Feedback control of juvenile hormone synthesis in cockroaches: possible role for ecdysterone.

Inactive female corpora allata implanted into adult males become active and continue to synthesize juvenile hormone at high rates. However, when an ovary is implanted together with the corpora allata, rates of juvenile hormone synthesis decline as the oocytes complete maturation. Injections of ecdysterone mimic the effect of an implanted ovary.

A putative farnesoic acid O-methyltransferase (FAMeT) orthologue in Drosophila melanogaster (CG10527): relationship to juvenile hormone biosynthesis?

Juvenile hormones (JHs) are key regulators of both metamorphosis and adult reproductive processes. Farnesoic acid O-methyltransferase (FAMeT) is thought to be an important enzyme in the JH biosynthetic pathway, catalyzing methylation of farnesoic acid (FA) to methyl farnesoate (MF). Previous evidence in other insects suggested that FAMeT is rate limiting and regulated by a neuropeptide family, the allatostatins. A full-length cDNA encoding a 296 amino acid putative FAMeT has been isolated. A recombinant (r)FAMeT was cloned, expressed and a specific antiserum generated. rFAMeT was assayed for enzymatic activity using a radiochemical assay. In this assay, no activity was detected either with rFAMeT alone or when added to a corpus allatum CA extract. Immunohistochemical analysis was used to confirm the presence of FAMeT in the CA of Drosophila melanogaster ring gland. Analysis of MF, JHIII and JHB3 release in wild type and mutant stocks in the presence and absence of Drome AST (PISCF-type) suggest that Drosophila FAMeT has little if any effect on sesquiterpenoid biosynthesis. Drome AST appears to have a select effect on JH bisepoxide biosynthesis and not MF or JHIII. Additional analysis of MF, JHIII and JHB3 release in strains with a deficiency or decrease of FAMeT compared to wild type shows no significant decrease in MF, JHIII or JH bisepoxide synthesis. Deficiency strains that reduce the level of FAMeT showed reduced longevity relative to wildtype but this result may be due to other genetic influences.

Isolation of cockroach Phe-Gly-Leu-amide allatostatins from the termite Reticulitermes flavipes and their effect on juvenile hormone synthesis.

Immunoreactivity to cockroach Diploptera punctata allatostatin-7 (Dippu AST-7) has been demonstrated previously in axons innervating the corpora allata of the termite Reticulitermes flavipes. This peptide and Dippu AST-11 inhibited juvenile hormone (JH) synthesis by corpora allata (CA) of brachypterous neotenic reproductives (secondary reproductives) of termites. The present study shows that R. flavipes CA are also inhibited by Dippu AST-2, AST-5, AST-8, and AST-9 at approximately the same rank order of potency as demonstrated in D. punctata. Another allatostatin from Periplaneta americana (Peram AST-12) also inhibits JH synthesis by R. flavipes CA. Sensitivity to the allatostatins is higher in glands with low rates of JH synthesis than in those with relatively high JH synthetic rates as has been demonstrated in CA from male and female secondary reproductives as well as in those from non-egg-laying and egg-laying females. The identical inhibitory effects of R. flavipes brain extract on CA from both D. punctata and R. flavipes and the isolation and identification of five cockroach allatostatins (Dippu AST-1, AST-2, AST-5, AST-8, and Peram AST-12) from termite brain extract reflect the close relationship between cockroaches and termites.

Endocrine and reproductive differences and genetic divergence in two populations of the cockroach Diploptera punctata.

The viviparous cockroach, Diploptera punctata, has been a valuable model organism for studies of the regulation of reproduction by juvenile hormone (JH) in insects. As a result of its truly viviparous mode of reproduction, precise regulation of JH biosynthesis and reproduction is required for production of offspring, providing a model system for the study of the relationship between JH production and oocyte growth and maturation. Most studies to date have focused on individuals isolated from a Hawaiian population of this species. A new population of this cockroach was found in Nakorn Pathom, Thailand, which demonstrated striking differences in cuticle pigmentation and mating behaviours, suggesting possible physiological differences between the two populations. To better characterize these differences, rates of JH release and oocyte growth were measured during the first gonadotrophic cycle. The Thai population was found to show significantly earlier increases in the rate of JH release, and oocyte development as compared with the Hawaiian population. Breeding experiments to determine the degree of interfertility between the two populations demonstrated greatly reduced fertility in crosses between the two populations. Additionally, levels of genetic divergence between the two populations estimated by sequencing a fragment of the mitochondrial 16S rRNA gene were surprisingly high. The significant differences in physiology and mating behaviours, combined with the reduced interfertility and high levels of sequence divergence, suggest that these two populations of D. punctata are quite distinct, and may even be in the process of speciation. Moreover, these studies have important implications for the study of JH function in the reproductive cycle of insects, as differences in timing of rates of JH biosynthesis may suggest a process of heterochrony in reproduction between the two populations.

