Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti

Glycoprotein hormone receptors mediate a diverse range of physiological functions in vertebrate and invertebrate organisms. The heterodimeric glycoprotein hormone GPA2/GPB5 and its receptor LGR1, constitute a recently discovered invertebrate neuroendocrine signaling system that remains to be functionally characterized. We previously reported that LGR1 is expressed in the testes of adult Aedes aegypti mosquitoes, where its immunoreactivity is particularly regionalized. Here, we show that LGR1 immunoreactivity is associated with the centriole adjunct of spermatids and is observed transiently during spermatogenesis in mosquitoes, where it may act to mediate the regulation of flagellar development. RNA interference to downregulate LGR1 expression was accomplished by feeding mosquito larvae with bacteria that produced LGR1-specific dsRNA, which led to defects in spermatozoa, characterized with shortened flagella. LGR1 knockdown mosquitoes also retained ∼60% less spermatozoa in reproductive organs and demonstrated reduced fertility compared to controls. To date, the endocrine regulation of spermatogenesis in mosquitoes remains an understudied research area. The distribution of LGR1 and detrimental effects of its knockdown on spermatogenesis in A. aegypti indicates that this heterodimeric glycoprotein hormone signaling system contributes significantly to the regulation of male reproductive biology in this important disease-vector

DataSheet_1_Molecular characterization, localization, and physiological roles of ITP and ITP-L in the mosquito, Aedes aegypti.pdf

The insect ion transport peptide (ITP) and its alternatively spliced variant, ITP-like peptide (ITP-L), belong to the crustacean hyperglycemic hormone family of peptides and are widely conserved among insect species. While limited, studies have characterized the ITP/ITP-L signaling system within insects, and putative functions including regulation of ion and fluid transport, ovarian maturation, and thirst/excretion have been proposed. Herein, we aimed to molecularly investigate Itp and Itp-l expression profiles in the mosquito, Aedes aegypti, examine peptide immunolocalization and distribution within the adult central nervous system, and elucidate physiological roles for these neuropeptides. Transcript expression profiles of both AedaeItp and AedaeItp-l revealed distinct enrichment patterns in adults, with AedaeItp expressed in the brain and AedaeItp-l expression predominantly within the abdominal ganglia. Immunohistochemical analysis within the central nervous system revealed expression of AedaeITP peptide in a number of cells in the brain and in the terminal ganglion. Comparatively, AedaeITP-L peptide was localized solely within the pre-terminal abdominal ganglia of the central nervous system. Interestingly, prolonged desiccation stress caused upregulation of AedaeItp and AedaeItp-l levels in adult mosquitoes, suggesting possible functional roles in water conservation and feeding-related activities. RNAi-mediated knockdown of AedaeItp caused an increase in urine excretion, while knockdown of both AedaeItp and AedaeItp-l reduced blood feeding and egg-laying in females as well as hindered egg viability, suggesting roles in reproductive physiology and behavior. Altogether, this study identifies AedaeITP and AedaeITP-L as key pleiotropic hormones, regulating various critical physiological processes in the disease vector, A. aegypti.</p

DataSheet_1_Protein localization of aquaporins in the adult female disease vector mosquito, Aedes aegypti.pdf

The female Aedes aegypti mosquito is a vector for several arboviral diseases, due to their blood feeding behavior and their association with urban communities. While ion transport in Ae. aegypti has been studied, much less is known about mechanisms of water transport. Rapid water and ion excretion occurs in the adult female mosquito post blood meal and involves a set of organs including the midgut, Malpighian tubules (MTs), and hindgut. The MTs are responsible for the formation of primary urine and are considered the most important site for active transport of ions. Within the cells of the MTs, along with various ion transporters, there are aquaporin water channels that aid in the transport of water across the tubule cell membrane. Six aquaporin genes have been molecularly identified in Ae. aegypti (AQP1–6) and found to be responsible for the transport of water and in some cases, small solutes such as glycerol. In this study, we used immunohistochemistry to localize AaAQP1, 2, 4, 5, and 6 in the adult female Ae. aegypti, in non-blood fed and post blood feeding (0.5 and 24hr) conditions. We further examined the main water transporting aquaporin, AaAQP1, using western blotting to determine protein abundance changes in isolated MTs pre- and post-blood feeding. Using fluorescence in situ hybridization, aqp1 mRNA was found exclusively in the principal cells of female MTs. Finally, we used immunogold staining with transmission electron microscopy to determine subcellular localization of AaAQP1 in the Malpighian tubules under non-blood fed conditions. Interestingly, AaAQP1 was found to be predominantly in the principal cells of the MTs, dispersed throughout the brush border; however, there was also evidence of some AaAQP1 localization in the stellate cells of the MTs.</p

Identification, functional characterization, and pharmacological profile of a serotonin type-2b receptor in the medically important insect, Rhodnius prolixus

