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Klaus W. Beyenbach - One of the best experts on this subject based on the ideXlab platform.
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the septate junction protein tetraspanin 2a is critical to the structure and function of Malpighian Tubules in drosophila melanogaster
American Journal of Physiology-cell Physiology, 2020Co-Authors: Klaus W. Beyenbach, Frederike Schone, Leonhard F Breitsprecher, Felix Tiburcy, Mikio Furuse, Yasushi Izumi, Heiko Meyer, Sima JonusaiteAbstract:Tetraspanin-2A (Tsp2A) is an integral membrane protein of smooth septate junctions in Drosophila melanogaster. To elucidate its structural and functional roles in Malpighian Tubules, we used the c42-GAL4/UAS system to selectively knock down Tsp2A in principal cells of the tubule. Tsp2A localizes to smooth septate junctions (sSJ) in Malpighian Tubules in a complex shared with partner proteins Snakeskin (Ssk), Mesh, and Discs large (Dlg). Knockdown of Tsp2A led to the intracellular retention of Tsp2A, Ssk, Mesh, and Dlg, gaps and widening spaces in remaining sSJ, and tumorous and cystic Tubules. Elevated protein levels together with diminished V-type H+-ATPase activity in Tsp2A knockdown Tubules are consistent with cell proliferation and reduced transport activity. Indeed, Malpighian Tubules isolated from Tsp2A knockdown flies failed to secrete fluid in vitro. The absence of significant transepithelial voltages and resistances manifests an extremely leaky epithelium that allows secreted solutes and water to leak back to the peritubular side. The tubular failure to excrete fluid leads to extracellular volume expansion in the fly and to death within the first week of adult life. Expression of the c42-GAL4 driver begins in Malpighian Tubules in the late embryo and progresses upstream to distal Tubules in third instar larvae, which can explain why larvae survive Tsp2A knockdown and adults do not. Uncontrolled cell proliferation upon Tsp2A knockdown confirms the role of Tsp2A as tumor suppressor in addition to its role in sSJ structure and transepithelial transport.
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cloning and functional characterization of inward rectifying potassium kir channels from Malpighian Tubules of the mosquito aedes aegypti
Insect Biochemistry and Molecular Biology, 2013Co-Authors: Peter M. Piermarini, Matthew F Rouhier, Matthew Schepel, Christin Kosse, Klaus W. BeyenbachAbstract: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+ > NH4+) 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+.
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a dynamic paracellular pathway serves diuresis in mosquito Malpighian Tubules
Annals of the New York Academy of Sciences, 2012Co-Authors: Klaus W. BeyenbachAbstract:Female mosquitoes gorge on vertebrate blood, a rich nutrient source for developing eggs. But gorging meals increase the risk of predation. Mosquitoes are quick to reduce the flight payload with a potent diuresis. Diuretic peptides of the insect kinin family induce a tenfold-reduction in the paracellular resistance of Malpighian Tubules and increase the paracellular permeation of Cl−, the counterion of the transepithelial secretion of Na+ and K+. As a result, the transepithelial secretion of NaCl and KCl and water increases. Insect kinins signal to the opening of the paracellular pathway via G protein-coupled receptors and the elevation of intracellular [Ca2+], which leads to the reorganization of the cytoskeleton associated with the septate junction. The reorganization may affect the septate junctional proteins that control the barrier and permselectivity properties of the paracellular pathway. The proteins involved in the embryonic formation of the septate junction and in epithelial polarization are largely known for ectodermal epithelia, but the proteins that form and mediate the dynamic functions of the septate junction in Malpighian Tubules remain to be determined.
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The Developmental, Molecular, and Transport Biology of Malpighian Tubules
Annual review of entomology, 2010Co-Authors: Klaus W. Beyenbach, Helen Skaer, Julian A. T. DowAbstract:Molecular biology is reaching new depths in our understanding of the development and physiology of Malpighian Tubules. In Diptera, Malpighian Tubules derive from ectodermal cells that evaginate from the primitive hindgut and subsequently undergo a sequence of orderly events that culminates in an active excretory organ by the time the larva takes its first meal. Thereafter, the Tubules enlarge by cell growth. Just as modern experimental strategies have illuminated the development of Tubules, genomic, transcriptomic, and proteomic studies have uncovered new tubule functions that serve immune defenses and the breakdown and renal clearance of toxic substances. Moreover, genes associated with specific diseases in humans are also found in flies, some of which, astonishingly, express similar pathophenotypes. However, classical experimental approaches continue to show their worth by distinguishing between -omic possibilities and physiological reality while providing further detail about the rapid regulation of the transport pathway through septate junctions and the reversible assembly of proton pumps.
