Mosquito Malpighian Tubules Research Articles

Effects of dietary calcium (Ca2+) and blood feeding on the immunochemical expression of the plasma membrane Ca2+-ATPase (PMCA) in Malpighian tubules of adult female mosquitoes (Aedes aegypti)

Insect Malpighian tubules contribute to Ca2+ homeostasis via Ca2+ storage in intracellular compartments, Ca2+ secretion into the tubule lumen, and Ca2+ reabsorption into the hemolymph. A plasma membrane Ca2+-ATPase (PMCA) is hypothesized to be a Ca2+-transporter involved in renal Ca2+ transport of insects, however few studies have investigated its immunochemical expression in Malpighian tubules. Here we characterized the abundance and localization of PMCA-like immunoreactivity in Malpighian tubules of adult female mosquitoes Aedes aegypti using an antibody against Drosophila melanogaster PMCA. Western blotting revealed expression of a relatively abundant 109 kDa isoform and a relatively sparse 115 kDa isoform. Feeding mosquitoes 10% sucrose with 50 mM CaCl2 for 7 days did not affect PMCA immunoreactivity. However, at 24, 48, and 96 h post-blood feeding (PBF), the relative abundance of the 109 kDa isoform decreased while that of the 115 kDa isoform increased. Immunolabeling of Malpighian tubules revealed PMCA-like immunoreactivity in both principal and stellate cells; principal cell labeling was intracellular, whereas stellate cell labeling was along the basal membrane. Blood feeding enhanced immunolabeling of PMCA in stellate cells but weakened that in principal cells. Moreover, a unique apicolateral pattern of PMCA-like immunolabeling occurred in principal cells of the proximal segment at 24 h PBF, suggesting potential trafficking to septate junctions. Our results suggest PMCA isoforms are differentially expressed and localized in mosquito Malpighian tubules where they contribute to redistributing tubule Ca2+ during blood meal processing.

The V-type H+-ATPase is targeted in antidiuretic hormone control of the Malpighian "renal" tubules.

Like other insects, secretion by mosquito Malpighian tubules (MTs) is driven by the V-type H+-ATPase (VA) localized in the apical membrane of principal cells. In Aedes aegypti, the antidiuretic neurohormone CAPA inhibits secretion by MTs stimulated by select diuretic hormones; however, the cellular effectors of this inhibitory signaling cascade remain unclear. Herein, we demonstrate that the VA inhibitor bafilomycin selectively inhibits serotonin (5HT)- and calcitonin-related diuretic hormone (DH31)-stimulated secretion. VA activity increases in DH31-treated MTs, whereas CAPA abolishes this increase through a NOS/cGMP/PKG signaling pathway. A critical feature of VA activation involves the reversible association of the cytosolic (V1) and membrane (Vo) complexes. Indeed, higher V1 protein abundance was found in membrane fractions of DH31-treated MTs, whereas CAPA significantly decreased V1 abundance in membrane fractions while increasing it in cytosolic fractions. V1 immunolocalization was observed strictly in the apical membrane of DH31-treated MTs, whereas immunoreactivity was dispersed following CAPA treatment. VA complexes colocalized apically in female MTs shortly after a blood meal consistent with the peak and postpeak phases of diuresis. Comparatively, V1 immunoreactivity in MTs was more dispersed and did not colocalize with the Vo complex in the apical membrane at 3 h post blood meal, representing a time point after the late phase of diuresis has concluded. Therefore, CAPA inhibition of MTs involves reducing VA activity and promotes complex dissociation hindering secretion. Collectively, these findings reveal a key target in hormone-mediated inhibition of MTs countering diuresis that provides a deeper understanding of this critical physiological process necessary for hydromineral balance.

Ornithine decarboxylase deficiency critically impairs nitrogen metabolism and survival in Aedes aegypti mosquitoes.

Ornithine decarboxylase (ODC; EC 4.1.1.17) catalyzes the conversion of ornithine to putrescine, the rate-limiting first step for de novo polyamine biosynthesis. Previously, we reported that genetic knockdown of xanthine dehydrogenase 1 (XDH1)-a gene encoding the enzyme involved in the last two steps of uric acid synthesis-causes an increase in ODC transcript levels in fat body of blood-fed Aedes aegypti mosquitoes, suggesting a crosstalk at molecular level between XDH1 and ODC during nitrogen disposal. To further investigate the role of ODC in nitrogen metabolism, we conducted several biochemical and genetic analyses in sugar- and blood-fed A. aegypti females. Distinct ODC gene and protein expression patterns were observed in mosquito tissues dissected during the first gonotrophic cycle. Both pharmacological and RNA interference-mediated knockdown of ODC negatively impacted mosquito survival, disrupted nitrogen waste disposal, delayed oviposition onset, and decreased fecundity in vitellogenic blood-fed females. A lag in the expression of two major digestive serine proteases, a reduction of blood meal digestion in the midgut, and a decrease in vitellogenin yolk protein uptake in ovarian follicles were observed by western blots in ODC-deficient females. Moreover, genetic silencing of ODC showed a broad transcriptional modulation of genes encoding proteins involved in multiple metabolic pathways in mosquito fat body, midgut, and Malpighian tubules prior to and after blood feeding. All together, these data demonstrate that ODC plays an essential role in mosquito metabolism, and that ODC crosstalks with multiple genes and proteins to prevent deadly nitrogen perturbations in A. aegypti females.

