Sulfakinin Receptors Research Articles

Activation of the G protein-coupled sulfakinin receptor inhibits blood meal intake in the mosquito Aedes aegypti.

Little is known about the blood-feeding physiology of arbovirus vector Aedes aegypti although this type of mosquito is known to transmit infectious diseases dengue, Zika, yellow fever, and chikungunya. Blood feeding in the female A. aegypti mosquito is essential for egg maturation and for transmission of disease agents between human subjects. Here, we identify the A. aegypti sulfakinin receptor gene SKR from the A. aegypti genome and show that SKR is expressed at different developmental stages and in varied anatomical localizations in the adult mosquito (at three days after eclosion), with particularly high expression in the CNS. Knockingdown sulfakinin and sulfakinin receptor gene expression in the female A. aegypti results in increased blood meal intake, but microinjection in the thorax of the sulfakinin peptide 1 and 2 both inhibits dose dependently blood meal intake (and delays the time course of blood intake), which is reversible with receptor antagonist. Sulfakinin receptor expressed ectopically in mammalian cells CHO-K1 responds to sulfakinin stimulation with persistent calcium spikes, blockable with receptor antagonist. These data together suggest that activation of the Gq protein-coupled (i.e., calcium-mobilizing) sulfakinin receptor inhibits blood meal intake in female A. aegypti mosquitoes and could serve as a strategic node for the future control of A. aegypti mosquito reproduction/population and disease transmission.

Identification of sulfakinin receptor regulating feeding behavior and hemolymph trehalose homeostasis in the silkworm, Bombyx mori

Feeding behavior, the most fundamental physiological activity, is controlled by two opposing groups of factors, orexigenic and anorexigenic factors. The sulfakinin family, an insect analogue of the mammalian satiety factor cholecystokinin (CCK), has been shown to suppress food intake in various insects. Nevertheless, the mechanisms through which sulfakinin regulates feeding behavior remain a biological question. This study aimed to elucidate the signaling pathway mediated by the anorexigenic peptide sulfakinin in Bombyx mori. We identified the Bombyx mori neuropeptide G protein-coupled receptor A9 (BNGR-A9) as the receptor for sulfakinin through functional assays. Stimulation with sulfakinin triggered a swift increase in intracellular IP3, Ca2+, and a notable enhancement of ERK1/2 phosphorylation, in a manner sensitive to a Gαq-specific inhibitor. Treatment with synthetic sulfakinin resulted in decreased food consumption and average body weight. Additionally, administering synthetic sulfakinin to silkworms significantly elevated hemolymph trehalose levels, an effect markedly reduced by pre-treatment with BNGR-A9 dsRNA. Consequently, our findings establish the sulfakinin/BNGR-A9 signaling pathway as a critical regulator of feeding behavior and hemolymph trehalose homeostasis in Bombyx mori, highlighting its roles in the negative control of food intake and the positive regulation of energy balance.

Identification and functional characterization of the sulfakinin and sulfakinin receptor in the Chinese white pine beetle Dendroctonus armandi

The sulfakinin (SK) is an important signal molecule. As a neuromodulator, it mediates a variety of behavioral processes and physiological functions in invertebrates through the interaction with G-protein-coupled receptors (GPCRs). However, there is no report on the functional role of SK in the Chinese white pine beetle, Dendroctonus armandi. We have cloned and characterized SK and SKR genes in the D. armandi and carried out bioinformatics predictions on the basis of the deduced amino acid sequences, which are very similar to those from Dendroctonus ponderosa. The expression levels of the two genes were different between male and female adults, and there were significant changes in different developmental stages, tissues, and between starvation and following re-feeding states. Additionally, RNA-interference (RNAi) using double-stranded RNA to knock down SK and SKR reduced the transcription levels of the target genes and increased their body weight. In parallel, injection of SK caused a significant reduction in body weight and increase in mortality of D. armandi and also led to an increase in trehalose and a decrease in glycogen and free fatty acid. The results show that the SK signal pathway plays a positive and significant role in feeding regulation and provides a potential molecular target for the control of this pest.