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Gap junctions in Malpighian Tubules of Aedes aegypti
Journal of Experimental Biology, 2008Co-Authors: Xing-he Weng, Peter M. Piermarini, Atsuko Yamahiro, Daniel J. Aneshansley, Klaus W. BeyenbachAbstract:SUMMARY We present electrical, physiological and molecular evidence for substantial electrical coupling of epithelial cells in Malpighian Tubules via gap junctions. Current was injected into one principal cell of the isolated Malpighian tubule and membrane voltage deflections were measured in that cell and in two neighboring principal cells. By short-circuiting the transepithelial voltage with the diuretic peptide leucokinin-VIII we largely eliminated electrical coupling of principal cells through the tubule lumen, thereby allowing coupling through gap junctions to be analyzed. The analysis of an equivalent electrical circuit of the tubule yielded an average gap-junction resistance ( R gj ) of 431 kΩ between two cells. This resistance would stem from 6190 open gap-junctional channels, assuming the high single gap-junction conductance of 375 pS found in vertebrate tissues. The addition of the calcium ionophore A23187 (2 μmol l –1 ) to the peritubular Ringer bath containing 1.7 mmol l –1 Ca 2+ did not affect the gap-junction resistance, but metabolic inhibition of the tubule with dinitrophenol (0.5 mmol l –1 ) increased the gap-junction resistance 66-fold, suggesting the regulation of gap junctions by ATP. Lucifer Yellow injected into a principal cell did not appear in neighboring principal cells. Thus, gap junctions allow the passage of current but not Lucifer Yellow. Using RT-PCR we found evidence for the expression of innexins 1, 2, 3 and 7 (named after their homologues in Drosophila ) in Malpighian Tubules. The physiological demonstration of gap junctions and the molecular evidence for innexin in Malpighian Tubules of Aedes aegypti call for the double cable model of the tubule, which will improve the measurement and the interpretation of electrophysiological data collected from Malpighian Tubules.
Peter M. Piermarini - One of the best experts on this subject based on the ideXlab platform.
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Malpighian Tubules as Novel Targets for Mosquito Control
International journal of environmental research and public health, 2017Co-Authors: Peter M. Piermarini, Carlos J. Esquivel, Jerod S. DentonAbstract:The Malpighian Tubules and hindgut are the renal excretory tissues of mosquitoes; they are essential to maintaining hemolymph water and solute homeostasis. Moreover, they make important contributions to detoxifying metabolic wastes and xenobiotics in the hemolymph. We have focused on elucidating the molecular mechanisms of Malpighian tubule function in adult female mosquitoes and developing chemical tools as prototypes for next-generation mosquitocides that would act via a novel mechanism of action (i.e., renal failure). To date, we have targeted inward rectifier potassium (Kir) channels expressed in the Malpighian Tubules of the yellow fever mosquito Aedes aegypti and malaria mosquito Anopheles gambiae. Inhibition of these channels with small molecules inhibits transepithelial K+ and fluid secretion in Malpighian Tubules, leading to a disruption of hemolymph K+ and fluid homeostasis in adult female mosquitoes. In addition, we have used next-generation sequencing to characterize the transcriptome of Malpighian Tubules in the Asian tiger mosquito Aedes albopictus, before and after blood meals, to reveal new molecular targets for potentially disrupting Malpighian tubule function. Within 24 h after a blood meal, the Malpighian Tubules enhance the mRNA expression of genes encoding mechanisms involved with the detoxification of metabolic wastes produced during blood digestion (e.g., heme, NH3, reactive oxygen species). The development of chemical tools targeting these molecular mechanisms in Malpighian Tubules may offer a promising avenue for the development of mosquitocides that are highly-selective against hematophagous females, which are the only life stage that transmits pathogens.
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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
PeerJ, 2016Co-Authors: Carlos J. Esquivel, Bryan J Cassone, Peter M. PiermariniAbstract: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.