The roles of Dippu-allatostatin in the modulation of hormone release in Locusta migratoria.

Dippu-allatostatins (ASTs) have pleiotropic effects in Locusta migratoria. Dippu-ASTs act as releasing factors for adipokinetic hormone I (AKH I) from the corpus cardiacum (CC) and also alter juvenile hormone (JH) biosynthesis and release from the corpus allatum (CA). Dippu-AST-like immunoreactivity is found within lateral neurosecretory cells (LNCs) of the brain and axons within the paired nervi corporis cardiaci II (NCC II) to the CC and the CA, where there are extensive processes and nerve endings over both of these neuroendocrine organs. There was co-localization of Dippu-AST-like and proctolin-like immunoreactivity within these regions. Dippu-ASTs increase the release of AKH I in a dose-dependent manner, with thresholds below 10(-11)M (Dippu-AST 7) and between 10(-13) and 10(-12)M (Dippu-AST 2). Both proctolin and Dippu-AST 2 caused an increase in the cAMP content of the glandular lobe of the CC. Dippu-AST 2 also altered the release of JH from the locust CA, but this effect depended on the concentration of peptide and the basal release rates of the CA. These physiological effects for Dippu-ASTs in Locusta have not been shown previously.

Comparative genomic analysis of allatostatin-encoding (Ast) genes in Drosophila species and prediction of regulatory elements by phylogenetic footprinting.

The role of the YXFGLa family of allatostatin (AST) peptides in dipterans is not well-established. The recent completion of sequencing of genomes for multiple Drosophila species provides an opportunity to study the evolutionary variation of the allatostatins and to examine regulatory elements that control gene expression. We performed comparative analyses of Ast genes from seven Drosophila species (Drosophila melanogaster, Drosophila simulans, Drosophila ananassae, Drosophila yakuba, Drosophila pseudoobscura, Drosophila mojavensis, and Drosophila grimshawi) and used phylogenetic footprinting methods to identify conserved noncoding motifs, which are candidates for regulatory regions. The peptides encoded by the Ast precursor are nearly identical across species with the exception of AST-1, in which the leading residue may be either methionine or valine. Phylogenetic footprinting predicts as few as 3, to as many as 17 potential regulatory sites depending on the parameters used during analysis. These include a Hunchback motif approximately 1.2 kb upstream of the open reading frame (ORF), overlapping motifs for two Broad-complex isoforms in the first intron, and a CF2-II motif located in the 3'-UTR. Understanding the regulatory elements involved in Ast expression may provide insight into the function of this neuropeptide family.

Evolutionary selective trends of insect/mosquito antimicrobial defensin peptides containing cysteine-stabilized alpha/beta motifs.

Insect defensins containing cysteine-stabilized alpha/beta motifs (Cs-alpha/beta defensin) are cationic, inducible antibacterial peptides involved in humoral defence against pathogens. To examine trends in molecular evolution of these antimicrobial peptides, sequences similar to the well-characterized Cs-alpha/beta defensin peptide of Anopheles gambiae, using six cysteine residues as landmarks, were retrieved from genomic and protein databases. These sequences were derived from different orders of insects. Genes of insect Cs-alpha/beta defensin appear to constitute a multigene family in which the copy number varies between insect species. Phylogenetic analysis of these sequences revealed two main lineages, one group comprising mainly lepidopteran insects and a second, comprising Hemiptera, Coleoptera, Diptera and Hymenoptera insects. Moreover, the topology of the phylogram indicated dipteran Cs-alpha/beta defensins are diverse, suggesting diversity in immune mechanisms in this order of insects. Overall evolutionary analysis indicated marked diversification and expansion of mature defensin isoforms within the species of mosquitoes relative to non-mosquito defensins, implying the presence of finely tuned immune responses to counter pathogens. The observed higher synonymous substitution rate relative to the nonsynonymous rate in almost all the regions of Cs-alpha/beta defensin of mosquitoes suggests that these peptides are predominately under purifying selection. The maximum-likelihood models of codon substitution indicated selective pressure at different amino acid sites in mosquito mature Cs-alpha/beta defensins is differ and are undergoing adaptive evolution in comparison to non-mosquito Cs-alpha/beta defensins, for which such selection was inconspicuous; this suggests the acquisition of selective advantage of the Cs-alpha/beta defensins in the former group. Finally, this study represents the most detailed report on the evolutionary strategies of Cs-alpha/beta defensins of mosquitoes in particular and insects in general, and indicates that insect Cs-alpha/beta defensins have evolved by duplication followed by divergence, to produce a diverse set of paralogues.