Analysis of rhodopsin G protein-coupled receptor orthologs reveals semiochemical peptides for parasite (Schistosoma mansoni) and host (Biomphalaria glabrata) interplay

Schistosomiasis is a medically significant disease caused by helminth parasites of the genus Schistosoma. The schistosome life cycle requires chemically mediated interactions with an intermediate (aquatic snail) and definitive (human) host. Blocking parasite development within the snail stage requires improved understanding of the interactions between the snail host and the Schistosoma water-borne free-living form (miracidium). Innovations in snail genomics and aquatic chemical communication provide an ideal opportunity to explore snail-parasite coevolution at the molecular level. Rhodopsin G protein-coupled receptors (GPCRs) are of particular interest in studying how trematode parasites navigate towards their snail hosts. The potential role of GPCRs in parasites makes them candidate targets for new antihelminthics that disrupt the intermediate host life-cycle stages, thus preventing subsequent human infections. A genomic-bioinformatic approach was used to identify GPCR orthologs between the snail Biomphalaria glabrata and miracidia of its obligate parasite Schistosoma mansoni. We show that 8 S. mansoni rhodopsin GPCRs expressed within the miracidial stage share overall amino acid similarity with 8 different B. glabrata rhodopsin GPCRs, particularly within transmembrane domains, suggesting conserved structural features. These GPCRs include an orphan peptide receptor as well as several with strong sequence homologies with rhabdomeric opsin receptors, a serotonin receptor, a sulfakinin (SK) receptor, an allatostatin-A (buccalin) receptor and an FMRFamide receptor. Buccalin and FMRFa peptides were identified in water conditioned by B. glabrata, and we show synthetic buccalin and FMRFa can stimulate significant rates of change of direction and turn-back responses in S. mansoni miracidia. Ortholog GPCRs were identified in S. mansoni miracidia and B. glabrata. These GPCRs may detect similar ligands, including snail-derived odorants that could facilitate miracidial host finding. These results lay the foundation for future research elucidating the mechanisms by which GPCRs mediate host finding which can lead to the potential development of novel anti-schistosome interventions.

Sulfakinins influence lipid composition and insulin-like peptides level in oenocytes of Zophobas atratus beetles

Insect sulfakinins are pleiotropic neuropeptides with the homology to vertebrate gastrin/cholecystokinin peptide family. They have been identified in many insect species and affect different metabolic processes. They have a strong influence on feeding and digestion as well as on carbohydrate and lipid processing. Our study reveals that sulfakinins influence fatty acids composition in Zophobas atratus oenocytes and regulate insulin-like peptides (ILPs) level in these cells. Oenocytes are cells responsible for maintenance of the body homeostasis and have an important role in the regulation of intermediary metabolism, especially of lipids. To analyze the lipid composition in oenocytes after sulfakinins injections we used gas chromatography combined with mass spectrometry and for ILPs level determination an immunoenzymatic test was used. Because sulfakinin peptides and their receptors are the main components of sulfakinin signaling, we also analyzed the presence of sulfakinin receptor transcript (SKR2) in insect tissues. We have identified for the first time the sulfakinin receptor transcript (SKR2) in insect oenocytes and found its distribution more widespread in the peripheral tissues (gut, fat body and haemolymph) as well as in the nervous and neuro-endocrine systems (brain, ventral nerve cord, corpora cardiaca/corpora allata CC/CA) of Z. atratus larvae. The presence of sulfakinin receptor transcript (SKR2) in oenocytes suggests that observed effects on oenocytes lipid and ILPs content may result from direction action of these peptides on oenocytes.

Cholecystokinin-like peptide mediates satiety by inhibiting sugar attraction.

