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Holometabolous insects stop feeding at the final larval instar stage and then undergo metamorphosis; however, the mechanism is unclear. In the present study, using the serious lepidopteran agricultural pest Helicoverpa armigera as a model, we revealed that 20-hydroxyecdysone (20E) binds to the dopamine receptor (DopEcR), a G protein-coupled receptor, to stop larval feeding and promote pupation. DopEcR was expressed in various tissues and its level increased during metamorphic molting under 20E regulation. The 20E titer was low during larval feeding stages and high during wandering stages. By contrast, the dopamine (DA) titer was high during larval feeding stages and low during the wandering stages. Injection of 20E or blocking dopamine receptors using the inhibitor flupentixol decreased larval food consumption and body weight. Knockdown of DopEcR repressed larval feeding, growth, and pupation. 20E, via DopEcR, promoted apoptosis; and DA, via DopEcR, induced cell proliferation. 20E opposed DA function by repressing DA-induced cell proliferation and AKT phosphorylation. 20E, via DopEcR, induced gene expression and a rapid increase in intracellular calcium ions and cAMP. 20E induced the interaction of DopEcR with G proteins αs and αq. 20E, via DopEcR, induced protein phosphorylation and binding of the EcRB1-USP1 transcription complex to the ecdysone response element. DopEcR could bind 20E inside the cell membrane or after being isolated from the cell membrane. Mutation of DopEcR decreased 20E binding levels and related cellular responses. 20E competed with DA to bind to DopEcR. The results of the present study suggested that 20E, via binding to DopEcR, arrests larval feeding and promotes pupation., Competing Interests: The authors have declared that no competing interests exist.

Cuticle tanning occurs in insects immediately after hatching or molting. During this process, the cuticle becomes dark and rigid due to melanin deposition and protein crosslinking. In insects, different from mammals, melanin is synthesized mainly from dopamine, which is produced from DOPA by the enzyme DOPA decarboxylase. In this work, we report that the silencing of the RpAadc-2 gene, which encodes the putative Rhodnius prolixus DOPA decarboxylase enzyme, resulted in a reduction in nymph survival, with a high percentage of treated insects dying during the ecdysis process or in the expected ecdysis period. Those treated insects that could complete ecdysis presented a decrease in cuticle pigmentation and hardness after molting. In adult females, the knockdown of AADC-2 resulted in a reduction in the hatching of eggs; the nymphs that managed to hatch failed to tan the cuticle and were unable to feed. Despite the failure in cuticle tanning, knockdown of the AADC-2 did not increase the susceptibility to topically applied deltamethrin, a pyrethroid insecticide. Additionally, our results showed that the melanin synthesis pathway did not play a major role in the detoxification of the excess (potentially toxic) tyrosine from the diet, an essential trait for hematophagous arthropod survival after a blood meal., (Copyright © 2019. Published by Elsevier Ltd.)

Insect cuticle is a composite made of chitin filaments embedded in a proteinaceous matrix, consisting mainly of structural cuticular proteins. In the present study, an endocuticle structural glycoprotein gene, LmAbd-9, was characterized based on the Locusta migratoria transcriptome. LmAbd-9 encodes a glycoprotein with a chitin binding domain 4, belonging to RR-1 subclass of the CPR family, which has two potential O-linked glycosylation sites (S115 and T137) at which glycosylation modification may occur. LmAbd-9 was highly expressed in the integument and showed periodic expression during molting. The expression levels of LmAbd-9 were significantly down-regulated after injection with 20-hydroxyecdysone (20E) for 6, 12, and 24 h, whereas it was upregulated after double-stranded RNA-mediated RNA interference of the 20E receptor gene LmEcR and LmFTZ-F1beta at day 2 of fifth instar nymphs for 48 h. After injection of dsLmAbd-9 on day 2 of fifth instar nymphs, the insects could normally molt to adults and showed no macroscopic phenotype; however, the cuticle of the adults was thinner, and there were significantly fewer endocuticular lamellae than in the control. Thus, LmAbd-9 that negatively regulated by the 20E signaling pathway was involved in the formation of the endocuticle in L. migratoria., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Peritrophic membrane (PM) is a chitin and protein-containing extracellular matrix that lines the midgut in most insect species, functioning as a barrier to exogenous toxins and pathogens. Midgut chitin deacetylases (CDAs) are chitin-modifying enzymes known to alter the mechanical property and permeability of PM. However, biochemical properties and specific roles of these enzymes remain elusive. In this study, the midgut-expressed CDAs (BmCDA6, BmCDA7 and BmCDA8) from Bombyx mori were cloned, recombinantly expressed and purified and their enzymatic activities toward PM chitin were determined. Of the three enzymes, BmCDA7 exhibited the highest activity (0.284 μmol/min/μmol), while BmCDA8 showed lower activity of 0.061 μmol/min/μmol. BmCDA6 was inactive towards PM chitin. Gene expression patterns indicated that although all three CDA genes were specifically expressed in the anterior midgut, they differed in their temporal expression patterns. BmCDA6 was expressed almost exclusively at the mid-molt stage, the stage when the PM was thick and with multiple chitin layers. Unlike BmCDA6, high expression levels of BmCDA7 and BmCDA8 were observed only at the feeding stage, the stage when the PM is thin and with fewer chitin layers. The different gene expression patterns and biochemical characteristics provide new information about the functional specialization among BmCDA6, BmCDA7 and BmCDA8 proteins., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The oriental fruit fly Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) is an important invasive pest with high reproductive capacity and invasiveness; it has shown remarkable range expansion and brings higher risk to the environment and agriculture. The insect cuticle serves as skin and skeleton, protecting insects against numerous harmful stresses. One gene named imaginal disc growth factor 4 (idgf4) which is involved in cuticle formation, plays an important role in organizing proteins in the chitin-matrix, as well as in adult molting. This gene in the poorly-described glycoside hydrolase 18 (GH 18) family was chosen to study the function of chitinases in insect defense barrier against heat and molting using quantitative real-time PCR (qRT-PCR) and RNA interference (RNAi). qRT- PCR showed that idgf4 was expressed in all nine developmental stages and was mainly expressed in the early and late pupal, as well as adult stages. Knocking down the idgf4 gene via RNAi in 3 rd instar larvae led to the decreased survival of larvae under high temperatures and malformed individuals as adults. The results indicated the function of the idgf4 gene in the fruit fly's defense barrier and development. It can provide new insights into understanding the function of one member in the GH 18 family, and may reveal a new potential gene for pest control.

