Calcitonin-like Diuretic Hormone Research Articles

Multiple neuropeptides produced by sex-specific neurons control activity of the male accessory glands and gonoducts in the silkworm Bombyx mori

The male accessory glands (AG) and gonoducts of moths develop during metamorphosis and are essential for successful fertilization of females. We found that these reproductive organs are innervated by a sex-specific cluster of peptidergic neurons in the posterior 9th neuromere of the terminal abdominal ganglion (TAG). This cluster of ~20 neurons differentiate during metamorphosis to innervate the accessory glands and sperm ducts. Using immunohistochemistry and in situ hybridization (ISH) we showed that these neurons express four neuropeptide precursors encoding calcitonin-like diuretic hormone (CT-DH), allatotropin (AT) and AT-like peptides (ATLI-III), allatostatin C (AST-C), and myoinhibitory peptides (MIPs). We used contraction bioassay in vitro to determine roles of these neuropeptides in the gonoduct and accessory gland activity. Spontaneous contractions of the seminal vesicle and AG were stimulated in a dose depended manner by CT-DH and AT, whereas AST-C and MIP elicited dose dependent inhibition. Using quantitative RT-PCR we confirmed expression of receptors for these neuropeptides in organs innervated by the male specific cluster of neurons. Our results suggest a role of these neuropeptides in regulation of seminal fluid movements during copulation.

Calcitonin-like peptide hormone (CT/DH) in the frontal ganglia as a feeding regulatory peptide of the silkworm, Bombyx mori

The frontal ganglion (FrG) in insects contributes to the modulation of feeding behavior via the regulation of foregut contraction and other neural networks. Profiling the peptides of the FrG is important to understand endocrine regulation of feeding behavior in insects. High-resolution spiral matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified an ion peak, corresponding to calcitonin-like diuretic hormone 31 (CT/DH) in the FrG of silkworm Bombyx mori larvae. RT-PCR confirmed that CT/DH is expressed in the FrG, as are other peptide hormones, including allatoregulatory peptides. A feeding latency assay using synthetic CT/DH revealed that it increases the time to the initiation of feeding in a dose-dependent manner. These data indicate that CT/DH is a candidate regulatory peptide that modulates the feeding behavior of B. mori.

Molecular cloning, expression and identification of the promoter regulatory region for the neuropeptide trissin in the nervous system of the silkmoth Bombyx mori.

Trissin has recently been identified as a conserved insect neuropeptide, but its cellular expression and function is unknown. We detected the presence of this neuropeptide in the silkworm Bombyx mori using in silico search and molecular cloning. In situ hybridisation was used to examine trissin expression in the entire central nervous system (CNS) and gut of larvae, pupae and adults. Surprisingly, its expression is restricted to only two pairs of small protocerebral interneurons and four to five large neurons in the frontal ganglion (FG). These neurons were further characterised by subsequent multiple staining with selected antibodies against insect neuropeptides. The brain interneurons innervate edges of the mushroom bodies and co-express trissin with myoinhibitory peptides (MIP) and CRF-like diuretic hormones (CRF-DH). In the FG, one pair of neurons co-express trissin with calcitonin-like diuretic hormone (CT-DH), short neuropeptide F (sNPF) and MIP. These neurons innervate the brain tritocerebrum and musculature of the anterior midgut. The other pair of trissin neurons in the FG co-express sNPF and project axons to the tritocerebrum and midgut. We also used the baculovirus expression system to identify the promoter regulatory region of the trissin gene for targeted expression of various molecular markers in these neurons. Dominant expression of trissin in the FG indicates its possible role in the regulation of foregut-midgut contractions and food intake.

Peptidomics of Neuropeptidergic Tissues of the Tsetse Fly Glossina morsitans morsitans.