Feeding is essential for animal survival and reproduction and is regulated by both internal states and external stimuli. However, little is known about how internal states influence the perception of external sensory cues that regulate feeding behavior. Here, we investigated the neuronal and molecular mechanisms behind nutritional state-mediated regulation of gustatory perception in control of feeding behavior in the brown planthopper and Drosophila. We found that feeding increases the expression of the cholecystokinin-like peptide, sulfakinin (SK), and the activity of a set of SK-expressing neurons. Starvation elevates the transcription of the sugar receptor Gr64f and SK negatively regulates the expression of Gr64f in both insects. Interestingly, we found that one of the two known SK receptors, CCKLR-17D3, is expressed by some of Gr64f-expressing neurons in the proboscis and proleg tarsi. Thus, we have identified SK as a neuropeptide signal in a neuronal circuitry that responds to food intake, and regulates feeding behavior by diminishing gustatory receptor gene expression and activity of sweet sensing GRNs. Our findings demonstrate one nutritional state-dependent pathway that modulates sweet perception and thereby feeding behavior, but our experiments cannot exclude further parallel pathways. Importantly, we show that the underlying mechanisms are conserved in the two distantly related insect species.

Identification of sulfakinin receptors (SKR) in Tenebrio molitor beetle and the influence of sulfakinins on carbohydrates metabolism

Sulfakinins (SKs) are pleiotropic neuropeptides commonly found in insects, structurally and functionally homologous to the mammalian gastrin/cholecystokinin (CCK) neuropeptides. SKs together with sulfakinin receptors (SKRs) are involved in sulfakinin signaling responsible for variety of biological functions, including food intake or fatty acid metabolism. In the present study, we determined the distribution of SKRs in Tenebrio molitor larvae and characterized the impact of nonsulfated and sulfated SKs on carbohydrates and insulin-like peptides (ILPs) level in beetle hemolymph. Our results indicate the presence of both sulfakinin receptors, SKR1 and SKR2, in the nervous system of T. molitor. The distribution of SKR2 in peripheral tissues was more widespread than SKR1, and their transcripts have been found in fat body, gut and hemolymph. This is also the first evidence for SKRs presence in insect hemocytes indicating immunotropic activity of SKs. Moreover, in the present study, we have demonstrated that SKs regulate ILPs and carbohydrates level in insect hemolymph, and that sulfation is not crucial for peptides activity. Our study confirms the role of SKs in maintaining energy homeostasis in beetles.

Identification, Functional Characterization, and Pharmacological Analysis of Two Sulfakinin Receptors in the Medically-Important Insect Rhodnius prolixus

The chordate gastrin/cholecystokinin and ecdysozoan sulfakinin (SK)-signaling systems are functionally and structurally homologous. In the present study, we isolated the cDNA sequences encoding the SK receptors in Rhodnius prolixus (Rhopr-SKR-1 and Rhopr-SKR-2). The Rhopr-SKRs have been functionally characterized and their intracellular signaling pathways analysed via a functional receptor assay. Both Rhopr-SKRs are exclusively activated via the two native R. prolixus sulfakinins, Rhopr-SK-1 and Rhopr-SK-2, but not via nonsulfated Rhopr-SK-1. The Rhopr-SKRs are each linked to the intracellular Ca2+ second messenger pathway, and not to the cyclic AMP pathway. Spatial transcript expression analyses revealed that each Rhopr-SKR is predominantly expressed in the central nervous system with lower expression throughout peripheral tissues. The critical importance of the SK-signaling pathway in the blood-feeding behaviour of R. prolixus was demonstrated by knockdown of the transcripts for Rhopr-SKs and Rhopr-SKRs, which results in an increase in the mass of blood meal taken. The parasite causing Chagas disease is transmitted to the host after R. prolixus has taken a blood meal, and characterization of the SKRs provides further understanding of the coordination of feeding and satiation, and ultimately the transmission of the parasite.

