Role in Diuresis of a Calcitonin Receptor (GPRCAL1) Expressed in a Distal-Proximal Gradient in Renal Organs of the Mosquito Aedes aegypti (L.)

Evolution of anthropophilic hematophagy in insects resulted in the coordination of various physiological processes for survival. In female mosquitoes, a large blood meal provides proteins for egg production and as a trade-off, rapid elimination of the excess water and solutes (Na⁺, Cl⁻) is critical for maintaining homeostasis and removing excess weight to resume flight and avoid predation. This post-prandial excretion is achieved by the concerted action of multiple hormones. Diuresis and natriuresis elicited by the calcitonin-like diuretic hormone 31 (DH₃₁) are believed to be mediated by a yet uncharacterized calcitonin receptor (GPRCAL) in the mosquito Malpighian tubules (MTs), the renal organs. To contribute knowledge on endocrinology of mosquito diuresis we cloned GPRCAL1 from MT cDNA. This receptor is the ortholog of the DH₃₁ receptor from Drosophila melanogaster that is expressed in principal cells of the fruit fly MT. Immunofluorescence similarly showed AaegGPRCAL1 is present in MT principal cells in A. aegypti, however, exhibiting an overall gradient-like pattern along the tubule novel for a GPCR in insects. Variegated, cell-specific receptor expression revealed a subpopulation of otherwise phenotypically similar principal cells. To investigate the receptor contribution to fluid elimination, RNAi was followed by urine measurement assays. In vitro, MTs from females that underwent AaegGPRcal1 knock-down exhibited up to 57% decrease in the rate of fluid secretion in response to DH₃₁. Live females treated with AaegGPRcal1 dsRNA exhibited 30% reduction in fluid excreted after a blood meal. The RNAi-induced phenotype demonstrates the critical contribution of this single secretin-like family B GPCR to fluid excretion in invertebrates and highlights its relevance for the blood feeding adaptation. Our results with the mosquito AaegGPRCAL1 imply that the regulatory function of calcitonin-like receptors for ion and fluid transport in renal organs arose early in evolution.     

The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster

Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH₄₄) and Drosophila melanogaster kinin (Drome-kinin, DK) in desiccation and starvation tolerance.Gene expression and labelled DH₄₄ ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH₄₄ in neurons of the pars intercerebralis and DH₄₄ receptor (DH₄₄-R2) in Malpighian tubule principal cells, indicate that suppression of DH₄₄ signalling improves desiccation tolerance of the intact fly.Drome-kinin receptor, encoded by the leucokinin receptor gene, LKR, is expressed in DH₄₄ neurons as well as in stellate cells of the Malpighian tubules. LKR knockdown in DH₄₄-expressing neurons reduces Malpighian tubule-specific LKR, suggesting interactions between DH₄₄ and LK signalling pathways.Finally, although a role for DK in desiccation tolerance was not defined, we demonstrate a novel role for Malpighian tubule cell-specific LKR in starvation tolerance. Starvation increases gene expression of epithelial LKR. Also, Malpighian tubule stellate cell-specific knockdown of LKR significantly reduced starvation tolerance, demonstrating a role for neuropeptide signalling during starvation stress.     

Calcium influx enhances neuropeptide activation of ecdysteroid hormone production by mosquito ovaries

A critical step in mosquito reproduction is the ingestion of a blood meal from a vertebrate host. In mosquitoes like Aedes aegypti, blood feeding stimulates the release of ovary ecdysteroidogenic hormone (OEH) and insulin-like peptide 3 (ILP3). This induces the ovaries to produce ecdysteroid hormone (ECD), which then drives egg maturation. In many immature insects, prothoracicotropic hormone (PTTH) stimulates the prothoracic glands to produce ECD that directs molting and metamorphosis. The receptors for OEH, ILP3 and PTTH are different receptor tyrosine kinases with OEH and ILP3 signaling converging downstream in the insulin pathway and PTTH activating the mitogen-activated protein kinase pathway. Calcium (Ca²⁺) flux and cAMP have also been implicated in PTTH signaling, but the role of Ca²⁺ in OEH, ILP3, and cAMP signaling in ovaries is unknown. Here, we assessed whether Ca²⁺ flux affects OEH, ILP3, and cAMP activity in A. aegypti ovaries and also asked whether PTTH stimulated ovaries to produce ECD. Results indicated that Ca²⁺ flux enhanced but was not essential for OEH or ILP3 activity, whereas cAMP signaling was dependent on Ca²⁺ flux. Recombinant PTTH from Bombyx mori fully activated ECD production by B. mori PTGs, but exhibited no activity toward A. aegypti ovaries. Recombinant PTTH from A. aegypti also failed to stimulate either B. mori PTGs or A. aegypti ovaries to produce ECD. We discuss the implications of these results in the context of mosquito reproduction and ECD biosynthesis by insects generally.     

