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Nineteen species of various families of the order Diptera and one species from the order Mecoptera are investigated with mass spectrometry for the presence and primary structure of putative adipokinetic hormones (AKHs). Additionally, the peptide structure of putative AKHs in other Diptera are deduced from data mining of publicly available genomic or transcriptomic data. The study aims to demonstrate the structural biodiversity of AKHs in this insect order and also possible evolutionary trends. Sequence analysis of AKHs is achieved by liquid chromatography coupled to mass spectrometry. The corpora cardiaca of almost all dipteran species contain AKH octapeptides, a decapeptide is an exception found only in one species. In general, the dipteran AKHs are order-specific- they are not found in any other insect order with two exceptions only. Four novel AKHs are revealed by mass spectrometry: two in the basal infraorder of Tipulomorpha and two in the brachyceran family Syrphidae. Data mining revealed another four novel AKHs: one in various species of the infraorder Culicumorpha, one in the brachyceran superfamily Asiloidea, one in the family Diopsidae and in a Drosophilidae species, and the last of the novel AKHs is found in yet another Drosophila . In general, there is quite a biodiversity in the lower Diptera, whereas the majority of the cyclorraphan Brachycera produce the octapeptide Phote-HrTH. A hypothetical molecular peptide evolution of dipteran AKHs is suggested to start with an ancestral AKH, such as Glomo-AKH, from which all other AKHs in Diptera to date can evolve via point mutation of one of the base triplets, with one exception., (Copyright © 2020 Gäde, Šimek and Marco.)

Seventeen species of the coleopteran series Cucujiformia are investigated for the presence and sequence of putative adipokinetic hormones (AKHs). Cucujiformia includes species from the major superfamilies, that is, Chrysomeloidea, Curculionoidea, Cucujoidea, and Tenebrionoidea. The clade Phytophaga in which the Chrysomeloidea and Curculionoidea reside, harbor very detrimental species for agriculture and forestry. Thus, this study aims not only to demonstrate the structural biodiversity of AKHs in these beetle species and possible evolutionary trends but also to determine whether the AKHs from harmful pest species can be used as lead substances for a future putative insecticide that is harmless to beneficial insects. Sequence analysis of AKHs is achieved by liquid chromatography coupled to mass spectrometry. Most of the investigated species contain AKH octapeptides in their corpora cardiaca, although previously published work also found a few decapeptides, which we comment on. The signature and sole AKH in cerambycidae Chrysomeloidea and Curculionoidea is Peram-CAH-I (pEVNFSPNW amide), which is also found in the majority of chrysomelidae Chrysomeloidea and in the one investigated species of Cucujoidea albeit in a few cases associated with a second AKH which can be either Peram-CAH-II (pELTFTPNW amide), Emppe-AKH (pEVNFTPNW amide), or Micvi-CC (pEINFTPNW amide). The most often encountered AKH in Tenebrionoidea, family Meloidae as well as family Tenebrionidae, is Tenmo-HrTH (pELNFSPNW amide) followed by Pyrap-AKH (pELNFTPNW amide) and a Tenmo-HrTH extended decapeptide (in Meloidae). Finally, we examine AKH sequences from 43 species of cucujiform beetles, including the superfamily Coccinelloidea for a possible lead compound for producing a cucujiform-specific pesticide., (© 2019 Wiley Periodicals, Inc.)

The aim of the current study is to identify the adipokinetic hormone(s) (AKHs) of a basal suborder of the species-rich Coleoptera, the Adephaga, and possibly learn more about the ancestral AKH of beetles. Moreover, we wanted to compare the ancestral AKH with AKHs of more advanced beetles, of which a number are pest insects. This would allow us to assess whether AKH mimetics would be suitable as insecticides, that is, be harmful to the pest species but not to the beneficial species. Nine species of the Adephaga were investigated and all synthesize only one octapeptide in the corpus cardiacum, as revealed by Edman degradation sequencing techniques or by mass spectrometry. The amino acid sequence pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp corresponds to Schgr-AKH-II that was first identified in the desert locust. It is assumed that Schgr-AKH-II-the peptide of a basal beetle clade-is the ancestral AKH for beetles. Some other beetle families, as well as some Hymenoptera (including honey bees) also contain this peptide, whereas most of the pest beetle species have different AKHs. This argues that those peptides and their receptors should be explored for developing mimetics with insecticidal properties. A scenario where Schgr-AKH-II (the only AKH of Adephaga) is used as basic molecular structure to derive almost all other known beetle AKHs via single step mutations is very likely, and supports the interpretation that Schgr-AKH-II is the ancestral AKH of Coleoptera., (© 2017 Wiley Periodicals, Inc.)

Six species of the order Mantodea (praying mantises) are investigated for the presence and sequence of putative adipokinetic hormones (AKHs). The selected species span a wide evolutionary range of various families and subfamilies of the clade Mantodea. The corpora cardiaca of the different species are dissected, methanolic extracts prepared, peptides separated by liquid chromatography, and AKHs detected and sequenced by ion trap mass spectrometry. All six species investigated contain an octapeptide with the primary structure pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp amide, which is code-named Emppe-AKH and had been found earlier in three other species of Mantodea. Conspecific bioassays with the species Creoboter sp. (family Hymenopodidae) reveal an adipokinetic but not a hypertrehalosemic function of Emppe-AKH. Comparison with other members of the Dictyoptera (cockroaches, termites) show that Emppe-AKH is only found in certain termites, which have been recently placed into the Blattaria (cockroaches) as sister group to the family Cryptocercidae. Termites and cockroaches both show biodiversity in the sequence of AKHs, and some cockroach species even contain two AKHs. In contrast, all praying mantises-irrespective of their phylogenetic position-synthesize uniformly only one and the same octapeptide Emppe-AKH., (© 2017 Wiley Periodicals, Inc.)

Olfaction is an important sensory modality that regulates a plethora of behavioural expressions in insects. Processing of olfactory information takes place in the primary olfactory centres of the brain, namely the antennal lobes (ALs). Neuropeptides have been shown to be present in the olfactory system of various insect species. In the present study, we analyse the distribution of tachykinin, FMRFamide-related peptides, allatotropin, allatostatin, myoinhibitory peptides and SIFamide in the AL of the male Egyptian cotton leafworm, Spodoptera littoralis. Immunocytochemical analyses revealed that most neuropeptides were expressed in different subpopulations of AL neurons. Their arborisation patterns within the AL suggest a significant role of neuropeptide signalling in the modulation of AL processing. In addition to local interneurons, our analysis also revealed a diversity of extrinsic peptidergic neurons that connected the antennal lobe with other brain centres. Their distributions suggest that extrinsic neurons perform various types of context-related modulation.

