Entomology

In insects, hydrocarbons waterproof the cuticle, protect the insect from the external environment, and serve as semiochemicals or their metabolic precursors. In the German cockroach, Blattella germanica, hydrocarbons are synthesized by the abdominal integument, but the precise site of biosynthesis is not known. We developed a method for separation of oenocytes from other cells in the abdominal integument using enzymatic dissociation followed by Percoll gradient centrifugation. Radiolabeled propionate was then used to monitor de novo synthesis of hydrocarbons by dissociated cells. Oenocyte-enriched cell suspensions of abdominal sternites synthesized hydrocarbons, whereas suspensions enriched with epidermal cells did not. Our results show conclusively that hydrocarbons are produced by oenocytes not only in insects whose oenocytes are localized within the hemocoel, but also in those insects whose oenocytes are within the abdominal integument. Furthermore, these data support a hemolymph pathway for transport and delivery of hydrocarbons to both external and internal tissues, including the epicuticle, fat body, and ovaries.

Abstract  The physiological and biochemical characteristics of the diapause larvae of Asian corn borer Ostrinia furnacalis, were investigated. The molecular weight of diapause associated protein in hemolymph was determined. The development of male reproductive system, the body water content, the tolerance to suffocation and the rates of respiration of the diapause and non-diapause larvae were compared. The results revealed that the diapause larvae have the following characteristics: 1. There is a diapause associated protein in heamolymoph; its molecular weights of two polypeptide chains are 72,000 and 84,000 Dal. respectively. 2. The male reproductive organ is underdeveloped. 3. The body water content is lowered. 4. A much higher tolerance to suffocation is exhibited after submergence under water at room temperature. 5. A low rate of respiration prevailed.

Cuticular hydrocarbons of arthropods serve multiple functions, including as barriers to water loss and as pheromones and pheromone precursors. In the oviparous German cockroach, Blattella germanica, long-chain hydrocarbons are produced by oenocytes within the abdominal integument and are transported by a blood lipoprotein, lipophorin, both to the cuticular surface and into vitellogenic oocytes. Using radiotracer approaches, we tracked the location and metabolic fate of 14 C-and 3 H-labeled hydrocarbons through vitellogenic females and their embryos and nymphs. A considerable amount (~50%) of radiolabeled maternal hydrocarbons was transferred to oocytes and persisted through a 20-day embryogenesis and the first two nymphal stadia. The maternal hydrocarbons were not degraded or lost during this protracted period, except for significant losses of cuticular hydrocarbons starting with the first-to-second instar molt. Thus, although embryos and nymphs can produce their own hydrocarbons, maternal hydrocarbons provide a significant fraction of the cuticular and hemolymph hydrocarbons of both stages. These results show, for the first time in any insect, that a mother provides a significant complement of her offspringʼs cuticular hydrocarbons. Further research will be needed to determine whether provisioning hydrocarbons to eggs is a general strategy among insects and other arthropods or if this strategy is limited to taxa where eggs and early instars are susceptible to desiccation.

Embryos of the viviparous cockroach Diploptera punctata accumulate large amounts of hydrocarbon (HC) of either maternal or embryonic origin. HC synthesis and its accumulation in maternal and embryonic tissues were measured over the course of gestation. Female abdominal integument was the only tissue that synthesized appreciable amounts of HC in vitro, and did so at an increasing rate from the time of mating to mid-pregnancy, when rates of synthesis declined. The embryos synthesized HC at rates o1% those of the female, showing that the majority of HC detected in and on embryos was of maternal origin. The brood sac that houses the developing embryos did not synthesize HC in vitro, indicating that HC must be transported from the female abdominal integument to the embryos. The mass of female epicuticular HC was constant at $183 mg, while her internal HC increased fourfold from mating to mid-pregnancy, then declined until parturition. The decline in internal HC reflected both declining HC synthesis in the female and greater export to the embryos, as embryonic internal HC increased 250-fold prior to parturition. An external HC coating over the oothecal covering and chorion of the embryos increased to mid-pregnancy, then declined. Unlike oviparous cockroaches, D. punctata females fed throughout the reproductive cycle, reflecting the nutritional demands of continuously provisioning the developing embryos. r