Anopheles gambiae devotes over 2% of its protein coding genes to its 298 structural cuticular proteins (CPs). This paper provides new LC-MS/MS data on two adult structures, proboscises and palps, as well as three larval samples - 4th instar larvae, just their terminal segment, and a preparation enriched in their tracheae. These data were combined with our previously published results of proteins from five other adult structures, whole adults, and two preparations chosen for their relatively clean cuticle, the larval head capsules left behind after ecdysis and the pupal cuticles left behind after adult eclosion. Peptides from 28 CPs were recovered in all adult structures; 24 CPs were identified for the first time, 6 of these were members of the TWDL family. Most newly identified proteins came from the larval sources. Based solely on peptide recovery, from our data and from other investigators, most available on VectorBase, there were only 4 CPs that were restricted to a single adult structure. More were restricted to a single metamorphic stage, 14 in larvae, 0 in pupae and 32 in adults. Expression data from our earlier RT-qPCR studies reduces these numbers. Charting restriction of CPs to stage or structure is a step forward in establishing their specific roles., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Fibroin modulator binding protein-1 (FMBP-1) is a novel DNA-binding protein containing a conserved score and three amino acid peptide repeat (STPR) domain. The roles of factors containing STPR domain are less known. Although multiple transcription factors are involved in the transcriptional regulation of silk protein genes during the development of silkworm, the mechanism of transcriptional repression of silk protein genes during molting remains unclear. Here, we found that FMBP-1 expression was contrary to that of fibroin heavy chain (fib-H) during the fourth molting period of Bombyx mori. FMBP-1 repressed fib-H promoter activity by directly binding to the -130 element in the fib-H promoter region. We also identified two proteins, Bmsage and Bmdimm, that interacted with FMBP-1 in the posterior silk gland of silkworm larvae, and further verified these interactions by far western blotting and microscale thermophoresis in vitro, as well as co-immunoprecipitation and bimolecular fluorescence complementation at the cellular level. The luciferase reporter assay showed that the interaction between FMBP-1 and Bmdimm antagonized the activation of Bmdimm on fib-H transcription, but did not affect FMBP-1-mediated transcriptional repression on fib-H gene. Therefore, we proposed the following mechanism of fib-H transcriptional repression by FMBP-1 during the molting of silkworm larvae: 1) FMBP-1 directly binds to the -130 element in the fib-H promoter to repress fib-H transcription; 2) FMBP-1 interacts with Bmdimm to antagonize the activation of Bmdimm on fib-H transcription. Our findings promote a better understanding of fib-H transcriptional regulation and provide novel insights into the transcriptional repression of fib-H by FMBP-1 and basic helix-loop-helix factors Bmdimm during the molting of silkworm larvae. Our study also provides valuable information regarding the biological function of factors containing STPR domain., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

ATP-binding cassette (ABC) transporters constitute a large superfamily of proteins that mediate transport of a diverse number of substrates including nutrients, lipids and xenobiotics across membranes serving a variety of developmental and physiological functions. Here, we report on the molecular properties and biological roles of the ABC transporter LmABCH-9C in the migratory locust Locusta migratoria. LmABCH-9C was expressed continuously during nymphal development in all tissues including the integument. Expression was highest just after molting. Suppression of LmABCH-9C transcript levels by RNA interference (RNAi) in nymphs provoked death during or soon after molting to the next stage. These nymphs lost weight within minutes after molting. Moreover, high humidity rescued the lethality of molted dsLmABCH-9C-injected nymphs. In histological experiments, we find that the amounts of inner-cuticular lipids are reduced in nymphs with suppressed LmABCH-9C expression. These data together indicate that LmABCH-9C is needed for lipid-dependent desiccation resistance, paralleling the function of ABCH-9C in Tribolium castaneum. Hence, the function of this ABC transporter seems to be conserved across insect species ranging from hemimetabolous (L. migratoria) to holometabolous (T. castaneum) species. In addition, we find that cuticle inward impermeability is compromised in nymphs with reduced LmABCH-9C function. In summary, consistent with the model that cuticular lipids are necessary to prevent desiccation and penetration of xenobiotics in insects, we hypothesize that LmABCH-9C is involved in the construction of a lipid-based barrier at the surface of the cuticle especially after molting to protect the animal against uncontrolled water loss and entry. Susceptibility of this ABC transporter to RNAi-mediated knockdown designates it as an excellent target for RNAi-based insect pest control., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the three-dimensional extracellular matrix of the insect cuticle, horizontally aligned microfibrils composed of the polysaccharide chitin and associated proteins are stacked either parallel to each other or helicoidally. The underlying molecular mechanisms that implement differential chitin organization are largely unknown. To learn more about cuticle organization, we sought to study the role of chitin deacetylases (CDA) in this process. In the body cuticle of nymphs of the migratory locust Locusta migratoria, helicoidal chitin organization is changed to an organization with unidirectional microfibril orientation when LmCDA2 expression is knocked down by RNA interference. In addition, the LmCDA2-deficient cuticle is less compact suggesting that LmCDA2 is needed for chitin packaging. Animals with reduced LmCDA2 activity die at molting, underlining that correct chitin organization is essential for survival. Interestingly, we find that LmCDA2 localizes only to the initially produced chitin microfibrils that constitute the apical site of the chitin stack. Based on our data, we hypothesize that LmCDA2-mediated chitin deacetylation at the beginning of chitin production is a decisive reaction that triggers helicoidal arrangement of subsequently assembled chitin-protein microfibrils., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Locusts jump by using a catapult mechanism in which energy produced by slow contractions of the extensor tibiae muscles of the hind legs is stored in distortions of the exoskeleton, most notably (1) the two semi-lunar processes at each knee joint and (2) the tendons of the extensor muscles themselves. The energy is then suddenly released from these stores to power the rapid, propulsive movements of the hind legs. The reliance on the mechanical storage of energy is likely to impact on jumping because growth occurs by a series of five moults, at each of which the exoskeleton is replaced by a new one. All developmental stages (instars) nevertheless jump as a means of forward locomotion, or as an escape movement. Here, I show that in each instar, resilin is added to the semi-lunar processes and to the core of the extensor tendons so that their thickness increases. As the next moult approaches, a new exoskeleton forms within the old one, with resilin already present in the new semi-lunar processes. The old exoskeleton, the tendons and their resilin are discarded at moulting. The resilin of the semi-lunar processes and tendons of the new instar is initially thin, but a similar pattern of deposition results in an increase of their thickness. In adults, resilin continues to be deposited so that at 4 weeks old the thickness in the semi-lunar processes has increased fourfold. These changes in the energy stores accompany changes in jumping ability and performance during each moulting cycle., (© 2016. Published by The Company of Biologists Ltd.)