A study of signal transduction for the two diuretic peptides of Diploptera punctata.

We investigated second messengers involved in the action of the CRF-related peptide Dippu-DH46 and the calcitonin-like peptide Dippu-DH31 in Diploptera punctata. Dippu-DH46 causes a dose-dependent increase in intracellular cAMP levels, its diuretic activity is mimicked by cAMP agonists, but is attenuated by Rp-cAMPS. Dippu-DH46 acts synergistically with kinins and thapsigargin; both mobilize intracellular Ca2+. Dippu-DH46 also acts synergistically with cAMP agonists, and its effect is inhibited by a PKC inhibitor, suggesting it also activates intracellular Ca2+. Dippu-DH31 has no effect on cAMP levels and its activity is not blocked by cAMP agonists. Neither peptide stimulated cGMP levels in a dose-dependent manner, nor does cGMP have any effect on fluid secretion.

Phe-Gly-Leu-amide allatostatin in the termite Reticulitermes flavipes: content in brain and corpus allatum and effect on juvenile hormone synthesis.

In the subterranean termite Reticulitermes flavipes, allatostatins (ASTs) with the C-terminus Phe-Gly Leu-amide were localized by immunocytochemistry with antibody against a cockroach AST, Dippu AST-7. AST-immunoreactivity occurred in the corpus cardiacum and corpus allatum and in the lateral and medial neurosecretory cells of the brain that innervate these organs as well as in many other nerve cells of the brain. This was observed in workers, nymphs, soldiers and secondary reproductives. A radioimmunoassay, using anti-Dippu AST-11, demonstrated about 40 fmole equivalents of AST in brains of soldiers and secondary reproductives. The product of the corpora allata in this species was determined to be juvenile hormone III. Its synthesis by corpora allata of secondary reproductives, determined by in vitro radiochemical assay, was inhibited in a dose-dependent fashion by two cockroach allatostatins, Dippu AST-7 and Dippu AST-11. Thus, as in cockroaches and crickets, allatostatin-containing nerves innervate the corpora allata of this termite species and their production of juvenile hormone is inhibited by these neuropeptides.

Anatomy and electrophysiology of neurons terminating in the corpora allata of the cockroach Diploptera punctata.

Intracellular recording and dye injection were used to study the structure and electrophysiological properties of individual neurons that project to the corpora allata of the cockroach, Diploptera punctata. Neurons in the pars intercerebralis generate long-duration, tetrodotoxin-sensitive action potentials. Dye injection revealed two cell types. One type extends axons to the contralateral nervi corporis cardiaci I, some of which innervate the corpora allata, and another type extends a major axon down each of the circumoesophageal connectives. Neurons in the pars lateralis also generate long-duration action potentials. These neurons extend axons to the ipsilateral nervi corporis cardiaci II, which continue on to terminate in the corpora cardiaca and the corpora allata. Small groups of all the above neuronal types are dye and electrically coupled. Penetration and dye injection into nerve terminals in the corpora allata and corpora cardiaca confirmed the innervation of the corpora allata by neurons located in the pars intercerebralis and pars lateralis and revealed a third class of neurons that have terminals in the corpora allata: intrinsic neurons of the corpora cardiaca.

In situ hybridization analysis of leucomyosuppressin mRNA expression in the cockroach, Diploptera punctata.