Rab proteins are small GTPases that play essential roles in vesicle transport. In this study, we examined the expression of Rab proteins and neuropeptide hormones in the brain of the silkworm, Bombyx mori. We produced antibodies against B. mori Rab1 and Rab14 in rabbits. Immunoblotting of samples of brain tissue from B. mori revealed a single band for each antibody. Rab1 and Rab14 immunohistochemical labeling in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Rab1, Rab7 and Rab14 co-localized with bombyxin. Rab1 and Rab7 co-localized with eclosion hormone. Rab1 co-localized with prothoracicotropic hormone. These results suggest that Rab1, Rab7 and Rab14 may be involved in neuropeptide transport in the brain of B. mori. This is the first report on the specificity of Rab proteins for the secretion of different neuropeptides in insects.

Studies about the sex pheromone of the mealybug Dysmicoccus grassii , a main pest of Canary Islands banana cultivars, showed strong evidence that (-)-(R)-lavandulyl propionate and acetate in a 6:1 ratio are principal components of it. Volatile collection and GC-MS analysis from aeration of virgin females allowed the structural elucidation of the compounds. The activity and stereochemistry of both substances were established by means of relative attraction of mealybug males to synthetic standards in competitive Petri dish bioassays. (R)-Lavandulyl propionate induced a stronger attractive effect when compared to (R)-lavandulyl acetate. The attractiveness of the mixture of the two compounds at the original source ratio showed no statistically significant difference from that of the sum of each of the single compounds alone, suggesting that both components are not synergistic but additive.

Human malaria, a major public health burden in tropical and subtropical countries, is transmitted exclusively by the bite of a female Anopheles mosquito. Malaria control strategies aimed at inducing sexual sterility in natural vector populations are an attractive alternative to the use of insecticides. However, despite their importance as disease vectors, limited information is available on the molecular mechanisms regulating fertility in Anopheles mosquitoes. In the major malaria vector, An. gambiae, the full complement of sperm and seminal fluid required for a female's lifelong egg production is obtained from a single mating event. This single mating has important consequences for the physiology and behavior of An. gambiae females: in particular, they become refractory to further insemination, and they start laying eggs. In other insects including Drosophila, similar post-copulatory changes are induced by seminal proteins secreted by the male accessory glands and transferred to the female during mating. In this review, we analyze the current state of knowledge on the function and characterization of male seminal proteins in An. gambiae, and provide a comparative assessment of the role of these male reproductive factors in other mosquito vectors of human disease in which female post-copulatory behavior has been studied. Knowledge of the factors and mechanisms regulating fertility in An. gambiae and other vectors can help the design of novel control strategies to fight the spread of disease.

Probing of a host and ingestion of a blood-meal in a fifth instar Rhodnius prolixus results in a cascade of tightly integrated events. The huge blood-meal is pumped into the anterior midgut during feeding, then modified by diuresis and stored until it is digested. While serotonin is known to be a diuretic hormone in R. prolixus, a peptidergic factor(s) was also known to play a role in diuresis. In the present study we employed molecular techniques and mass spectrometry to determine the sequence of a native CRF-like peptide from R. prolixus (Rhopr DH). In addition, we confirmed the distribution and localization of Rhopr DH using in situ hybridization and immunohistochemistry, and demonstrated its potent biological activity on both the anterior midgut and Malpighian tubules.

We show a straightforward workflow combining homology search in Rhodnius prolixus genome sequence with cloning by rapid amplification of cDNA ends and mass spectrometry. We have identified 32 genes and their transcripts that encode a number of neuropeptide precursors leading to 194 putative peptides. We validated by mass spectrometry 82 of those predicted neuropeptides in the brain of R. prolixus to achieve the first comprehensive genomic, transcriptomic and neuropeptidomic analysis of an insect disease vector. Comparisons of available insect neuropeptide sequences revealed that the R. prolixus genome contains most of the conserved neuropeptides in insects, many of them displaying specific features at the sequence level. Some gene families reported here are identified for the first time in the order Hemiptera, a highly biodiverse group of insects that includes many human, animal and plant disease agents., (Insect Molecular Biology © 2010 The Royal Entomological Society. No claim to original US government works.)

Juvenile hormone (JH) III is responsible for control of a variety of insect physiological and developmental states, including caste differentiation of the Formosan subterranean termite (Coptotermes formosanus Shiraki). We report here a simplified, efficient sample preparation and an optimized LC-ESI-MS method for quantifying JH III in whole body extracts. Sample preparation comprises hexane extraction (from termite whole bodies) and C18 cartridge purification. Previous LC-ESI-MS protocols exhibited the following two problems: (1) ion fragmentation differed when comparing spectra from insect samples and authentic JH III and (2) a JH III monitoring ion was not resolved from other unknown compounds in whole body samples from termites. To overcome these problems, we used a pentafluorophenyl LC column and water/acetonitrile containing ammonium acetate as solvent. In a mass chromatogram (m/z 235) of termite samples, a peak was detected at the retention time of authentic JH III, and MS(2) of this peak confirmed that the ion is a fragment of JH III, [M-CH(3)OH+H](+), being the base peak in both termites and authentic JH III samples. The protocol enables quantification of JH III in a single termite with signal/noise >10:1 and the limit of quantification is 21pg.

Recently, the peptidomic analysis of neuropeptides from the retrocerebral complex and abdominal perisympathetic organs of polyphagous stinkbugs (Pentatomidae) revealed the group-specific sequences of pyrokinins, CAPA peptides (CAPA-periviscerokinins/PVKs and CAPA-pyrokinin), myosuppressin, corazonin, adipokinetic hormone, and short neuropeptide F. In this study, we used mass spectrometric profiling of nervous tissue from the species-rich taxon Hemiptera to identify products of two previously unobserved neuropeptide genes from these species, namely allatotropin-related peptide (ATRP) and tachykinin-related peptides (TKRPs). Since neither TKRPs nor allatotropin are accumulated in neurohemal organs, immunocytochemical data were analyzed to find potential accumulation sites within the central nervous system. By mass spectrometry, TKRPs were found to be accumulated in the antennal lobes, and ATRP was identified in the most posterior region of the abdominal ventral nerve cord and fourth abdominal nerves. In addition to neuropeptides from stink bugs, TKRPs and ATRP were also identified from the distantly related bugs Oncopeltus fasciatus (Lygaeidae) and Pyrrhocoris apterus (Pyrrhocoridae). In total, six TKRPs and one ATRP from each species could be elucidated by tandem mass spectrometry. The ATRP of all species is sequence-identical with Locusta migratoria accessory gland myotropin-1 (Lom-AG-MT-1), a member of the highly conserved insect allatotropin family.

The mite Varroa destructor Anderson & Trueman is a parasite of the honeybee Apis mellifera L. and represents a major threat for apiculture in the Western world. Reproduction takes place only inside bee brood cells that are invaded just before sealing; drone cells are preferred over worker cells, whereas queen cells are not normally invaded. Lower incidence of mites in queen cells is at least partly due to the deterrent activity of royal jelly. In this study, the repellent properties of royal jelly were investigated using a lab bioassay. Chemical analysis showed that octanoic acid is a major volatile component of royal jelly; by contrast, the concentration is much lower in drone and worker larval food. Bioassays, carried out under lab conditions, demonstrated that octanoic acid is repellent to the mite. Field studies in bee colonies confirmed that the compound may interfere with the process of cell invasion by the mite.