Olfaction is one of the main sensory modalities that allow insects to interpret their environment. Several proteins, including odorant-binding proteins (OBPs) and odorant receptors (ORs), are involved in this process. Odorant receptors are ion channels formed by a binding unit OR and an odorant receptor co-receptor (Orco). The main goal of this study was to characterize the Orco gene of Rhodnius prolixus (RproOrco) and to infer its biological functions using gene silencing. The full-length RproOrco gene sequence was downloaded from VectorBase. This gene has 7 introns and is located in the genome SuperContig GL563069: 1,017,713-1,023,165. RproOrco encodes a protein of 473 amino acids, with predicted 7 transmembrane domains, and is highly expressed in the antennae during all R. prolixus developmental stages. The RNAi technique effectively silenced RproOrco, reducing the gene's expression by approximately 73%. Interestingly, the effect of gene silencing persisted for more than 100 days, indicating a prolonged effect of dsRNA that was maintained even after molting. The phenotypic effects of silencing involved the following: (1) loss of the ability to find a vertebrate host in a timely manner, (2) decreased ingested blood volume, (3) delayed and decreased molt rate, (4) increased mortality rate, and (5) decreased egg laying. Our data strongly suggest that dsOrco disrupts R. prolixus host-finding behavior, which is further reflected in the blood ingestion, molting, mortality, and egg laying data. This study clearly demonstrates that Orco is an excellent target for controlling triatomine populations. Thus, the data presented here open new possibilities for the control of vector-borne diseases., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Silkworm (Bombyx mori) is a lepidopteran-holometabolic model organism. To understand its developmental biochemistry, we characterized the larval hemolymph metabonome from the third instar to prepupa stage using (1)H NMR spectroscopy whilst hemolymph fatty acid composition using GC-FID/MS. We unambiguously assigned more than 60 metabolites, among which tyrosine-o-β-glucuronide, mesaconate, homocarnosine, and picolinate were reported for the first time from the silkworm hemolymph. Phosphorylcholine was the most abundant metabolite in all developmental stages with exception for the periods before the third and fourth molting. We also found obvious developmental dependence for the hemolymph metabonome involving multiple pathways including protein biosyntheses, glycolysis, TCA cycle, the metabolisms of choline amino acids, fatty acids, purines, and pyrimidines. Most hemolymph amino acids had two elevations during the feeding period of the fourth instar and prepupa stage. Trehalose was the major blood sugar before day 8 of the fifth instar, whereas glucose became the major blood sugar after spinning. C16:0, C18:0 and its unsaturated forms were dominant fatty acids in hemolymph. The developmental changes of hemolymph metabonome were associated with dietary nutrient intakes, biosyntheses of cell membrane, pigments, proteins, and energy metabolism. These findings offered essential biochemistry information in terms of the dynamic metabolic changes during silkworm development.

Chemosensory proteins (CSPs) have been predicted to be involved in development; however, direct evidence for their involvement is lacking, and genetic basis is largely unknown. To determine the function of the chemosensory protein 9 (Si-CSP9) gene in Solenopsis invicta, we used RNA interference to silence Si-CSP9 in 3rd-instar larvae. The 3rd-instar larvae failed to shed their cuticle after being fed Si-CSP9-directed siRNA, and expression profiling of RNAi-treated and untreated control larvae showed that 375 genes were differentially expressed. Pathway enrichment analysis revealed that 4 pathways associated with larval development were significantly enriched. Blast analysis revealed that one fatty acid amide hydrolase (FAAH) gene was up-regulated and 4 fatty acid synthase (FAT) genes and one protein kinase DC2 gene (PKA) were down-regulated in the enriched pathways. Significantly higher expression of these genes was found in 4th-instar larvae, and Pearson correlation analysis of the expression patterns revealed significant relationships among Si-CSP9, PKA, FAAH, and FAT1-4. Moreover, we confirmed that expression levels of Si-CSP9, FAAH, and FAT1-4 were significantly reduced and that the development of 3rd-instar larvae was halted with PKA silencing. These results suggest that Si-CSP9 and PKA may be involved in the network that contributes to development of 3rd-instar larvae.

Genes encoding cuticle proteins are helpful subjects to study the molecular mechanisms of insect molting and metamorphosis. In this study, we isolated and characterized a novel cuticle protein R&R gene, referred to as AccCPR1, from Apis cerana cerana. The open reading frame of AccCPR1 has a length of 573 nt and encodes a protein of 190 amino acids that contains a chitin binding region and is a typical cuticle R&R-2 protein. Five putative E74 binding sites and four BR-C binding sites were predicted in the 5'-flanking region, which suggests a potential function in molting and metamorphosis. RT-qPCR showed that AccCPR1 transcript occurred as the ecdysteroid titer decreased after reaching a peak, which suggests AccCPR1 expression requires a "pulse" regimen of ecdysteroids. This hypothesis was tested using different experimental strategies. When larvae were reared with different concentrations of 20E in their diet, the ecdysteroid peak repressed AccCPR1 expression. Exposure of the thoracic integument of the pupae in vitro to different concentration of 20E repressed AccCPR1 expression, which recovered after the removal of 20E. These results suggest that AccCPR1 is a typical cuticle R&R-2 protein that plays an important role in development, and an ecdysteroid pulse is critical for high AccCPR1 gene expression., (© 2013.)