Emergence of a cholecystokinin/sulfakinin signalling system in Lophotrochozoa

Chordate gastrin/cholecystokinin (G/CCK) and ecdysozoan sulfakinin (SK) signalling systems represent divergent evolutionary scenarios of a common ancestral signalling system. The present article investigates for the first time the evolution of the CCK/SK signalling system in a member of the Lophotrochozoa, the second clade of protostome animals. We identified two G protein-coupled receptors (GPCR) in the oyster Crassostrea gigas (Mollusca), phylogenetically related to chordate CCK receptors (CCKR) and to ecdysozoan sulfakinin receptors (SKR). These receptors, Cragi-CCKR1 and Cragi-CCKR2, were characterised functionally using a cell-based assay. We identified di- and mono-sulphated forms of oyster Cragi-CCK1 (pEGAWDY(SO3H)DY(SO3H)GLGGGRF-NH2) as the potent endogenous agonists for these receptors. The Cragi-CCK genes were expressed in the visceral ganglia of the nervous system. The Cragi-CCKR1 gene was expressed in a variety of tissues, while Cragi-CCKR2 gene expression was more restricted to nervous tissues. An in vitro bioassay revealed that different forms of Cragi-CCK1 decreased the frequency of the spontaneous contractions of oyster hindgut. Expression analyses in oysters with contrasted nutritional statuses or in the course of their reproductive cycle highlighted the plausible role of Cragi-CCK signalling in the regulation of feeding and its possible involvement in the coordination of nutrition and energy storage in the gonad. This study confirms the early origin of the CCK/SK signalling system from the common bilaterian ancestor and delivers new insights into its structural and functional evolution in the lophotrochozoan lineage.

The 5-amino acid N-terminal extension of non-sulfated drosulfakinin II is a unique target to generate novel agonists

The ability to design agonists that target peptide signaling is a strategy to delineate underlying mechanisms and influence biology. A sequence that uniquely characterizes a peptide provides a distinct site to generate novel agonists. Drosophila melanogaster sulfakinin encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH2) and nsDSK II (GGDDQFDDYGHMRF-NH2). Drosulfakinin is typical of sulfakinin precursors, which are conserved throughout invertebrates. Non-sulfated DSK II is structurally related to DSK I, however, it contains a unique 5-residue N-terminal extension; drosulfakinins signal through G-protein coupled receptors, DSK-R1 and DSK-R2. Drosulfakinin II distinctly influences adult and larval gut motility and larval locomotion; yet, its structure-activity relationship was unreported. We hypothesized substitution of an N-terminal extension residue may alter nsDSK II activity. By targeting the extension we identified, not unexpectedly, analogs mimicking nsDSK II, yet, surprisingly, we also discovered novel agonists with increased (super) and opposite (protean) effects. We determined [A3] nsDSK II increased larval gut contractility rather than, like nsDSK II, decrease it. [N4] nsDSK II impacted larval locomotion, although nsDSK II was inactive. In adult gut, [A1] nsDSK II, [A2] nsDSKII, and [A3] nsDSK II mimicked nsDSK II, and [A4] nsDSK II and [A5] nsDSK II were more potent; [N3] nsDSK II and [N4] nsDSK II mimicked nsDSK II. This study reports nsDSK II signals through DSK-R2 to influence gut motility and locomotion, identifying a novel role for the N-terminal extension in sulfakinin biology and receptor activation; it also led to the discovery of nsDSK II structural analogs that act as super and protean agonists.

Flexibility and extracellular opening determine the interaction between ligands and insect sulfakinin receptors.

Despite their fundamental importance for growth, the mechanisms that regulate food intake are poorly understood. Our previous work demonstrated that insect sulfakinin (SK) signaling is involved in inhibiting feeding in an important model and pest insect, the red flour beetle Tribolium castaneum. Because the interaction of SK peptide and SK receptors (SKR) initiates the SK signaling, we have special interest on the structural factors that influence the SK-SKR interaction. First, the three-dimensional structures of the two T. castaneum SKRs (TcSKR1 and TcSKR2) were generated from molecular modeling and they displayed significance in terms of the outer opening of the cavity and protein flexibility. TcSKR1 contained a larger outer opening of the cavity than that in TcSKR2, which allows ligands a deep access into the cavity through cell membrane. Second, normal mode analysis revealed that TcSKR1 was more flexible than TcSKR2 during receptor-ligand interaction. Third, the sulfated SK (sSK) and sSK-related peptides were more potent than the nonsulfated SK, suggesting the importance of the sulfate moiety.