The invertebrate neuropeptide Y (NPY) homolog, neuropeptide F (NPF), has been characterized for a wide range of invertebrate phyla, including platyhelminthes, molluscs, and arthropods. Current hypotheses suggest that NPF may be capable of regulating responses to diverse external cues related to nutritional status and feeding. The qualitative and quantitative distribution of an NPF-like peptide in fifth instar Rhodnius prolixus was undertaken using an antiserum raised against Drosophila NPF. Immunohistochemistry reveals NPF-like immunoreactive neurons and processes in the central nervous system, stomatogastric nervous system and peripheral nervous system. The distribution of NPF-like immunoreactivity within the medial neurosecretory cells of the brain and neurohemal areas of the corpus cardiacum and dorsal vessel, suggests NPF may act as a neurohormone. Immunoreactive processes are present over the surface of the hindgut and the immunoreactivity in these processes is greatly reduced in intensity 24h post-feeding. The quantification of partially purified NPF-like material in the CNS of R. prolixus was conducted by HPLC fractionation and radioimmunoassay. The results suggest that NPF-like material is present in fifth instar R. prolixus and likely released into the hemolymph following a blood meal.

Small neuropeptides of the adipokinetic/red pigment-concentrating hormone (AKH/RPCH) family regulate energy metabolism in insects. Within lepidopterans, the nonapeptide Manduca sexta AKH (Manse-AKH) represents a widely occurring AKH, whereas the decapeptide Helze-HrTH (at first isolated from Helicoverpa zea) seems to be restricted to moths. Here we report the identification of the Manse-AKH-like Spofr-AKH 1 and the Helze-HrTH-like Spofr-AKH 2 prohormone precursors from the fall armyworm, Spodoptera frugiperda. Moreover, by PCR screening of a random primer cDNA library and by RACE, three 668, 835 and 1008 bp cDNA sequences were obtained, which encode putative translation products of 67-74 amino acids, each containing one copy of a peptide sequence that in its processed form has the sequence of QLTFSSGW-amide (Spofr-AKH 3). Another cDNA sequence of 634 bp encodes a putative translation product of 40 amino acids, potentially leading to one copy of an elongated, non-amidated Helze-HrTH (pQLTFSSGWGNCTS-OH; Spofr-AKH 4). Q-RT-PCR analysis showed that the Spofr-AKH mRNAs are expressed in 2d-old female brain/corpora cardiaca complexes, but also in ovaries, midgut, fat body, accessory glands and muscle tissues. Expression was also found in the ovaries of 4d-old females. Whole-mount in situ RT-PCR analysis with ovaries from 2d-old females showed that the Spofr-AKH 2 and Spofr-AKH 4 were mainly localized in the germarium (phase 3), whereas the Spofr-AKH 1, and the three mRNA isoforms of Spofr-AKH 3 were localized at the end of the vitellarium and in the fully developed oocytes (phase 1 and 2). The results suggest that Spofr-AKH genes play a role in the regulation of oocyte maturation in S. frugiperda.

Antisera against a variety of vertebrate and invertebrate neuropeptides were used to characterize neurons with somata in the pars intercerebralis (PI), pars lateralis (PL), and subesophageal ganglion (SEG), designated as PI neurons, PL neurons, and SEG neurons, respectively, all of which project to the retrocerebral complex in the blow fly, Protophormia terraenovae. Immunocytochemistry combined with backfills through the cardiac-recurrent nerve revealed that at least two pairs of PI and SEG neurons for each were FMRFamide-immunoreactive. Immunoreactivity against [Arg7]-corazonin, beta-pigment-dispersing hormone (beta-PDH), cholecystokinin8, or FMRFamide was observed in PL neurons. Immunoreactive colocalization of [Arg7]-corazonin with beta-PDH, [Arg7]-corazonin with cholecystokinin8, or beta-PDH with FMRFamide was found in two to three somata in the PL of a hemisphere. Based on their anatomical and immunocytochemical characteristics, PI neurons were classified into two types, PL neurons into six types, and SEG neurons into two types. Fibers in the retrocerebral complex showed [Arg7]-corazonin, beta-PDH, cholecystokinin8, and FMRFamide immunoreactivity. Cholecystokinin8 immunoreactivity was also detected in intrinsic cells of the corpus cardiacum. The corpus allatum was densely innervated by FMRFamide-immunoreactive varicose fibers. These results suggest that PI, PL, and SEG neurons release [Arg7]-corazonin, beta-PDH, cholecystokinin8, or FMRFamide-like peptides from the corpus cardiacum or corpus allatum into the hemolymph, and that some PL neurons may simultaneously release several neuropeptides.

Neuropeptides and neurohormones are among the more diverse and functionally important classes of cell-to-cell signaling molecules involved in animal development and behavior. Less is known about the hormones and neuropeptides of the red flour beetle, Tribolium castaneum, than many other insects. However, the genomic information becoming available from this organism presents an opportunity to identify multiple neuropeptide and hormone genes, and hence their associated protein precursors. Using similarity-based prediction, we report new neuropeptides and hormone precursors from T. castaneum, bringing the number of annotated precursors to 37. We identified one prohormone (SVDPIDGDLIG-containing) having little similarity to other insect prohormones. The conversion of the protein precursors into bioactive peptides requires a suite of processing enzymes and a number of enzymatic steps; using the web-based NeuroPred application and similarity-based bioinformatics approaches, we predict 132 likely peptides that may result from the enzymatic processing of these gene products.

Neuropeptides are a diverse widespread class of signaling substances in the nervous system. As a basis for the analysis of peptidergic neurotransmission in the insect olfactory system, we have studied the distribution of neuropeptides in the antennal lobe of the moth Heliothis virescens. Immunocytochemical experiments with antisera recognizing A-type allatostatins (AST-As), Manduca sexta allatotropin (Mas-AT), FMRFamide-related peptides (FaRPs), and tachykinin-related peptides (TKRPs) have shown that members of all four peptide families are present in local interneurons of the antennal lobe. Whereas antisera against AST-As, Mas-AT, and FaRPs give similar staining patterns characterized by dense meshworks of processes confined to the core of all antennal-lobe glomeruli, TKRPs are present only in neurons with blebby processes distributed throughout each glomerulus. In addition to local neurons, a pair of centrifugal neurons with cell bodies in the lateral subesophageal ganglion, arborizations in the antennal lobe, and projections in the inner antenno-cerebral tracts exhibits tachykinin immunostaining. Double-label immunofluorescence has detected the co-localization of AST-As, Mas-AT, and FaRPs in certain local interneurons, whereas TKRPs occurs in a distinct population. MALDI-TOF mass spectrometry has revealed nearly 50 mass peaks in the antennal lobe. Seven of these masses (four AST-As, two N-terminally extended FLRFamides, and Mas-AT) match known moth neuropeptides. The data thus show that local interneurons of the moth antennal lobe are highly differentiated with respect to their neuropeptide content. The antennal lobe therefore represents an ideal preparation for the future analysis of peptide signaling in insect brain.