Insects require molting fluids to shed the old cuticle during molting. β-N-acetyl-D-hexosaminidase, known as Hex1, together with various chitinases, is responsible for degrading the chitin component of the old cuticle. This study showed that another β-N-acetyl-D-hexosaminidase, termed OfHex3, interacted with Hex1 and functioned in the molting fluid, although the homolog of OfHex3 was known as a sperm-plasma enzyme functioning in egg-sperm recognition. OfHex3 is an enzyme cloned from the insect Asian corn borer, Ostrinia furnacalis, which is one of the most destructive pests of maize. The enzymatic activity analysis indicated that OfHex3 was able to degrade chitooligosaccharides, but at a lower rate than that of OfHex1. Because OfHex3 did not have substrate inhibition, we deduced that the presence of OfHex3 might help OfHex1 relieve substrate inhibition during chitin degradation during molting. The expression patterns of OfHex3 during O. furnacalis development were studied by real-time PCR as well as western blot. The results showed that both gene transcription and protein translation levels of OfHex3 were up-regulated during larval-larval molting. The tissue-specific expression pattern analysis indicated that OfHex3 was mostly localized in the fat body and testis. All these data further supported that Hex3 was involved in molting as well as in fertilization. This study may help to understand the complexity of cuticle degradation during insect molting, and may provide a possible target for pest control.

Molting, or the replacement of the old exoskeleton with a new cuticle, is a complex developmental process that all insects must undergo to allow unhindered growth and development. Prior to each molt, the developing new cuticle must resist the actions of potent chitinolytic enzymes that degrade the overlying old cuticle. We recently disproved the classical dogma that a physical barrier prevents chitinases from accessing the new cuticle and showed that the chitin-binding protein Knickkopf (Knk) protects the new cuticle from degradation. Here we demonstrate that, in Tribolium castaneum, the protein Retroactive (TcRtv) is an essential mediator of this protective effect of Knk. TcRtv localizes within epidermal cells and specifically confers protection to the new cuticle against chitinases by facilitating the trafficking of TcKnk into the procuticle. Down-regulation of TcRtv resulted in entrapment of TcKnk within the epidermal cells and caused molting defects and lethality in all stages of insect growth, consistent with the loss of TcKnk function. Given the ubiquity of Rtv and Knk orthologs in arthropods, we propose that this mechanism of new cuticle protection is conserved throughout the phylum., Competing Interests: The authors have declared that no competing interests exist.

The functional characterization of an entire class of 17 genes from the red flour beetle, Tribolium castaneum, which encode two families of Cuticular Proteins Analogous to Peritrophins (CPAPs) has been carried out. CPAP genes in T. castaneum are expressed exclusively in cuticle-forming tissues and have been classified into two families, CPAP1 and CPAP3, based on whether the proteins contain either one (CPAP1), or three copies (CPAP3) of the chitin-binding domain, ChtBD2, with its six characteristically spaced cysteine residues. Individual members of the TcCPAP1 and TcCPAP3 gene families have distinct developmental patterns of expression. Many of these proteins serve essential and non-redundant functions in maintaining the structural integrity of the cuticle in different parts of the insect anatomy. Three genes of the TcCPAP1 family and five genes of the TcCPAP3 family are essential for insect development, molting, cuticle integrity, proper locomotion or fecundity. RNA interference (RNAi) targeting TcCPAP1-C, TcCPAP1-H, TcCPAP1-J or TcCPAP3-C transcripts resulted in death at the pharate adult stage of development. RNAi for TcCPAP3-A1, TcCPAP3-B, TcCPAP3-D1 or TcCPAP3-D2 genes resulted in different developmental defects, including adult/embryonic mortality, abnormal elytra or hindwings, or an abnormal 'stiff-jointed' gait. These results provide experimental support for specialization in the functions of CPAP proteins in T. castaneum and a biological rationale for the conservation of CPAP orthologs in other orders of insects. This is the first comprehensive functional analysis of an entire class of cuticular proteins with one or more ChtBD2 domains in any insect species.

During each molting cycle of insect development, synthesis of new cuticle occurs concurrently with the partial degradation of the overlying old exoskeleton. Protection of the newly synthesized cuticle from molting fluid enzymes has long been attributed to the presence of an impermeable envelope layer that was thought to serve as a physical barrier, preventing molting fluid enzymes from accessing the new cuticle and thereby ensuring selective degradation of only the old one. In this study, using the red flour beetle, Tribolium castaneum, as a model insect species, we show that an entirely different and unexpected mechanism accounts for the selective action of chitinases and possibly other molting enzymes. The molting fluid enzyme chitinase, which degrades the matrix polysaccharide chitin, is not excluded from the newly synthesized cuticle as previously assumed. Instead, the new cuticle is protected from chitinase action by the T. castaneum Knickkopf (TcKnk) protein. TcKnk colocalizes with chitin in the new cuticle and organizes it into laminae. Down-regulation of TcKnk results in chitinase-dependent loss of chitin, severe molting defects, and lethality at all developmental stages. The conservation of Knickkopf across insect, crustacean, and nematode taxa suggests that its critical roles in the laminar ordering and protection of exoskeletal chitin may be common to all chitinous invertebrates.

Insect intestinal mucins (McIIM2-4) expressed in the midgut of feeding, starved and moulting Mamestra configurata larvae were identified. McIIM2 and McIIM4 were associated with the peritrophic matrix (PM). PMs from feeding and starved larvae were translucent and contained organized chitin bundles perpendicular to their long axis, whereas PM from moulting larvae consisted of an inner opaque mass surrounded by an outer translucent sleeve. Serine protease genes (McSP1, McSP2, McSP25 and McSP29) were also expressed in these larvae and several serine proteases were associated with the PM. Serine protease activity was also detected in the midgut of feeding, starved and moulting larvae.

In Drosophila melanogaster, Vestigial (Vg) and Scalloped (Sd) form a transcription factor complex and play a crucial role in wing development. To extend our knowledge of insect wing formation, we isolated vg and sd homologues from two ladybird beetle species, Henosepilachna vigintioctopunctata and Harmonia axyridis. Although the ladybird beetle vg homologues had only low homology with D. melanogaster vg, ectopic expression of H. vigintioctopunctata vg induced wing-like tissues in antennae and legs of D. melanogaster. Subsequent larval RNA interference (RNAi) analysis in H. vigintioctopunctata demonstrated conserved functions of vg and sd in wing development, and an unexpected novel function of sd in pupal ecdysis. Furthermore, our results can be applied to the production of a flightless ladybird beetle for biological control purposes using larval RNAi.