The activity of the nonsulfated sulfakinin Zopat-SK-1 in the neck-ligated larvae of the beetle Zophobas atratus

Insect sulfakinins (SKs) are multifunctional neuropeptides structurally and functionally homologous to the mammalian gastrin/cholecystokinin (CCK). It has been proposed that SKs play a role in modulating energy management in insects by interacting with adipokinetic hormone (AKH), the principle hormone controlling insect intermediary metabolism. To exclude head factors (including AKH) that influence the activity of the nonsulfated sulfakinin Zopat-SK-1 in the larvae of the beetle Zophobas atratus, ligature and in vitro bioassays were used. Our study showed that in the neck-ligated larvae, Zopat-SK-1 evoked a much more pronounced glycogenolytic effect in fat body tissue and a significantly higher hypertrahelosemic effect in hemolymph than in larvae without ligation. We found that the concentration of the sugar trehalose increased under hormonal treatment but no changes in glucose levels were observed. Under in vitro conditions, the maximal glycogenolytic effect of Zopat-SK-1 in fat body was observed at 10pmol of hormone. Ligature and in vitro bioassays indicated that Zopat-SK-1 activity in the Z. atratus larvae is modulated by head signals and/or factors from the gastrointestinal tract. Our data indicate the existence of a brain–gastrointestinal axis that has a role in controlling of energy (carbohydrate) metabolism in the insect body. Moreover, these results, together with immunological evidence of a cholecystokinin-like (sulfakinin) receptor in the Z. atratus fat body, help us to better understand the SK signaling pathways and its physiological role in insect biology.

CCK(-like) and receptors: Structure and phylogeny in a comparative perspective

Cholecystokinin (CCK) and gastrin are regulatory peptides in vertebrates. Their homologues are widely present in metazoan animals, in form of cionin in tunicates, neuropeptide-like protein 12 in nematodes and sulfakinin (SK) in arthropods. CCK(-like) peptides exert diverse physiological effects through binding their corresponding receptors, which are important members of the hormone-binding G-protein-coupled receptors.In this paper, CCK(-like) peptides and receptors are reviewed in a comparative way at levels of molecular structure, physiological functions and phylogeny. CCK signalling system is widely involved in the regulation of satiety, gastric acid secretion, pancreatic secretion, anxiety and memory processes in vertebrates. Its counterpart SK in arthropods is also found with similar functions on regulation of satiety and gastrointestinal motility. Co-evolution of peptide and receptor has been recognized through metazoans. The CCK(-like) receptors seem to be evolved from a common ancestor based on the phylogenetic analysis, with species-specific events in arthropods. In addition, tetraploidization has been brought up to study the evolution of receptors. There are 2 receptors in chordates and nematodes, whereas, the number of sulfakinin receptor varies in arthropods from 0 to 2. We discussed here that the presence or absence of the SK signalling system is likely to be related to feeding behaviour.

Signaling Properties and Pharmacological Analysis of Two Sulfakinin Receptors from the Red Flour Beetle, Tribolium castaneum