Isolation and Functional Characterization of Calcitonin-Like Diuretic Hormone Receptors in Rhodnius prolixus

Several families of diuretic hormones exist in insects, one of which is the calcitonin-like diuretic hormone (CT/DH) family. CT/DH mediates its effects by binding to family B G-protein coupled receptors (GPCRs). Here we isolate and functionally characterize two R. prolixus CT/DH receptor paralogs (Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2) using a novel heterologous assay utilizing a modified human embryonic kidney 293 (HEK293) cell line. Rhopr-CT/DH-R1 is orthologous to the previously characterized D. melanogaster CT/DH receptor (CG17415) while Rhopr-CT/DH-R2 is orthologous to the D. melanogaster receptor (CG4395), an orphan receptor whose ligand was unknown until now. We determine the cDNA sequences of three splice variants encoding Rhopr-CT/DH-R1 (Rhopr-CT/DH-R1-A, Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C) and two splice variants encoding Rhopr-CT/DH-R2 (Rhopr-CT/DH-R2-A and Rhopr-CT/DH-R2-B). Rhopr-CT/DH-R1-A and Rhopr-CT/DH-R2-A encode truncated receptors that lack six and seven of the characteristic seven transmembrane domains, respectively. Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C, which only differ by 2 amino acids in their C-terminal domain, can both be activated by Rhopr-CT/DH at equal sensitivities (EC₅₀ = 200-300nM). Interestingly, Rhopr-CT/DH-R2-B is much more sensitive to Rhopr-CT/DH (EC₅₀ = 15nM) compared to Rhopr-CT/DH-R1-B/C and also yields a much greater response (amplitude) in our heterologous assay. This is the first study to reveal that insects possess at least two CT/DH receptors, which may be functionally different. Quantitative PCR demonstrates that Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2 have distinct expression patterns, with both receptors expressed centrally and peripherally. Moreover, the expression analysis also identified novel target tissues for this neuropeptide, including testes, ovaries and prothoracic glands, suggesting a possible role for Rhopr-CT/DH in reproductive physiology and development.     

Investigations of the signaling cascade involved in diuretic hormone stimulation of Malpighian tubule fluid secretion in Rhodnius prolixus

In insects, the excretory system is comprised of the Malpighian tubules (MTs) and the hindgut, which collectively function to maintain ionic and osmotic balance of the haemolymph and rid the organism of toxic compounds or elements in excess. Secretion by the Malpighian tubules of insects is regulated by a variety of hormones including peptidergic factors as well as biogenic amines. In Rhodnius prolixus, two endogenous diuretic hormones have been identified; the biogenic amine serotonin (5-hydroxytryptamine, 5-HT) and the corticotropin releasing factor-related peptide, RhoprCRF. Both factors significantly increase secretion by MTs and are known to elevate intracellular levels of cAMP. Interestingly, applying sub-maximal doses of these two diuretic factors in combination on isolated MTs in vitro reveals synergistic effects as rates of fluid secretion are significantly higher than would be expected if rates of secretion from MTs treated with each factor alone were summed. This observed synergism suggests that different downstream targets may be activated by the two diuretic factors, but that some cellular elicitors may be shared since cAMP is elevated in response to either diuretic hormone.     

Nutrient-mediated juvenile hormone secretion in mosquitoes

Our objective was to determine whether environmental conditions affect the juvenile hormone (J.H.) regulated phase of ovarian development of mosquitoes. Primary ovarian follicles of nutrient-deprived adult Aedes aegypti reared in non-crowded cultures remain in an early, teneral stage of development. After a meal of sucrose or blood, follicles of crowded mosquitoes develop to a stage competent to deposit yolk directly following blood ingestion; a topically-applied analogue of J.H. stimulates the ovaries to develop to a similar stage. A. aegypti collected in nature are of a size suggesting that their follicles would remain in the teneral stage when adults are nutrient-deprived.     