Neuropeptides and peptide hormones are essential signaling molecules that regulate nearly all physiological processes. The recent release of the tsetse fly genome allowed the construction of a detailed in silico neuropeptide database (International Glossina Genome Consortium, Science 344, 380-386 (2014)), as well as an in-depth mass spectrometric analysis of the most important neuropeptidergic tissues of this medically and economically important insect species. Mass spectrometric confirmation of predicted peptides is a vital step in the functional characterization of neuropeptides, as in vivo peptides can be modified, cleaved, or even mispredicted. Using a nanoscale reversed phase liquid chromatography coupled to a Q Exactive Orbitrap mass spectrometer, we detected 51 putative bioactive neuropeptides encoded by 19 precursors: adipokinetic hormone (AKH) I and II, allatostatin A and B, capability/pyrokinin (capa/PK), corazonin, calcitonin-like diuretic hormone (CT/DH), FMRFamide, hugin, leucokinin, myosuppressin, natalisin, neuropeptide-like precursor (NPLP) 1, orcokinin, pigment dispersing factor (PDF), RYamide, SIFamide, short neuropeptide F (sNPF) and tachykinin. In addition, propeptides, truncated and spacer peptides derived from seven additional precursors were found, and include the precursors of allatostatin C, crustacean cardioactive peptide, corticotropin releasing factor-like diuretic hormone (CRF/DH), ecdysis triggering hormone (ETH), ion transport peptide (ITP), neuropeptide F, and proctolin, respectively. The majority of the identified neuropeptides are present in the central nervous system, with only a limited number of peptides in the corpora cardiaca-corpora allata and midgut. Owing to the large number of identified peptides, this study can be used as a reference for comparative studies in other insects. Graphical Abstract ᅟ.

The contribution of the genomes of a termite and a locust to our understanding of insect neuropeptides and neurohormones.

The genomes of the migratory locust Locusta migratoria and the termite Zootermopsis nevadensis were mined for the presence of genes encoding neuropeptides, neurohormones, and their G-protein coupled receptors (GPCRs). Both species have retained a larger number of neuropeptide and neuropeptide GPCRs than the better known holometabolous insect species, while other genes that in holometabolous species appear to have a single transcript produce two different precursors in the locust, the termite or both. Thus, the recently discovered CNMa neuropeptide gene has two transcripts predicted to produce two structurally different CNMa peptides in the termite, while the locust produces two different myosuppressin peptides in the same fashion. Both these species also have a calcitonin gene, which is different from the gene encoding the calcitonin-like insect diuretic hormone. This gene produces two types of calcitonins, calcitonins A and B. It is also present in Lepidoptera and Coleoptera and some Diptera, but absent from mosquitoes and Drosophila. However, in holometabolous insect species, only the B transcript is produced. Their putative receptors were also identified. In contrast, Locusta has a highly unusual gene that codes for a salivation stimulatory peptide. The Locusta genes for neuroparsin and vasopressin are particularly interesting. The neuroparsin gene produces five different transcripts, of which only one codes for the neurohormone identified from the corpora cardiaca. The other four transcripts code for neuroparsin-like proteins, which lack four amino acid residues, and that for that reason we called neoneuroparsins. The number of transcripts for the neoneuroparsins is about 200 times larger than the number of neuroparsin transcripts. The first exon and the putative promoter of the vasopressin genes, of which there are about seven copies in the genome, is very well-conserved, but the remainder of these genes is not. The relevance of these findings is discussed.

The excretion of NaCl and KCl loads in mosquitoes. 1. Control data.

