Peter M. Piermarini

Eliciting Renal Failure in Mosquitoes with a Small- Molecule Inhibitor of Inward-Rectifying Potassium Channels

Mosquito-borne diseases such as malaria and dengue fever take a large toll on global health. The primary chemical agents used for controlling mosquitoes are insecticides that target the nervous system. However, the emergence of resistance in mosquito populations is reducing the efficacy of available insecticides. The development of new insecticides is therefore urgent. Here we show that VU573, a small-molecule inhibitor of mammalian inward-rectifying potassium (Kir) channels, inhibits a Kir channel cloned from the renal (Malpighian) tubules of Aedes aegypti (AeKir1). Injection of VU573 into the hemolymph of adult female mosquitoes (Ae. aegypti) disrupts the production and excretion of urine in a manner consistent with channel block of AeKir1 and renders the mosquitoes incapacitated (flightless or dead) within 24 hours. Moreover, the toxicity of VU573 in mosquitoes (Ae. aegypti) is exacerbated when hemolymph potassium levels are elevated, suggesting that Kir channels are essential for maintenance of whole-animal potassium homeostasis. Our study demonstrates that renal failure is a promising mechanism of action for killing mosquitoes, and motivates the discovery of selective small-molecule inhibitors of mosquito Kir channels for use as insecticides.

Cloning and Functional Characterization of Inward-Rectifying Potassium (Kir) Channels from Malpighian Tubules of the Mosquito Aedes aegypti

Inward-rectifying K(+) (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K(+) currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na(+). Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl(+) > K(+) > Rb(+) > NH(4)(+)) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K(+).

Transcriptomic Evidence for a Dramatic Functional Transition of the Malpighian Tubules after a Blood Meal in the Asian Tiger Mosquito Aedes albopictus

Background The consumption of a vertebrate blood meal by adult female mosquitoes is necessary for their reproduction, but it also presents significant physiological challenges to mosquito osmoregulation and metabolism. The renal (Malpighian) tubules of mosquitoes play critical roles in the initial processing of the blood meal by excreting excess water and salts that are ingested. However, it is unclear how the tubules contribute to the metabolism and excretion of wastes (e.g., heme, ammonia) produced during the digestion of blood. Methodology/Principal Findings Here we used RNA-Seq to examine global changes in transcript expression in the Malpighian tubules of the highly-invasive Asian tiger mosquito Aedes albopictus during the first 24 h after consuming a blood meal. We found progressive, global changes in the transcriptome of the Malpighian tubules isolated from mosquitoes at 3 h, 12 h, and 24 h after a blood meal. Notably, a DAVID functional cluster analysis of the differentially-expressed transcripts revealed 1) a down-regulation of transcripts associated with oxidative metabolism, active transport, and mRNA translation, and 2) an up-regulation of transcripts associated with antioxidants and detoxification, proteolytic activity, amino-acid metabolism, and cytoskeletal dynamics. Conclusions/Significance The results suggest that blood feeding elicits a functional transition of the epithelium from one specializing in active transepithelial fluid secretion (e.g., diuresis) to one specializing in detoxification and metabolic waste excretion. Our findings provide the first insights into the putative roles of mosquito Malpighian tubules in the chronic processing of blood meals.

Discovery and characterization of a potent and selective inhibitor of Aedes aegypti inward rectifier potassium channels.

