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In the yellow fever mosquito, Aedes aegypti (L.), hemolymph-circulating diuretic hormones act upon the renal organs (Malpighian tubules) to regulate primary urine composition and secretion rate; however, the molecular endocrine mechanisms underlying rapid water elimination upon adult eclosion and blood feeding are not fully understood. Bioinformatic analysis of the current Aedes aegypti genome assembly reveals only a single predicted corticotropin releasing factor (CRF)-like diuretic hormone 44 (DH44) gene, but two DH44 receptor genes. The tissue expression profiles of the DH44 receptor(s), and specifically the identity of the DH44 receptor(s) in the Malpighian tubule, are undetermined in any mosquito species. This dissertation shows that Vectorbase gene ID AAEL008292 encodes a DH44 receptor (AaegGPRdih1) transcribed in Malpighian tubules. Sequence analysis and transcript localization indicate that AaegGPRdih1 is the co-ortholog of the Drosophila melanogaster DH44 receptor (CG12370-PA). The presence of conserved amino acid residues between AaegGPRdih1 and vertebrate CRF receptors suggests this mosquito receptor modulates multiple G protein-dependent intracellular signaling pathways. Quantitative PCR analysis of a time course of Malpighian tubule cDNA reveals AaegGPRdih1 abundance increases paralleling periods of observed urination. This suggests that target tissue receptor biology is linked to the known periods of release of diuretic hormones from the nervous system, pointing to a common up-stream regulatory mechanism. Higher relative abundance of AaegGPRdih1 transcript in female Malpighian tubules 24 hours after blood feeding suggests a role for AaegGPRdih1 in the excretion of nitrogen waste. RNA-mediated silencing to establish the significance of AaegGPRdih1 to mosquito Malpighian tubule physiology was inconclusive.

<p>DH31 and drosokinin are known promoters of fluid secretion in <em>Drosophila</em> Malpighian tubules, while the effect of allatostatin A on Malpighian tubule fluid secretion is unknown. The expression of these peptides and their receptors is widespread in the larval gut and central nervous system. The scanning ion-selective electrode technique (SIET) was used to measure changes in epithelial K⁺ flux in the gut as a proxy for the flow of osmotically-obliged water between the gut lumen and the haemolymph. The primary goal of this study was to investigate the effects of DH31 and drosokinin on the gut as an indicator of coordination of diuretic activity between this tissue and the Malpighian tubules. Such coordination, whereby Malpighian tubule fluid secretion is stimulated concomitantly with fluid uptake by the gut, would be physiologically essential for the maintenance of haemolymph volume and osmolarity. Secondarily, this study sought to characterize the function of allatostatin A with respect to its effect on gut K⁺ transport. DH31 stimulated K⁺ absorption across the anterior midgut epithelium and reduced K⁺ absorption in the middle midgut copper cell zone. Drosokinin increased K⁺ absorption across the anterior midgut epithelium and was also stimulatory in the posterior midgut neutral zone. Allatostatin A stimulated K⁺ absorption across the epithelia of the anterior midgut and middle midgut copper cell zone, but was inhibitory in the large flat cell zone. The larger surface area of the anterior midgut relative to the middle midgut means that all three peptides were likely net stimulators of K⁺ uptake. The reduction in K⁺ absorption in the middle midgut by DH31 and allatostatin A may relate to a redistribution of fluid uptake among the regions of the midgut to preserve lumenal pH. DH31 and drosokinin also independently increased the contraction frequency of the anterior midgut, while the contraction frequency of the pyloric sphincter was increased by combined application of both peptides. Stimulation of gut contractions has previously been attributed to these and other diuretic factors in insects. The findings of this investigation implicate DH31, drosokinin, and allatostatin A in the stimulation of midgut K⁺ absorption, which may suggest a role for these peptides in altering fluid transport across this epithelium in larval <em>Drosophila</em>.</p> / Master of Science (MSc)