The handling of Na(+) and K(+) loads was investigated in isolated Malpighian tubules and in whole mosquitoes of Aedes aegypti. Isolated Malpighian tubules bathed in Na(+)-rich Ringer solution secreted Na(+)-rich fluid, and tubules bathed in K(+)-rich Ringer solution secreted K(+)-rich fluid. Upon Na(+) loading the hemolymph, the mosquito removed 77% the injected Na(+) within the next 30 min. The rapid onset and magnitude of this diuresis and the excretion of more Na(+) than can be accounted for by tubular secretion in vitro is consistent with the release of the calcitonin-like diuretic hormone in the mosquito to remove the Na(+) load from the hemolymph. Downstream, K(+) was reabsorbed with water in the hindgut, which concentrated Na(+) in excreted urine hyperosmotic to the hemolymph. Upon K(+) loading the hemolymph, the mosquito took 2 h to remove 100% of the injected K(+) from the hemolymph. The excretion of K(+)-rich isosmotic urine was limited to clearing the injected K(+) from the hemolymph with a minimum of Cl(-) and water. As a result, 43.3% of the injected Cl(-) and 48.1% of the injected water were conserved. The cation retained in the hemolymph with Cl(-) was probably N-methyl-d-glucamine, which replaced Na(+) in the hemolymph injection of the K(+) load. Since the tubular secretion of K(+) accounts for the removal of the K(+) load from the hemolymph, the reabsorption of K(+), Na(+), Cl(-), and water must be inhibited in the hindgut. The agents mediating this inhibition are unknown.

Structure–activity relationships of two Rhodnius prolixus calcitonin-like diuretic hormone analogs

The calcitonin-like diuretic hormone (CT/DH) in Rhodnius prolixus influences various tissues associated with feeding-related physiological events. The receptors for this peptide have also been identified and shown to be expressed in these tissues. In the present study, we have investigated the effects of two R. prolixus CT/DH analogs (full-length form and N-terminal truncated form) on hindgut contractions and in a heterologous receptor expression system. The analogs contained the amino acid methyl-homoserine in place of methionine in order to prevent them from being oxidized and thus increase their stability. The full-length form of the analog retained all of its activity in our assays when compared to the endogenous peptide. Truncated analog displayed no activity in our assays.

Isolation and Functional Characterization of Calcitonin-Like Diuretic Hormone Receptors in Rhodnius prolixus

Several families of diuretic hormones exist in insects, one of which is the calcitonin-like diuretic hormone (CT/DH) family. CT/DH mediates its effects by binding to family B G-protein coupled receptors (GPCRs). Here we isolate and functionally characterize two R. prolixus CT/DH receptor paralogs (Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2) using a novel heterologous assay utilizing a modified human embryonic kidney 293 (HEK293) cell line. Rhopr-CT/DH-R1 is orthologous to the previously characterized D. melanogaster CT/DH receptor (CG17415) while Rhopr-CT/DH-R2 is orthologous to the D. melanogaster receptor (CG4395), an orphan receptor whose ligand was unknown until now. We determine the cDNA sequences of three splice variants encoding Rhopr-CT/DH-R1 (Rhopr-CT/DH-R1-A, Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C) and two splice variants encoding Rhopr-CT/DH-R2 (Rhopr-CT/DH-R2-A and Rhopr-CT/DH-R2-B). Rhopr-CT/DH-R1-A and Rhopr-CT/DH-R2-A encode truncated receptors that lack six and seven of the characteristic seven transmembrane domains, respectively. Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C, which only differ by 2 amino acids in their C-terminal domain, can both be activated by Rhopr-CT/DH at equal sensitivities (EC50 = 200-300nM). Interestingly, Rhopr-CT/DH-R2-B is much more sensitive to Rhopr-CT/DH (EC50 = 15nM) compared to Rhopr-CT/DH-R1-B/C and also yields a much greater response (amplitude) in our heterologous assay. This is the first study to reveal that insects possess at least two CT/DH receptors, which may be functionally different. Quantitative PCR demonstrates that Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2 have distinct expression patterns, with both receptors expressed centrally and peripherally. Moreover, the expression analysis also identified novel target tissues for this neuropeptide, including testes, ovaries and prothoracic glands, suggesting a possible role for Rhopr-CT/DH in reproductive physiology and development.

Calcitonin receptor 1 (AedaeGPCRCAL1) hindgut expression and direct role in myotropic action in females of the mosquito Aedes aegypti (L.)