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transcriptomic evidence for a dramatic functional transition of the Malpighian Tubules after a blood meal in the asian tiger mosquito aedes albopictus
PLOS Neglected Tropical Diseases, 2014Co-Authors: Carlos J. Esquivel, Bryan J Cassone, Peter M. PiermariniAbstract: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.
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cloning and functional characterization of inward rectifying potassium kir channels from Malpighian Tubules of the mosquito aedes aegypti
Insect Biochemistry and Molecular Biology, 2013Co-Authors: Peter M. Piermarini, Matthew F Rouhier, Matthew Schepel, Christin Kosse, Klaus W. BeyenbachAbstract: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+ > NH4+) 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+.
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Gap junctions in Malpighian Tubules of Aedes aegypti
Journal of Experimental Biology, 2008Co-Authors: Xing-he Weng, Peter M. Piermarini, Atsuko Yamahiro, Daniel J. Aneshansley, Klaus W. BeyenbachAbstract:SUMMARY We present electrical, physiological and molecular evidence for substantial electrical coupling of epithelial cells in Malpighian Tubules via gap junctions. Current was injected into one principal cell of the isolated Malpighian tubule and membrane voltage deflections were measured in that cell and in two neighboring principal cells. By short-circuiting the transepithelial voltage with the diuretic peptide leucokinin-VIII we largely eliminated electrical coupling of principal cells through the tubule lumen, thereby allowing coupling through gap junctions to be analyzed. The analysis of an equivalent electrical circuit of the tubule yielded an average gap-junction resistance ( R gj ) of 431 kΩ between two cells. This resistance would stem from 6190 open gap-junctional channels, assuming the high single gap-junction conductance of 375 pS found in vertebrate tissues. The addition of the calcium ionophore A23187 (2 μmol l –1 ) to the peritubular Ringer bath containing 1.7 mmol l –1 Ca 2+ did not affect the gap-junction resistance, but metabolic inhibition of the tubule with dinitrophenol (0.5 mmol l –1 ) increased the gap-junction resistance 66-fold, suggesting the regulation of gap junctions by ATP. Lucifer Yellow injected into a principal cell did not appear in neighboring principal cells. Thus, gap junctions allow the passage of current but not Lucifer Yellow. Using RT-PCR we found evidence for the expression of innexins 1, 2, 3 and 7 (named after their homologues in Drosophila ) in Malpighian Tubules. The physiological demonstration of gap junctions and the molecular evidence for innexin in Malpighian Tubules of Aedes aegypti call for the double cable model of the tubule, which will improve the measurement and the interpretation of electrophysiological data collected from Malpighian Tubules.
Andrew Donini - One of the best experts on this subject based on the ideXlab platform.
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A mosquito entomoglyceroporin, Aedes aegypti AQP5, participates in water transport across the Malpighian Tubules of larvae.
The Journal of Experimental Biology, 2017Co-Authors: Lidiya Misyura, Gil Y. Yerushalmi, Andrew DoniniAbstract:ABSTRACT The mosquito Aedes aegypti is the primary vector for arboviral diseases such as Zika fever, dengue fever, chikungunya and yellow fever. The larvae reside in hypo-osmotic freshwater habitats, where they face dilution of their body fluids from osmotic influx of water. The Malpighian Tubules help maintain ionic and osmotic homeostasis by removing excess water from the hemolymph; however, the transcellular pathway for this movement remains unresolved. Aquaporins are transmembrane channels thought to permit transcellular transport of water from the hemolymph into the Malpighian tubule lumen. Immunolocalization of A . aegypti aquaporin 5 (AaAQP5) revealed expression by Malpighian tubule principal cells of the larvae, with localization to both the apical and basolateral membranes. Knockdown of AaAQP5 with double-stranded RNA decreased larval survival, reduced rates of fluid, K + and Na + secretion by the Malpighian Tubules, and reduced Cl − concentrations in the hemolymph. These findings indicate that AaAQP5 participates in transcellular water transport across the Malpighian Tubules of larval A . aegypti where global AaAQP5 expression is important for larval survival.