The transport of lipids constitutes a vital function in insects and requires the plasma lipoprotein lipophorin. In all insects examined to date, cuticular hydrocarbons are also transported through the hemolymph by lipophorin, and in social insects they play important roles not only in water proofing the cuticle but also in nestmate recognition. High-density lipophorin (HDLp), isolated from Reticulitermes flavipes plasma by KBr gradient ultracentrifugation, contains 66.2% protein and 33.8% lipids; hydrocarbons constitute its major neutral lipid (20.4% of total lipids). Anti-lipophorin serum was generated in rabbit and its specific association with lipophorin, and not with any other plasma proteins, was verified with Western blotting. Immunoprecipitation also confirmed that this antibody specifically recognizes lipophorin, because all hemolymph hydrocarbons of the termites R. flavipes and R. lucifugus and the cockroach Supella longipalpa, which associate only with lipophorin, were recovered in the immunoprecipitated protein. Cross-reactivity of the antiserum with lipophorin from related species was investigated by double immunodiffusion with 10 termite species in the genera Reticulitermes, Coptotermes, Zootermopsis, and Kalotermes, and with five cockroach species. Involvement of lipophorin in hydrocarbon transport was shown by injecting HDLp antiserum into Zootermopsis nevadensis and then monitoring the de novo biosynthesis of hydrocarbons and their transport to the cuticular surface; the antiserum significantly disrupted hydrocarbon transport. ELISA revealed a gradual increase in the lipophorin titer in successively larger R. flavipes workers, and differences among castes in lipophorin titers were highest between nymphs and first instar larvae. #

The ability to distinguish between members of a social group and unfamiliar individuals is a critical element of social behaviour. Social insects can differentiate between nestmates and non-nestmates via recognition cues, which in most species are cuticular hydrocarbons. Cuticular hydrocarbon patterns are altered by genotype and environmental conditions, but it is unclear whether colony social conditions can also affect nestmate interactions. Honeybee queens produce pheromones that regulate many aspects of worker behaviour, physiology and colony organization. A five-component blend, queen mandibular pheromone (QMP), produces many of the effects of a live queen. We found that QMP treatment alters how resident bees interact with intruder bees, based on standard nestmate recognition assays. However, QMP does not appear to alter the ability of bees to distinguish between nestmates and non-nestmates, or general aggression levels. Rather, QMP exposure significantly alters cuticular hydrocarbon patterns of worker bees, suggesting that QMP-treated nestmates are no longer recognized as nestmates by untreated bees, and vice versa. Thus, queen pheromone can have significant effects on nestmate recognition and interactions in honeybees, which may be important for colony cohesion. Ó

Assessing interactions between the associations of common genetic susceptibility variants, reproductive history and body mass index with breast cancer risk in the Breast Cancer Association Consortium: a combined case-control study. Breast cancer research, 12 (6). R110.

Background: Regulation of worker behavior by dominant queens or workers is a hallmark of insect societies, but the underlying molecular mechanisms and their evolutionary conservation are not well understood. Honey bee and bumble bee colonies consist of a single reproductive queen and facultatively sterile workers. The queens' influences on the workers are mediated largely via inhibition of juvenile hormone titers, which affect division of labor in honey bees and worker reproduction in bumble bees. Studies in honey bees identified a transcription factor, Krüppel-homolog 1 (Kr-h1), whose expression in worker brains is significantly downregulated in the presence of a queen or queen pheromone and higher in forager bees, making this gene an ideal candidate for examining the evolutionary conservation of socially regulated pathways in Hymenoptera. Results: In contrast to honey bees, bumble bees foragers do not have higher Kr-h1 levels relative to nurses: in one of three colonies levels were similar in nurses and foragers, and in two colonies levels were higher in nurses. Similarly to honey bees, brain Kr-h1 levels were significantly downregulated in the presence versus absence of a queen. Furthermore, in small queenless groups, Kr-h1 levels were downregulated in subordinate workers with undeveloped ovaries relative to dominant individuals with active ovaries. Brain Kr-h1 levels were upregulated by juvenile hormone treatment relative to a vehicle control. Finally, phylogenetic analysis indicates that KR-H1 orthologs are presence across insect orders. Though this protein is highly conserved between honey bees and bumble bees, there are significant differences between orthologs of insects from different orders. Conclusions: Our results suggest that Kr-h1 is associated with juvenile hormone mediated regulation of reproduction in bumble bees. The expression of this transcription factor is inhibited by the queen and associated with endocrine mediated regulation of social organization in two species of bees. Thus, KR-H1 may transcriptionally regulate a conserved genetic module that is part of a pathway that has been co-opted to function in social behavior, and adjusts the behavior of workers to their social environmental context.

