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.

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.

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.

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.

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.

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.

Localization and physiological effects of RFamides in the corpora allata of the cockroach Diploptera punctata in relation to allatostatins.

The distribution of FMRFamide immunoreactivity in the brain-retrocerebral complex of adult female Diploptera punctata was examined. Immunoreactivity was observed in the brain and corpus allatum as well as in the corpus cardiacum. Immunoreactivity co-localized with allatostatin immunoreactivity within several lateral neurosecretory cells of the brain and in their endings within the corpus allatum. By in vitro radiochemical assay of juvenile hormone release, the effect of two native D. punctata RFamides, an FLRFamide (Leucomyosuppressin) and an FIRFamide were examined. The latter, for which the sequence (SKPANFIRFamide) is reported here, stimulated juvenile hormone release but acted only on corpora allata from females at the end of vitellogenesis (day 6). The interaction of these two RFamides and three D. punctata allatostatins, Dippu-AST 2, 5, and 7 were similarly examined. Only Dippu-AST 2 stimulated release of RFamides from the corpora allata and only on day 6 whereas both RFamides were able to attenuate the inhibitory activity of Dippu-AST 2.

Comparative immunohistochemistry and cellular distribution of farnesoic acid O-methyltransferase in the shrimp and the crayfish.

Farnesoic acid O-methyltransferase (FAMeT) catalyzes the conversion of farnesoic acid (FA) to methylfarnesoate (MF) by the mandibular organ (MO) of crustaceans. Here we report the cellular localization of FAMeT and radiochemical assay of endogenous FAMeT activity in shrimp (Metapenaeus ensis) and crayfish (Procambarus clarkii) tissues. As in the eyestalk (ES), FAMeT is concentrated in specific neurosecretory cells of the ventral nerve cord (VNC) whereas only weak FAMeT immunoreactivity was observed in the MO. FAMeT was also detected in the ventral nerve cord, heart (HET), eyestalk, and muscle of the juvenile shrimp. Although the VNC shows the greatest FAMeT immunoreactivity, the heart extract exhibited the highest FAMeT enzymatic activity. These results suggest that FAMeT in the VNC may be inactive or inactivated at the stages of development tested. Contrary to the previous reports in other crustaceans, MO extract in shrimp shows only low FAMeT activity. The eyestalk, epidermis, ovary and testis show appreciable FAMeT activity. The presence of FAMeT in neurosecretory cells of VNC and eyestalk of shrimp and crayfish implies a possible interaction of FAMeT with the eyestalk CHH-family of neuropeptides. The widespread activity of FAMeT suggests that it has a wide spectrum of action in many tissues that contribute to the function and regulation of MF synthesis in shrimp and crayfish.

Characterization of an additional molt inhibiting hormone-like neuropeptide from the shrimp Metapenaeus ensis.

We have identified a second form of the type-II neuropeptide encoding a molt inhibiting hormone-like (MeeMIH-B) neuropeptide. MeeMIH-B showed only a 70% amino acid identity to the MIH-A (formerly MIH) isolated from the same species, suggesting a possible different function of the deduced neuropeptide. Like other neuropeptide members of the CHH family, the MIH-B gene consists of three exons separated by two introns. The levels of MIH-B mRNA transcript in the eyestalk decrease in the initial phase of gonad maturation and increase towards the end of maturation. The drop in MIH-B level suggests an inhibitory role for this neuropeptide in the initiation of vitellogenesis. MIH-B transcripts can also be detected in the brain, thoracic ganglion and ventral nerve cord. Together with the CHH-B peptide that we have previously described, this is the second peptide of the CHH family that can also be identified in the ventral nerve cord and in the XOSG complex. A recombinant MIH-B was produced and a polyclonal antibody against rMIH-B was subsequently generated. Specific anti-rMIH-B antiserum recognized the presence of MIH-B in the sinus gland, X-organs, as well as a giant neuron of the ventral nerve cord. Injection of rMIH-B delayed the molting cycle of the maturing female. Taken together, the results of this study suggest that a drop in MIH-B level may be required for the delay in the molting of the maturing females.

Methyl farnesoate and juvenile hormone production in embryos of Diploptera punctata in relation to innervation of corpora allata and their sensitivity to allatostatin.

Corpora allata (CA) of embryos of Diploptera punctata have been previously shown to produce JH III. We have re-examined sesquiterpenoid biosynthesis throughout embryonic development and have found that early embryos produce both methyl farnesoate (MF) and JH III; as development proceeds, less MF and more JH is produced. The cockroach allatostatin peptide Dippu-allatostatin (AST) 7 inhibits sesquiterpenoid production by CA of mid to late embryos whereas it exerts a dose-dependent stimulatory effect in early embryos. This stimulatory effect is particularly apparent on MF biosynthesis. CA become innervated by allatostatin-containing nerves in early embryos (35% development). Shortly thereafter, the allatostatin-containing innervation of the CA appears complete.

