Evidence for D1 Dopamine Receptor Activation by a Paracrine Signal of Dopamine in Tick Salivary Glands

Ticks that feed on vertebrate hosts use their salivary secretion, which contains various bioactive components, to manipulate the host's responses. The mechanisms controlling the tick salivary gland in this dynamic process are not well understood. We identified the tick D1 receptor activated by dopamine, a potent inducer of the salivary secretion of ticks. Temporal and spatial expression patterns examined by immunohistochemistry and reverse transcription polymerase chain reaction suggest that the dopamine produced in the basal cells of salivary gland acini is secreted into the lumen and activates the D1 receptors on the luminal surface of the cells lining the acini. Therefore, we propose a paracrine function of dopamine that is mediated by the D1 receptor in the salivary gland at an early phase of feeding. The molecular and pharmacological characterization of the D1 receptor in this study provides the foundation for understanding the functions of dopamine in the blood-feeding of ticks

Neuropeptide Receptor Transcriptome Reveals Unidentified Neuroendocrine Pathways

Neuropeptides are an important class of molecules involved in diverse aspects of metazoan development and homeostasis. Insects are ideal model systems to investigate neuropeptide functions, and the major focus of insect neuropeptide research in the last decade has been on the identification of their receptors. Despite these vigorous efforts, receptors for some key neuropeptides in insect development such as prothoracicotropic hormone, eclosion hormone and allatotropin (AT), remain undefined. In this paper, we report the comprehensive cloning of neuropeptide G protein-coupled receptors from the silkworm, Bombyx mori, and systematic analyses of their expression. Based on the expression patterns of orphan receptors, we identified the long-sought receptor for AT, which is thought to stimulate juvenile hormone biosynthesis in the corpora allata (CA). Surprisingly, however, the AT receptor was not highly expressed in the CA, but instead was predominantly transcribed in the corpora cardiaca (CC), an organ adjacent to the CA. Indeed, by using a reverse-physiological approach, we purified and characterized novel allatoregulatory peptides produced in AT receptor-expressing CC cells, which may indirectly mediate AT activity on the CA. All of the above findings confirm the effectiveness of a systematic analysis of the receptor transcriptome, not only in characterizing orphan receptors, but also in identifying novel players and hidden mechanisms in important biological processes. This work illustrates how using a combinatorial approach employing bioinformatic, molecular, biochemical and physiological methods can help solve recalcitrant problems in neuropeptide research

Identification of a complex peptidergic neuroendocrine network in the hard tick, Rhipicephalus appendiculatus

Neuropeptides are crucial regulators of development and various physiological functions but little is known about their identity, expression and function in vectors of pathogens causing serious diseases, such as ticks. Therefore, we have used antibodies against multiple insect and crustacean neuropeptides to reveal the presence of these bioactive molecules in peptidergic neurons and cells of the ixodid tick Rhipicephalus appendiculatus. These antibodies have detected 15 different immunoreactive compounds expressed in specific central and peripheral neurons associated with the synganglion. Most central neurons arborize in distinct areas of the neuropile or the putative neurohaemal periganglionic sheath of the synganglion. Several large identified neurons in the synganglion project multiple processes through peripheral nerves to form elaborate axonal arborizations on the surface of salivary glands or to terminate in the lateral segmental organs (LSO). Additional neuropeptide immunoreactivity has been observed in intrinsic secretory cells of the LSO. We have also identified two novel clusters of peripheral neurons embedded in the cheliceral and paraspiracular nerves. These neurons project branching axons into the synganglion and into the periphery. Our study has thus revealed a complex network of central and peripheral peptidergic neurons, putative neurohaemal and neuromodulatory structures and endocrine cells in the tick comparable with those found in insect and crustacean neuroendocrine systems. Strong specific staining with a large variety of antibodies also indicates that the tick nervous system and adjacent secretory organs are rich sources of diverse neuropeptides related to those identified in insects, crustaceans or even vertebrates

A Command Chemical Triggers an Innate Behavior by Sequential Activation of Multiple Peptidergic Ensembles

Background: At the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system.Results: To elucidate neural substrates underlying the ecdysis sequence, we identified neurons expressing ETH receptors (ETHRs) in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence.Conclusions: Our findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central eptidergic ensembles

Enteroendocrine peptides regulate feeding behavior via controlling intestinal contraction of the silkworm Bombyx mori.

Our previous study demonstrated that predominant feeding inhibitory effects were found in the crude extracts of foregut and midgut of the silkworm Bombyx mori larvae. To address the entero-intestinal control crucial for the regulation of insect feeding behavior, the present study identified and functionally characterized feeding inhibitory peptides from the midgut of B. mori larvae. Purification and structural analyses revealed that the predominant inhibitory factors in the crude extracts were allatotropin (AT) and GSRYamide after its C-terminal sequence. In situ hybridization revealed that AT and GSRYamide were expressed in enteroendocrine cells in the posterior and anterior midgut, respectively. Receptor screening using Ca2+-imaging technique showed that the B. mori neuropeptide G protein-coupled receptor (BNGR)-A19 and -A22 acted as GSRYamide receptors and BNGR-A5 acted as an additional AT receptor. Expression analyses of these receptors and the results of the peristaltic motion assay indicated that these peptides participated in the regulation of intestinal contraction. Exposure of pharynx and ileum to AT and GSRYamide inhibited spontaneous contraction in ad libitum-fed larvae, while exposure of pharynx to GSRYamide did not inhibit contraction in non-fed larvae, indicating that the feeding state changed their sensitivity to inhibitory peptides. These different responses corresponded to different expression levels of their receptors in the pharynx. In addition, injection of AT and GSRYamide decreased esophageal contraction frequencies in the melamine-treated transparent larvae. These findings strongly suggest that these peptides exert feeding inhibitory effects by modulating intestinal contraction in response to their feeding state transition, eventually causing feeding termination

Ecdysis triggering hormone signaling in arthropods

Ecdysis triggering hormones (ETHs) from endocrine Inka cells initiate the ecdysis sequence through action on central neurons expressing ETH receptors (ETHR) in model moth and dipteran species. We used various biochemical, molecular and BLAST search techniques to detect these signaling molecules in representatives of diverse arthropods. Using peptide isolation from tracheal extracts, cDNA cloning or homology searches, we identified ETHs in a variety of hemimetabolous and holometabolous insects. Most insects produce two related ETHs, but only a single active peptide was isolated from the cricket and one peptide is encoded by the eth gene of the honeybee, parasitic wasp and aphid. Immunohistochemical staining with antiserum to Manduca PETH revealed Inka cells on tracheal surface of diverse insects. In spite of conserved ETH sequences, comparison of natural and the ETH-induced ecdysis sequence in the honeybee and beetle revealed considerable species-specific differences in pre-ecdysis and ecdysis behaviors. DNA sequences coding for putative ETHR were deduced from available genomes of several hemimetabolous and holometabolous insects. In all insects examined, the ethr gene encodes two subtypes of the receptor (ETHR-A and ETHR-B). Phylogenetic analysis showed that these receptors fall into a family of closely related GPCRs. We report for the first time the presence of putative ETHs and ETHRs in genomes of other arthropods, including the tick (Arachnida) and water flea (Crustacea). The possible source of ETH in ticks was detected in paired cells located in all pedal segments. Our results provide further evidence of structural and functional conservation of ETH-ETHR signaling