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salinity alters snakeskin and mesh transcript abundance and permeability in midgut and Malpighian Tubules of larval mosquito aedes aegypti
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2017Co-Authors: Sima Jonusaite, Andrew Donini, Scott P KellyAbstract:This study examined the distribution and localization of the septate junction (SJ) proteins snakeskin (Ssk) and mesh in osmoregulatory organs of larval mosquito (Aedes aegypti), as well as their response to altered environmental salt levels. Ssk and mesh transcripts and immunoreactivity were detected in tissues of endodermal origin such as the midgut and Malpighian Tubules of A. aegypti larvae, but not in ectodermally derived hindgut and anal papillae. Immunolocalization of Ssk and mesh in the midgut and Malpighian Tubules indicated that both proteins are concentrated at regions of cell-cell contact between epithelial cells. Transcript abundance of ssk and mesh was higher in the midgut and Malpighian Tubules of brackish water (BW, 30% SW) reared A. aegypti larvae when compared with freshwater (FW) reared animals. Therefore, [3H]polyethylene glycol (MW 400 Da, PEG-400) flux was examined across isolated midgut and Malpighian tubule preparations as a measure of their paracellular permeability. It was found that PEG-400 flux was greater across the midgut of BW versus FW larvae while the Malpighian Tubules of BW-reared larvae had reduced PEG-400 permeability in conjunction with increased Cl− secretion compared to FW animals. Taken together, data suggest that Ssk and mesh are found in smooth SJs (sSJs) of larval A. aegypti and that their abundance alters in association with changes in epithelial permeability when larvae reside in water of differing salt content. This latter observation suggests that Ssk and mesh play a role in the homeostatic control of salt and water balance in larval A. aegypti.
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differential actions of diuretic factors on the Malpighian Tubules of rhodnius prolixus
The Journal of Experimental Biology, 2008Co-Authors: Andrew Donini, Michael J Odonnell, Ian OrchardAbstract:SUMMARY The effects of corticotropin-releasing factor (CRF)-related (ZooneDH), calcitonin (CT)-related (RhoprDH 31 ) and kinin-related (leucokinin I) peptides on the ion composition of fluid secreted by upper Rhodnius prolixus Malpighian Tubules and on KCl reabsorption by the lower Tubules were assessed. ZooneDH stimulated fluid secretion while increasing the [Na + ] of secreted fluid at the expense of [K + ]. Upper Tubules responded to ZooneDH with a characteristic triphasic change in the transepithelial potential (TEP), reminiscent of the response to 5-hydroxytryptamine (5HT). RhoprDH 31 produced a small (∼9 mV) lumen-positive shift in TEP of the upper tubule but had no effect on the rate of fluid secretion or ion composition of the secreted fluid. In contrast to 5HT, both peptides failed to activate KCl reabsorption by the lower tubule. Leucokinin I had no effect on the ion composition of fluid secreted by whole or upper Malpighian Tubules. We propose that: (1) 5HT and a native CRF-related peptide similar to ZooneDH activate the same second messenger systems and ion transporters in the upper tubule cells; (2) CRF-related peptide is utilized to maintain high rates of fluid secretion during the post-feeding diuresis and is additionally used at times when KCl reabsorption is unnecessary or detrimental. The differential actions of multiple diuretic factors allows for intricate control of ionic and osmotic balance in R. prolixus .
Carlos J. Esquivel - One of the best experts on this subject based on the ideXlab platform.
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Malpighian Tubules as Novel Targets for Mosquito Control
International journal of environmental research and public health, 2017Co-Authors: Peter M. Piermarini, Carlos J. Esquivel, Jerod S. DentonAbstract:The Malpighian Tubules and hindgut are the renal excretory tissues of mosquitoes; they are essential to maintaining hemolymph water and solute homeostasis. Moreover, they make important contributions to detoxifying metabolic wastes and xenobiotics in the hemolymph. We have focused on elucidating the molecular mechanisms of Malpighian tubule function in adult female mosquitoes and developing chemical tools as prototypes for next-generation mosquitocides that would act via a novel mechanism of action (i.e., renal failure). To date, we have targeted inward rectifier potassium (Kir) channels expressed in the Malpighian Tubules of the yellow fever mosquito Aedes aegypti and malaria mosquito Anopheles gambiae. Inhibition of these channels with small molecules inhibits transepithelial K+ and fluid secretion in Malpighian Tubules, leading to a disruption of hemolymph K+ and fluid homeostasis in adult female mosquitoes. In addition, we have used next-generation sequencing to characterize the transcriptome of Malpighian Tubules in the Asian tiger mosquito Aedes albopictus, before and after blood meals, to reveal new molecular targets for potentially disrupting Malpighian tubule function. Within 24 h after a blood meal, the Malpighian Tubules enhance the mRNA expression of genes encoding mechanisms involved with the detoxification of metabolic wastes produced during blood digestion (e.g., heme, NH3, reactive oxygen species). The development of chemical tools targeting these molecular mechanisms in Malpighian Tubules may offer a promising avenue for the development of mosquitocides that are highly-selective against hematophagous females, which are the only life stage that transmits pathogens.