Aspartate 1-decarboxylase (ADC) and 3,4-dihydroxyphenylalanine decarboxylase (DDC) provide beta-alanine and dopamine used in insect cuticle tanning. beta-Alanine is conjugated with dopamine to yield N-beta-alanyldopamine (NBAD), a substrate for the phenol oxidase laccase that catalyzes the synthesis of cuticle protein cross-linking agents and pigment precursors. We identified ADC and DDC genes in the red flour beetle, Tribolium castaneum (Tc), and investigated their functions. TcADC mRNA was most abundant prior to the pupal-adult molt. Injection of TcADC double-stranded (ds) RNA (dsTcADC) into mature larvae resulted in depletion of NBAD in pharate adults, accumulation of dopamine, and abnormally dark pigmentation of the adult cuticle. Injection of beta-alanine, the expected product of ADC, into dsTcADC-treated pupae rescued the pigmentation phenotype, resulting in normal rust-red color. A similar pattern of catechol content consisting of elevated dopamine and depressed NBAD was observed in the genetic black mutants of Tribolium, in which levels of TcADC mRNA were drastically reduced. Furthermore, from the Tribolium black mutant and dsTcADC-injected insects both exhibited similar changes in material properties. Dynamic mechanical analysis of elytral cuticle from beetles with depleted TcADC transcripts revealed diminished cross-linking of cuticular components, further confirming the important role of oxidation products of NBAD as cross-linking agents during cuticle tanning. Injection of dsTcDDC into larvae produced a lethal pupal phenotype, and the resulting grayish pupal cuticle exhibited many small patches of black pigmentation. When dsTcDDC was injected into young pupae, the resulting adults had abnormally dark brown body color, but there was little mortality. Injection of dsTcDDC resulted in more than a 5-fold increase in levels of DOPA, indicating that lack of TcDDC led to accumulation of its substrate, DOPA.

Fatty acid transport protein (FATP) is an evolutionarily conserved membrane-bound protein that facilitates the uptake of extracellular long chain fatty acids. In humans and mice, six FATP isoforms have been identified and their tissue-specific distributions suggest that each plays a discrete role in lipid metabolism in association with fatty acid uptake. While the presence of FATP homologs in insects has been demonstrated, their functional role remains to be characterized. Pheromonogenesis is defined as the dynamic period in which all machinery required for sex pheromone biosynthesis is generated and organized within the pheromone gland (PG) cells. By exploiting this unique system in the PG of the silkmoth, Bombyx mori, we found that BmFATP is predominantly expressed in the PG and undergoes up-regulation 1 day prior to eclosion. Before eclosion, B. mori PG cells accumulate cytoplasmic lipid droplets (LDs), which play a role in storing the pheromone (bombykol) precursor fatty acid in the form of triacylglycerol. RNAi-mediated gene silencing of BmFATP in vivo significantly suppressed LD accumulation by preventing the synthesis of triacylglycerols and resulted in a significant reduction in bombykol production. These results, in conjunction with the findings that BmFATP stimulates the uptake of extracellular long-chain fatty acids and BmFATP knockdown reduces cellular long-chain acyl-CoA synthetase activity, suggest that BmFATP plays an essential role in bombykol biosynthesis by stimulating both LD accumulation and triacylglycerol synthesis via a process called vectorial acylation that couples the uptake of extracellular fatty acids with activation to CoA thioesters during pheromonogenesis.

Postembryonic development of holometabolous and hemimetabolous insects occurs through successive molts triggered by 20-hydroxyecdysone (20E). The molecular action of 20E has been extensively studied in holometabolous insects, but data on hemimetabolous are scarce. We have demonstrated that during the nymphal development of the hemimetabolous insect Blattella germanica, 20E binds to the heterodimeric receptor formed by the nuclear receptors BgEcR-A and BgRXR activating a cascade of gene expression, including the nuclear receptors BgE75 and BgHR3. Herein, we report the characterization of BgFTZ-F1, another nuclear hormone receptor involved in 20E action. BgFTZ-F1 is activated at the end of each instar, and RNAi has demonstrated that BgHR3 is needed for BgFTZ-F1 activation, and that BgFTZ-F1 has critical functions of during the last nymphal instar. Nymphs with silenced BgFTZ-F1 cannot ecdyse, arrest development, and show structures of ectodermal origin duplicated. BgFTZ-F1 also controls the timing of the ecdysteroid molting pulse., (Copyright (c) 2008 Wiley-Liss, Inc.)

During posteclosion, insects undergo sequential processes of wing expansion and cuticle tanning. Bursicon, a highly conserved neurohormone implicated in regulation of these processes, was characterized recently as a heterodimeric cystine knot protein in Drosophila melanogaster. Here we report the predicted precursor sequences of bursicon subunits (Masburs and Maspburs) in the moth Manduca sexta. Distinct developmental patterns of mRNA transcript and subunit-specific protein labeling of burs and pburs as well as crustacean cardioactive peptide in neurons of the ventral nervous system were observed in pharate larval, pupal, and adult stages. A subset of bursicon neurons located in thoracic ganglia of larvae expresses ecdysis-triggering hormone (ETH) receptors, suggesting that they are direct targets of ETH. Projections of bursicon neurons within the CNS and to neurohemal secretory sites are consistent with both central signaling and circulatory hormone functions. Intrinsic cells of the corpora cardiaca contain pburs transcripts and pburs-like immunoreactivity, whereas burs transcripts and burs-like immunoreactivity were absent in these cells. Recombinant bursicon induces both wing expansion and tanning, whereas synthetic eclosion hormone induces only wing expansion., ((c) 2007 Wiley-Liss, Inc.)

During the last larval molt in Manduca sexta, in response to an increasing, then decreasing ecdysteroid titer, a number of transcription factors such as E75B, MHR3, MHR4, and betaFTZ-F1 appear and disappear in the abdominal epidermis leading to dopa decarboxylase (DDC) expression. Messenger RNAs for both the 20E-induced transcription factors, MHR3 and E75B, are maximal near the peak of the ecdysteroid titer with MHR4 mRNA appearing as the titer declines followed by betaFTZ-F1 and DDC mRNAs. E75B and MHR4 mRNA were not expressed in Manduca GV1 cells, either during exposure to 20E or after its removal. When either MHR3 dsRNA was transfected or E75B was constitutively expressed in these cells, MHR4 mRNA appeared in response to 20E by 6h. E75B was found to form a heterodimer with MHR3 using the BacterioMatch II two-hybrid assay. We conclude that MHR3 apparently suppresses MHR4 expression in the presence of 20E; the appearance of E75B then removes MHR3 by dimerization, allowing MHR4 to be expressed. Because of significant basal activity of the ddc promoter in the GV1 cells, we could perform rescue experiments by adding various factors. Constitutive expression of either E75B or MHR4 in the cells suppressed the significant basal activity of the 3.2kb ddc promoter in the GV1 cells, but 20E had no effect on this activity. Thus, E75B and MHR4 are 20E-induced inhibitory factors that suppress ddc expression and therefore act as ecdysteroid-regulated timers to coordinate the onset of ddc expression at the end of the molt.