This is the published version of an article published by Frontiers Media.In the Chagas disease vector, Rhodnius prolixus, two diuretic hormones act synergistically to dramatically increase fluid secretion by the Malpighian tubules (MTs) during the rapid diuresis that is initiated upon engorgement of vertebrate blood. One of these diuretic hormones is the biogenic amine, serotonin (5-hydroxytryptamine, 5-HT), which controls a variety of additional activities including cuticle plasticization, salivary gland secretion, anterior midgut absorption, cardioacceleratory activity, and myotropic activities on a number of visceral tissues. To better understand the regulatory mechanisms linked to these various physiological actions of serotonin, we have isolated and characterized a serotonin type 2b receptor in R. prolixus, Rhopr5HTR2b, which shares sequence similarity to the vertebrate serotonin type 2 receptors. Rhopr5HTR2b transcript is enriched in well-recognized physiological targets of serotonin, including the MTs, salivary glands and dorsal vessel (i.e., insect heart). Notably, Rhopr5HTR2b was not enriched in the anterior midgut where serotonin stimulates absorption and elicits myotropic control. Using a heterologous functional receptor assay, we examined Rhopr5HTR2b activation characteristics and its sensitivity to potential agonists, antagonists, and other biogenic amines. Rhopr5HTR2b is dose-dependently activated by serotonin with an EC50 in the nanomolar range. Rhopr5HTR2b is sensitive to alpha-methyl serotonin and is inhibited by a variety of serotonin receptor antagonists, including propranolol, spiperone, ketanserin, mianserin, and cyproheptadine. In contrast, the cardioacceleratory activity of serotonin revealed a unique pharmacological profile, with no significant response induced by alpha-methyl serotonin and insensitivity to ketanserin and mianserin. This distinct agonist/antagonist profile indicates that a separate serotonin receptor type may mediate cardiomodulatory effects controlled by serotonin in R. prolixus

Data from: Anti-diuretic activity of a CAPA neuropeptide can compromise Drosophila chill tolerance

For insects, chilling injuries that occur in the absence of freezing are often related to a systemic loss of ion and water balance that leads to extracellular hyperkalemia, cell depolarization, and the triggering of apoptotic signalling cascades. The ability of insect ionoregulatory organs (e.g. the Malpighian tubules) to maintain ion balance in the cold has been linked to improved chill tolerance, and many neuroendocrine factors are known to influence ion transport rates of these organs. Injection of micromolar doses of CAPA (an insect neuropeptide) have been previously demonstrated to improve Drosophila cold tolerance, but the mechanisms through which it impacts chill tolerance are unclear, and low doses of CAPA have been previously demonstrated to cause anti-diuresis in insects, including dipterans. Here, we provide evidence that low (fM) and high (uM) doses of CAPA impair and improve chill tolerance, respectively, via two different effects on Malpighian tubule ion and water transport. While low doses of CAPA are anti-diuretic, reduce tubule K+ clearance rates and reduce chill tolerance, high doses facilitate K+ clearance from the haemolymph and increase chill tolerance. By quantifying CAPA peptide levels in the central nervous system, we estimated the maximum achievable hormonal titres of CAPA, and found further evidence that CAPA may function as an anti-diuretic peptide in Drosophila. We provide the first evidence of a neuropeptide that can negatively affect cold tolerance in an insect, and further evidence of CAPA functioning as an anti-diuretic peptide in this ubiquitous insect model

Complete data set for all experiments

An Excel file containing several sheets. Each sheet contains the complete dataset for each experiment, with notes provided on each sheet for interpretation

Identification, functional characterization and pharmacological profile of a serotonin type-2b receptor in the medically important insect, Rhodnius prolixus

In the Chagas disease vector, Rhodnius prolixus, two diuretic hormones act synergistically to dramatically increase fluid secretion by the Malpighian tubules (MTs) during the rapid diuresis that is initiated upon engorgement of vertebrate blood. One of these diuretic hormones is the biogenic amine, serotonin (5-hydroxytryptamine, 5-HT), which controls a variety of additional activities including cuticle plasticization, salivary gland secretion, anterior midgut absorption, cardioacceleratory activity, and myotropic activities on a number of visceral tissues. To better understand the regulatory mechanisms linked to these various physiological actions of serotonin, we have isolated and characterized a serotonin type 2b receptor in R. prolixus, Rhopr5HTR2b, which shares sequence similarity to the vertebrate serotonin type 2 receptors. Rhopr5HTR2b transcript is enriched in well-recognized physiological targets of serotonin, including the MTs, salivary glands and dorsal vessel (i.e. insect heart). Notably, Rhopr5HTR2b was not enriched in the anterior midgut where serotonin stimulates absorption and elicits myotropic control. Using a heterologous functional receptor assay, we examined Rhopr5HTR2b activation characteristics and its sensitivity to potential agonists, antagonists, and other biogenic amines. Rhopr5HTR2b is dose-dependently activated by serotonin with an EC50 in the nanomolar range. Rhopr5HTR2b is sensitive to alpha-methyl serotonin and is inhibited by a variety of serotonin receptor antagonists, including propranolol, spiperone, ketanserin, mianserin and cyproheptadine. In contrast, the cardioacceleratory activity of serotonin revealed a unique pharmacological profile, with no significant response induced by alpha-methyl serotonin and insensitivity to ketanserin and mianserin. This distinct agonist/antagonist profile indicates that a separate serotonin receptor type may mediate cardiomodulatory effects controlled by serotonin in R. prolixus