Mechanisms controlling haemolymph circulation under resting conditions in the chagas disease vector Rhodnius Prolixus (Stål).

Chagas disease vectors can ingest several times their own volume in blood with each meal. This ad libitum feeding causes an intense process of diuresis inducing the insect to eliminate a large quantity of urine and faeces. To ensure diuresis, the speed of circulation of the haemolymph is increased. Triatominae circulatory system is quite simple, including the dorsal vessel which pumps haemolymph in an anterograde direction. The return is caused by peristaltic contractions of the anterior midgut. Triatominae insects can spend several weeks without feeding, meaning that during most of the time, the insect is in a resting condition. While the mechanisms controlling the circulation of the haemolymph during post-prandial diuresis was largely analysed, the mechanisms controlling it during resting conditions are poorly understood. In this study we analyse several canonical pathways (i.e. L-type VGCC; GPCR; RyR; IP3R) and a novel system represented by the recently characterized Piezo proteins. Our results show that during the resting condition haemolymph circulation depends on a cross-talk between myogenic activity, inhibitory and stimulatory cellular messengers, and also Piezo proteins. This report also unveils for the first time the existence of a putative Piezo protein in Hemiptera.

G protein-coupled receptor signal transduction and Ca2+ signaling pathways of the allatotropin/orexin system in Hydra.

Allatotropin is a pleiotropic peptide originally characterized in insects. The existence of AT neuropeptide signaling was proposed in other invertebrates. In fact, we previously proposed the presence of an AT-like system regulating feeding behavior in Hydra sp. Even in insects, the information about the AT signaling pathway is incomplete. The aim of this study is to analyze the signaling cascade activated by AT in Hydra plagiodesmica using a pharmacological approach. The results show the involvement of Ca2+ and IP3 signaling in the transduction pathway of the peptide. Furthermore, we confirm the existence of a GPCR system involved in this pathway, that would be coupled to a Gq subfamily of Gα protein, which activates a PLC, inducing an increase in IP3 and cytosolic Ca2+. To the best of our knowledge, this work represents the first in vivo approach to study the overall signaling pathway and intracellular events involved in the myoregulatory effect of AT in Hydra sp.

The Evolutionary History of The Orexin/Allatotropin GPCR Family: from Placozoa and Cnidaria to Vertebrata.

Peptidic messengers constitute a highly diversified group of intercellular messengers widely distributed in nature that regulate a great number of physiological processes in Metazoa. Being crucial for life, it seem that they have appeared in the ancestral group from which Metazoa evolved, and were highly conserved along the evolutionary process. Peptides act mainly through G-protein coupled receptors (GPCRs), a family of transmembrane molecules. GPCRs are also widely distributed in nature being present in metazoan, but also in Choanoflagellata and Fungi. Among GPCRs, the Allatotropin/Orexin (AT/Ox) family is particularly characterized by the presence of the DRW motif in the second intracellular loop (IC Loop 2), and seems to be present in Cnidaria, Placozoa and in Bilateria, suggesting that it was present in the common ancestor of Metazoa. Looking for the evolutionary history of this GPCRs we searched for corresponding sequences in public databases. Our results suggest that AT/Ox receptors were highly conserved along evolutionary process, and that they are characterized by the presence of the E/DRWYAI motif at the IC Loop 2. Phylogenetic analyses show that AT/Ox family of receptors reflects evolutionary relationships that agree with current phylogenetic understanding in Actinopterygii and Sauropsida, including also the largely discussed position of Testudines.

Allatoregulatory-like systems and changes in cytosolic Ca2+ modulate feeding behavior in Hydra.