Background: For holometabolous insects there has been an explosion of proteomic and peptidomic information thanks to large genome sequencing projects. Heterometabolous insects, although comprising many important species, have been far less studied. The migratory locust Locusta migratoria, a heterometabolous insect, is one of the most infamous agricultural pests. They undergo a well-known and profound phase transition from the relatively harmless solitary form to a ferocious gregarious form. The underlying regulatory mechanisms of this phase transition are not fully understood, but it is undoubtedly that neuropeptides are involved. However, neuropeptide research in locusts is hampered by the absence of genomic information., Results: Recently, EST (Expressed Sequence Tag) databases from Locusta migratoria were constructed. Using bioinformatical tools, we searched these EST databases specifically for neuropeptide precursors. Based on known locust neuropeptide sequences, we confirmed the sequence of several previously identified neuropeptide precursors (i.e. pacifastin-related peptides), which consolidated our method. In addition, we found two novel neuroparsin precursors and annotated the hitherto unknown tachykinin precursor. Besides one of the known tachykinin peptides, this EST contained an additional tachykinin-like sequence. Using neuropeptide precursors from Drosophila melanogaster as a query, we succeeded in annotating the Locusta neuropeptide F, allatostatin-C and ecdysis-triggering hormone precursor, which until now had not been identified in locusts or in any other heterometabolous insect. For the tachykinin precursor, the ecdysis-triggering hormone precursor and the allatostatin-C precursor, translation of the predicted neuropeptides in neural tissues was confirmed with mass spectrometric techniques., Conclusion: In this study we describe the annotation of 6 novel neuropeptide precursors and the neuropeptides they encode from the migratory locust, Locusta migratoria. By combining the manual annotation of neuropeptides with experimental evidence provided by mass spectrometry, we demonstrate that the genes are not only transcribed but also translated into precursor proteins. In addition, we show which neuropeptides are cleaved from these precursor proteins and how they are post-translationally modified.

The mating-induced increase in juvenile hormone (JH) biosynthesis in Heliothis virescens females may be stimulated by production and/or release of stimulatory neuropeptides such as allatotropins (AT). Although there is evidence that H. virescens allatotropin may be structurally related to Manduca sexta allatotropin (Manse-AT), little is known of its occurrence and distribution in H. virescens. An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against Manse-AT was used to quantify concentrations of Manse-AT immunoreactivity in tissue extracts of H. virescens. In mated females, the highest concentrations of Manse-AT-like material occurred in the brain. The ventral nervous system and the accessory glands also contained considerable amounts of Manse-AT-like material, whereas concentrations were very low in ovaries, fat body, and flight muscle. The Manse-AT antibody was used for whole-mount immunocytochemistry to localize Manse-AT-immunoreactivity in the central nervous system. Several groups of Manse-AT-immunoreactive cells were discovered in the brain, subesophageal ganglion, and thoracic and abdominal ganglia of H. virescens females and males. Strong immunoreactivity was detected in axons going through the corpora cardiaca and branching out over the surface of the corpora allata. The presence of Manse-AT-like material in various locations in the central nervous system suggests that these peptides may have other as yet unknown functions. At the posterior margin of the terminal ganglion of males, a group of large immunoreactive cells was observed that was not present in females. Other than that, there were no obvious differences between virgin and mated females or males. The lack of differences in AT distribution in mated and virgin females suggests that mating-induced differences in female JH biosynthesis rates may be caused by changes in cellular response to AT at the level of the CA, rather than by changes in the amounts of AT acting on the CA., (2006 Wiley-Liss, Inc.)

The cDNA encoding eclosion hormone (EH), which plays an integral role in triggering ecdysis behavior at the end of each molt, was cloned from the cotton bollworm, Helicoverpa armigera (Har) (Lepidoptera: Noctuidae). The EH polyprotein precursor contains a 26-amino acid signal peptide and a single 62-amino acid mature EH. Compared the mature Har-EH with other known EHs, it shows 94%, 84%, and 59% identities to Manduca sexta, Bombyx mori, and Drosophila melanogaster, respectively. Har-EH mRNA is expressed only in the brain by Northern blot and RT-PCR, but not in other tissues. By in situ hybridization and immunocytochemistry, both Har-EH mRNA and protein are localized in two pairs of neurosecretory cells of the brain. Prior to a molt, expression level of Har-EH gene reaches the highest point, and then drops after molt. EH release is detected both centrally, within the ganglia, and peripherally, into the hemolymph. A peak of the EH titer in hemolymph measured by ELISA presents at ecdysis. These results are consistent with the biological function of Har-EH associated with ecdysis. Furthermore, Har-EH gene is expressed throughout all of the developmental stages examined, implicating that the EH gene may possess other biological functions in post-embryonic development other than triggering ecdysis behavior.

In colonies of the queenless ant Streblognathus peetersi, dominance interactions produce a reproductive hierarchy in which one individual, the alpha, is capable of producing offspring while her subordinates remain infertile. Based on differences between behaviour and cuticular hydrocarbon profiles, the subordinates can be further divided into high and low ranking workers. Although it had been shown previously that alphas treated with a juvenile hormone analog lose their reproductive status, little was known of the endocrinological basis of dominance in this species. To elucidate the underlying endocrinology of these three ranks, we measured the individual in vitro rate of juvenile hormone (JH) production of excised corpora allata, and the ecdysteroid titer of pooled hemolymph samples. Production of JH was highest in low-ranking workers, intermediate in high rankers, and almost undetectable in alphas. Ecdysteroid titers were low for low rankers, but were more than twice as high for both high rankers and alphas. The results support the hypothesis that JH suppresses ovarian function in these queenless ants, and suggest that ecdysteroids may be responsible for stimulating vitellogenin production. The possible role of these hormones as behavioural modulators is also discussed.

The first peptidergic neurohormone from the ticks Ixodes ricinus and Boophilus microplus has been identified by using a combination of immunocytochemistry and mass spectrometric analysis of single cells. The novel peptide (Ixori-PVK, PALIPFPRV-NH2) shows a high sequence homology with members of the insect periviscerokinin/CAP2b peptides that in insects are involved in the regulation of water balance. The function of this peptide in ticks is still unknown, but these pests consume large amounts of blood in a single blood meal which is a challenge for the regulation of diuretic processes. Thus, the novel peptide may be involved in one of the key physiological processes in ticks. High energy collision-induced dissociation was successfully used to distinguish between Leu/Ile ambiguities in single cell preparations. This is the first successful de novo sequencing of a peptide from single cell preparations of arthropods.