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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
PeerJ, 2016Co-Authors: Carlos J. Esquivel, Bryan J Cassone, Peter M. PiermariniAbstract: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.
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transcriptomic evidence for a dramatic functional transition of the Malpighian Tubules after a blood meal in the asian tiger mosquito aedes albopictus
PLOS Neglected Tropical Diseases, 2014Co-Authors: Carlos J. Esquivel, Bryan J Cassone, Peter M. PiermariniAbstract: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.
Michael J Odonnell - One of the best experts on this subject based on the ideXlab platform.
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the Malpighian Tubules and cryptonephric complex in lepidopteran larvae
2019Co-Authors: Dennis Kolosov, Michael J OdonnellAbstract:Abstract Lepidopterans (butterflies and moths) are an ecologically and agriculturally important group of holometabolous insects. Their larvae and adults exhibit trophic partitioning, which is reflected by the various modifications of their digestive and excretory systems. Adults are capable of flight and feed mostly on the nectar of plants, acting as pollinators. Larvae are voracious leaf eaters whose extremely alkaline midgut (pH ≈ 11) is an adaptation to high tannin levels in the host plants. Morphologically and functionally regionalized Malpighian Tubules of the larva modify fluid as it flows through them. The larvae also exhibit the so-called cryptonephric condition, where the distal end of the tubule is juxtaposed to the rectum and enveloped by the perinephric membrane. The distal part of the free tubule adjacent to the ileum, termed the distal ileac plexus, is characterized by a high density of secondary cells. Recent studies have identified several unusual aspects of ion transport physiology of the distal ileac plexus: (i) gap junctional coupling of principal and secondary cells that allows them to transport ions in opposite directions, (ii) the ability to switch between K+ secretion and K+ reabsorption depending on the input from the cryptonephric tubule, (iii) the presence of voltage-gated, ligand-gated and mechanosensitive ion channels and (iv) coordinated regulation of water and septate junction permeability during the reversal from secretion to reabsorption (presumably aimed at retention of water and solute content in the distal ileac plexus lumen, while Na+ and K+ are being reabsorbed). We describe recent advances in understanding ion-transporting and regulatory mechanisms in the Malpighian Tubules of larval Lepidoptera with a special emphasis on the distal ileac plexus segment.
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physiological and molecular characterization of methotrexate transport by Malpighian Tubules of adult drosophila melanogaster
Journal of Insect Physiology, 2009Co-Authors: Sarah Chahine, Michael J OdonnellAbstract:Abstract A radioisotope tracer technique and quantitative PCR were used to study the mechanisms and regulation of transepithelial transport of the type II organic anion methotrexate (MTX) by the Malpighian Tubules of Drosophila melanogaster. Transport of MTX was saturable and Na+-independent; the kinetic parameters Jmax and Kt were 437 fmol min−1 and 23.5 μM, respectively. The transport of MTX was competitively inhibited by phenol red and probenecid; non-competitively inhibited by salicylate, verapamil and MK-571; and uncompetitively inhibited by Texas Red. Dietary exposure to 0.1 mM MTX led to dramatic increases in gene expression for several members of the ABC family of transporters in both the Malpighian Tubules and the gut. Our results suggest that multiple transporters are upregulated in response to dietary exposure to MTX. Increased levels of the protein products which may result from expression of these genes may enhance elimination of toxic compounds such as MTX or its metabolites.