Two stages of juvenile hormone-mediated growth of secondary follicles in Culex pipiens

To investigate the effect of juvenile hormone deprivation on the growth of secondary follicles during the second gonotrophic cycle, female mosquitoes, Culex pipiens L., were allatectomized daily after the first blood meal. Allatectomy on days 1–3 suppressed growth of secondary follicles indicating that juvenile hormone was required for a second gonotrophic cycle. When allatectomy was performed 4 days or more after the first blood meal, secondary follicles grew, indicating the presence of juvenile hormone. However, if mosquitoes were allatectomized before oviposition, only 25% developed a second batch of eggs after a second blood meal.Allatectomies performed 1 and 24 h after oviposition indicated that additional juvenile hormone was released after deposition of the first batch of eggs, and that this second release was needed for secondary follicles to complete previtellogenic growth. Thus, in C. pipiens, secondary follicles undergo two periods of juvenile hormone-mediated growth-one before and one after oviposition.     

Activation of vitellogenin synthesis in the mosquito Aedes aegypti by ecdysone

Injected β-ecdysone was found to induce the synthesis of yolk protein (vitellogenin) in adult female Aedes aegypti without a blood meal. After injection of 5 μg ecdysone per mosquito, vitellogenin constituted 80 per cent of the total protein secreted by explanted fat body, a proportion comparable to that produced by fat body from blood-fed females. Moreover, the time course of induction of vitellogenin synthesis in ecdysone-injected mosquitoes was similar to that triggered by a blood meal. Response to ecdysone is dosedependent: 0·5 μg per female was required to stimulate synthesis to 50 per cent of the level found 18 hr after a blood meal. Ecdysone was effective in decapitated or ovariectomized mosquitoes, and also when applied directly to fat body preparations in vitro. Thus it appears that ecdysone acts directly on the fat body to induce specific protein synthesis, as does the vitellogenin stimulating hormone (VSH) from the ovary of blood-fed mosquitoes. These results suggest that ecdysone can replace VSH in inducing vitellogenin synthesis in the unfed mosquito.     

Juvenile hormone regulation of the second biting cycle in Culex pipiens

Juvenile hormone regulation of the second biting cycle was studied in Culex pipiens by allatectomizing mosquitoes at daily intervals after the first blood meal. Mosquitoes oviposited and were tested for biting 2 days later. Allatectomy on days 1 through 4 prevented biting in a high percentage of females, indicating that juvenile hormone was required for a second biting cycle. When allatectomy was delayed 6 or more days after the first blood meal, the mosquitoes took a second blood meal after oviposition even though eggs were retained at the time of allatectomy. Thus, egg retention did not prevent mosquitoes from releasing juvenile hormone for a second biting cycle. Unoperated mosquitoes also bit when they were forced to retain eggs after the first blood meal. However, these mosquitoes only fed on blood if they were confined on or near the host; they would not feed in cages where host seeking was required to locate the host. Based on these findings, it appeared that oviposition was necessary to initiate host-seeking behaviour, whereas juvenile hormone release initiated biting. These results, in conjunction with earlier work demonstrating juvenile hormone induction of the first biting cycle, indicated that alternating periods of juvenile hormone production and oviposition regulate the cyclic patterns of biting and host-seeking behaviour throughout the life of the mosquito.     

Behavioral and molecular responses of Aedes aegypti to ultrasound

Mosquito-borne infectious diseases cause mortality and global infectious disease burden worldwide. There are several electronic mosquito repellents (EMRs) based on ultrasound have been developed and commercialized to reduce human-mosquito contacts. However, the efficacy of EMRs against mosquitoes is still unclear. In this study, we present experimental evidence that ultrasound of different frequency and sound pressure differentially affects the host-seeking behavior of Aedes aegypti females. Behavioral tests were accompanied by molecular experiments to check whether mosquitoes respond to ultrasound and are there any changes in specific mRNA expression. Experiments in bioassays revealed that the ultrasound of 100 kHz frequency and 90–110 dB pressure significantly disrupted CO?-oriented olfactory behaviors and blocked indoor invasion. Furthermore, a long time (>24 h) exposure to 100 kHz frequency/90 dB pressure of ultrasound decreased attractive behaviors to human skin. At the molecular level, there was no change in expression of odorant receptor co-receptor (AaOrco) in ultrasound treated animals, while one of the CO₂ receptor genes, AaGr3, and putative hearing-related gene, AAEL009258, were down-regulated and up-regulated, respectively. Our study indicates that high frequency (100 kHz) and pressure (90–110 dB) of the ultrasound has repellent effects to olfactory–driven behaviors of mosquitoes.     