Vector-borne diseases such as dengue fever and malaria, which are transmitted by infected female mosquitoes, affect nearly half of the worlds population. The emergence of insecticide-resistant mosquito populations is reducing the effectiveness of conventional insecticides and threatening current vector control strategies, which has created an urgent need to identify new molecular targets against which novel classes of insecticides can be developed. We previously demonstrated that small molecule inhibitors of mammalian Kir channels represent promising chemicals for new mosquitocide development. In this study, high-throughput screening of approximately 30,000 chemically diverse small-molecules was employed to discover potent and selective inhibitors of Aedes aegypti Kir1 (AeKir1) channels heterologously expressed in HEK293 cells. Of 283 confirmed screening ‘hits’, the small-molecule inhibitor VU625 was selected for lead optimization and in vivo studies based on its potency and selectivity toward AeKir1, and tractability for medicinal chemistry. In patch clamp electrophysiology experiments of HEK293 cells, VU625 inhibits AeKir1 with an IC50 value of 96.8 nM, making VU625 the most potent inhibitor of AeKir1 described to date. Furthermore, electrophysiology experiments in Xenopus oocytes revealed that VU625 is a weak inhibitor of AeKir2B. Surprisingly, injection of VU625 failed to elicit significant effects on mosquito behavior, urine excretion, or survival. However, when co-injected with probenecid, VU625 inhibited the excretory capacity of mosquitoes and was toxic, suggesting that the compound is a substrate of organic anion and/or ATP-binding cassette (ABC) transporters. The dose-toxicity relationship of VU625 (when co-injected with probenecid) is biphasic, which is consistent with the molecule inhibiting both AeKir1 and AeKir2B with different potencies. This study demonstrates proof-of-concept that potent and highly selective inhibitors of mosquito Kir channels can be developed using conventional drug discovery approaches. Furthermore, it reinforces the notion that the physical and chemical properties that determine a compounds bioavailability in vivo will be critical in determining the efficacy of Kir channel inhibitors as insecticides.

A de novo transcriptome of the Malpighian tubules in non-blood-fed and blood-fed Asian tiger mosquitoes Aedes albopictus: insights into diuresis, detoxification, and blood meal processing

Background. In adult female mosquitoes, the renal (Malpighian) tubules play an important role in the post-prandial diuresis, which removes excess ions and water from the hemolymph of mosquitoes following a blood meal. After the post-prandial diuresis, the roles that Malpighian tubules play in the processing of blood meals are not well described. Methods. We used a combination of next-generation sequencing (paired-end RNA sequencing) and physiological/biochemical assays in adult female Asian tiger mosquitoes (Aedes albopictus) to generate molecular and functional insights into the Malpighian tubules and how they may contribute to blood meal processing (3–24 h after blood ingestion). Results/Discussion. Using RNA sequencing, we sequenced and assembled the first de novo transcriptome of Malpighian tubules from non-blood-fed (NBF) and blood-fed (BF) mosquitoes. We identified a total of 8,232 non-redundant transcripts. The Malpighian tubules of NBF mosquitoes were characterized by the expression of transcripts associated with active transepithelial fluid secretion/diuresis (e.g., ion transporters, water channels, V-type H+-ATPase subunits), xenobiotic detoxification (e.g., cytochrome P450 monoxygenases, glutathione S-transferases, ATP-binding cassette transporters), and purine metabolism (e.g., xanthine dehydrogenase). We also detected the expression of transcripts encoding sodium calcium exchangers, G protein coupled-receptors, and septate junctional proteins not previously described in mosquito Malpighian tubules. Within 24 h after a blood meal, transcripts associated with active transepithelial fluid secretion/diuresis exhibited a general downregulation, whereas those associated with xenobiotic detoxification and purine catabolism exhibited a general upregulation, suggesting a reinvestment of the Malpighian tubules’ molecular resources from diuresis to detoxification. Physiological and biochemical assays were conducted in mosquitoes and isolated Malpighian tubules, respectively, to confirm that the transcriptomic changes were associated with functional consequences. In particular, in vivo diuresis assays demonstrated that adult female mosquitoes have a reduced diuretic capacity within 24 h after a blood meal. Moreover, biochemical assays in isolated Malpighian tubules showed an increase in glutathione S-transferase activity and the accumulation of uric acid (an end product of purine catabolism) within 24 h after a blood meal. Our data provide new insights into the molecular physiology of Malpighian tubules in culicine mosquitoes and reveal potentially important molecular targets for the development of chemical and/or gene-silencing insecticides that would disrupt renal function in mosquitoes.