In anautogenous mosquitoes such as Aedes aegypti females the calcitonin-like diuretic hormone 31 (DH31) stimulates natriuretic fluid excretion from the Malpighian tubules (MTs) after a blood meal. We previously cloned and functionally characterized AedaeGPCRcal1 from A. aegypti, the ortholog of the Drosophila melanogaster DH31 receptor and immunolocalized it in the MTs. However, localization of the calcitonin receptor-like receptor 1 (GPCRCAL1) in the hindgut of any insect is unknown, and specifically, knowledge on its role in hindgut contraction in response to Aedae-DH31 peptide is lacking. We analyzed the expression of AedaeGPCRCAL1 in hindgut by western blot and immunohistochemisty, and evaluated its role in hindgut contractility by application of Aedae-DH31 before and after receptor RNA interference (RNAi). The receptor was detected as a 73 kDa band in western blots of hindgut and immunofluorescence revealed the receptor was expressed in hindgut circular and longitudinal muscles but not in the hindgut epithelial cells. In vitro, incubation in 1 μM solution of Aedae-DH31 peptide significantly increased the hindgut contraction frequency in normal mosquitoes. Hindguts from females treated with AedaeGPCRcal1 dsRNA and incubated with DH31 showed a reduction of 50% percent in their contraction frequency with respect to controls. These results suggest that DH31 hormone released from the brain post-blood meal has a direct and coordinative action on the excretory system, MTs and hindgut, by which AedaeGPCRCAL1 signaling stimulates MT primary urine secretion and hindgut contraction resulting in rapid postprandial fluid excretion.

Role in diuresis of a calcitonin receptor (GPRCAL1) expressed in a distal-proximal gradient in renal organs of the mosquito Aedes aegypti (L.).

Evolution of anthropophilic hematophagy in insects resulted in the coordination of various physiological processes for survival. In female mosquitoes, a large blood meal provides proteins for egg production and as a trade-off, rapid elimination of the excess water and solutes (Na+, Cl−) is critical for maintaining homeostasis and removing excess weight to resume flight and avoid predation. This post-prandial excretion is achieved by the concerted action of multiple hormones. Diuresis and natriuresis elicited by the calcitonin-like diuretic hormone 31 (DH31) are believed to be mediated by a yet uncharacterized calcitonin receptor (GPRCAL) in the mosquito Malpighian tubules (MTs), the renal organs. To contribute knowledge on endocrinology of mosquito diuresis we cloned GPRCAL1 from MT cDNA. This receptor is the ortholog of the DH31 receptor from Drosophila melanogaster that is expressed in principal cells of the fruit fly MT. Immunofluorescence similarly showed AaegGPRCAL1 is present in MT principal cells in A. aegypti, however, exhibiting an overall gradient-like pattern along the tubule novel for a GPCR in insects. Variegated, cell-specific receptor expression revealed a subpopulation of otherwise phenotypically similar principal cells. To investigate the receptor contribution to fluid elimination, RNAi was followed by urine measurement assays. In vitro, MTs from females that underwent AaegGPRcal1 knock-down exhibited up to 57% decrease in the rate of fluid secretion in response to DH31. Live females treated with AaegGPRcal1 dsRNA exhibited 30% reduction in fluid excreted after a blood meal. The RNAi-induced phenotype demonstrates the critical contribution of this single secretin-like family B GPCR to fluid excretion in invertebrates and highlights its relevance for the blood feeding adaptation. Our results with the mosquito AaegGPRCAL1 imply that the regulatory function of calcitonin-like receptors for ion and fluid transport in renal organs arose early in evolution.

Calcitonin-like diuretic hormones in insects

Insect neuropeptides control various biological processes including growth, development, homeostasis and reproduction. The calcitonin-like diuretic hormone (CT/DH) is one such neuropeptide that has been shown to affect salt and water transport by Malpighian tubules of several insects. With an increase in the number of sequenced insect genomes, CT/DHs have been predicted in several insect species, making it easier to characterize the gene encoding this hormone and determine its function in the species in question. This mini review summarizes the current knowledge on insect CT/DHs, focusing on mRNA and peptide structures, distribution patterns, physiological roles, and receptors in insects.