Physiological studies in heterozygous calcium sensing receptor (CaSR) gene-ablated mice confirm that the CaSR regulates calcitonin release <Emphasis Type="Italic">in vivo</Emphasis>

Background  

The calcium sensing receptor (CaSR) regulates serum calcium by suppressing secretion of parathyroid hormone; it also regulates renal tubular calcium excretion. Inactivating mutations of CaSR raise serum calcium and reduce urine calcium excretion. Thyroid C-cells (which make calcitonin) express CaSR and may, therefore, be regulated by it. Since calcium stimulates release of calcitonin, the higher blood calcium caused by inactivation of CaSR should increase serum calcitonin, unless CaSR mutations alter the responsiveness of calcitonin to calcium.     

Gamma-melanocyte stimulating hormone regulates the expression and cellular localization of epithelial sodium channel in inner medullary collecting duct cells

Gamma₂-melanocyte-stimulating hormone (γ₂MSH) is a peptide hormone released by the pituitary gland which is thought to act directly on the renal inner medulla to promote increased sodium excretion into urine (natriuresis). The aim of this study was to determine if a stable analog, [Nle³, D-Phe⁶]-γ₂MSH (NDP-γ₂MSH), of the native peptide regulated the activity, expression and cellular localization of epithelial sodium channel (ENaC) in a murine inner medullary collecting duct (mIMCD-3) cell line. Our results indicate that expression of the γ₂MSH receptor, melanocortin receptor 3 receptor (MC3R), is up-regulated by culturing the cells in media with an increased osmolality (∼400 mOsm/kg). Furthermore, stimulation of cAMP signaling and sodium transport by 1 nM NDP-γ₂MSH occurs only in cells cultured in the high osmolality media. Finally, treatment of mIMCD-3 cells cultured in high osmolality medium for 1 h with 1 nM NDP-γ₂MSH causes a reduction in expression of serum- and glucocorticoid-induced kinase (sgk1) and a reduction in expression and cell surface abundance of the alpha subunit of ENaC. Collectively, this data suggest that γ₂MSH directly regulates both ENaC expression and cellular localization in the inner medulla to exert its natriuretic effect.     

Metallothionein-like proteins and zinc-copper interaction in the hindgut of Porcellio scaber (crustacea: Isopoda) exposed to zinc

Metallothioneins (MTs) are ubiquitous low-molecular-weight metalbinding proteins, with a variety of functions in metal metabolism ascribed to them. Among terrestrial invertebrates, MTs have been studied in nematodes, insects, snails, and earthworms. The aim of this study was the characterization of MT-like proteins in the terrestrial isopod crustacean Porcellio scaber in order to analyze their probable role in the metaboliss of copper (Cu) and zinc (Zn). Dietary Zn supplementation (793 μg Zn/g dry food, 6 d) was applied to stimulate MT synthesis. After separation of the hindgut postmicrosomic supernatant (cytosol) of Zn-exposed animals by gel filtration on a Sephadex G-75 column, a Cu- and Zn-containing peak was detected in the position of V _c/V_o≈2, where MTs are expected to elute. Rechromatography of these fractions by size-exclusion chromatography-high-performance liquid chromatography revealed that the 215-nm absorbance peak coincided with the absorbance peak of the rabbit MT II standard. These low-molecular-weight Cu- and Zn-binding compounds, detected in the cytosol of the hindgut cells in Zn-exposed P. scaber. are considered to be Cu, Zn-MT-like proteins. To our knowledge, this is the first report on the characterization of MT-like proteins in isopod crustaceans. These results also indicate that both Zn and Cu dynamics in P. scaber hindgut are affected at the given dietary Zn supplementation and that MT-like proteins are involved in this Zn-Cu interaction.     