In the cockroach Diploptera punctata, sequencing of the cDNA for the insect myoinhibitory neuropeptide, leucomyosuppressin (LMS), has demonstrated that LMS is the only Phe-Met-Arg-Phe-amide (NH2) (FMRFamide)-related peptide to be encoded by this gene (Donly et al. [1996] Insect Biochem. Mol. Biol. 26:627-637). However, in the present study, high performance liquid chromatography analysis of brain extracts showed six discrete FMRFamide-like immunoreactive fractions, one of which co-eluted with LMS. This study compared the distribution of FMRFamide-related peptides visualized by immunohistochemistry with LMS mRNA expression demonstrated by in situ hybridization in D. punctata. Immunohistochemistry with a polyclonal antiserum generated against FMRFamide, but which recognizes extended RFamide peptides, demonstrated numerous RFamide-like immunoreactive cells and processes in both nervous and nonnervous tissues. RFamide-like immunoreactivity was found in cells and processes of the brain and optic lobes, the stomatogastric nervous system, including the frontal and ingluvial ganglia, and the suboesophageal ganglion. Immunoreactivity was also present in all ganglia of the ventral nerve cord and in the alimentary canal. Within the alimentary canal, positively stained processes were found in the crop, midgut, and hindgut, and immunoreactive endocrinelike cells were located in the midgut. In situ hybridization with a digoxigenin-labeled RNA probe spanning the entire LMS coding region showed cell bodies containing LMS mRNA in all ganglia studied, other than the ingluvial ganglion. Expression was most abundant in the brain and optic lobes and in the frontal and suboesophageal ganglia. LMS mRNA was also apparent, although less intensely, in all other ganglia of the ventral nerve cord. Within the alimentary canal, LMS mRNA-positive cells were only visible in the anterior portion of the midgut, in the endocrinelike cells. The appearance of LMS mRNA in the central nervous system, stomatogastric nervous system, and midgut suggests that LMS may play a central role in Diploptera and may be associated with feeding and digestion.

Signal transduction in the inhibition of juvenile hormone biosynthesis by allatostatins: roles of diacylglycerol and calcium.

The effects of pharmacological agents that interfere with the 1,4,5-inositol trisphosphate (IP3)/diacylglycerol (DAG) pathway on juvenile hormone (JH) biosynthesis by corpora allata (CA) of the cockroach Diploptera punctata have been investigated. These effects were assessed in the presence of the inhibitory neuropeptides, allatostatins, with a view to elucidating the pathway for signal transduction in the inhibition of JH biosynthesis. Treatment of CA with inhibitors of DAG kinase to elevate the concentration of DAG within the CA cells, resulted in a significant, dose-dependent decrease in JH biosynthesis. Simultaneous treatment of glands with both DAG kinase inhibitors and allatostatins further enhanced this effect, suggesting that DAG is an intermediate in the allatostatin-induced inhibition of JH production. The inhibitory actions of the phorbol ester activator of PKC, PDBu, or of allatostatin on JH biosynthesis were partially blocked by pre-incubating the CA with PKC inhibitors. Treatment of CA with the calcium-mobilizing drug thapsigargin resulted in a significant stimulation in JH biosynthesis in glands from mated females producing JH at high rates. Thapsigargin was also able to reverse the effect of allatostatins in high-activity mated CA. This suggests an involvement of the other product of phosphoinositide hydrolysis, IP3, in the modulation of JH biosynthesis at specific developmental times and in glands showing specific levels of activity.

Assessment of the role of cyclic nucleotides in allatostatin-induced inhibition of juvenile hormone biosynthesis in Diploptera punctata.

In an effort to identify the signal transduction mechanism associated with the inhibition of juvenile hormone (JH) biosynthesis by the neuropeptides allatostatins, levels of the cyclic nucleotides cAMP and cGMP were measured in corpora allata (CA) of virgin and mated Diploptera punctata females using radioimmunoassays. Treatment of isolated CA with varying concentrations of synthetic allatostatins 1, 2, 3 or 4 did not elicit significant changes in the levels of either cAMP or cGMP in any of the test glands, suggesting that these compounds do not act as second messengers for the four allatostatins tested. Simultaneous treatment of CA with allatostatin 4 and the adenylate cyclase activator forskolin did not increase the degree of inhibition of juvenile hormone biosynthesis relative to that obtained with forskolin (5 or 50 microM) alone. We interpret these results as lending further support to the suggestion that cyclic nucleotides do not play a role in the signal transduction of allatostatins 1-4 in cockroach CA.

Evidence for an inhibitory role of cyclic AMP in the control of juvenile hormone biosynthesis by cockroach corpora allata.

High levels of cyclic AMP were found in the corpora allata of adult female Diploptera punctata. The adenylate cyclase activator forskolin caused a rapid, reversible and dose-dependent accumulation of cAMP in the corpora allata in vitro. The sensitivity of the corpora allata to forskolin was low when juvenile hormone (JH) synthetic activity was high, and vice versa. Incubation of corpora allata with compounds which cause or mimic elevated intracellular cAMP levels (forskolin, 3-isobutyl-1-methylxanthine, 8-bromo-cAMP) led to a rapid and dose-dependent inhibition of juvenile hormone synthesis. Glands from day 5 virgin females were more sensitive to forskolin than glands from mated females of the same age. The results suggest that a cAMP second messenger system may be responsible for the intracellular transduction of inhibitory signals to the corpora allata of D. punctata.