Sex peptide (SP) and Ductus ejaculatorius peptide (Dup) 99B are synthesized in the retrogonadal complex of adult male Drosophila melanogaster, and are transferred in the male seminal fluid to the female genital tract during mating. They have been sequenced and shown to exhibit a high degree of homology in the C-terminal region. Both affect subsequent mating and oviposition by female D. melanogaster. SP also increases in vitro juvenile hormone (JH) biosynthesis in excised corpora allata (CA) of D. melanogaster and Helicoverpa armigera. We herein report that the partial C-terminal peptides SP 8-36 and SP 21-36 of D. melanogaster, and the truncated N-terminal SP 6-20 do not stimulate JH biosynthesis in vitro in CA of both species. Both of these C-terminal peptides reduce JH-III biosynthesis significantly. Dup99B, with no appreciable homology to SP in the N-terminal region, similarly lacks an effect on JH production by H. armigera CA. In contrast, the N-terminal peptides-SP 1-11 and SP 1-22-do significantly activate JH biosynthesis of both species in vitro. We conclude that the first five N-terminal amino acid residues at the least, are essential for allatal stimulation in these disparate insect species. We have previously shown that the full-length SP 1-36 depresses pheromone biosynthesis in H. armigera in vivo and in vitro. We now show that full-length Dup99B and the C-terminal partial sequence SP 8-36 at low concentrations strongly depress (in the range of 90% inhibition) PBAN-stimulated pheromone biosynthesis of H. armigera. In addition, the N-terminal peptide SP 1-22 , the shorter N-terminal peptide SP 1-11 and the truncated N-terminal SP 6-20 strongly inhibit pheromone biosynthesis at higher concentrations.

The maturation of corpora allata (CA) and the competence of pheromone glands in the adult moth Helicoverpa armigera, are both age-related and appear to be correlated. Sex pheromone glands of pharate adults do not produce sex pheromone independently, nor do they respond to exogenous PBAN. Newly emerged moths produce significantly less pheromone than day one moths. JH (juvenile hormone) II was found to be the main JH form produced by CA in vitro. JH II primed pheromone glands of pharate adults to respond to PBAN. In addition, injection or topical application of JH II to newly-emerged females induced pheromone production in the presence of PBAN. Our findings suggest that JH is involved in the initiation of pheromone production of Helicoverpa armigera.

The German cockroach, Blattella germanica (L.), produces several potent protein aeroallergens, including Bla g 4, a ∼ ∼ ∼ ∼ 20 kDa lipocalin. RT-PCR, Northern analyses and in situ hybridization showed that Bla g 4 is expressed only in the adult male reproductive system. Western blotting and ELISA with rBla g 4 antiserum detected immunoreactivity in the utricles and the conglobate gland, but not in other tissues of the male reproductive system. The Bla g 4 protein content of males increased from adult emergence to day 14, but during copulation Bla g 4 was depleted in the male and transferred to the female within the spermatophore. Topical application of juvenile hormone III stimulated Bla g 4 production by both conglobate gland and utricles.

Mated female Heliothis virescens and H. subflexa were induced to produce sex pheromone during the photophase by injection of pheromone biosynthesis activating neuropeptide (PBAN). When injected with 1 pmol Hez-PBAN, the total amount of pheromone that could be extracted from glands of mated females during the photophase was similar to that extracted from virgin females in the scotophase. The PBAN-induced profile of pheromone components was compared between mated, PBAN-injected females and virgin females during spring and fall. Virgin females exhibited some differences in the relative composition of the pheromone blend between spring and fall, but no such temporal differences were detected in PBAN-injected, mated females. Because the temporal variation in pheromone blend composition was greater for virgin females than for PBAN-injected females, PBAN can be used to determine a female's native pheromone phenotype. This procedure has the advantages that pheromone glands can be extracted during the photophase, from mated females that have already oviposited.