In Rhodnius prolixus, testes from both pharate adult and adult males are shown to produce and release ecdysteroids in vitro. Proteinaceous brain extracts from these stages caused stimulation of ecdysteroid production by testes of unfed adults. Therefore, the brain of Rhodnius contains peptides with testis ecdysiotropic activity. The Lymantria testis ecdysiotropin (LTE) also stimulated the in vitro production of ecdysteroids by unfed adult testis but had no stimulatory effect on prothoracic glands. Western blot analysis of brain peptides using anti-LTE revealed the presence of several medium to small size immunoreactive peptides. Two of these peptides with sizes of 16.8 and 11.0 kDa were present only during pharate adult development and the adult stage. Immunohistochemical analysis using confocal laser scanning microscopy revealed abundant LTE-immunoreactive material in cytoplasmic granules of specific neurosecretory cells in the brain and suboesophageal ganglion and the epithelium of the testis sheath. Clusters of two cytologically distinct cell types were seen within the medial neurosecretory cells (MNC) and also a pair of neurons in the posterior protocerebrum. Feeding in both larvae and adult males resulted in massive release of LTE-immunoreactive material from the MNC cells, suggesting a role of LTE-related peptides in both larval-adult development and in male reproductive development. Release from the MNC cells of LTE-immunoreactive material exhibited a clear daily cycling during larval-adult development, which was synchronous with the rhythms of release of prothoracicotropic hormone and bombyxin reported previously. The testis sheath exhibited intense immunofluorescence in pharate adults and unfed adults, which disappeared following a blood meal. It is concluded that LTE-related peptides are developmentally regulated in several locations and may act as ecdysiotropins in Rhodnius. Those in the MNC cells are very probably classical hormones, i.e. are transported to their target sites via the insect haemolymph.

A novel member of the AKH/RPCH family of peptides has been identified from the corpus cardiacum of an, as yet, unidentified species of the newly discovered insect order Mantophasmatodea from Namibia. The primary sequence of the peptide, which is denoted Manto-CC, was deduced from multiple MS(N) electrospray mass data to be an octapeptide: pGlu-Val-Asn-Phe-Ser-Pro-Gly-Trp amide. Synthetic Manto-CC co-elutes on reversed-phase HPLC with the natural peptide from the gland of the insect. Interestingly, Manto-CC is structurally very closely related (only one point mutation) to the AKH/RPCH peptides previously identified in mostly more basal insect taxa (Odonata, Blattodea, and Ensifera) and in Crustacea, the sister group of insects, whereas larger structural differences occur with peptides from Mantodea and Phasmatodea, which are thought to be close relatives of Mantophasmatodea. Functionally, Manto-CC may be employed to activate glycogen phosphorylase to mobilize carbohydrates.

Peptides are the largest class of signalling molecules found in animals. Nevertheless, in most proteomic studies peptides are overlooked since they literally fall through the mazes of the net. In analogy with proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomic approach aims at the simultaneous visualization and identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their post-translational modifications. In this paper we describe the analysis of the larval fruit fly central nervous system using two-dimensional capillary liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS/MS. Using the central nervous systems of only 50 larval Drosophila as starting material, we identified 38 peptides in a single analysis, 20 of which were not detected in a previous study that reported on the one-dimensional capillary LC/MS/MS analysis of the same tissue. Among the 38 sequenced peptides, some originate from precursors, such as the tachykinin and the IFamide precursor that were entirely missed in the first study. This clearly demonstrates that the two-dimensional capillary LC approach enhances the coverage of the peptidomic analysis., (Copyright 2005 John Wiley & Sons, Ltd.)

Gene expression and immunolocalisation studies have determined that the helicostatins are brain-gut peptides in larvae of the lepidopteran, Helicoverpa armigera. Mapping of the distribution of these peptides in the nervous system and alimentary canal has provided evidence for multifunctional regulatory roles. In situ hybridisation studies have shown that the helicostatin precursor gene is expressed in neurones of the central and stomatogastric nervous systems, and endocrine cells of the midgut demonstrating that the helicostatins are true brain-gut peptides. Antisera raised against Leu-callatostatin 3 (ANRYGFGL-NH(2)), a peptide isolated from the blowfly, Calliphora vomitoria was used to map the distribution of allatostatin-like immunoreactive (Ast-ir) material in H. armigera to elucidate possible functions of the helicostatins. In situ hybridisation studies verified that the helicostatin precursor gene is expressed in neurones shown to contain Ast-ir, providing strong evidence that the Ast-ir material is helicostatins. Extensive immunoreactive axonal projections into complex regions of neuropile indicate that the helicostatins may have a neuromodulatory role in the brain and segmental ganglia of the ventral nerve cord. The presence of large amounts of immunoreactive material in axons within the corpora cardiaca (CC) and transverse nerves of the perisympathetic nervous system, two known neurohaemal organs, provides evidence for a neurohormonal role. The corpora allata (CA) were innervated only sparsely by Ast-ir axons suggesting that the CA are not a neurohaemal release site or a target. Thus, it is unlikely that the helicostatins regulate juvenile hormone (JH) biosynthesis or release. Ast-ir axons extended from the frontal ganglion through the recurrent nerve and many branches were closely associated with muscles of the foregut, stomodeal valve, and anterior midgut, implicating helicostatins in regulation of foregut motility. Ast-ir material was also present in nerves associated with muscles of the pyloric valve and rectum, and in endocrine cells of the midgut., (Copyright 2004 Wiley-Liss, Inc.)

Circadian locomotor activity rhythms of the cockroach Leucophaea maderae are driven by two bilaterally paired and mutually coupled pacemakers that reside in the optic lobes of the brain. Transplantation studies have shown that this circadian pacemaker is located in the accessory medulla (AMe), a small neuropil of the medulla of the optic lobe. The AMe is densely innervated by about 12 anterior pigment-dispersing-hormone-immunoreactive (PDH-ir) medulla (PDHMe) neurons. PDH-ir neurons are circadian pacemaker candidates in the fruitfly and cockroach. A subpopulation of these neurons also appears to connect both optic lobes and may constitute at least one of the circadian coupling pathways. To determine whether PDHMe neurons directly connect both accessory medullae, we injected rhodamine-labeled dextran as neuronal tracer into one AMe and performed PDH immunocytochemistry. Double-labeled fibers in the anterior, shell, and internodular neuropil of the AMe contralaterally to the injection site showed that PDH-ir fibers directly connect both accessory medullae. This connection is formed by three anterior PDHMe neurons of each optic lobe, which, thus, fulfill morphological criteria for a direct circadian coupling pathway. Our double-label studies also showed that all except one of the midbrain projection areas of anterior PDHMe neurons were innervated ipsilaterally and contralaterally. Thus, anterior PDHMe neurons seem to play multiple roles in generating circadian rhythms. They also deliver timing information output and perform mutual pacemaker coupling in L. maderae.