Pharmacological Validation of an Inward-Rectifier Potassium (Kir) Channel as an Insecticide Target in the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are important disease vectors that transmit a wide variety of pathogens to humans, including those that cause malaria and dengue fever. Insecticides have traditionally been deployed to control populations of disease-causing mosquitoes, but the emergence of insecticide resistance has severely limited the number of active compounds that are used against mosquitoes. Thus, to improve the control of resistant mosquitoes there is a need to identify new insecticide targets and active compounds for insecticide development. Recently we demonstrated that inward rectifier potassium (Kir) channels and small molecule inhibitors of Kir channels offer promising new molecular targets and active compounds, respectively, for insecticide development. Here we provide pharmacological validation of a specific mosquito Kir channel (AeKir1) in the yellow fever mosquito Aedes aegypti. We show that VU590, a small-molecule inhibitor of mammalian Kir1.1 and Kir7.1 channels, potently inhibits AeKir1 but not another mosquito Kir channel (AeKir2B) in vitro. Moreover, we show that a previously identified inhibitor of AeKir1 (VU573) elicits an unexpected agonistic effect on AeKir2B in vitro. Injection of VU590 into the hemolymph of adult female mosquitoes significantly inhibits their capacity to excrete urine and kills them within 24 h, suggesting a mechanism of action on the excretory system. Importantly, a structurally-related VU590 analog (VU608), which weakly blocks AeKir1 in vitro, has no significant effects on their excretory capacity and does not kill mosquitoes. These observations suggest that the toxic effects of VU590 are associated with its inhibition of AeKir1.

An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria

Insecticide resistance is a growing threat to mosquito control programs around the world, thus creating the need to discover novel target sites and target-specific compounds for insecticide development. Emerging evidence suggests that mosquito inward rectifier potassium (Kir) channels represent viable molecular targets for developing insecticides with new mechanisms of action. Here we describe the discovery and characterization of VU041, a submicromolar-affinity inhibitor of Anopheles (An.) gambiae and Aedes (Ae.) aegypti Kir1 channels that incapacitates adult female mosquitoes from representative insecticide-susceptible and -resistant strains of An. gambiae (G3 and Akron, respectively) and Ae. aegypti (Liverpool and Puerto Rico, respectively) following topical application. VU041 is selective for mosquito Kir channels over several mammalian orthologs, with the exception of Kir2.1, and is not lethal to honey bees. Medicinal chemistry was used to develop an analog, termed VU730, which retains activity toward mosquito Kir1 but is not active against Kir2.1 or other mammalian Kir channels. Thus, VU041 and VU730 are promising chemical scaffolds for developing new classes of insecticides to combat insecticide-resistant mosquitoes and the transmission of mosquito-borne diseases, such as Zika virus, without harmful effects on humans and beneficial insects.

Slc4-like anion transporters of the larval mosquito alimentary canal

Mosquito larvae exhibit luminal pH extremes along the axial length of their alimentary canal that range from very alkaline (pH>10) in the anterior midgut to slightly acid in the hindgut. The principal buffer in the system is thought to be bicarbonate and/or carbonate, because the lumen is known to contain high levels of bicarbonate/carbonate and is surrounded by various epithelial cell types which express a variety of carbonic anhydrases. However, the precise mechanisms responsible for the transport of bicarbonate/carbonate into and out of the lumen are unclear. In the present study, we test the hypothesis that SLC4-like anion transporters play a role in bicarbonate/carbonate accumulation in the larval mosquito alimentary canal. Molecular, physiological and immnuohistochemical characterizations of Slc4-like transporters in the gut of larval mosquitoes (Aedes aegypti and Anopheles gambiae) demonstrate the presence of both a Na(+)-independent chloride/bicarbonate anion exchanger (AE) as well as a Na(+)-dependent anion exchanger (NDAE). Notably, immunolocalization experiments in Malpighian tubules show that the two proteins can be located in the same tissue, but to different cell types. Immunolabeling experiments in the gastric caecae show that the two proteins can be found in the same cells, but on opposite sides (basal vs. apical). In summary, our results indicate that the alimentary canal of larval mosquitoes exhibits robust expression of two SLC4-like transporters in locations that are consistent with a role in the regulation of luminal pH. The precise physiological contributions of each transporter remain to be determined.