In hemimetabolous insects, the molecular basis of the 20-hydroxyecdysone (20E)-triggered genetic hierarchy is practically unknown. In the cockroach Blattella germanica, we had previously characterized one isoform of the ecdysone receptor, BgEcR-A, and two isoforms of its heterodimeric partner, BgRXR-S and BgRXR-L. One of the early-late genes of the 20E-triggered genetic hierarchy, is HR3. In the present paper, we report the discovery of three isoforms of HR3 in B. germanica, that were named BgHR3-A, BgHR3-B(1) and BgHR3-B(2). Expression studies in prothoracic gland, epidermis and fat body indicate that the expression of the three isoforms coincides with the peak of circulating ecdysteroids at each nymphal instar. Experiments in vitro with fat body tissue have shown that 20E induces the expression of BgHR3 isoforms, and that incubation with 20E and the protein inhibitor cycloheximide does not inhibit the induction, which indicates that the effect of 20E on BgHR3 activation is direct. This has been further confirmed by RNAi in vivo of BgEcR-A, which has shown that this nuclear receptor is required to fully activate the expression of BgHR3. RNAi has been also used to demonstrate the functions of BgHR3 in ecdysis. Nymphs with silenced BgHR3 completed the apolysis but were unable to ecdyse (they had duplicated and superimposed the mouth parts, the hypopharinge, the tracheal system and the cuticle layers). This indicates that BgHR3 is directly involved in ecdysis. Finally, RNAi of specific isoforms has showed that they are functionally redundant, at least regarding the ecdysis process.

Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents.

Cytochrome P450 enzymes are involved in a number of steps in ecdysteroid (moulting hormone) homeostasis in insects. We report the cloning and characterization of an ecdysteroid agonist-responsive cytochrome P450, CYP18A1, from the cotton leafworm, Spodoptera littoralis. Northern blot analysis showed that the mRNA transcript was expressed at times of increasing ecdysteroid titre in final instar S. littoralis larvae and was induced by the ecdysteroid receptor agonist, RH-5992, in midgut and fat body. In addition, transcript expression was also detected in the prothoracic glands, a major ecdysteroid biosynthetic tissue, in both S. littoralis and the tobacco hornworm, Manduca sexta, at a time of increasing ecdysteroid titre. The exact significance of the temporal and spatial expression of CYP18A1 is unclear. The characterization of a P450 that is ecdysteroid agonist-responsive may provide a future target for exploitation in the development of novel insect control strategies.

In insects, continuous growth requires the periodic replacement of the exoskeleton during the moult. A moulting insect displays a stereotypical set of behaviours that culminate in the shedding of the old cuticle at ecdysis. Moulting is an intricate process requiring tightly regulated physiological changes and behaviours to allow integration of environmental cues and to ensure the proper timing and sequence of its components. This is under complex hormonal regulation, and is an important point of interaction between endocrine and neural control. Here, we focus on the locust frontal ganglion (FG), an important player in moulting behaviour, as a previously unexplored target for ecdysis peptides. We show that application of 10(-7) mol l(-1) ecdysis-triggering hormone (ETH) or 10(-7) mol l(-1) and 10(-6) mol l(-1) Pre-ecdysis-triggering hormone (PETH) to an isolated FG preparation caused an increase in bursting frequency in the FG, whereas application of 10(-6) mol l(-1) eclosion hormone (EH) caused an instantaneous, though temporary, total inhibition of all FG rhythmic activity. Crustacean cardioactive peptide (CCAP), an important peptide believed to turn on ecdysis behaviour, caused a dose-dependent increase of FG burst frequency. Our results imply a novel role for this peptide in generating air-swallowing behaviour during the early stages of ecdysis. Furthermore, we show that the modulatory effects of CCAP on the FG motor circuits are dependent on behavioural state and physiological context. Thus, we report that pre-treatment with ETH caused CCAP-induced effects similar to those induced by CCAP alone during pre-ecdysis. Thus, the action of CCAP seems to depend on pre-exposure to ETH, which is thought to be released before CCAP in vivo.

The insect molting hormone 20-hydroxyecdysone (20E) plays a central role in regulating gene expression during development and metamorphosis. In many Lepidoptera, the pro-hormone 3-dehydroecdysone (3DE), synthesized from cholesterol in the prothoracic gland, is rapidly converted to ecdysone (E) by a hemolymph reductase, and E is subsequently converted to 20E in various peripheral target tissues. Recently, four Drosophila melanogaster P450 enzymes, encoded by specific Halloween genes, were cloned and functionally characterized as mediating the last hydroxylation steps leading to 20E. We extended this work to the tobacco hornworm Manduca sexta, an established model for endocrinological and developmental studies. cDNA clones were obtained for three Manduca orthologs of CYP306A1 (phantom; phm, the 25-hydroxylase), CYP302A1 (disembodied; dib, the 22-hydroxylase) and CYP315A1 (shadow; sad, the 2-hydroxylase), expressed predominantly in the prothoracic gland during the fifth (final) larval instar and during pupal-adult development, with fifth instar mRNA levels closely paralleling the hemolymph ecdysteroid titer. The data indicate that transcriptional regulation of phm, dib and sad plays a role in the developmentally varying steroidogenic capacities of the prothoracic glands during the fifth instar. The consistent expression of the Halloween genes confirms the importance of the prothoracic glands in pupal-adult development. These studies establish Manduca as an excellent model for examining the regulation of the Halloween genes.

