The Peptide Hormone CNMa Influences Egg Production in the Mosquito Aedes aegypti.

Mosquito reproduction is regulated by a suite of hormones, many acting through membrane-bound receptor proteins. The Aedes aegypti G protein-coupled receptors AAEL024199 (AeCNMaR-1a) and AAEL018316 (AeCNMaR-1b) were identified as orthologs of the Drosophila melanogaster CNMa receptor (DmCNMaR). The receptor was duplicated early in the evolution of insects, and subsequently in Culicidae, into what we refer to as CNMaR-1a and CNMaR-1b. AeCNMaR-1a is only detected in male mosquito antennae while AeCNMaR-1b is expressed at high levels in mosquito ovaries. Using a heterologous cell assay, we determined that AeCNMa activates AeCNMaR-1a with a ~10-fold lower concentration than it does AeCNMaR-1b, though both receptors displayed half maximal effective concentrations of AeCNMa in the low nanomolar range. Finally, we show that injections of AeCNMa into blood-fed mated female Ae. aegypti resulted in fewer eggs laid.

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito.

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

Receptor Characterization and Functional Activity of Pyrokinins on the Hindgut in the Adult Mosquito, Aedes aegypti.

Pyrokinins are structurally related insect neuropeptides, characterized by their myotropic, pheromonotropic and melanotropic roles in some insects, but their function is unclear in blood-feeding arthropods. In the present study, we functionally characterized the pyrokinin-1 and pyrokinin-2 receptors (PK1-R and PK2-R, respectively), in the yellow fever mosquito, Aedes aegypti, using a heterologous cell system to characterize their selective and dose-responsive activation by members of two distinct pyrokinin subfamilies. We also assessed transcript-level expression of these receptors in adult organs and found the highest level of PK1-R transcript in the posterior hindgut (rectum) while PK2-R expression was enriched in the anterior hindgut (ileum) as well as in reproductive organs, suggesting these to be prominent target sites for their peptidergic ligands. In support of this, PRXa-like immunoreactivity (where X = V or L) was localized to innervation along the hindgut. Indeed, we identified a myoinhibitory role for a PK2 on the ileum where PK2-R transcript was enriched. However, although we found that PK1 did not influence myoactivity or Na+ transport in isolated recta, the PRXa-like immunolocalization terminating in close association to the rectal pads and the significant enrichment of PK1-R transcript in the rectum suggests this organ could be a target of PK1 signaling and may regulate the excretory system in this important disease vector species.

CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti.

Insect CAPA neuropeptides are homologs of mammalian neuromedin U and are known to influence ion and water balance by regulating the activity of the Malpighian 'renal' tubules (MTs). Several diuretic hormones are known to increase primary fluid and ion secretion by insect MTs and, in adult female mosquitoes, a calcitonin-related peptide (DH31) called mosquito natriuretic peptide, increases sodium secretion to compensate for the excess salt load acquired during blood-feeding. An endogenous mosquito anti-diuretic hormone was recently described, having potent inhibitory activity against select diuretic hormones, including DH31. Herein, we functionally deorphanized, both in vitro and in vivo, a mosquito anti-diuretic hormone receptor (AedaeADHr) with expression analysis indicating highest enrichment in the MTs where it is localized within principal cells. Characterization using a heterologous in vitro system demonstrated the receptor was highly sensitive to mosquito CAPA neuropeptides while in vivo, AedaeADHr knockdown abolished CAPA-induced anti-diuretic control of DH31-stimulated MTs. CAPA neuropeptides are produced within a pair of neurosecretory cells in each of the abdominal ganglia, whose axonal projections innervate the abdominal neurohaemal organs, where these neurohormones are released into circulation. Lastly, pharmacological inhibition of nitric oxide synthase (NOS) and protein kinase G (PKG) signaling eliminated anti-diuretic activity of CAPA, highlighting the role of the second messenger cGMP and NOS/PKG in this anti-diuretic signaling pathway.