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.

Rhodnius prolixus is a blood-gorging insect and a vector for human Chagas disease. The insect transmits the disease following feeding, when it excretes urine and feces contaminated with the Trypanosoma cruzi parasite. A corticotropin-releasing factor-like peptide acts as a diuretic hormone in R. prolixus (Rhopr-CRF/DH); however, its distribution throughout the insect's central nervous system (CNS) and the expression of its receptor in feeding-related tissue as well as the female reproductive system suggests a multifaceted role for the hormone beyond that of diuresis. Here we investigate the involvement of Rhopr-CRF/DH in feeding and reproduction in R. prolixus. Immunohistochemistry of the CNS showed diminished CRF-like staining in neurosecretory cells (NSCs) of the mesothoracic ganglionic mass (MTGM) immediately following feeding, and partial restocking of those same cells two hours later, indicating Rhopr-CRF/DH stores in this regions are involved in feeding. The results of the temporal qPCR analysis were consistent with the immunohistochemical findings, showing an increase in Rhopr-CRF/DH transcript expression in the MTGM immediately after feeding, presumably capturing the restocking of Rhopr-CRF/DH in the lateral NSCs following release of the peptide during feeding. Elevating haemolymph Rhopr-CRF/DH titres by injection of Rhopr-CRF/DH prior to feeding resulted in the intake of a significantly smaller blood meal in 5th instars and adults without an apparent effect on the rate of short-term diuresis. When adult females were injected with Rhopr-CRF/DH, they also produced and laid significantly fewer eggs. Finally, in vitro oviduct contraction assays illustrate that Rhopr-CRF/DH inhibits the amplitude of contractions of the lateral oviducts, highlighting a potential mechanism via which the hormone diminishes reproductive capacity. To conclude, the study of the Rhopr-CRF/DH pathway, its components and mechanisms of action, has implications for vector control by highlighting targets to alter feeding, diuresis, and reproduction of this disease vector.

Allatostatins (ASTs) are neuropeptides that were first identified as inhibitors of juvenile hormone biosynthesis by the corpora allata of some insect species. The FGLamide-related ASTs (FGLa/ASTs) belong to one of three families of insect ASTs. Previously, we showed that Rhodnius prolixus FGLa/ASTs (Rhopr-FGLa/ASTs) are present throughout the R. prolixus central nervous system and are associated with 5 dorsal unpaired median (DUM) neurons in the mesothoracic ganglionic mass. A similar set of neurons contain serotonin which is a diuretic hormone in R. prolixus. Rhopr-FGLa/ASTs inhibit both spontaneous contractions of the anterior midgut and leucokinin-1-induced hindgut contractions. Since these tissues are involved with post-feeding diuresis, these data suggest a possible role for FGLa/ASTs in events associated with feeding, and a possible interaction with serotonin. To investigate this possibility, we have examined the DUM neurons in more detail with regard to their peptide content, examined the potential release of Rhopr-FGLa/ASTs into the haemolymph following feeding, and further investigated the effects of Rhopr-FGLa/ASTs on feeding-related tissues. There are 10 DUM neurons in the abdominal neuromeres, 5 of which express serotonin-like immunoreactivity and the other 5 express FGLa/AST-like immunoreactivity. FGLa/AST-like immunoreactivity is reduced in the 5 DUM neuron cell bodies and their neurohaemal sites on abdominal nerves at 3-5 h post feeding. Rhopr-FGLa/ASTs do not inhibit serotonin-stimulated anterior midgut absorption or Malpighian tubule secretion but do inhibit hindgut contractions induced by an endogenous kinin, suggesting that they may only indirectly affect post-feeding diuresis in R. prolixus.

Within invertebrates, the kinin family of neuropeptides is responsible for the modulation of a host of physiological and behavioural processes. In Rhodnius prolixus, kinins are primarily responsible for eliciting myotropic effects on various feeding and diuresis-related tissues. Here, the R. prolixus kinin receptor (RhoprKR) has been identified, cloned and sequenced from the central nervous system (CNS) and hindgut of R. prolixus. Sequence analyses show high similarity and identity between RhoprKR and other cloned invertebrate kinin receptors. The expression profile of RhoprKR shows the RhoprKR transcript throughout the R. prolixus gut, with highest expression in the hindgut, suggesting a role of Rhopr-kinins in various aspects of feeding and digestion. RNA interference (RNAi)-mediated knockdown of the RhoprKR transcript resulted in a significant reduction of hindgut contractions in response to Rhopr-kinin 2 and an Aib-containing kinin analog. dsRhoprKR- injected insects also consumed a significantly larger meal, suggesting a role of Rhopr-kinins in satiety.

Two structurally related peptides, arginine vasotocin (AVT) and mesotocin (MT), are reported to regulate many physiological processes, such as anti-diuresis and oviposition in birds, and their actions are likely mediated by four AVT/MT receptors (AVPR1A, AVPR1B, MTR and AVPR2b), which are orthologous/paralogous to human AVPR1A, AVPR1B, OXTR and AVPR2 respectively. However, our knowledge regarding the functions of these avian AVT/MT receptors has been limited. Here, we examined the functionality and expression of these receptors in chickens and investigated the roles of AVT in the anterior pituitary. Our results showed that 1) AVPR1A, AVPR1B and AVPR2b could be preferentially activated by AVT, monitored by cell-based luciferase reporter assays and/or Western blot, indicating that they are AVT-specific receptors (AVPR1A; AVPR1B) or AVT-preferring receptor (AVPR2b) functionally coupled to intracellular calcium, MAPK/ERK and cAMP/PKA signaling pathways. In contrast, MTR could be activated by AVT and MT with similar potencies, indicating that MTR is a receptor common for both peptides; 2) Using qPCR, differential expression of the four receptors was found in chicken tissues including the oviduct and anterior pituitary. In particular, only AVPR1A is abundantly expressed in the uterus, suggesting its involvement in mediating AVT-induced oviposition. 3) In cultured chick pituitary cells, AVT could stimulate ACTH and PRL expression and secretion, an action likely mediated by AVPR1B and/or AVPR1A abundantly expressed in anterior pituitary. Collectively, our data helps to elucidate the roles of AVT/MT in birds, such as the 'oxytocic action' of AVT, which induces uterine muscle contraction during oviposition.