Previous studies demonstrate that virgin female adult Helicoverpa armigera (Lepidoptera: Noctuidae) moths exhibit calling behaviour and produce sex pheromone in scotophase from the day after emergence, and that mating turns off both of these pre-mating activities. In the fruit fly Drosophila melanogaster, a product of the male accessory glands, termed sex peptide (SP), has been identified as being responsible for suppressing female receptivity after transfer to the female genital tract during mating. Juvenile hormone (JH) production is activated in the D. melanogaster corpus allatum (CA) by SP in vitro. We herein demonstrate crossreactivity of D. melanogaster SP in the H. armigera moth: JH production in photophase virgin female moth CA in vitro is directly activated in a dose-dependent manner by synthetic D. melanogaster SP, and concurrently inhibits pheromone biosynthesis activating neuropeptide (PBAN)-activated pheromone production by isolated pheromone glands of virgin females. Control peptides (locust adipokinetic hormone, AKH-I, and human corticotropin, ACTH) do not inhibit in vitro pheromone biosynthesis. Moreover, SP injected into virgin H. armigera females, decapitated 24 h after eclosion, or into scotophase virgin females, suppresses pheromone production. In the light of these results, we hypothesize the presumptive existence of a SP-like factor among the peptides transmitted to female H. armigera during copulation, inducing an increased level of JH production and depressing the levels of pheromone produced thereafter.

The adrenergicagonistsoctopamine,tyramine and clonidineinhibitedthe normal pheromonotropic action due to PBAN (pheromone biosynthesisactivating neuropeptide) in incubations of intersegmentaltissues that are situated between the 8th and 9th abdominalsegments of the moth ovipositortip. This inhibitionwas reversedin the presence of the adrenergicantagonists phentolamine,yohimbine and chlorpromazine.Incubations of 8th segments alone, which do not produce pheromone, resulted in elevated levels of intracellularcAMP in the presence of octopamine.The physiologicalsignificanceof this phenomenonis unclear. However, clonidine (an a, selective agonist) did not duplicate octopamine stimulation of intracellular cAMP in 8th segment cultures. In intersegmentalmembrane cultures clonidine successfully duplicated the octopamineinhibitionof both pheromoneand intracellularcAMP production.The physiological significanceof octopaminergicreceptors mediatingthe inhibitory response of intersegments was investigated by experiments i v i When PBAN was injected into photophase females the normal pheromonotropicactivity due to the injected PBAN dropped after 2h. In the presence of clonidine, normal peak stimulator levels were never attained and a faster decline was observed. Clonidine also inhibited the pheromonotropicresponse of 24h-decapitated females to PBAN. Adrenergic antagonistssuccessfully reversed the inhibitory effect of clonidine in decapitated females, but did not reverse the effect of clonidine in photophase females. In addition, when clonidine was injected into female moths during the scotophase normal peak pheromone titers were reduced although no effect on calling behavior was observed.

A key characteristic of eusocial species is reproductive division of labour. Honey bee colonies typically have a single reproductive queen and thousands of sterile workers. Adult queens differ dramatically from workers in anatomy, physiology, behaviour and lifespan. Young female workers can activate their ovaries and initiate egg laying; these 'reproductive' workers differ from sterile workers in anatomy, physiology, and behaviour. These differences, however, are on a much smaller scale than those observed between the queen and worker castes. Here, we use microarrays to monitor expression patterns of several thousand genes in the brains of same-aged virgin queens, sterile workers, and reproductive workers. We found large differences in expression between queens and both worker groups (~2000 genes), and much smaller differences between sterile and reproductive workers (221 genes). The expression patterns of these 221 genes in reproductive workers are more queen-like, and may represent a core group of genes associated with reproductive physiology. Furthermore, queens and reproductive workers preferentially up-regulate genes associated with the nurse bee behavioural state, which supports the hypothesis of an evolutionary link between worker division of labour and molecular pathways related to reproduction. Finally, several functional groups of genes associated with longevity in other species are significantly up-regulated in queens. Identifying the genes that underlie the differences between queens, sterile workers, and reproductive workers will allow us to begin to characterize the molecular mechanisms underlying the evolution of social behaviour and largescale remodelling of gene networks associated with polyphenisms.