To initiate moulting and metamorphosis, 20-hydroxyecdysone (20E) binds to its nuclear receptors and the ligand-receptor complex then mediates changes in gene expression. Phosphorylation of the receptors is required for their function. The intracellular signal transduction pathway that is involved in receptor phosphorylation remains elusive. This study provides evidence that the receptor of activated C kinase 1 (RACK1) and protein kinase C (PKC) signal transduction cascade is involved in the 20E-induced expression of the moult-associated transcription factor CHR3. A cDNA clone encoding a receptor of activated C kinase 1 was isolated from Choristoneura fumiferana (CfRACK1). This single copy gene coded a 36 kDa protein and was expressed ubiquitously in all of the developmental stages and the tissues tested, including the midgut, epidermis, fat body, head, Malpighian tubules, ovary and testis of larvae. High levels of the transcripts were also detected in a midgut-derived CF-203 cell line. We noticed that the green fluorescence protein-fused CfRACK1 protein was distributed in the cytosol surrounding the nuclei in stably transformed cells. Interference of CfRACK1 mRNA suppressed the 20E-induced expression of the transcription factor CHR3. Dequalinium-14; 1,1'-decamethylenebis-4-aminoquinaldinium diiodide (DECA), an inhibitor of RACK1 binding to protein kinase C, blocked the 20E-induced expression of CHR3 and accumulation of the ecdysone receptor (EcR) in the nuclei. All of these data together suggest that 20E-induced expression of CHR3 may involve phosphorylation of the ecdysone receptor component through the PKC/RACK1 signal transduction cascade, which facilitates the import of the receptor into the nuclei of cells.

Corazonin is a highly conserved neuropeptide hormone of wide-spread occurrence in insects yet is associated with no universally recognized function. After discovery of the corazonin receptor in Drosophila, we identified its ortholog in the moth, Manduca sexta, as a prelude to physiological studies. The corazonin receptor cDNA in M. sexta encodes a protein of 436 amino acids with seven putative transmembrane domains and shares common ancestry with its Drosophila counterpart. The receptor exhibits high sensitivity and selectivity for corazonin when expressed in Xenopus oocytes (EC(50) approximately 200 pM) or Chinese hamster ovary cells (EC(50) approximately 75 pM). Northern blot analysis locates the receptor in peripheral endocrine Inka cells, the source of preecdysis- and ecdysis-triggering hormones. Injection of corazonin into pharate larvae elicits release of these peptides from Inka cells, which induce precocious preecdysis and ecdysis behaviors. In vitro exposure of isolated Inka cells to corazonin (25-100 pM) induces preecdysis- and ecdysis-triggering hormone secretion. Using corazonin receptor as a biosensor, we show that corazonin concentrations in the hemolymph 20 min before natural preecdysis onset range from 20 to 80 pM and then decline over the next 30-40 min. These findings support the role of corazonin signaling in initiation of the ecdysis behavioral sequence. We propose a model for peptide-mediated interactions between Inka cells and the CNS underlying this process in insect development.

We have isolated and characterised a Triatoma infestans cDNA encoding a lysozyme. A 174-bp fragment was amplified by PCR using degenerate oligodeoxyribonucleotide primers derived from the known amino acid sequences of lysozyme from other insects. This PCR fragment was used to screen a cDNA gut library of T. infestans. A clone containing the 3'-end of the lysozyme cDNA (219 bp) was isolated and sequenced. RACE was used to amplify the 5'-end of the lysozyme cDNA. After sequencing the complete lysozyme cDNA, the deduced 417 amino acid sequence showed high identity (40-50%) with other chicken-type lysozymes. The amino acid residues responsible for the catalytic activity and the binding of the substrate were essentially conserved. The expression pattern of the lysozyme gene in bugs at different molting and feeding states showed that this gene was upregulated in the digestive tract directly after the molt and after feeding. Additionally, this lysozyme gene was expressed differently in the different regions of the digestive tract, strongly in the cardia and stomach, the anterior regions of the midgut, and only traces of lysozyme mRNA could be detected in the small intestine, the posterior region of the midgut., (Copyright 2003 Wiley-Liss, Inc.)

A novel ecdysteroid-inducible gene was isolated from the anterior silk gland of the silkworm by mRNA differential display and named Bombyx mori chitinase-related gene 1 (BmChiR1). cDNA for BmChiR1 is 3.7 kbp encoding 1080 amino acids. Its predicted protein sequence consists of two tandem-repeated sequences, both showing high similarities to arthropod chitinases but lacking the active site glutamate essential for catalytic activity, suggesting that BmChiR1 protein has no chitinolytic activity. BmChiR1 mRNA was expressed simultaneously with chitinase mRNA in the anterior silk gland at the ends of the penultimate and last larval instar. Injection of 20-hydroxyecdysone (20E) into feeding last instar larvae induced accumulation of BmChiR1 mRNA. Topical application of a juvenile hormone analog, fenoxycarb, just after the 20E injection, suppressed this induction. BmChiR1 expression is therefore upregulated by ecdysteroid and downregulated by juvenile hormone.

FTZ-F1, a member of the nuclear receptor superfamily, has been implicated in the activation of the segmentation gene fushi tarazu during early embryogenesis of Drosophila melanogaster. We found that an isoform of FTZ-F1, betaFTZ-F1, is expressed in the nuclei of almost all tissues slightly before the first and second larval ecdysis and before pupation. Severely affected ftz-f1 mutants display an embryonic lethal phenotype, but can be rescued by ectopic expression of betaFTZ-F1 during the period of endogenous betaFTZ-F1 expression in the wild type. The resulting larvae are not able to molt, but this activity is rescued again by forced expression of betaFTZ-F1, allowing progression to the next larval instar stage. On the other hand, premature expression of betaFTZ-F1 in wild-type larvae at mid-first instar or mid-second instar stages causes defects in the molting process. Sensitive periods were found to be around the time of peak ecdysteroid levels and slightly before the start of endogenous betaFTZ-F1 expression. A hypomorphic ftz-f1 mutant that arrests in the prepupal stage can also be rescued by ectopic, time-specific expression of betaFTZ-F1. Failure of salivary gland histolysis, one of the phenotypes of the ftz-f1 mutant, is rescued by forced expression of the ftz-f1 downstream gene BR-C during the late prepupal period. These results suggest that betaFTZ-F1 regulates genes associated with ecdysis and metamorphosis, and that the exact timing of its action in the ecdysone-induced gene cascade is important for proper development.

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA-binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)-containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA-binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly-emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA-binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability., (Copyright 2000 John Wiley & Sons, Inc.)

