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 juvenile hormone described in Rhodnius prolixus by Wigglesworth is juvenile hormone III skipped bisepoxide.

Juvenile hormones (JHs) are sesquiterpenoids synthesized by the corpora allata (CA). They play critical roles during insect development and reproduction. The first JH was described in 1934 as a "metamorphosis inhibitory hormone" in Rhodnius prolixus by Sir Vincent B. Wigglesworth. Remarkably, in spite of the importance of R. prolixus as vectors of Chagas disease and model organisms in insect physiology, the original JH that Wigglesworth described for the kissing-bug R. prolixus remained unidentified. We employed liquid chromatography mass spectrometry to search for the JH homologs present in the hemolymph of fourth instar nymphs of R. prolixus. Wigglesworth's original JH is the JH III skipped bisepoxide (JHSB3), a homolog identified in other heteropteran species. Changes in the titer of JHSB3 were studied during the 10-day long molting cycle of 4th instar nymph, between a blood meal and the ecdysis to 5th instar. In addition we measured the changes of mRNA levels in the CA for the 13 enzymes of the JH biosynthetic pathway during the molting cycle of 4th instar. Almost 90 years after the first descriptions of the role of JH in insects, this study finally reveals that the specific JH homolog responsible for Wigglesworth's original observations is JHSB3.

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.

Allatostatin-C antagonizes the synergistic myostimulatory effect of allatotropin and serotonin in Rhodnius prolixus (Stal).

Haematophagous insects can ingest large quantities of blood in a single meal producing a large quantity of urine in the following hours to eliminate the excess of water and mineral ions incorporated. The excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation as a result of the intensification of aorta contractions, combined with an increase of anterior midgut peristaltic waves. We have recently shown that haemolymph circulation during post-prandial diuresis is modulated by the synergistic activity of allatotropin (AT) and serotonin, resulting in an increase in aorta and crop contraction rates. In the present study we describe the antagonistic effect of allatostatin-C (AST-C) on the increase of aorta frequency of contractions induced by serotonin/AT in Rhodnius prolixus. The administration of AST-C counteracted the increase in the frequency induced by the treatment with serotonin/AT, but did not affect the increase in frequency induced by the administration of serotonin alone, suggesting that AST-C is altering the synergism between serotonin and AT. Furthermore, the administration of AST-C during post-prandial diuresis decreases the number of peristaltic waves of the anterior midgut. The AST-C putative receptor is expressed in the hindgut, midgut and dorsal vessel, three critical organs involved in post-prandial diuresis. All together these findings provide evidence that AST-C plays a key role as a myoregulatory and cardioregulatory peptide in R. prolixus.

Genomic and functional characterization of a methoprene-tolerant gene in the kissing-bug Rhodnius prolixus.

Metamorphosis, which depends upon a fine balance between two groups of lipid-soluble hormones such as juvenile hormones (JHs) and ecdysteroids, is an important feature in insect evolution. While it is clear that the onset of metamorphosis depends on the decrease of JH levels, the way in which these hormones exert their activities is not fully understood in Triatominae species. The discovery of a Drosophila melanogaster mutant resistant to the treatment with the JH analog methoprene, led finally to the description of the methoprene-tolerant gene in Tribolium castaneum (TcMet) as a putative JH receptor. Here we present the genomic and functional characterization of an ortholog of the methoprene-tolerant gene in the hemimetabolous insect Rhodnius prolixus (RpMet). The analysis of the R. prolixus gene showed that the exonic structure is different from that described for holometabolous species, although all the critical protein motifs are well conserved. Expression analysis showed the presence of RpMet mRNA in all the tested tissues: ovary, testis, rectum, Malpighian tubules and salivary glands. When juvenile individuals were treated with RpMet specific double strand RNA (dsRNA), we observed abnormal molting events that resulted in individuals with morphological alterations (adultoids). Similarly, treatment of newly emerged fed females with dsRNA resulted in an abnormal development of the ovaries, with eggs revealing anomalies in size and accumulation of yolk, as well as a decrease in the amount of heme-binding protein. Altogether, our results validate that RpMet is involved in the transduction of JH signaling, controlling metamorphosis and reproduction in R. prolixus.

Allatotropin modulates myostimulatory and cardioacceleratory activities in Rhodnius prolixus (Stal).

Haematophagous insects can ingest large quantities of blood in a single meal and eliminate high volumes of urine in the next few hours. This rise in diuresis is possible because the excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation as a result of intensification of aorta contractions combined with an increase of the anterior midgut peristaltic waves. It has been previously described that haemolymph circulation during post-prandial diuresis is stimulated by the synergistic activity of allatotropin (AT) and serotonin in the kissing bug Triatoma infestans; resulting in an increase in aorta contractions. In the same species, AT stimulates anterior midgut and rectum muscle contractions to mix urine and feces and facilitate the voiding of the rectum. Furthermore, levels of AT in midgut and Malpighian tubules increased in the afternoon when insects are getting ready for nocturnal feeding. In the present study we describe the synergistic effect of AT and serotonin increasing the frequency of contractions of the aorta in Rhodnius prolixus. The basal frequency of contractions of the aorta in the afternoon is higher that the observed during the morning, suggesting the existence of a daily rhythmic activity. The AT receptor is expressed in the rectum, midgut and dorsal vessel, three critical organs involved in post-prandial diuresis. All together these findings provide evidence that AT plays a role as a myoregulatory and cardioacceleratory peptide in R. prolixus.