Mas-allatotropin (Mas-AT) and Lom-accessory gland-myotropin I (Lom-AG-MTI) are two members of a conserved family of insect neuropeptides, collectively termed allatotropins, which have diverse functions, ranging from stimulation of juvenile hormone secretion to myotropic effects on heart and hindgut. In addition, allatotropins appear to be abundant within the nervous system, suggesting neuroactive roles. To identify neurons in the insect brain suitable for a neurophysiological analysis of the roles of allatotropins, we used antisera against Mas-AT and Lom-AG-MTI to map allatotropin-immunoreactive neurons in the brain of a suitable insect, the locust Schistocerca gregaria. Both antisera revealed basically identical staining patterns throughout the locust brain with more than 12,500 immunostained interneurons per brain hemisphere. Neurosecretory cells were not labeled, and the retrocerebral complex was devoid of immunostaining. Prominent immunoreactive cell types include about 9,600 lamina monopolar neurons, medulla to lobula interneurons, local neurons of the antennal lobe, a giant interneuron of the mushroom body, projection neurons of the glomerular lobe to the mushroom body, and three systems of tangential neurons of the central complex. Several groups of neurons showed colocalization of Mas-AT- and gamma-aminobutyric acid immunostaining. Mass spectrometric analysis identified a peptide with a molecular mass identical to Lom-AG-MTI in all major parts of the locust brain but not in the retrocerebral complex. This study strongly suggests that Lom-AG-MTI is highly abundant in the locust brain, and is likely to play a neuroactive role in many brain circuits including all stages of sensory processing, learning and memory, and higher levels of motor control.

Helicoverpa assulta suboesophageal ganglion neuropeptide I (Has-SGNP I) is a 24-amino acids peptide amide, which shows 62.5% similarity with the diapause hormone of Bombyx mori (Bom-DH). It has been demonstrated that embryonic diapause is induced by DH in B. mori. Injection of synthetic amidated Has-SGNP I terminated pupal diapause in a dose-dependent manner. Therefore, Has-SGNP I might be referred to a "diapause termination hormone" in H. assulta (Has-DTH). The maximal dose of Has-DTH for diapause termination was 1.0 microg and the half-maximal dose 0.4 microg. The time required for diapause termination of Has-DTH was 2-3 days longer than that of 20-hydroxyecdysone. During the pupal stage, DTH mRNA content in the SGs of nondiapausing pupae was always higher than in diapausing pupae using the combined method of quantitative RT-PCR and Southern blot. DTH gene also expressed at a low level while diapausing pupae were chilled at 4 degrees C, but increased rapidly and largely after being transferred to 25 degrees C. Using a competitive ELISA, Has-DTH-like immunoreactivity in the haemolymph showed the same pattern as that of Has-DTH gene expression. Those results indicated that Has-DTH gene expression was related to diapause development and could be activated by low temperature. Has-DTH might be useful to elucidate the mechanism of diapause termination in pupal diapause species.

In many social insects the relationship between reproductive dominance and physiological correlates is poorly understood. Recent evidence now strongly suggests that cuticular hydrocarbons are important in reproductive differentiation in these societies where they are used as signals of ovarian activity in reproductive females. In this study we investigated the relationship between reproductive dominance, size of the corpora allata (CA, producer of Juvenile Hormone, JH) and the proportions of cuticular hydrocarbons present on the cuticle in overwintering foundresses and both associative (polygynous) and solitary (monogynous) pre-emergence colonies of the social wasp Polistes dominulus. Size of the CA was positively correlated with ovarian development in polygynous colonies. In contrast, solitary foundresses possessed significantly smaller CAs than dominant foundresses from polygynous nests, yet ovarian activity was similar for both female types. CA size variation was associated with variation in cuticular hydrocarbon proportions. Overwintering, solitary, dominant and subordinate (from associative nests) females all possessed distinctive cuticular chemical profiles revealed by multivariate discriminant analyses. Our data indicate that the social environment strongly affects reproductive physiology in this wasp, and we discuss the role of cuticular hydrocarbons in reproductive signaling in P. dominulus and other social insects.

The pars intercerebralis-corpora cardiaca complex in insects is the functional equivalent of the vertebrate brain-pituitary axis. During the past few decades more than 40 neuropeptides have been isolated from the locust brain-corpus cardiacum complex. Tedious and time-consuming successive purification rounds of large tissue extracts were necessary to achieve the purification and sequencing of most of these signal molecules. Nowadays, the combination of nanoscale liquid chromatography and the very sensitive tandem mass spectrometry allows us to identify and sequence peptides in very low concentration directly from tissue extracts. In this manuscript, we review previous data on the peptidome analysis of the locust corpora cardiaca, with emphasis on AKH processing. In addition, we report the peptide profiling of a single corpus cardiacum from Locusta migratoria. 23 peptides were isolated and sequenced in a single nano-LC-MS/MS experiment, demonstrating the sensitivity and effectiveness of mass spectrometry in peptide research.

In insects primary urine is produced by the Malpighian tubules under hormonal control. Here we have analysed the effects of the peptide locustatachykinin I (Lom-TK-I) on secretion in isolated Malphigian tubules. We also mapped the distribution of Lom-TK immunoreactivity in the gut in comparison with Locusta diuretic hormone (Lom-DH) and serotonin, two other factors that are active on locust tubules. Lom-TK-I produces an immediate, potent and long-lasting stimulation of fluid secretion. Furthermore, we show that Lom-TK-I acts synergistically with Lom-DH on fluid secretion and demonstrate that Lom-TKs are co-localised with Lom-DH in endocrine cells of the midgut ampullae. Thus, the two peptides might be released together to act synergistically on fluid secretion. Also serotonin and Lom-DH act synergistically and we can demonstrate a plexus of serotonin-containing axon processes over the midgut.

Originally named for its ability to stimulate juvenile hormone production by lepidopteran corpora allata, allatotropin has emerged as a neuropeptide with multiple neural, endocrine and myoactive roles. This paper describes the experimental evidence for allatotropin action, its localization in several species of insects, and its multiple effects on a variety of different tissues that lead to increased hemolymph circulation and gut motility. The overall physiological effects may also include species-specific effects such as the regulation of nutrient absorption, modulation of the circadian cycle and migratory preparedness. In addition, we present evidence suggesting that allatotropins are members of a family of myoactive peptides found in several invertebrate phyla. Finally, we speculate that the myoactive properties of allatotropins are basal and it is likely that the stimulatory action of allatotropins on juvenile hormone synthesis evolved secondarily.

The peptidomes of the corpora allata of Locusta migratoria and Schistocerca gregaria were investigated by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography quadrupole time-of-flight tandem mass spectrometry (nanoLC-Q-TOF MSMS). The pyrokinin (-FXPRLamide) family seems to be predominant. In addition to the known pyrokinins, we de novo sequenced four pyrokinins in L. migratoria and five in S. gregaria. In addition, one pyrokinin-like peptide (-PRLamide) was identified in S. gregaria. Besides the -(FX)PRLamides, FLRFamide-1, the allatostatins (A family) and numerous as yet unidentified peptides are also present in the corpora allata.