Proteins were extracted from the cuticle of mid-instar nymphs of locusts, Locusta migratoria, and cockroaches, Blaberus craniifer. Seven proteins were purified from the locust extract and five from the cockroach extract, and their amino acid sequences were determined. Polyacrylamide gel electrophoresis indicates that the proteins are present only in the post-ecdysially deposited layer of the nymphal cuticles. One of the locust and one of the cockroach nymphal proteins contain a 68-residue motif, the RR-2 sequence, which has been reported for several proteins from the solid cuticles of other insect species. Two of the cockroach proteins contain a 75-residue motif, which is also present in a protein from the larval/pupal cuticle of a beetle, Tenebrio molitor, and in proteins from the exoskeletons of a lobster, Homarus americanus, and a spider, Araneus diadematus. The motif contains a variant of the Rebers-Riddiford consensus sequence, and is called the RR-3 motif. One of the locust and three of the cockroach post-ecdysial proteins contain one or more copies of an 18-residue motif, previously reported in a protein from Bombyx mori pupal cuticle. The nymphal post-ecdysial proteins from both species have features in common with pre-ecdysial proteins (pharate proteins) in cuticles destined to be sclerotised; they show little similarity to the post-ecdysial cuticular proteins from adult locusts or to proteins from soft, pliable cuticles. Possible roles for post-ecdysial cuticular proteins are discussed in relation to the reported structures.

Proteins in pupal abdominal cuticle of the tobacco hornworm, Manduca sexta, were characterized during the pre-ecdysial and post-ecdysial periods of sclerotization and endocuticle formation. Protein extractability decreased dramatically as the cuticle became sclerotized through 6 h post-ecdysis, but increased rapidly from 9 to 48 h as endocuticular layers were secreted. Nearly 100 proteins that were extracted from pre-ecdysial cuticle became largely insoluble during sclerotization. Three major proteins in this group destined to become exocuticle had apparent molecular masses (Mapp) of 20, 27 and 36 kDa, and were designated MS-PCP20, MS-PCP27, and MS-PCP36. Amino acid analysis revealed glycine to predominate in all three proteins, and alanine, aspartate, glutamate, proline and serine were also relatively abundant. Histidine residues, which provide sites for adduct and cross-link formation with quinone metabolites of N-beta-alanyldopamine during sclerotization of pupal cuticle, ranged from 2 to 3 mol %. N-Terminal amino acid analysis of MSPC-20 and MSPC-36 also revealed some sequence similarities indicating they may be related. An almost entirely new group of proteins appeared by 9 h as endocuticule secretion began, and these increased in abundance through 48 h post-ecdysis. Two of these were major proteins with Mapps of 33 and 34 kDa, and they also had close similarities in their N-terminal amino acid sequences. This study showed that the large number of proteins secreted into the presumptive exocuticle of the pupa before ecdysis are involved in sclerotization reactions and as a consequence become largely insoluble. The epidermis then switches to the secretion of an entirely new group of proteins that are involved in formation of the endocuticle.

We identified of a set of neuropeptide-expressing cells sited along the respiratory system of Drosophila melanogaster using an antibody to the molluscan neuropeptide myomodulin. The number and positions of these 'peritracheal' myomodulin (PM) cells were reminiscent of the epitracheal Inka cells in the moth Manduca sexta. These Inka cells release the peptide ecdysis-triggering hormone, which helps elicit ecdysial behavior at the molt, and we show that they are also recognized by the myomodulin (MM) antibody. In both D. melanogaster and M. sexta, the PM and Inka cells are the only MM-positive cells outside the central nervous system. In both insects, MM immunoreactivity disappears at the end of the molt. In D. melanogaster, we have monitored the PM cells throughout development using two enhancer trap lines; the PM cells persist throughout development, but at larval, pupal and adult ecdyses, they display a loss of MM immunoreactivity. This transient loss occurs at a predictable time, just prior to ecdysis. In contrast, MM-positive neurons in the central nervous system do not show these changes. The PM cells also reveal a concomitant loss of immunostaining for an enzyme contained in secretory granules. The results are consistent with the hypothesis that the PM cells release MM-like peptides just prior to each ecdysis. In addition, we demonstrate that peritracheal cells of five widely divergent insect orders show a myomodulin phenotype. The peritracheal cell size, morphology, numbers and distribution vary in these different orders. These data suggest that peritracheal cells release MM-like peptides as part of a conserved feature of the endocrine regulation of insect ecdysis.

The distribution of integument peptides in relation to chitin and structural features has been studied in the surface epidermis of the caterpillar of Calpodes ethlius by immunoblotting and immunogold labelling using antibodies prepared to peptides isolated from lamellate endocuticle or from hemolymph. The intermoult cuticle consists of an epicuticle, an endocuticle of many chitin containing lamellae, and a chitin containing assembly zone directly above the apical epidermal microvilli and the perimicrovillar space. During the intermoult, the epidermis secretes peptides constitutively, that is, secretory vesicles containing peptides exocytose without accumulating, traverse the perimicrovillar space and form lamellae in the assembly zone. At moulting, the epidermis deposits ecdysial droplets in addition. These interrupt the last few lamellae which later go on to become the perforated ecdysial membrane. The integument is involved with four routing classes of peptide. Secretion is apical into the cuticle (C), basal into the hemolymph (H), bidirectional (BD), or transported to the cuticle across the epidermis from the hemolymph (T). Some peptides change their routing at moulting. There are several patterns of localization. (1) C and BD cuticular peptides occur mainly in chitin containing lamellate cuticle. (2) Some are also present in epicuticle, and are therefore not obligatorily linked to chitin or matrix between chitin fibers. Cuticular peptides that also occur in the hemolymph are glycosylated, whereas most that are only secreted apically into the cuticle are not. All BD but few C peptides carry alpha-D-glucose/alpha-D-mannose. Some C and BD peptides carry N-acetyl glucosamine. (3) C36 extracted from cuticle has most N-acetyl glucosamine and colocalizes with chitin rather than the protein matrix. It is therefore probably the main link between chitin fibers and the matrix. (4) H235 is barely detectable at the apical cell surface during the intermoult but is abundant at moulting around and below the ecdysial droplets. (5) T66 occurs in intermoult lamellate cuticle. At moulting, alone among the peptides examined, it is in ecdysial droplets. Intermoult C and BD peptides are not in ecdysial droplets but continue to be present in the ecdysial membrane, suggesting that constitutive secretion is independent from the exocytosis of transported moult peptides. T66 differs from most hemolymph peptides in that it does not carry N-acetyl glucosamine or alpha-D-glucose/alpha-D-mannose. (6) Weakly reacting BD peptides (and some H peptides barely detectable in cuticle) localize near the apical surface. Their distribution therefore favours apical secretion and retrieval as a mechanism for basal secretion.