Molecular cloning of the precursor cDNA for schistostatins, locust allatostatin-like peptides with myoinhibiting properties.

The cDNA encoding the precursor polypeptide for schistostatins, allatostatin-like peptides which have been shown to inhibit peristaltic movements of the lateral oviducts of Schistocerca gregaria, has been cloned and sequenced. Translation of this sequence reveals the presence of a pre-proschistostatin consisting of 283 amino acids. It contains ten different peptide sequences which are flanked by dibasic cleavage sites and C-terminal amidation signals. Eight of these peptides were identical to the schistostatins (or Scg-ASTs) that were previously purified from Schistocerca gregaria brain extracts. Two novel peptide sequences were discovered. One of these is the first AST-like peptide which has a C-terminal valine residue. Two peptides contain within their sequence an internal dibasic site which suggests a possible role for alternative processing and/or degradation. The schistostatin precursor differs from cockroach pre-proallatostatins in size, in sequence and in organization. It contains a lower number of peptides (10 versus 13 or 14) which are interrupted only once by an acidic spacer region (versus four in Diploptera punctata and Periplaneta americana). Northern analysis showed the presence of a 2.4 kb mRNA band in the locust central nervous system and midgut. This indicates that schistostatins, like other ASTs, are a good example of insect brain/gut peptides.

Isolation and characterization of eight myoinhibiting peptides from the desert locust, Schistocerca gregaria: new members of the cockroach allatostatin family.

Eight myoinhibiting peptides were purified by high performance liquid chromatography from a methanolic extract of 7000 brains of the desert locust, Schistocerca gregaria. Complete sequences were obtained via a novel, combined approach employing: (1) chemical microsequencing and (2) post-source decay analysis on a reflectron time-of-flight mass spectrometer using matrix-assisted laser desorption/ionisation. Each of the peptides shows C-terminal amino acid sequence similarity to cockroach and cricket allatostatins and to blowfly callatostatins. Therefore, these novel peptides were designated Schistocerca gregaria allatostatins (Scg-ASTs) or schistostatins and their primary structures were determined to be: Ala-Tyr-Thr-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu- NH2 (Scg-AST-2), Ala-Thr-Gly-Ala-Ala-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-3), Gly-Pro-Arg-Thr-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-4), Gly-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-5), Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-6), Ala-Gly-Pro-Ala-Pro-Ser-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-7), Glu-Gly-Arg-Met-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-8), and Ala-Pro-Ala-Glu-His-Arg-Phe-Ser-Phe-Gly-Leu-NH2 (Scg-AST-10). Synthetic Scg-AST peptides inhibit the peristaltic movements of the oviduct of S. gregaria. Although all eight peptides show potent inhibitory effects on juvenile hormone (JH) biosynthesis by corpora allata (CA) of the cockroach Diploptera punctata, no allatostatic effects were observed on CA of the desert locust (S. gregaria).

Terminal steps in JH biosynthesis in the honey bee (Apis mellifera L. ): developmental changes in sensitivity to JH precursor and allatotropin.

Juvenile hormone (JH) is considered the prime endogenous signal for the induction of queen development in honey bees (Apis mellifera L.). At the beginning of the last (5th) larval stadium, worker corpora allata synthesize less JH than queen corpora allata as a consequence of a limited production of JH precursors and a caste- and stage-specific block of the terminal step in JH biosynthesis. As previously shown, the Manduca sexta allatotropin stimulates JH biosynthesis in honey bee corpora allata in a dose-dependent and reversible manner, but can not overcome the stage-specific block in the terminal step of JH biosynthesis that is typical for worker early 5th instars. In experiments with M. sexta allatotropin and with the JH precursor farnesoic acid, we found characteristic stage-specific differences in their effects on JH biosynthesis. From the end of the spinning stage on, corpora allata could be stimulated by farnesoic acid to a much higher extent than in earlier developmental stages, suggesting a sudden increase in epoxidase activity. Manduca sexta allatotropin, however, stimulated corpora allata activity until the end of the spinning stage, at which time the corpora allata become suddenly insensitive. These data suggest that in worker larvae, important changes in the regulation of the terminal enzymatic steps in JH biosynthesis occur at the transition from the spinning stage to the prepupal stage. However, the analysis of in vitro activities of the involved enzymes, O-methyltransferase and methyl farnesoate epoxidase, remained inconclusive.