Expression of allatostatin in the oviducts of the cockroach Diploptera punctata.

The cockroach allatostatins (Y/FXFGL/Ia ASTs) are a ubiquitous family of peptides in the invertebrates. They affect numerous physiological processes including the inhibition of juvenile hormone III (JH) biosynthesis, inhibition of muscle contraction, inhibition of ovarian ecdysteroid biosynthesis and inhibition of vitellogenin (Vg) release from the fat body. We have developed and optimized a sensitive and specific quantitative competitive reverse transcriptase polymerase chain reaction (QC-RT-PCR) method to quantify Diploptera punctata AST (Dippu-AST) expression. Using this technique we show that tissues of both lateral and common oviducts and the ovary express message for Dippu-AST. Moreover, the pattern of expression observed in the oviducts and ovary is strikingly similar with significant changes occurring during the reproductive cycle. Specifically, expression of AST is drastically reduced during the time of maximal vitellogenin (Vg) uptake, with higher levels measured prior to and following vitellogenesis. Furthermore, using immunocytochemistry, we have shown Dippu-AST-like-immunoreactivity in the terminal abdominal ganglion, as well as in ventral nerve 7, some branches of which innervate the common and lateral oviducts with other branches innervating the bursa copulatrix and brood sac of mated female D. punctata. The pattern of Dippu-AST expression and immunocytochemical staining suggests that ASTs function, in part, to regulate the cycle of vitellogenesis in mated female D. punctata.

Enhanced haemolymph circulation by insect ventral nerve cord: hormonal control by Pseudaletia unipuncta allatotropin and serotonin.

The ventral diaphragm (VD) in many insects is a muscular membrane that essentially partitions a perineural sinus from the rest of the abdomen. In the true armyworm moth Pseudaletia unipuncta (Lepidoptera: Noctuidae) we describe how the VD is characterized by a series of aliform muscles inserted into a tissue matrix that is fused to the dorsal surface of the ventral nerve cord (VNC) itself. Because of this arrangement, the abdominal VNC can attain high rates of lateral oscillation, and is capable of directing haemolymph flow. We have previously demonstrated Manduca sexta allatotropin (Manse-AT)-like immunoreactivity throughout the central nervous system (CNS) in P. unipuncta, and that both Manse-AT and serotonin (5-HT) are dose-dependent stimulators of the dorsal vessel. Here we describe both Manse-AT- and 5-HT-like immunoreactivity associated with the VD. Furthermore, both Manse-AT and 5-HT are dose-dependent stimulators of the rates of VNC oscillation, and together are capable of maintaining highly elevated rates of VNC oscillation for extended periods of time. These data indicate that both the dorsal vessel and the VD/VNC are similarly modulated by both Manse-AT and 5-HT, and that VNC oscillations play a more active role in overall haemolymph circulation than previously recognized.

Function and cellular localization of farnesoic acid O-methyltransferase (FAMeT) in the shrimp, Metapenaeus ensis.

The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT (rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti-FAMeT serum. FAMeT was localized in the neurosecretory cells of the X-organ-sinus gland complex of the eyestalk. As shown by RT-PCR, FAMeT mRNA is constitutively expressed throughout the molt cycle in the eyestalk and the ventral nerve cord. To show that our cloned gene product had FAMeT activity, we demonstrated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay. The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp. We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland.

Cardioacceleratory effects of Manduca sexta allatotropin in the true armyworm moth, Pseudaletia unipuncta.

Manduca sexta allatotropin (Manse-AT), a peptide originally isolated on the basis of its ability to stimulate juvenile hormone (JH) biosynthesis in the tobacco hornworm, is a potent in vitro stimulator of the corpora allata (CA) in Pseudaletia unipuncta (Lepidoptera: Noctuidae). At 10(-6)M, Manse-AT stimulated in vitro rates of JH biosynthesis by CA of day 0 and 6 adult females 15- and 10-fold respectively. Both Manse-AT and serotonin were also shown to be dose-dependent stimulators of heart rate in day 0, 3 and 6 adult males and females. Furthermore, analysis suggests that there are differences in both resting and Manse-AT-stimulated heart rates depending on age and rearing conditions.

Characterization and baculovirus-directed expression of a myosuppressin encoding cDNA from the true armyworm, Pseudaletia unipuncta.