Sulfakinin is an insect neuropeptide that constitutes an important component of the complex network of hormonal and neural factors that regulate feeding and digestion. The key modulating functions of sulfakinin are mediated by binding and signaling via G-protein coupled receptors. Although a substantial amount of functional data have already been reported on sulfakinins in different insect species, only little information is known regarding the properties of their respective receptors. In this study, we report on the molecular cloning, functional expression and characterization of two sulfakinin receptors in the red flour beetle, Tribolium castaneum. Both receptor open reading frames show extensive sequence similarity with annotated sulfakinin receptors from other insects. Comparison of the sulfakinin receptor sequences with homologous vertebrate cholecystokinin receptors reveals crucial conserved regions for ligand binding and receptor activation. Quantitative reverse transcriptase PCR shows that transcripts of both receptors are primarily expressed in the central nervous system of the beetle. Pharmacological characterization using 29 different peptide ligands clarified the essential requirements for efficient activation of these sulfakinin receptors. Analysis of the signaling pathway in multiple cell lines disclosed that the sulfakinin receptors of T. castaneum can stimulate both the Ca2+ and cyclic AMP second messenger pathways. This in depth characterization of two insect sulfakinin receptors may provide useful leads for the further development of receptor ligands with a potential applicability in pest control and crop protection.

Development of cell-based bioassay with Sf9 cells expressing TcSKR1 and TcSKR2 and differential activation by sulfated and non-sulfated SK peptides

Insect sulfakinin receptors (SKRs) are G-protein-coupled receptors (GPCRs) that interact with sulfakinins (SKs) to modulate diverse biological processes. One of the indispensable roles of SKs is in the regulation of food intake in insects. In this project we report on the development of a cell-based receptor assay system with insect Sf9 cells, expressing TcSKR1 and TcSKR2 from the red flour beetle Tribolium castaneum, a model and important pest insect in agriculture. In this system, a stable presence of the two TcSKRs was supported by Western blotting. The expressed TcSKRs were coupled to Gαs-protein upon activation and stimulated cAMP accumulation in Sf9 cells. Exposure of the transfected cell lines to sulfated SK (sSK) activated TcSKR1 at 1nM; the EC50 of sSK to obtain 50% of receptor activation was similar for both receptors. In contrast, μM concentrations of non-sulfated SK were necessary to activate both TcSKRs. In conclusion, this cell-based TcSKR assay system is useful to screen SK-related peptides and mimetics and to better document ligand-receptor structure-activity relationships. Given the importance of SK signaling system in insects, the present study may provide new insights on the development of new methods to control pest insects.

Characterization of sulfakinin receptor 2 and its role in food intake in the red flour beetle, Tribolium castaneum

Insects acquire essential nutrients from their food to support a diverse range of biological processes such as development and reproduction. An important role in the food intake regulation is attributed to the neurohormone sulfakinin signaling pathway. Sulfakinins (SKs) elicit satiety together with sulfakinin receptors (SKRs). In this project, the gene coding a second putative SKR, namely skr2, was cloned, characterized and functionally studied in the red flour beetle Tribolium castaneum, with the purpose to understand its role in food intake regulation. The gene skr2 encoded a seven-transmembrane SKR2 protein with 420 amino acids and is evolutionarily close to the two SKRs in Drosophila. A distribution analysis by means of quantitative real-time PCR revealed a tissue- and stage-specific expression pattern with skr2 being dominantly expressed in head, and in the pupal and adult stages. Besides, its expression in gut was notable. With use of dsRNA of skr2, larval food intake was stimulated in an injection RNAi assay. Based on the data obtained, skr2 is considered as an indispensable component in the regulation of food intake in T. castaneum.

Identification of G protein-coupled receptors in the pea aphid, Acyrthosiphon pisum

GPCRs play crucial roles in the growth, development and reproduction of organisms. In insects, a large number of GPCRs have been reported for Holometabola but not Hemimetabola. The recently sequenced pea aphid genome provides us with the opportunity to analyze the evolution and potential functions of GPCRs in Hemimetabola. 82 GPCRs were identified from the representative model hemimetabolous insect Acyrthosiphon pisum, 37 of which have ESTs evidence, and 73 are annotated for the first time. A striking difference between A. pisum, Drosophila melanogaster and Tribolium castaneum is the duplication of the kinin and SIFamide receptors in A. pisum. Another divergence is the loss of the sulfakinin receptor in A. pisum. These duplications/losses are likely involved in the osmoregulation, reproduction and energy metabolism of A. pisum. Moreover, this work will promote functional analyses of GPCRs in A. pisum and may advance new drug target discovery for biological control of the aphid.

