rhodnius females possess abdominal neuro-
secretory organs which produce an antigonadotropic hormone (oostatic h ~ r m o n e ) .Ovaries ~~.~~
containing mature oocytes in the pedicels stimulate the release of the antigonadotropin, which
counters the effect of JH on the vitellogenin uptake by the oocytes. The changes in patency
induced by JH are inhibited in the presence of antigonadotr~pin.~~
Ten neurosecretory cells have been identified in the pars intercerebralis of the brain, which
produce a peptidergic myotropic factor that initiates ovulation (movement of the fully devel-
oped oocyte from the ovary) and subsequently o v i p o s i t i ~ n . Unmated
~ ~ . ~ ~ females retain eggs
in the ovarian pedicel for long periods, resulting in a much lower oviposition rate than mated
females, and it is the presence of a spermathecal factor in mated insects that facilitates the
release of the oviposition hormone.53However, it appears to be the production of ecdysteroids
by the ovary, 5 days after a blood meal, that induces the release of the myotropic ovulation
hormone from axon terminals in the corpora ~ a r d i a c a .The ~ ~ ecdysteroids
-~~ do not act directly
on the neurosecretory cells but indirectly via aminergic neuronss7while more recent investiga-
tions indicate that there is also a circadian influence over these endocrine events.58

4. Homopterans
Aphids form one of the few groups of insects that commonly alternate between partheno-
genetic and sexual reproduction (cyclical parthenogene~is).~~
While there are species (andlor
100 Insect Reproduction

clones) that will reproduce only by parthenogenesis, there are none that reproduce solely by
sexual means. In a number of species, both the mode of reproduction and ovarian development
in the parthenogenetic females is is controlled by the same hormone, JH. Photoperiodic cues
are most frequently found to determine the reproductive type: long days result in the formation
of parthenogenetic females (virginoparae), while short days promote the development of the
sexual forms (oviparae and male^).^
Work in the late 1970s indicated that the medial neurosecretory cells of the protocerebrum
(called the group I cells in aphid^)^' were involved in the determination of female embryos as
virginoparae or o ~ i p a r a eAblation
.~~ of these cells by radiocautery, in long-day reared vetch
aphids, Megoura viciae, induced the production of "short-day" oviparae rather than the
expected virginoparae. The observations indicated that a factor produced by these protocerebral
cells promoted the appearance of virginoparae, and it was proposed that the factor acted
directly upon the developing embryos in the ovarioles. The factor was termed "virginoparin"
by In contemporary studies, topical application of natural juvenile hormones or JH
analogues was found to modify embryonic development such that female embryos that were
photoperiodically determined as sexual females (oviparae) were redirected to develop as
parthenogenetic females (~irginoparae).~"-" In addition to the switch induced in ovary type,
there was a concommitant induction of winged or partially winged forms, many of which were
sterile, but judicious application of JH produced seemingly normal virginoparous females that
were capable of normal reprod~ction."~~' Thus, "virginoparin" and JH had the same effect on
the development of female embryos and promoted their development as virginoparae; they
were "long-day" hormones.
It was later shown that "virginoparin" and JH were part of the endocrine pathway for the
induction of ~irginoparae.~~ It was shown that corpus allatum volume (this is a single fused
gland in the aphid) differed, from day 10 onwards, between short-day- and long-day-reared
females that were producing oviparae or virginoparae, respectively. When the group I cells
were ablated in long-day insects, and the progeny switched from virginoparae to oviparae,
there was a concomittant change in the corpus allatum volume such that it became equal in
size to the short-day-reared insect corpora allata. Aphids that underwent sham cautery re-
mained similar to untreated controls; the corpora allata were the same size, and they continued
to produce virginoparae. It appears that the group I cells regulate JH synthesis via the corpus
allatum. Assessment of JH titers in the aphids showed only JH I11 present and, although the
levels were low, there were higher JH titers in the long-day- than the short-day reared aphids.69
There are other subtleties to aphid reproduction in that the parthenogenetic females un-
dergo precocious ovarian development which results in the telescoping of generations.'O The
two ovaries of adult virginoparae (mother) comprise a number of ovarioles containing em-
bryos (daughters) in various stages of development. The most mature of these embryos already
contain ovaries with developing embryos (granddaughters). Parthenogenetic aphids are also
viviparous and give birth to live young which, at birth, already have ovaries with ovarioles that
contain one or two developing embryos. In the black bean aphid Aphis fabae, oocyte devel-
opment and embryogenesis (of the granddaughter generation) within the embryonic (daughter
generation) ovaries begins during the mother's fourth in~tar.~' Decapitation, which included
the removal of the corpora allata, drastically reduced the rate of oocyte differentiation, but this
could be restored by JH application. In addition, the growth of the terminal (daughter)
embryos is reduced almost completely by decapitation, but again is restored by JH treatment.
However, this stimulation of embryogenesis is not reflected in a shortening of the pre-
reproductive period or in the initial rate of reproduction, possibly because other endocrine
factors are involved in part~rition.~~
It appears that JH has both a role in determining the parthenogenetic aphid morph and in
the regulation of ovarian development in that morph. Additionally, as in other insects, it
regulates metarnorph~sis.'~ However, in most insects the metamorphic role of JH terminates
Hormones and Reproduction 101

at the final moult, when JH often takes on a reproductive role. Perhaps uniquely in aphids,
adult form is determined by low JH titers in the third (penultimate) instar larva and JH can then
take on a reproductive role, precociously, in the last larval instar. Control of ovarian devel-
opment in the sexual females has not been researched, but it will differ.