Insect myosuppressins are a highly conserved sub-family of peptides which are primarily characterized by the ability to suppress contraction of visceral muscles in a variety of insect species. We have isolated a cDNA from the true armyworm, Pseudaletia unipuncta, that encodes a prohormone containing a peptide identical to ManducaFLRFamide. We have shown that this myosuppressin gene appears to be expressed in late larval and adult insects. In Manduca sexta, a number of extended-FLRFamide peptides have previously been purified including ManducaFLRFamide, F7D (DPSFLRFamide), F7G (GNSFLRFamide) and two larger peptides F24 and F39 that contain the shorter ManducaFLRFamide sequence at their C-terminus. Comparison with the true armyworm prepropeptide characterized here identifies F24 and F39 as partially processed products from the same precursor. Expression in the true armyworm was shown by in situ hybridization to occur in over 150 cells throughout the adult brain and nerve cord, and also to occur in both open and closed endocrine type cells of the gut. Overexpression of the P. unipuncta FLRFamide cDNA from a baculovirus vector in cabbage looper caterpillars was used to assess the potential for myosuppressin expression as a means of enhancing virus efficacy. Viral expression of the armyworm prohormone cDNA resulted in raised levels of RFamide-like products in the hemolymph of infected insects, but the products were found to be chemically distinguishable from authentic mature peptide and probably represent partially processed hormone.

Biosynthetic pathway of insect juvenile hormone III in cell suspension cultures of the sedge Cyperus iria.

In most insect species, juvenile hormones regulate critical physiological processes such as metamorphosis and reproduction. In insects, these sesquiterpenoids are synthesized by retrocerebral endocrine organs, the corpora allata, via the classical mevalonate (MVA) pathway. One of these compounds, juvenile hormone III (JH III), has also been identified in the sedge Cyperus iria. In higher plants, biosynthesis of the sesquiterpenoid backbone may proceed through two distinct pathways: the MVA pathway or the 2C-methyl erythritol 4-phosphate pathway or through a combination of both pathways. Cell suspension cultures of C. iria were used to elucidate the biosynthetic pathway of JH III in the plant. Enzyme inhibition and labeling studies conclusively demonstrated that the biosynthesis of the sesquiterpenoid backbone of JH III proceeds via the MVA pathway. Inhibitor and precursor feeding studies also suggest that later steps of JH III biosynthesis in C. iria are similar to the insect pathway and that the final enzymatic reaction in JH III biosynthesis is catalyzed by a cytochrome P(450) monooxygenase.

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules.

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic hormone was identified in a BLAST search of the Drosophila genome database. The predicted 31-residue amidated peptide (D. melanogaster DH31; Drome-DH31) was synthesised and tested for activity on fruit fly Malpighian tubules. It increases tubule secretion by approximately 35 % of the response obtained with a myokinin from the housefly Musca domestica (muscakinin; Musdo-K) and has an EC50 of 4.3 nmol x l(-1). The diuretic activities of Drome-DH31 and Musdo-K were additive when tested at threshold and supra-maximal concentrations, which suggests that they target different transport processes. In support of this, Drome-DH31 increased the rate of secretion by tubules held in bathing fluid with a reduced Cl- concentration, whereas Musdo-K did so only in the presence of Drome-DH31. Stimulation with Drome-DH31 increased the lumen-positive transepithelial potential in the main secretory segment of the tubule. This was attributed to activation of an apical electrogenic proton-translocating V-ATPase in principal cells, since it was associated with hyperpolarisation of the apical membrane potential and acidification of secreted urine by 0.25 pH units. Exogenous 8-bromo-cyclic AMP and cyclic GMP increased tubule secretion to the same extent as Drome-DH31 and, when tested together with the diuretic peptide, their activities were not additive. Stimulation with Drome-DH31 resulted in a dose-dependent increase in cyclic AMP production by tubules incubated in saline containing 0.5 mmol x l(-1) 3-isobutyl-1-methylxanthine, whereas cyclic GMP production was unchanged. Taken together, the data are consistent with Drome-DH31 activating an apical membrane V-ATPase via cyclic AMP. Since the K+ concentration of the secreted urine was unchanged, it is likely that Drome-DH31 has an equal effect on K+ and Na+ entry across the basolateral membrane.

Flights of fancy: possible roles of allatostatin and allatotropin in migration and reproductive success of Pseudaletia unipuncta.

Many invertebrate neuropeptides have recently been identified and there is evidence that the same compound may serve different roles in different species and/or multiple functions within a given species. However, until the relevant receptors or 'knock out' animals, lacking the neuropeptide of interest, become available it will be difficult to clarify the precise inter- and intraspecific functions of these neuropeptides. In the present paper, we argue that until these tools are available a more meaningful understanding of the roles of neuropeptides could be obtained by carrying out experiments within an ecological context. Furthermore, this approach would allow us to generate hypotheses that could be rigorously tested when more sophisticated techniques are developed. We discuss these ideas using our interdisciplinary research on the reproductive biology of the true armyworm, Pseudaletia unipuncta, as a case study.