Genomic analyses of the Daphnia pulex peptidome

Genome mining has provided a valuable tool for peptide discovery in many species, yet no crustacean has undergone this analysis. Currently, the only crustacean with a sequenced genome is the cladoceran Daphnia pulex, a model organism in many fields of biology. Here, we have mined the D. pulex genome for peptide-encoding genes. For each gene identified, the encoded precursor protein was deduced, and its mature peptides predicted. Twenty-four peptide-encoding genes were identified, including ones predicted to produce members of the A-type allatostatin, B-type allatostatin, C-type allatostatin, allatotropin (ATR), bursicon α, bursicon β, calcitonin-like diuretic hormone, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone, ecdysis-triggering hormone, eclosion hormone (EH), insulin-like peptide (ILP), molt-inhibiting hormone, neuropeptide F, orcokinin (two genes), pigment-dispersing hormone, proctolin, red pigment concentrating hormone/adipokinetic hormone (RPCH/AKH), short neuropeptide F, SIFamide, sulfakinin, and tachykinin-related peptide (TRP) families/subfamilies. In total, 96 peptides were predicted from these genes. Our identification of isoforms of corazonin, EH, ILP, proctolin, RPCH/AKH, sulfakinin and TRP are the first for D. pulex, while our prediction of ATR from this species is the first from any crustacean. The number of peptides predicted in our study shows the power of genome mining for peptide discovery, and provides a model for future genomic analyses of the peptidomes of other crustaceans. In addition, the data presented in our study provide foundations for future molecular, biochemical, anatomical, and physiological investigation of peptidergic signaling in D. pulex and other cladoceran species.

Bioinformatic prediction of arthropod/nematode-like peptides in non-arthropod, non-nematode members of the Ecdysozoa

The Onychophora, Priapulida and Tardigrada, along with the Arthropoda, Nematoda and several other small phyla, form the superphylum Ecdysozoa. Numerous peptidomic studies have been undertaken for both the arthropods and nematodes, resulting in the identification of many peptides from each group. In contrast, little is known about the peptides used as paracrines/hormones by species from the other ecdysozoan taxa. Here, transcriptome mining and bioinformatic peptide prediction were used to identify peptides in members of the Onychophora, Priapulida and Tardigrada, the only non-arthropod, non-nematode members of the Ecdysozoa for which there are publicly accessible expressed sequence tags (ESTs). The extant ESTs for each phylum were queried using 106 arthropod/nematode peptide precursors. Transcripts encoding calcitonin-like diuretic hormone and pigment-dispersing hormone (PDH) were identified for the onychophoran Peripatopsis sedgwicki, with transcripts encoding C-type allatostatin (C-AST) and FMRFamide-like peptide identified for the priapulid Priapulus caudatus. For the Tardigrada, transcripts encoding members of the A-type allatostatin, C-AST, insect kinin, orcokinin, PDH and tachykinin-related peptide families were identified, all but one from Hypsibius dujardini (the exception being a Milnesium tardigradum orcokinin-encoding transcript). The proteins deduced from these ESTs resulted in the prediction of 48 novel peptides, six onychophoran, eight priapulid and 34 tardigrade, which are the first described from these phyla.