Characterization of sulfakinin and sulfakinin receptor and their roles in food intake in the red flour beetle Tribolium castaneum

Sulfakinins (SK) are multifunctional neuropeptides widely found in insects that are structurally and functionally homologous to the mammalian gastrin/cholecystokinin (CCK) neuropeptides. CCK is involved in various biological processes such as the feeding regulation where it induces satiety. In this project we characterized SK and SK receptor (SKR) of an important pest and model beetle insect, the red flour beetle Tribolium castaneum, with the aim to better understand the SK signaling pathway and its function in food intake.The sk gene encoded a SK precursor with 113 amino acids and the skr gene a seven-transmembrane SKR with 554 amino acids. Both genes were expressed in the larval, pupal and adult stages with different expression levels in tested tissues. By RNA interference, sk dsRNA and skr dsRNA reduced the expression of the corresponding target gene by 80–90% and 30–50%, respectively, and stimulated food intake in the larvae. In parallel, we injected insects with a SK analog reducing food intake.In conclusion, the data are discussed in relation to the SK signaling pathway and its physiological-endocrinological role in regulating food intake and potential usage in the control of important pest insects.

Neuropeptide signaling sequences identified by pyrosequencing of the American dog tick synganglion transcriptome during blood feeding and reproduction

Ticks are important vectors of numerous pathogens that impact human and animal health. The tick central nervous system represents an understudied area in tick biology and no tick synganglion-specific transcriptome has been described to date. Here we characterize whole or partial cDNA sequences of fourteen putative neuropeptides (allatostatin, insulin-like peptide, ion-transport peptide, sulfakinin, bursicon alpha/beta, eclosion hormone, glycoprotein hormone alpha/beta, corazonin, four orcokinins) and five neuropeptide receptors (gonadotropin receptor, leucokinin-like receptor, sulfakinin receptor, calcitonin receptor, pyrokinin receptor) translated from cDNA synthesized from the synganglion of unfed, partially fed and replete female American dog ticks, Dermacentor variabilis. Their homology to the same neuropeptides in other taxa is discussed. Many of these neuropeptides such as an allatostatin, insulin-like peptide, eclosion hormone, bursicon alpha and beta and glycoprotein hormone alpha and beta have not been previously described in the Chelicerata. An insulin-receptor substrate protein was also found indicating that an insulin signaling network is present in ticks. A putative type-2 proprotein processing convertase was also sequenced that may be involved in cleavage at monobasic and dibasic endoproteolytic cleavage sites in prohormones. The possible physiological role of the proteins discovered in adult tick blood feeding and reproduction will be discussed.

Discovery of a Cholecystokinin-Gastrin-Like Signaling System in Nematodes

Members of the cholecystokinin (CCK)/gastrin family of peptides, including the arthropod sulfakinins, and their cognate receptors, play an important role in the regulation of feeding behavior and energy homeostasis. Despite many efforts after the discovery of CCK/gastrin immunoreactivity in nematodes 23 yr ago, the identity of these nematode CCK/gastrin-related peptides has remained a mystery ever since. The Caenorhabditis elegans genome contains two genes with high identity to the mammalian CCK receptors and their invertebrate counterparts, the sulfakinin receptors. By using the potential C. elegans CCK receptors as a fishing hook, we have isolated and identified two CCK-like neuropeptides encoded by neuropeptide-like protein-12 (nlp-12) as the endogenous ligands of these receptors. The neuropeptide-like protein-12 peptides have a very limited neuronal expression pattern, seem to occur in vivo in the unsulfated form, and react specifically with a human CCK-8 antibody. Both receptors and ligands share a high degree of structural similarity with their vertebrate and arthropod counterparts, and also display similar biological activities with respect to digestive enzyme secretion and fat storage. Our data indicate that the gastrin-CCK signaling system was already well established before the divergence of protostomes and deuterostomes.