In the tobacco hornworm, Manduca sexta, pupal diapause can be induced by exposure of fifth-instar larvae to a short-day photoperiod. We studied the effect of surgical ablation of tissues containing the neurosecretory cells of the brain of fifth-instar larvae on the photoperiodic induction of pupal diapause. At the end of the experiments, we immunostained the neurosecretory cells to determine the success of the ablations. Under long-day conditions (LD 16:8 at 22 degrees C), all intact larvae, most of the sham-operated larvae, and control-operated larvae developed into nondiapausing pupae. Under short-day conditions (LD 10:14 at 22 degrees C), most intact, sham-operated, and control-operated larvae developed into diapausing pupae. Removal of type-II cells did not interfere with the photoperiodic response. Under long-day conditions, elimination of type-Ia(1) cells did not affect the incidence of nondiapausing pupae. When type-Ia(1) cells were removed under short-day conditions, however, the incidence of nondiapausing pupae was higher (51%, n = 41) than that of the intact (16%, n = 75), sham-operated (24%, n = 88), control-operated larvae (5%, n = 40), and larvae with type-II cells removed (11%, n = 27). Thus, removal of type-Ia(1) cells can impede induction of diapause. These results indicate that the type-Ia(1) neurosecretory cells have an important role in the induction of pupal diapause., (Copyright 2003 Wiley-Liss, Inc.)

Small neuropeptides of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family regulate energy metabolism in insects. Within lepidopterans, the nonapeptide Manduca sexta AKH (Manse-AKH) represents a widely occurring AKH, whereas the decapeptide Helze-HrTH (at first isolated from Heliothis zea) seems to be restricted to moths. Here we show that Vanca-AKH, a non-amidated undecapeptide which we recently found in the painted lady butterfly, Vanessa cardui, is also present in the retrocerebral complex of several other butterflies (Danaus plexippus, Precis coenia, Aglais urticae) and a moth (Spodoptera frugiperda). This study also demonstrates the power of modern nano-electrospray-quadrupole TOF tandem mass spectrometry in the sequence confirmation of peptides from minute amounts of small neuropeptides.

A new procedure for the measurement of adipokinetic hormone (AKH) concentrations in locust (Schistocerca gregaria) haemolymph is described: Haemolymph is extracted with chloroform/methanol/water and the aqueous layer is fractionated with reverse-phase cartridges and HPLC. The fractions corresponding to AKH-I (Lom-AKH-I) and AKH-II (Scg-AKH-II) are then measured in a competitive binding assay using specific antibodies and [3H]AKHs. The procedures could be applied to any peptides containing N-terminal pyroglutamate residues including all members of the adipokinetic/hyperglycaemic/red pigment concentrating hormone family. Results show that the concentrations of both AKH-I and AKH-II increase within 5 min of initiation of flight and are maintained at approx. 15-fold (AKH-I) and 6-fold (AKH-II) the resting levels over flights of at least 60 min. Poisoning of locusts with either the insecticide deltamethrin or with potassium chloride also caused release of hormones. Starvation for 6 h caused elevation of hormone levels in 5th instar nymphs, but starvation for 6 or 20 h had little effect on hormones in adults, despite an increase in haemolymph diacylglycerols at 20 h.

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.

Microsporidia (M) is a phylum of protists parasitizing obligatory in animal cells. Long way of adaptation of M to intracellular parasitism resulted in establishment of quite close relationships between the parasite and its host. Different species of M induce in their hosts symptoms similar to those caused by misbalance of juvenile hormone (JH) and ecdysone. M infection leads to pathology of different hormone-dependent functions such as cell differentiation and specialization, molting, metamorphosis, diapause and reproduction of insects. The signs of hormonal dysfunction evidence for elevated titer of JH in M-infected insects. Two possible explanation of this could be offered: JH secretion by M or specific influence of the parasites on the insect endocrine systems. Impact on insect endogenous JH titer by M could be mediated by affection of secretory activity of corpora allata or by suppression of enzymatic degradation of JH. According to different hypotheses, insect hormonal status during microsporidiosis could be modified by a) insect host stress-reaction, b) exhaustion of insect host reserves, characteristic for acute phase of the disease, c) destruction of infected insect cells and tissues during mass sporogenesis of M. Data found in literature and provided by our experiments evidence for presence of JH analogues or juvenilizing substance in the extracts of M spores. From detailed examination of pathological process it is also seen that juvenilizing effect of M infection is usually restricted to the invaded regions of tissues (i.e. expressed locally) but not a systemic one. Ability of M to modify morpho-functional features of infected tissues at the level of hormonal regulation is undoubtfully a prominent adaptation for stabilizing "microsporidia-insect" parasite-host systems.

General features of the changes in hemolymph PTTH titers during larval-pupal and pupal-adult development of the silkworm Bombyx mori were analyzed by comparing the patterns of the titer changes between different races and between silkworms reared under different environmental conditions. In common to all types of the silkworms tested, we observed low PTTH titers during the phagoperiod of the final instar, a small rise in PTTH titer on the day before wandering, two middle-sized peaks of the titer at the wandering and prepupal stages, high PTTH titers during early pupal-adult development, and a gradual titer increase shortly before adult eclosion. Increases in hemolymph PTTH titer were closely correlated with increases in ecdysteroid titers and with subsequent occurrences of morphological and behavioral changes characteristic of the initiation or progression of metamorphosis. The timing of the increase in hemolymph PTTH titer on the day of wandering was photoperiodically controlled, but that timing at the later stages seemed not to be influenced by the light-dark cycle., (Copyright 2002 Elsevier Science (USA))

An ultrastructural study of the antennae of the yellow fever mosquito, Aedes aegypti, revealed that as in the salt marsh mosquito, Culex salinarius, the first flagellar segment of both sexes of A. aegypti contain neuroendocrine cells. These cells not only extend an axon via the antennal nerve to the antennal lobe of the deutocerebrum, but project collaterals to the periphery of the antennae, where they modulate the antennal sensory neurons by forming synapses with the dendrites of these afferent neurons. To our knowledge, this is the first report in any animal of neurites of neuroendocrine cells forming axo-dendritic synapses with sensory neurons.

A Microwave-Assisted Process (MAP is a Trade-Mark of Her Majesty the Queen in Right of Canada as represented by the Minister of the Environment) solvent extraction procedure was used in conjunction with GC-MS analysis to investigate the chemical composition of dried and live cockroaches. The main components extracted were classified into four groups: sterols. fatty acids and their esters, long chain alkanes and fused aromatic hydrocarbons.