Bioinformatic analyses of the publicly accessible crustacean expressed sequence tags (ESTs) reveal numerous novel neuropeptide-encoding precursor proteins, including ones from members of several little studied taxa

ESTs have been generated for many crustacean species, providing an invaluable resource for peptide discovery in members of this arthropod subphylum. Here, these data were mined for novel peptide-encoding transcripts, with the mature peptides encoded by them predicted using a combination of online peptide prediction programs and homology to known arthropod sequences. In total, 70 mature full-length/partial peptides representing members of 16 families/subfamilies were predicted, the vast majority being novel; the species from which the peptides were identified included members of the Branchiopoda ( Daphnia carinata and Triops cancriformis), Maxillopoda ( Caligus clemensi, Caligus rogercresseyi, Lepeophtheirus salmonis and Lernaeocera branchialis) and Malacostraca ( Euphausia superba, Marsupenaeus japonicus, Penaeus monodon, Homarus americanus, Petrolisthes cinctipes, Callinectes sapidus and Portunus trituberculatus). Of particular note were the identifications of an intermediate between the insect adipokinetic hormones and crustacean red pigment concentrating hormone and a modified crustacean cardioactive peptide from the daphnid D. carinata; Arg 7-corazonin was also deduced from this species, the first identification of a corazonin from a non-decapod crustacean. Our data also include the first reports of members of the calcitonin-like diuretic hormone, FMRFamide-related peptide (neuropeptide F subfamily) and orcokinin families from members of the Copepoda. Moreover, the prediction of a bursicon α from the euphausid E. superba represents the first peptide identified from any member of the basal eucaridean order Euphausiacea. In addition, large collections of insect eclosion hormone- and neuroparsin-like peptides were identified from a variety of species, greatly expanding the number of known members of these families in crustaceans.

Identification of a calcitonin-like diuretic hormone that functions as an intrinsic modulator of the American lobster,Homarus americanus, cardiac neuromuscular system

In insects, a family of peptides with sequence homology to the vertebrate calcitonins has been implicated in the control of diuresis, a process that includes mixing of the hemolymph. Here, we show that a member of the insect calcitonin-like diuretic hormone (CLDH) family is present in the American lobster, Homarus americanus, serving, at least in part, as a powerful modulator of cardiac output. Specifically, during an ongoing EST project, a transcript encoding a putative H. americanus CLDH precursor was identified; a full-length cDNA was subsequently cloned. In silico analyses of the deduced prepro-hormone predicted the mature structure of the encoded CLDH to be GLDLGLGRGFSGSQAAKHLMGLAAANFAGGPamide (Homam-CLDH), which is identical to a known Tribolium castaneum peptide. RT-PCR tissue profiling suggests that Homam-CLDH is broadly distributed within the lobster nervous system, including the cardiac ganglion (CG), which controls the movement of the neurogenic heart. RT-PCR analysis conducted on pacemaker neuron- and motor neuron-specific cDNAs suggests that the motor neurons are the source of the CLDH message in the CG. Perfusion of Homam-CLDH through the isolated lobster heart produced dose-dependent increases in both contraction frequency and amplitude and a dose-dependent decrease in contraction duration, with threshold concentrations for all parameters in the range 10(-11) to 10(-10) mol l(-1) or less, among the lowest for any peptide on this system. This report is the first documentation of a decapod CLDH, the first demonstration of CLDH bioactivity outside the Insecta, and the first detection of an intrinsic neuropeptide transcript in the crustacean CG.

Biological activity of diuretic factors on the anterior midgut of the blood-feeding bug, Rhodnius prolixus