Hormones in the insulin superfamily initiate a wide range of physiological processes in higher eukaryotic organisms. Although insulin-like peptides (ILPs) are likely present in all arthropods, they have been characterized for only a few insect species, including Locusta migratoria (Orthoptera [28]); three species of Lepidoptera [57]; and two species of Diptera, the fruit fly, Drosophila melanogaster [7] and the mosquito, Anopheles gambiae [26,41]. Most insect ILP genes encode a single prepropeptide with a signal peptide and contiguous B, C, and A peptides. The propeptide is processed into an active form by linkage of the A and B peptides by disulfide bridges followed by proteolytic cleavage of the C peptide, as with insulin and related peptides in vertebrates. An exception is the insulin growth factors (IGFs), which possess a short C peptide p e p t i d e s x x x (2 0 0 6) x x x-x x x a r t i c l e i n f o Article history:

Large amounts of hydrocarbons accumulate during vitellogenesis in the developing basal oocytes of the German cockroach Blattella germanica (L.), and all ovarian hydrocarbons are deposited into an egg case (ootheca) during oviposition. Hydrocarbons are not synthesized by the ovaries, but are delivered by hemolymph lipoproteins and accumulate within the basal oocytes. A native B. germanica hydrocarbon, [(3)H]3,11-dimethylnonacosane, injected into adult females of various ages, was taken up by the ovaries in relation to oocyte growth. Ovarian uptake of the hydrocarbon was low in day 0-1 females, increased dramatically between days 3 and 6 and declined sharply through oviposition on day 8-9; ovarian uptake of the hydrocarbon was low during a 21-day pregnancy that followed. [1-(14)C]Propionate, which becomes incorporated into methyl-branched hydrocarbons, was injected into 5-day-old vitellogenic females to monitor the de novo biosynthesis of hydrocarbons and the time course of hydrocarbo...

Hormones in the insulin superfamily initiate a wide range of physiological processes in higher eukaryotic organisms. Although insulin-like peptides (ILPs) are likely present in all arthropods, they have been characterized for only a few insect species, including Locusta migratoria (Orthoptera [28]); three species of Lepidoptera [57]; and two species of Diptera, the fruit fly, Drosophila melanogaster [7] and the mosquito, Anopheles gambiae [26,41]. Most insect ILP genes encode a single prepropeptide with a signal peptide and contiguous B, C, and A peptides. The propeptide is processed into an active form by linkage of the A and B peptides by disulfide bridges followed by proteolytic cleavage of the C peptide, as with insulin and related peptides in vertebrates. An exception is the insulin growth factors (IGFs), which possess a short C peptide p e p t i d e s x x x (2 0 0 6) x x x-x x x a r t i c l e i n f o Article history:

The adrenergicagonistsoctopamine,tyramine and clonidineinhibitedthe normal pheromonotropic action due to PBAN (pheromone biosynthesisactivating neuropeptide) in incubations of intersegmentaltissues that are situated between the 8th and 9th abdominalsegments of the moth ovipositortip. This inhibitionwas reversedin the presence of the adrenergicantagonists phentolamine,yohimbine and chlorpromazine.Incubations of 8th segments alone, which do not produce pheromone, resulted in elevated levels of intracellularcAMP in the presence of octopamine.The physiologicalsignificanceof this phenomenonis unclear. However, clonidine (an a, selective agonist) did not duplicate octopamine stimulation of intracellular cAMP in 8th segment cultures. In intersegmentalmembrane cultures clonidine successfully duplicated the octopamineinhibitionof both pheromoneand intracellularcAMP production.The physiological significanceof octopaminergicreceptors mediatingthe inhibitory response of intersegments was investigated by experiments i v i When PBAN was injected into photophase females the normal pheromonotropicactivity due to the injected PBAN dropped after 2h. In the presence of clonidine, normal peak stimulator levels were never attained and a faster decline was observed. Clonidine also inhibited the pheromonotropicresponse of 24h-decapitated females to PBAN. Adrenergic antagonistssuccessfully reversed the inhibitory effect of clonidine in decapitated females, but did not reverse the effect of clonidine in photophase females. In addition, when clonidine was injected into female moths during the scotophase normal peak pheromone titers were reduced although no effect on calling behavior was observed.