Calcitonin receptor 1 (AedaeGPCRCAL1) hindgut expression and direct role in myotropic action in females of the mosquito Aedes aegypti (L.)

• AedaeGPCRCAL1 was immunolocalized in circular and longitudinal hindgut muscles.
• Aedae-DH31, calcitonin-like diuretic hormone 31, significantly increased hindgut contractions.
• RNAi of AedaeGPCRCAL1 reduced the hindgut contraction frequency by 50% with respect to control mosquitoes after application of Aedae-DH31.

In anautogenous mosquitoes such as Aedes aegypti females the calcitonin-like diuretic hormone 31 (DH31) stimulates natriuretic fluid excretion from the Malpighian tubules (MTs) after a blood meal. We previously cloned and functionally characterized AedaeGPCRcal1 from A. aegypti, the ortholog of the Drosophila melanogaster DH31 receptor and immunolocalized it in the MTs. However, localization of the calcitonin receptor-like receptor 1 (GPCRCAL1) in the hindgut of any insect is unknown, and specifically, knowledge on its role in hindgut contraction in response to Aedae-DH31 peptide is lacking. We analyzed the expression of AedaeGPCRCAL1 in hindgut by western blot and immunohistochemisty, and evaluated its role in hindgut contractility by application of Aedae-DH31 before and after receptor RNA interference (RNAi). The receptor was detected as a 73 kDa band in western blots of hindgut and immunofluorescence revealed the receptor was expressed in hindgut circular and longitudinal muscles but not in the hindgut epithelial cells. In vitro, incubation in 1 μM solution of Aedae-DH31 peptide significantly increased the hindgut contraction frequency in normal mosquitoes. Hindguts from females treated with AedaeGPCRcal1 dsRNA and incubated with DH31 showed a reduction of 50% percent in their contraction frequency with respect to controls. These results suggest that DH31 hormone released from the brain post-blood meal has a direct and coordinative action on the excretory system, MTs and hindgut, by which AedaeGPCRCAL1 signaling stimulates MT primary urine secretion and hindgut contraction resulting in rapid postprandial fluid excretion.

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