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.

Expression of a neuropeptide similar to allatotropin in free living turbellaria (platyhelminthes).

Mechanisms coordinating cell-cell interaction have appeared early in evolution. Allatotropin (AT), a neuropeptide isolated based on its ability to stimulate the synthesis of juvenile hormones (JHs) in insects has also been found in other invertebrate phyla. Despite this function, AT has proved to be myotropic. In the present study we analyze its expression in two groups of Turbellaria (Catenulida, Macrostomida), and its probable relationship with muscle tissue. The results show the presence of an AT-like peptide in the free living turbellaria analyzed. The analysis of the expression of the peptide together with phalloidin, suggests a functional relationship between the peptide and muscle tissue, showing that it could be acting as a myoregulator. The finding of immunoreactive fibers associated with sensory organs like ciliated pits in Catenulida and eyes in Macrostomida makes probable that AT could play a role in the physiological mechanisms controlling circadian activities. Furthermore, the existence of AT in several phyla of Protostomata suggests that this peptide could be a synapomorphic feature of this group. Indeed, the presence in organisms that do not undergo metamorphosis, could be signaling that it was first involved in myotropic activities, being the stimulation of the synthesis of JHs a secondary function acquired by the phylum Arthropoda.

Cardioacceleratory and myostimulatory activity of allatotropin in Triatoma infestans.

Haematophagous insects incorporate a large quantity of blood with each meal, producing a big quantity of urine in a few hours. The activity of the Malpighian tubules (MTs) is facilitated by the increase of the circulation of the haemolymph produced by the increase of the aorta contractions as well as, of the peristaltic waves of the anterior midgut. MTs of Triatoma infestans secrete an allatotropin-like peptide, which has a myostimulatory effect on the hindgut, inducing the mixing and voiding of the content during post-prandial diuresis. We are reporting now the activity of allatotropin (AT) as a cardioacceleratory and a myostimulatory peptide at the level of the anterior midgut. The peptide induced the increase of the rate of contractions of the anterior midgut and the aorta in a wide range of concentrations. The cardioacceleratory effect of AT was dependent on the feeding state of the insects and on the presence of serotonin. The response showed the existence of a differential behavior between sexes, inducing a higher increase on the frequency of contractions, as well as, the width of the aorta in males than in females. Finally, our results suggest that AT interacts with serotonin to facilitate the circulation of haemolymph during post-prandial diuresis.

Allatotropin-like peptide in Malpighian tubules: insect renal tubules as an autonomous endocrine organ.

Malpighian tubules (MTs) are recognised as the main excretory organ in insects, ensuring water and mineral balance. Haematophagous insects incorporate with each meal a large quantity of blood, producing a particularly large volume of urine in a few hours. In the present study, we report the presence of an allatotropin-like (AT-like) peptide in MTs of Triatoma infestans (Klug). The AT-like content in MTs decreased during the first hours after blood-intake, correlating with the post-prandial diuresis. In vivo artificial dilution of haemolymph showed a similar effect. Isolated MTs challenged with a diluted saline solution resulted in an autonomous and reversible response of the organ regulating the quantity of peptide released to the medium, and suggesting that MTs synthesise the AT-like peptide. While MTs are recognised as the target for several hormones, our results corroborate that they also have the ability to produce and secrete a hormone in an autonomous way.

Allatotropin-like peptide released by Malpighian tubules induces hindgut activity associated with diuresis in the Chagas disease vector Triatoma infestans (Klug).

Haematophagous insects incorporate a large amount of blood with each meal, producing a big quantity of urine in a few hours to eliminate the excess water and Na(+). Malpighian tubules (MTs) have traditionally been seen as a system that responds to neuroendocrine stimulus. In a related paper, we demonstrated that MTs of Triatoma infestans produce an autonomous endocrine secretion of an allatotropin-like (AT-like) peptide. In the present study, we report a myostimulatory activity of AT at the level of the hindgut (HG), associated with endocrine mechanisms regulating post-prandial diuresis. Allatotropin induced an increase in frequency and intensity of peristaltic contractions at the level of the HG. The release of the HG content in MTs-HG in vitro preparations undergoing an osmotic shock occurred at different times, depending on the number of MTs present, and there was no release in treatments without MTs. The application of an AT-antiserum to MTs-HG preparations undergoing osmotic shock produced a delay or a long-term blockade of diuresis, depending on the antiserum dilution applied. Similar results were obtained when AT-antiserum was applied in vivo prior to blood intake, decreasing the volume of urine eliminated during the first 2 h. Our results allow us to assign a specific endocrine function to the AT-like peptide released by MTs that is linked to the elimination of urine after blood meals.