Functional characterization of CCHamides and deorphanization of their receptors in the yellow fever mosquito, Aedes aegypti

Abstract

As a widely distributed anthropophilic mosquito species and vector of various arboviruses, Aedes aegypti poses a significant threat to human health on a global scale. Investigating mosquito neuropeptides allows us to better understand their physiology. The neuropeptides CCHamide1 (CCHa-1) and CCHamide-2 (CCHa-2) and their associated G protein-coupled receptors (CCHa-1R and CCHa-2R) were recently identified and studied across insects. However, expression profiles and physiological roles of CCHamides and their receptors in many other insects, including A. aegypti, remain unclear. This research aimed to quantify and localize the expression of CCHamides along with their receptors and to elucidate their physiological function in the yellow fever mosquito. RT-qPCR analysis revealed transcript abundance of CCHamides and receptors changes over development. Differential expression was also observed in tissues/organs of adult mosquitoes indicating CCHa-1 and CCHa-2 transcripts are most highly enriched in the midgut, while receptors are expressed across various tissues. Further, CCHamides were immunolocalized in neurons in the brain and ventral nerve cord along with enteroendocrine cells in the posterior midgut adjacent to the midgut-hindgut junction, corroborating their transcript expression profiles. Using different mass spectrometrical approaches, presence of CCHamides were confirmed in the brain of both sexes, including the pars intercerebralis of female mosquitoes, as well as in the gut of adult mosquitoes. For chemical identification of predicted CCHamides, we analyzed brain and gut extracts by ESI-Q Exactive Orbitrap MS and resulting fragmentations confirmed CCHa1 and CCHa2 in brain and midgut samples of both male and female mosquitoes. A heterologous functional assay was used to confirm the specificity and sensitivity of the two CCHamide receptors by assessing their activation in response to diverse mosquito peptides, which confirmed CCHa-1 and CCHa-2 as the natural ligands. Finally, using a capillary feeder (CAFE) bioassay, our results suggest that CCHa-2 modulates feeding behaviour in female mosquitoes.