Allatotropin (AT) and allatostatin-C (AST-C) are neuropeptides originally characterized by their ability to modulate the secretion of juvenile hormones in insects. Beyond the allatoregulatory function, these neuropeptides are pleiotropic acting as myoregulators not only in insects, but also in other groups of invertebrates. We have previously proposed the existence of AT and AST-C like systems in Hydra sp., a member of the phylum Cnidaria, which is a basal group of Metazoa, sharing a common ancestor with Bilateria. In the present study we analyze the regulatory effects of both peptides on the activity of the hypostome during feeding in Hydra sp. Furthermore, the importance of changes in the cytosolic Ca2+ levels involved in the response of the hypostome were analyzed. Physiological assays showed that while the presence of food or treatment with AT stimulates the extrusion of the hypostome, AST-C has an inhibitory effect on the behavior induced by both, food and AT. These facts suggest that both systems participate in the regulatory mechanisms associated with feeding and, as in insects, AST-C and AT may exert opposite effects. The use of thapsigargin (TG) and nifedipine, two compounds that modify the levels of cytosolic Ca2+, showed that changes in the levels of this ion are involved in the regulation of the activity of the hypostome. Indeed, these results suggest that the two basic mechanisms operating to increase the cytosolic levels of Ca2+ (i.e. the influx from the extracellular space and the release from endoplasmic reticulum) are relevant for the extrusion of the hypostome. Like in insects, the treatment with TG counteracted the effect of AST-C, suggesting that this peptide acts by reducing cytosolic Ca2+ levels. Furthermore, nifedipine prevented the myostimulatory effect of AT, showing that the effect of this peptide depends on the influx of Ca2+ throughout voltage-gated calcium channels. Altogether, these results suggest that the Allatotropin/Orexin and Allatostatin/Somatostatin regulatory systems could represent an ancestral mechanisms regulating hypostome activity and feeding behavior in Cnidaria.

Somatostatin signaling system as an ancestral mechanism: Myoregulatory activity of an Allatostatin-C peptide in Hydra.

The coordination of physiological processes requires precise communication between cells. Cellular interactions allow cells to be functionally related, facilitating the maintaining of homeostasis. Neuropeptides functioning as intercellular signals are widely distributed in Metazoa. It is assumed that neuropeptides were the first intercellular transmitters, appearing early during the evolution. In Cnidarians, neuropeptides are mainly involved in neurotransmission, acting directly or indirectly on epithelial muscle cells, and thereby controlling coordinated movements. Allatostatins are a group of chemically unrelated neuropeptides that were originally characterized based on their ability to inhibit juvenil hormone synthesis in insects. Allatostatin-C has pleiotropic functions, acting as myoregulator in several insects. In these studies, we analyzed the myoregulatory effect of Aedes aegypti Allatostatin-C in Hydra sp., a member of the phylum Cnidaria. Allatostatin-C peptide conjugated with Qdots revealed specifically distributed cell populations that respond to the peptide in different regions of hydroids. In vivo physiological assays using Allatostatin-C showed that the peptide induced changes in shape and length in tentacles, peduncle and gastrovascular cavity. The observed changes were dose and time dependent suggesting the physiological nature of the response. Furthermore, at highest doses, Allatostatin-C induced peristaltic movements of the gastrovascular cavity resembling those that occur during feeding. In silico search of putative Allatostatin-C receptors in Cnidaria showed that genomes predict the existence of proteins of the somatostatin/Allatostatin-C receptors family. Altogether, these results suggest that Allatostatin-C has myoregulatory activity in Hydra sp, playing a role in the control of coordinated movements during feeding, indicating that Allatostatin-C/Somatostatin based signaling might be an ancestral mechanism.

Allatotropin: an ancestral myotropic neuropeptide involved in feeding.

BACKGROUND: Cell-cell interactions are a basic principle for the organization of tissues and organs allowing them to perform integrated functions and to organize themselves spatially and temporally. Peptidic molecules secreted by neurons and epithelial cells play fundamental roles in cell-cell interactions, acting as local neuromodulators, neurohormones, as well as endocrine and paracrine messengers. Allatotropin (AT) is a neuropeptide originally described as a regulator of Juvenile Hormone synthesis, which plays multiple neural, endocrine and myoactive roles in insects and other organisms. METHODS: A combination of immunohistochemistry using AT-antibodies and AT-Qdot nanocrystal conjugates was used to identify immunoreactive nerve cells containing the peptide and epithelial-muscular cells targeted by AT in Hydra plagiodesmica. Physiological assays using AT and AT- antibodies revealed that while AT stimulated the extrusion of the hypostome in a dose-response fashion in starved hydroids, the activity of hypostome in hydroids challenged with food was blocked by treatments with different doses of AT-antibodies. CONCLUSIONS: AT antibodies immunolabeled nerve cells in the stalk, pedal disc, tentacles and hypostome. AT-Qdot conjugates recognized epithelial-muscular cell in the same tissues, suggesting the existence of anatomical and functional relationships between these two cell populations. Physiological assays indicated that the AT-like peptide is facilitating food ingestion. SIGNIFICANCE: Immunochemical, physiological and bioinformatics evidence advocates that AT is an ancestral neuropeptide involved in myoregulatory activities associated with meal ingestion and digestion.