Probing of a host and ingestion of a blood-meal in a fifth instar Rhodnius prolixus results in a cascade of tightly integrated events, including salivary gland secretion, plasticisation of the abdominal cuticle, increased ion and water movement across the anterior midgut (crop) and Malpighian tubules (which rapidly produce urine) and the regular expulsion of urine from the hindgut. In this study we have focussed on the role of the anterior midgut during the rapid postprandial diuresis. The huge blood-meal is pumped into the anterior midgut, during feeding, then modified by diuresis and stored until it is digested. Changes in the anterior midgut activity are rapid. Within minutes of the commencement of feeding there is an increase in the frequency of anterior midgut contractions and diuresis begins with the movement of salt and water across the epithelium of the anterior midgut into the haemolymph. While serotonin, a diuretic hormone in R. prolixus, is known to play a role in the physiological activity of the anterior midgut, we were interested in exploring further the role of serotonin, and other diuretic peptides. We have tested the activity of several peptides, including R. prolixus calcitonin-like diuretic hormone (Rhopr-DH 31), corticotropin-releasing factor (CRF)-like peptide from Zootermopsis nevadensis DH (Zoone-DH) and a kinin from Leucophaea maderae, Leucokinin 1 (LK1). These peptides families are known to be present in the central nervous system of R. prolixus, are putative neurohormones released into the haemolymph after the start of feeding, and have been shown to have activity on a variety of tissues involved in post-feeding diuresis. We show here that both serotonin and Zoone-DH increase the cAMP content of the anterior midgut and that serotonin, Zoone-DH and cAMP analogues increase absorption of water from the anterior midgut, increase the short circuit current and voltage, while decreasing the resistance across the epithelium. While LK1 and Rhopr-DH 31 do not significantly increase absorption, or short circuit current, LK1 does significantly decrease the resistance and transepithelial voltage of the anterior midgut epithelium. All of the factors studied increase the frequency of contractions of the anterior midgut.

Identification of putative peptide paracrines/hormones in the water flea Daphnia pulex (Crustacea; Branchiopoda; Cladocera) using transcriptomics and immunohistochemistry

The cladoceran crustacean Daphnia pulex has emerged as a model species for many biological fields, in particular environmental toxicology and toxicogenomics. Recently, this species has been the subject of an extensive transcriptome project, resulting in the generation and public deposition of over 150,000 expressed sequence tags (ESTs). This resource makes D. pulex an excellent model for protein discovery using bioinformatics. Here, in silico searches of the D. pulex EST database were conducted to identify transcripts encoding putative peptide precursors. Moreover, the mature peptides contained within the deduced prepro-hormones were predicted using online peptide processing programs and homology to known arthropod isoforms. In total, 63 putative peptide-encoding ESTs were identified encompassing 14 distinct peptide families/subfamilies: A-type allatostatin, B-type allatostatin, C-type allatostatin, bursicon (both α and β subunit peptides), crustacean cardioactive peptide (CCAP), crustacean hyperglycemic hormone (CHH)/ion transport peptide (both CHH- and moult-inhibiting hormone-like subfamilies), diuretic hormone (calcitonin-like), ecdysis-triggering hormone (ETH), FMRFamide (both neuropeptide F and short neuropeptide F subfamilies), orcokinin and pigment dispersing hormone. From these transcripts, the structures of 76 full-length/partial peptides were predicted, which included the first C-type allatostatin-like peptide identified from a crustacean, the first crustacean calcitonin-like diuretic hormone, an undescribed CCAP isoform, two hitherto unknown ETH variants, and two new orcokinins. Neuronal localization of several of the identified peptide families was confirmed using immunohistochemitry ( i.e. A-type allatostatin, CCAP, FMRFamide and PDH). In addition, immunohistochemical analyses identified other putative neuropeptides for which no ESTs had been found ( i.e. corazonin, insect kinin, proctolin, red pigment concentrating hormone, SIFamide, sulfakinin and tachykinin-related peptide). Collectively, the data presented here not only catalog an extensive array of putative D. pulex peptide paracrines/hormones, but also provide a strong foundation for future investigations of the effects of environmental/anthropogenic stressors on peptidergic control in this model organism.