The molecular and immunochemical expression of innexins in the yellow fever mosquito, Aedes aegypti: insights into putative life stage- and tissue-specific functions of gap junctions.

Gap junctions (GJ) mediate direct intercellular communication by forming channels through which certain small molecules and/or ions can pass. Connexins, the proteins that form vertebrate GJ, are well studied and known to contribute to neuronal, muscular and epithelial physiology. Innexins, the GJ proteins of insects, have only recently received much investigative attention and many of their physiological roles remain to be determined. Here we characterize the molecular expression of six innexin (Inx) genes in the yellow fever mosquito Aedes aegypti (AeInx1, AeInx2, AeInx3, AeInx4, AeInx7, and AeInx8) and the immunochemical expression of one innexin protein, AeInx3, in the alimentary canal. We detected the expression of no less than four innexin genes in each mosquito life stage (larva, pupa, adult) and tissue/body region from adult males and females (midgut, Malpighian tubules, hindgut, head, carcass, gonads), suggesting a remarkable potential molecular diversity of GJ in mosquitoes. Moreover, the expression patterns of some innexins were life stage and/or tissue specific, suggestive of potential functional specializations. Cloning of the four full-length cDNAs expressed in the Malpighian tubules of adult females (AeInx1, AeInx2, AeInx3, and AeInx7) revealed evidence for 1) alternative splicing of AeInx1 and AeInx3 transcripts, and 2) putative N-glycosylation of AeInx3 and AeInx7. Finally, immunohistochemistry of AeInx3 in the alimentary canal of larval and adult female mosquitoes confirmed localization of this innexin to the intercellular regions of Malpighian tubule and hindgut epithelial cells, suggesting that it is an important component of GJ in these tissues.

Molecular identification and expression analysis of a diapause hormone receptor in the corn earworm, Helicoverpa zea.

Diapause hormone (DH) is an insect neuropeptide that is highly effective in terminating the overwintering pupal diapause in members of the Helicoverpa/Heliothis complex of agricultural pests, thus DH and related compounds have promise as tools for pest management. To augment our development of effective DH analogs and antagonists that could be used as diapause disruptors this study focuses on the cloning and identification of the DH receptor (DHR) in the corn earworm, Helicoverpa zea. The full-length dhr cDNA contains 2153 nucleotides encoding 511 amino acids. Our results suggest there are at least two splicing variants of Hezea-DHR. Hydrophobicity analysis and sequence alignment indicate that Hezea-DHR has 7 transmembrane regions and a highly conserved C-terminal region that is also present in related receptors. Hezea-DHR has 95%, 82% and 79% identity to a partial DHR sequence from Heliothis virescens, a full-length DHR in Orgyia thyellina, and DHR-1 in Bombyx mori, but only 45-49% identity to pheromone biosynthesis activating neuropeptide receptor (PBANR). Expression of dhr mRNA remained low in whole body extracts throughout diapause and in young nondiapausing pupae, but was distinctly elevated as development ensued in pharate adults 7 days after pupation. The highest expression of dhr mRNA we noted was in the ovary. A DHR fusion protein with enhanced-green fluorescent protein was successfully expressed heterologously in X. laevis oocytes, as verified by fluorescent imaging and Western blots, but an electrophysiological assay failed to detect receptor-ligand binding activity, which suggests that an essential cofactor and/or accessory protein is required for functional activity of the DHR.