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differential actions of diuretic factors on the Malpighian Tubules of rhodnius prolixus
The Journal of Experimental Biology, 2008Co-Authors: Andrew Donini, Michael J Odonnell, Ian OrchardAbstract:SUMMARY The effects of corticotropin-releasing factor (CRF)-related (ZooneDH), calcitonin (CT)-related (RhoprDH 31 ) and kinin-related (leucokinin I) peptides on the ion composition of fluid secreted by upper Rhodnius prolixus Malpighian Tubules and on KCl reabsorption by the lower Tubules were assessed. ZooneDH stimulated fluid secretion while increasing the [Na + ] of secreted fluid at the expense of [K + ]. Upper Tubules responded to ZooneDH with a characteristic triphasic change in the transepithelial potential (TEP), reminiscent of the response to 5-hydroxytryptamine (5HT). RhoprDH 31 produced a small (∼9 mV) lumen-positive shift in TEP of the upper tubule but had no effect on the rate of fluid secretion or ion composition of the secreted fluid. In contrast to 5HT, both peptides failed to activate KCl reabsorption by the lower tubule. Leucokinin I had no effect on the ion composition of fluid secreted by whole or upper Malpighian Tubules. We propose that: (1) 5HT and a native CRF-related peptide similar to ZooneDH activate the same second messenger systems and ion transporters in the upper tubule cells; (2) CRF-related peptide is utilized to maintain high rates of fluid secretion during the post-feeding diuresis and is additionally used at times when KCl reabsorption is unnecessary or detrimental. The differential actions of multiple diuretic factors allows for intricate control of ionic and osmotic balance in R. prolixus .
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ion selective microelectrode analysis of salicylate transport by the Malpighian Tubules and gut of drosophila melanogaster
The Journal of Experimental Biology, 2005Co-Authors: Michael J Odonnell, Mark R RheaultAbstract:SUMMARY Transport of the organic anion salicylate by the Malpighian Tubules and gut of larval and adult fruit flies was studied using two salicylate-selective microelectrode methods. The first method combined the high selectivity of tridodecylmethylammonium-based electrodes for salicylate with the self-referencing ion-selective microelectrode technique for non-invasive spatial and temporal analysis of salicylate flux. Measurements with this technique revealed secretion of salicylate across the main and distal segments of the Malpighian tubule as well as the midgut, ileum and rectum. The second method used a salicylate-selective microelectrode to measure the concentration of salicylate in fluid droplets secreted by isolated Drosophila Malpighian Tubules set up in a Ramsay secretion assay. Transepithelial salicylate flux was calculated as the product of fluid secretion rate and secreted fluid salicylate concentration. Measurements with this method revealed that salicylate transport was active and saturable; the kinetic parameters Jmax and Kt were 2.72 pmol min-1 tubule-1 and 0.046 mmol l-1, respectively. Measurements of transepithelial salicylate flux determined by both microelectrode methods were in good agreement. Transepithelial flux measurements measured by microelectrodes were also validated by comparing them with measurements of radiolabelled salicylate levels in secreted droplets. Salicylate concentrations in haemolymph samples were measured with salicylate-selective microelectrodes after injection of salicylate into the haemocoel or after insects were fed salicylate-rich diets. The rate of salicylate secretion by Malpighian Tubules in vitro was sufficient to account for the measured rate of decline of salicylate concentration in the haemolymph in vivo.
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analysis of epithelial k transport in Malpighian Tubules of drosophila melanogaster evidence for spatial and temporal heterogeneity
The Journal of Experimental Biology, 2001Co-Authors: Mark R Rheault, Michael J OdonnellAbstract:Transport of K+ by the lower, main and distal segments of the Malpighian Tubules of Drosophila melanogaster was analyzed using self-referencing K+-selective microelectrodes. Transport properties of the Malpighian Tubules of Drosophila melanogaster change along their length. Self-referencing ion-selective (SeRIS) microelectrode measurements (relative to the bath concentration of 20 mmoll−1) showed a 1% reduction ( P <0.05) of [K+] in the unstirred layer adjacent to the main segment of the Malpighian Tubules, confirming secretion of K+ from the bath to the tubule lumen. Conversely, SeRIS measurements showed a 0.7% increase ( P <0.05) in [K+] in the unstirred layer adjacent to the lower segment of Malpighian Tubules, confirming reabsorption of K+ from the luminal fluid to the bath. Measurements using SeRIS also showed that the distal segment neither secreted nor reabsorbed K+. There was pronounced spatial heterogeneity in K+ transport by the lower segment and the main segment; not all morphologically similar cells participated equally in K+ transport, nor did all main segment cells respond equally to stimulation of K+ transport by cyclic AMP. Pronounced temporal heterogeneity in K+ reabsorption by the lower Malpighian Tubules was also observed. We suggest that this reflects periodic reduction in K+ reabsorption due to retention of fluid within the lower segment when the ureter contracts.