Two new allatostatins from the brains of Diploptera punctata.

Allatostatins VI and VII have been isolated from saline extracts of the brain of the viviparous cockroach Diploptera punctata. Active fractions, obtained by successive reverse-phase high pressure liquid chromatography separations, inhibited juvenile hormone (JH) III production by corpora allata (CA) in vitro. The primary structures, Y-P-Q-E-H-R-F-S-F-G-L-amide (VI) and D-G-R-M-Y-S-F-G-L-amide (VII), clearly identify them as members of a family of D. punctata allatostatins, five of which have been identified previously and shown to have F-G-L-amide at the C terminus. Synthetic allatostatins VI and VII coeluted with the native allatostatins on two successive high pressure liquid chromatography separations. Allatostatins VI and VII have the same capacity to inhibit JH production by CA of 2 day virgin females as does allatostatin I, previously shown to be a potent inhibitor similar in activity to allatostatin V. Allatostatins VI and VII also resemble the other allatostatins in their ability to affect both larval and adult CA, in the reversibility of their effect, and in the abolition of their effect by addition of farnesoic acid (a late precursor of JH) to CA in vitro.

Injection of Dip-allatostatin or Dip-allatostatin pseudopeptides into mated female Diploptera punctata inhibits endogenous rates of JH biosynthesis and basal oocyte growth.

Studies on the catabolism of allatostatins (ASTs) provided the rationale for the design of a series of Dip-allatostatin-derived pseudopeptide mimetic analogues. In vitro, the Dip-ASTs and pseudopeptides show varying degrees of resistance to catabolism and all show significant inhibition of juvenile hormone (JH) biosynthesis. This study was undertaken to determine whether potent Dip-ASTs and/or their pseudopeptide mimetic counterparts caused 'allatostatic' effects in vivo following injection into mated female Diploptera punctata. Animals injected with aqueous solvent or Dip-AST 7(1-7) N-terminal fragment, which excludes the active core region of the ASTs, were used as controls. An in vitro radiochemical assay revealed that injection of Dip-AST 5, 7 or pseudopeptide analogues 397-2 or AST(b)φ2 significantly inhibited the biosynthesis of JH (P<0.05). The results also indicate that basal oocyte growth was significantly inhibited by injection of these same compounds, with the exception of Dip-AST 7 (P<0.05). Analogues 396-1 and 419 did not significantly inhibit rates of JH biosynthesis but did significantly inhibit the growth of basal oocytes. Analyses of feeding, excretion and food absorption/utilization patterns of these same animals suggested that these compounds are not toxic to the insect; rather they directly inhibit the biosynthesis of JH by the corpora allata, and reduce the rate of growth of basal oocytes. Disruption of critical reproductive and/or developmental processes by pseudopeptide analogues of the ASTs could provide novel and selective strategies for future insect pest management.

Molecular characterization of a cDNA from Pseudaletia unipuncta encoding the Manduca sexta allatostatin peptide (Mas-AST).

A 15-residue neuropeptide, Manduca sexta allatostatin (Mas-AST), strongly inhibits juvenile hormone (JH) biosynthesis in vitro by corpora allata (CA) from Manduca fifth-stadium larvae and adult females as well as Helicoverpa zea adult females (Kramer et al., 1991 Proc. Natl. Acad. Sci (USA) 88, 9458-9462). In contrast, this study found that 1.0 microM Mas-AST has no JH biosynthesis inhibitory activity in Pseudaletia unipuncta sixth instar larvae or newly-emerged (day 0) adults but inhibited CA of 5-day-old adult females by 60%. From a P. unipuncta brain cDNA library, was isolated a cDNA that encodes a 125 amino acid polypeptide containing the Mas-AST sequence. Within the precursor, Mas-AST is situated at the carboxy terminus and is flanked by different dibasic proteolytic cleavage signals. The Pseudaletia gene specifying the Mas-AST peptide is present as a single copy per haploid genome. Expression of this gene was low in Pseudaletia sixth instar larvae, prepupae and early pupae but was relatively high in late pupae, and day 1 and 3 adults of both sexes. In day 5 adults, the relative transcript level appears to be maintained in females but declines in males. This pattern of Mas-AST expression does not correlate well with the profile of JH biosynthesis in Pseudaletia, which increases during the first 5 days of adult life, suggesting additional or alternative functions for this peptide.