Master of Science / Department of Entomology / Yoonseong Park / Neuropeptides are diffusible signal molecules mediating vital physiological processes. We have been interested in a group of neuropeptides and their receptors involved in osmoregulatory neuroendocrine system which has been suggested as a possible target for development of new biopesticides. Since the genome sequence of the T. castaneum has recently been completed, we were able to identify the respective genes encoding three peptide hormones from T. castaneum that were characterized for their diuretic activities in other insects: one calcitonin-like (CT-like DH31) and two corticotropin releasing factor-like (CRF-like DH37 and DH47, the numbers indicates the number of amino acid residues). This peptide is expressed at all developmental stages and in the central nervous system (CNS), Malpighian tubules (MT) and gut. The synthetic peptide TricaDH31 also has been show to be biologically active, inducing significant excretions in adults beetles. When Tcdh31 was silenced using RNAi, adults had deformed wings and abnormal body shape. Mortality in adults was high, the number of eggs laid was reduced as well as the hatchability of the eggs. The two biologically active CRF-like peptides in T. castaneum, are encoded by one gene which undergoes alternative splicing. When Tcdh47 was knocked down, high mortality occurred as well as low oviposition and egg hatchability. Similar effects were observed with silencing of both CRF-like genes. However, RNAi of Tcdh37 transcripts had similar, but less severe effects. Adults also had deformed wings when both CRF-like genes were silenced, but not when just one of them was knocked down. These results indicate that CRF-like genes could have additional biological functions to their roles in dieresis. We tested the in vivo activity of these peptides. TenmoDH47 induced high excretions in adults, whereas TenmoDH37 induces smaller excretions. We identified the respective genes encoding two putative receptors for TricaDH31 as Glean_13321 and Glean_02694 (Trica-ctr1 and Trica-ctr2, respectively) and two receptors for CRF-like peptide as Glean_12799 and Glean_07104 (Trica-crfr1 and Trica-crfr2, respectively). The CT-like receptors are expressed at all developmental stages, in the CNS and MT. RNAi of the receptors revealed that only Trica-ctr2 silencing caused significant mortality and reduction in the number of eggs laid. The CRF-like receptors are expressed at all developmental stages. Adults also had deformed wings and laid fewer eggs after RNAi of Trica-crfr1. RNAi of Trica-crf2 also caused significant mortality. These peptides and receptors seem to fine tune the beetle physiology and may have functions not yet known.

G protein-coupled receptors (GPCRs) are composed of seven transmembrane domains and play an essential role in regulating physiological functions and mediating responses to environmental stimuli, biogenic amines, neurotransmitters, peptides, lipids, and hormones. The calcitonin-like diuretic hormone 31 (DH31) is known to elicit natriuresis from the Malpighian tubules (MTs) of mosquitoes Anopheles gambiae and Aedes aegypti upon blood feeding. However, the contribution of DH31 cognate receptor, calcitonin receptor 1 (GPRCAL1), has not been evaluated with respect to postprandial fluid regulation or myostimulatory activity in blood feeding insects. Thus, this dissertation has investigated potential roles of AedaeGPRCAL1 in the regulation of fluid homeostasis and hindgut muscle contraction in female A. aegypti mosquito. The full length cDNA encoding AedaeGPRCAL1 was cloned and sequenced. The receptor expression in the MTs and hindgut from female mosquito was analyzed by western blot and immunohistochemistry using anti-AedaeGPRCAL1 affinity purified antibodies, and subsequently its role in fluid transport and hindgut contraction was evaluated by RNA interference (RNAi). The mosquitoes that underwent knock-down of the AedaeGPRcal1 exhibited up to 57% lower rate of MT fluid secretion in presence of Aedae-DH31 in the in vitro assay and a ~30% reduction in the fluid excreted from live females upon blood feeding. The receptor was immunolocalized in principal cells, predominantly towards the distal end of MTs. Analyses of receptor signal probability indicate the receptor is expressed in a gradient-like fashion along the length of the MTs. A striking discovery was the fact that not all principal cells express the receptor, contrary to previous belief. Immunolocalization revealed the AedaeGPRCAL1 is expressed in hindgut circular and longitudinal muscles. The application of DH31 increased the frequency of hindgut contractions in all female mosquitoes, those injected with AedaeGPRcal1 dsRNA and controls, as compared to their basal contraction rate, but the percent change in frequency of hindgut contraction from AedaeGPRcal1 knock-down females was about 2-fold lower than the controls after application of Aedae-DH31. To my knowledge, this is first evidence of RNAi-induced phenotypes in any invertebrate that allowed the quantification of the contribution of single family B GPCR to fluid loss and muscle contractility.