Amino acid sequence and biological activity of a calcitonin-like diuretic hormone (DH31) from Rhodnius prolixus

Diuresis in the blood-gorging hemipteran Rhodnius prolixus is under neurohormonal control and involves a variety of processes and tissues. These include ion and water movement across the epithelium of the crop and the Malpighian tubules, and muscle contractions of the crop, hindgut and dorsal vessel, which facilitate mixing of the blood-meal, mixing of the haemolymph, as well as the expulsion of waste. One of the neurohormones that might play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH(31)) family of insect peptides. Previously we have demonstrated the presence of DH(31)-like peptides in the central nervous system (CNS) and gut of R. prolixus 5th instars. In the present work, a DH(31) from the CNS of 5th instar R. prolixus was isolated using reversed-phase liquid chromatography (RPLC), monitored with an enzyme-linked immunosorbent assay (ELISA) combined with matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, and sequenced using tandem mass spectrometry and Edman degradation. This neuropeptide is the first to be sequenced in R. prolixus and has a sequence identical to that found previously for Dippu-DH(31) from the cockroach Diploptera punctata. In previous studies testing Rhopr/Dippu-DH(31) in Malpighian tubule secretion assays, we demonstrated increases in the rate of secretion that were small, relative to that induced by serotonin, but nevertheless 14-fold over baseline. In the present study, we investigated second messenger pathways in response to Rhopr/Dippu-DH(31) and found no increase or decrease in cyclic adenosine monophosphate (cyclic AMP) content of the Malpighian tubules. DH(31)-like immunoreactivity is present over the dorsal hindgut, anterior dorsal vessel and dorsal diaphragm, and bioassays of the R. prolixus dorsal vessel and hindgut indicate that Rhopr/Dippu-DH(31) increases the frequency of muscle contractions of both tissues. Second messenger pathways were also investigated for the dorsal vessel and hindgut.

Distribution and activity of a Dippu DH 31-like peptide in the large milkweed bug Oncopeltus fasciatus

The milkweed bug, Oncopeltus fasciatus, is a plant feeding hemipteran. While there has been much research done on the neurohormonal control of the post-feeding diuresis in the blood-feeding hemipteran, Rhodnius prolixus, little is known about the control of the post-feeding diuresis in O. fasciatus. One of the neurohormones that may play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH 31) family of insect peptides. In this study we demonstrate the presence of DH 31-like immunoreactivity in the central nervous system (CNS) and gut of O. fasciatus 5th instars. As well, DH 31-like material was quantified and partially purified from the CNS of 5th instar O. fasciatus using reversed-phase liquid chromatography (RPLC) and monitored with an enzyme-linked immunosorbent assay (ELISA). When tested on O. fasciatus 5th instar Malpighian tubules, DH 31-like peptides significantly increased the rate of secretion over saline controls. The results suggest that there is a DH 31-like peptide(s) present in the CNS of O. fasciatus and that this peptide may play a role in the control of Malpighian tubule secretion.

Central peptidergic ensembles associated with organization of an innate behavior

At the end of each developmental stage, insects perform the ecdysis sequence, an innate behavior necessary for shedding the old cuticle. Ecdysis triggering hormones (ETHs) initiate these behaviors through direct actions on the CNS. Here, we identify the ETH receptor (ETHR) gene in the moth Manduca sexta, which encodes two subtypes of GPCR (ETHR-A and ETHR-B). Expression of ETHRs in the CNS coincides precisely with acquisition of CNS sensitivity to ETHs and behavioral competence. ETHR-A occurs in diverse networks of neurons, producing both excitatory and inhibitory neuropeptides, which appear to be downstream signals for behavior regulation. These peptides include allatostatins, crustacean cardioactive peptide (CCAP), calcitonin-like diuretic hormone, CRF-like diuretic hormones (DHs) 41 and 30, eclosion hormone, kinins, myoinhibitory peptides (MIPs), neuropeptide F, and short neuropeptide F. In particular, cells L(3,4) in abdominal ganglia coexpress kinins, DH41, and DH30, which together elicit the fictive preecdysis rhythm. Neurons IN704 in abdominal ganglia coexpress CCAP and MIPs, whose joint actions initiate the ecdysis motor program. ETHR-A also is expressed in brain ventromedial cells, whose release of EH increases excitability in CCAP/MIP neurons. These findings provide insights into how innate, centrally patterned behaviors can be orchestrated via recruitment of peptide cotransmitter neurons.