Release of egg development neurosecretory hormone in Aedes aegypti and Aedes taeniorhynchus induced by an ovarian factor

Ovariectomized Aedes aegypti do not synthesize vitellogenin after a blood meal, unless an ovary from a blood-fed donor is implanted. Decapitation, however, prior to implantation inhibits vitellogenin synthesis. A female ovariectomized and decapitated 6 hr after a blood meal, synthesizes vitellogenin if an ovary from a blood-fed donor is implanted. On the other hand, females that are fed on blood and immediately decapitated can not be stimulated to synthesize vitellogenin with implanted ovaries removed from blood-fed donors. These experiments led to the hypothesis that the blood meal stimulates the ovary to secrete a corpus cardiacum stimulating factor, that in turn promotes release of egg development neurosecretory hormone stored in the corpus cardiacum.Injection of 20-hydroxy-ecdysone or ovarian extract prepared from ovaries removed from unfed females does not release egg development neurosecretory hormone. Thus corpus cardiacum stimulating factor is not 20-hydroxy-ecdysone, and ovaries removed from unfed females do not store it.The rate of inactivation of egg development neurosecretory hormone released from the corpus cardiacum after a blood meal was investigated by implanting an ovary into females that were blood fed for various intervals than decapitated and ovariectomized. Seventy per cent of implants grow when the operation is done 18 hr after feeding, and 30% when the operation is done between 18 and 24 hr after feeding, indicating that egg development neurosecretory hormone is stable for the first 18 hr after a blood meal.Aedes taeniorhynchus females ovariectomized 24 hr after adult emergence do not synthesize vitellogenin. When such a female is implanted with an ovary removed from a sugar-fed or blood-fed Aedes aegypti donor vitellogenin synthesis is initiated, and the implant grows. Decapitation prior to implantation inhibit vitellogenin synthesis and implants do not grow. These results indicate that corpus cardiacum stimulating factor is not species specific.     

Isolation,identification and synthesis of locustapyrokinin II from Locusta migratoria,another member of the FXPRL-amide peptide family

1. A blocked decapeptide was isolated from brain corpora cardiaca-corpora allata sub-oesophageal ganglion extracts of the locust, Locusta migratoria. Biological activity was monitored during HPLC purification by observing the myotropic effect of column fractions on the isolated hindgut of Leucophaea maderae.2. The primary structure of this myotropic peptide was established as: pGlu-Ser-Val-Pro-Thr-Phe-Thr-Pro-Arg-Leu-NH₂.3. The Chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis.4. This decapeptide is the sixth natural analog of a series of locust peptides with a Phe-X-Pro-Arg-Leu-NH₂ carboxyterminus. This carboxyl terminal sequence is also found in other peptides identified in other insects and it is the biological active core sequence for diverse biological activities: muscle contraction, pheromone production, pigment synthesis and diapauze.5. Like the locustamyotropins and locustapyrokinin I, locustapyrokinin II stimulates contractions of the oviduct in Locusta.     

The BB2 receptor antagonist BW2258U89 attenuates the feeding responses evoked by exogenous gastrin releasing peptide-29

This confirmatory work is aimed to test that the hypothesis that the gastrin releasing peptide (GRP) receptor – the BB₂ receptor – is necessary for reduction of meal size (MS) and prolongation of the intermeal interval (IMI) by the small and the large forms of GRP in the rat, GRP-10 and GRP-29, and to confirm the sites of action regulating such responses – the vascular bed of the celiac artery (CA, supplying stomach and upper duodenum). To pursue these aims we measured first MS and IMI length in response to GRP-10 and GRP-29 (0, 0.5 nmol/kg) infused in the CA (n = 8 rats) and the cranial mesenteric artery (CMA, supplying the small and part of the large intestine, n = 8 rats) in near spontaneously free feeding rats pretreated with the BB₂ receptor antagonist BW2258U89 (0.1 mg/kg) in the same arteries prior to the onset of the dark cycle. We found that GRP-29, but not GRP-10, infused by the CA reduced MS and prolonged the IMI by decreasing meal latency and meal duration and the BB₂ receptor antagonist BW2258U89 infused in the same artery attenuated these responses. These results suggest that the BB₂ receptor is necessary for reduction of MS and prolongation of the IMI by exogenous GRP-29, and the vascular bed of the CA, stomach and upper duodenum, contains sites of action regulating these feeding responses.