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.昆虫嗜血性的演化进程促使各类生理过程协同调控以维持生存。对于雌性蚊子而言，足量吸血可为卵子发生提供蛋白质，而作为相应的权衡机制，快速清除体内多余水分与溶质（Na+、Cl−）对维持体内稳态、减轻体重以恢复飞行并规避捕食均至关重要。这种餐后排泄过程需依托多种激素的协同作用方能完成。由降钙素样利尿激素31（calcitonin-like diuretic hormone 31, DH31）诱发的利尿与排钠效应，被认为是通过蚊子肾脏器官——马尔皮基小管（Malpighian tubules, MTs）中尚未鉴定的降钙素受体（GPRCAL）介导实现的。为丰富蚊子利尿内分泌学的研究认知，我们从马尔皮基小管的cDNA中克隆得到了GPRCAL1。该受体是黑腹果蝇（Drosophila melanogaster）DH31受体的同源基因，后者在果蝇马尔皮基小管的主细胞中表达。免疫荧光实验同样证实，埃及伊蚊（Aedes aegypti）体内的AaegGPRCAL1表达于其马尔皮基小管主细胞中，且呈现出沿小管分布的整体梯度模式——这在昆虫G蛋白偶联受体（G protein-coupled receptor, GPCR）中尚属首次报道。这种不均一的细胞特异性受体表达模式，揭示了一组表型相似的主细胞亚群。为探究该受体对体液清除的调控作用，我们采用RNA干扰（RNA interference, RNAi）结合尿液测量实验开展研究。体外实验显示，经AaegGPRcal1敲低的雌性蚊子马尔皮基小管，在受到DH31刺激时的体液分泌速率最多下降57%。经AaegGPRcal1双链RNA（double-stranded RNA, dsRNA）处理的活体雌性蚊子，吸血后排出的体液量减少30%。RNA干扰诱导的表型证实，这一属于分泌素样B家族的G蛋白偶联受体，在无脊椎动物的体液排泄过程中发挥关键作用，并凸显其与吸血适应性的相关性。我们针对埃及伊蚊AaegGPRCAL1的研究结果表明，降钙素样受体在肾脏器官中调控离子与体液转运的功能，在演化早期便已出现。