A very sensitive time-resolved fluoroimmunoassay for the prothoracicotropic hormone (PTTH) of the silkworm Bombyx mori has been established. The lower limit of detection in this assay was 0.1 pg. With this assay method, the amounts of PTTH in the central nervous system and hemolymph were quantified. PTTH was detected only in the brain within the central nervous system, and, in the fifth instar, its content in the brain increased gradually with larval growth and decreased rapidly after the beginning of wandering. A substantial amount of PTTH was also found in the retrocerebral complex of day-3 fifth instar larvae, accounting for 28% of total PTTH. The PTTH titer in hemolymph changed dramatically during Bombyx development, with a small peak in the middle of the fourth instar, medium-sized peaks at the wandering and prepupal stages in the fifth instar, and a large prolonged peak during early pupal-adult development. The changes were overall closely correlated with those in hemolymph ecdysteroid titer. However, some unexpected aspects of PTTH dynamics in hemolymph have also been disclosed. Based on these observations, the significance of PTTH secretion in the control of insect development is discussed.

A diuretic hormone (DH) was isolated from extracts of heads of Zootermopsis nevadensis, a dampwood termite. The peptide has 46 residues, M(r) = 5,328.2 Da, with the sequence TGAVPSLSIVNPLDVLRQRLLLEIARRRMRQSQDQIQANREMLQTI-NH(2,) showing it to be a CRF-related DH. This peptide increases cyclic AMP production in Malpighian tubules of Manduca sexta. We detected another factor in the head extracts which behaved as a more basic peptide on ion exchange chromatography. The latter factor also stimulated cyclic AMP production in the bioassay, but two large scale attempts to isolate this peptide were unsuccessful. We believe the second peptide is acid labile.

[His(7)]-corazonin has recently been identified in the corpora cardiaca (CC) of two locust species, the migratory locust, Locusta migratoria and the desert locust, Schistocerca gregaria, as the dark colour inducing neurohormone. Here, we investigate whether [His(7)]-corazonin occurs in the brain-CC axis of a Schistocerca albino strain. From data obtained by immunocytochemistry, injection experiments, chromatographic and mass spectrometric analysis of brain and CC tissues, it could be concluded that an albino strain of S. gregaria from Denmark contains authentic [His(7)]-corazonin. This was unequivocally demonstrated by sequencing the [His(7)]-corazonin-immunoreactive factor in albino Schistocerca brain-CC extracts with ESI-Qq-oa-TOF mass spectrometry. Albinism in this strain is hence not caused by the deficiency of authentic [His(7)]-corazonin in the brain-CC axis, nor by defects in release. Conversely to L. migratoria albinos, injection of [His(7)]-corazonin failed to induce dark pigmentation in Schistocerca albinos. Therefore, albinism in the investigated Schistocerca strain is likely to be situated at the level of the receptor, signal transduction mechanisms or of pigment biosynthesis.

The triple co-localisation of peptidergic material immunoreactive to antisera raised against allatostatins of the Y/FXFGL-NH2 type, Manduca sexta allatostatin (Mas-AS), and allatotropin has been demonstrated in a single pair of anterodorsal neurones in the frontal ganglion of the tomato moth, Lacanobia oleracea (Noctuidae). Another pair of posterior neurones contain only Y/FXFGL-NH2-type allatostatin immunoreactivity. The neurites of all four cells trifurcate, and axons project to the brain in the frontal connectives and to the foregut in the recurrent nerve. Axons from the anterior neurones, within the recurrent nerve, have prominent lateral branches supplying muscles of the crop, and axons from both anterior and posterior cells show profuse branching and terminal arborisations in the region of the stomodeal valve. The brain contributes Y/FXFGL-NH2-immunoreactive material, but not allatotropin or Mas-AS, to the recurrent nerve via NCC 1+2 and NCC 3. All three peptides have a reversible effect on the spontaneous (peristaltic) contractions of the foregut (crop) in vitro. Thus, both types of allatostatin are inhibitory at 10(-12) to 10(-7) M, whereas allatotropin is strongly myostimulatory at 10(-14) M. This is the first demonstration of the gut myoinhibitory effects of Mas-AS and, taken together with the effects of Y/FXFGL-NH2-type allatostatins and allatotropin, reveals a different functional aspect to that normally attributed to these three peptides, i.e. control of juvenile hormone synthesis by the corpus allatum.

Anatomical study of neurons projecting to the retrocerebral complex of the adult blow fly, Protophormia terraenovae, was done by NiCl2 filling and immunocytochemistry. Retrograde filling through the cardiac-recurrent nerve labeled three groups of neurons in the brain/subesophageal ganglion: (1) paramedial clusters of the pars intercerebralis, (2) neurons in each pars lateralis, and (3) neurons in the subesophageal ganglion. The pars intercerebralis neurons send prominent axons into the median bundle and exit from the brain via the contralateral nervus corporis cardiaci. Based on the projection pattern, two types of the pars lateralis neurons can be distinguished: the most lateral pairs of neurons contralaterally extend through the posterior lateral tract and the remainder ipsilaterally extend through the posterior lateral tract. The neurons in the subesophageal ganglion run through the contralateral nervus corporis cardiaci. The dendritic arborization of the pars intercerebralis and pars lateralis neurons is restricted to the superior protocerebral neuropil and to the anterior neuropil of the subesophageal ganglion where the neurons in the subesophageal ganglion also project. Retrograde filling from the corpus allatum indicated that the pars lateralis neurons and a few pars intercerebralis neurons project to the corpus allatum, but that the neurons in the subesophageal ganglion do not. Orthograde filling from the pars intercerebralis and staining by paraldehyde-thionin/paraldehyde-fuchsin indicated that the pars intercerebralis neurons project primarily to the corpus cardiacum/hypocerebral ganglion complex. Immunostaining with a polyclonal antiserum against diapause hormone, a member of the FXPRLamide family, suggests that some of the subesophageal ganglion neurons contain FXPRLamide-like peptides.

Knowledge of the structures of neuropeptides that regulate development, metabolism, and behaviour in insects is extensive, but nothing is known of the identity of regulatory peptides in the aphid neuroendocrine system. The present study applies a radioimmunoassay to reveal the existence of at least two allatostatin-like peptides in the aphid, Megoura viciae. Immunocytochemistry using antibodies recognising cockroach and dipteran allatostatins (Dip-AST-7 and Cav-AST-1) revealed the presence of allatostatin-like peptides in the protocerebrum of the brain, in the supraoesophageal ganglion, and in the fused thoracic ganglia. Both the corpora cardiaca and the corpus allatum, as well as the nervi corporis cardiaci I, stained strongly with the allatostatin antibodies. AKH/ HrTH-like peptides were detected in extracts of M. viciae using conspecific bioassays for hypertrehalosaemic and hyperlipaemic activity. Endocrine cells of the corpora cardiaca contained AKH-like material that reacted to antibodies directed to the N- and C-terminus of Lom-AKH-I. Antibodies specific for the C-terminus of Lom-AKH-I gave extensive staining in the brain and immunoreactive fibres were also found in the suboesophageal and fused thoracic ganglia. In contrast, staining with antibodies recognising the N-terminus of Lom-AKH-I was restricted to the corpora cardiaca and a region of the pars intercerebralis. There was no difference between apterous and alate morphs of M. viciae in the distribution of both AKH-like and allatostatin-like peptides. These results suggest an endocrine role for AKH/HrTH and allatostatin-like peptides in aphids.