Discovery and Characterization of 2-Nitro-5-(4-(phenylsulfonyl)piperazin-1-yl)-N-(pyridin-4-ylmethyl)anilines as Novel Inhibitors of the Aedes aegypti Kir1 (AeKir1) Channel

Mosquito-borne arboviral diseases such as Zika, dengue fever, and chikungunya are transmitted to humans by infected adult female Aedes aegypti mosquitoes and affect a large portion of the world's population. The Kir1 channel in Ae. aegypti ( AeKir1) is an important ion channel in the functioning of mosquito Malpighian (renal) tubules and one that can be manipulated in order to disrupt excretory functions in mosquitoes. We have previously reported the discovery of various scaffolds that are active against the AeKir1 channel. Herein we report the synthesis and biological characterization of a new 2-nitro-5-(4-(phenylsulfonyl) piperazin-1-yl)- N-(pyridin-4-ylmethyl)anilines scaffold as inhibitors of AeKir1. This new scaffold is more potent in vitro compared to the previously reported scaffolds, and the molecules kill mosquito larvae.

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.

Transcriptomic evidence for a dramatic functional transition of the malpighian tubules after a blood meal in the Asian tiger mosquito Aedes albopictus.

BackgroundThe 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 FindingsHere 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/SignificanceThe 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.

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.

Actin redistribution in mosquito Malpighian tubules after a blood meal and cyclic AMP stimulation

Fluid secretion by mosquito Malpighian tubules is critical to maintaining fluid and electrolyte balance after a blood meal. Endogenous cAMP levels increase in Malpighian tubules after a blood meal. Here, we determined if corresponding changes in intracellular actin distribution occur after a blood meal or dibutyryl-cAMP (db-cAMP) stimulation and whether altering actin turnover inhibits secretion. In untreated Malpighian tubules, β-actin immunostaining was more intense in the apical region of adult Malpighian tubules than in the cytoplasm. Stimulation by a blood meal or db-cAMP significantly decreased β-actin immunostaining in the non-apical region of the cell. Db-cAMP had similar effects in larvae and pupae Malpighian tubules. In contrast, no detectable shift in F-actin distribution was detected; however, F-actin bundles within the cytoplasm increased in size after treatment with db-cAMP. Pretreatment of Malpighian tubules with agents perturbing actin fiber assembly and disassembly decreased basal secretion rates and inhibited the stimulatory effects of db-cAMP. Our results show (1) β-actin redistributes toward the apical membrane after a blood meal and this correlates temporally with increase urine flow rate and intracellular cAMP levels, (2) Malpighian tubules from all developmental stages exhibit this same response to db-cAMP-stimulation, and (3) dynamic assembly and disassembly of β-actin is required for db-cAMP-stimulated secretion.

MALARIA IN EXPERIMENTAL BIOLOGY

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Phylogeny and cloning of ion transporters in mosquitoes

Membrane transport in insect epithelia appears to be energized through proton-motive force generated by the vacuolar type proton ATPase (V-ATPase). However, secondary transport mechanisms that are coupled to V-ATPase activity have not been fully elucidated. Following a blood meal, the female mosquito regulates fluid and ion homeostasis through a series of characteristic behaviors that require brain-derived factors to regulate ion secretion. Despite the knowledge on the behaviors of the mosquito, little is known of the targets of several factors that have been implicated in cellular changes following a blood meal. This review discusses current models of membrane transport in insects and specific data on mosquito ion regulation together with the molecular aspects of membrane transport systems that are potentially linked to V-ATPase activity, which collectively determine the functioning of mosquito midgut and Malpighian tubules. Ion transport mechanisms will be discussed from a comparative physiology perspective to gain appreciation of the exquisite mechanisms of mosquito ion regulation.

Cloning and tissue distribution of two Na+/H+ exchangers from the Malpighian tubules of Aedes aegypti.

A Na(+)/H(+) exchanger (NHE) on the apical membrane of mosquito Malpighian tubule (MT) cells is believed to participate in the blood-feeding mosquito's vital secretion of Na(+) and fluid. This study presents the molecular cloning, primary structure, and tissue distribution of two cDNAs encoding Aedes aegypti mosquito MT NHEs. The cDNA sequences were obtained from mosquito MT total RNA using reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE). The two sequences encode proteins of 678 and 1,179 amino acids with calculated molecular weights of 74,473 and 130,276, respectively. When comparing the 678 amino acid protein to the first 678 amino acids of the other protein, the two clones show 98% identity to each other. They also exhibit high identity to Drosophila melanogaster NHEs. Hydropathy analysis reveals that while both clones have 10-13 transmembrane segments, the 1,179 amino acid protein has an extensive carboxy terminus while the 678 amino acid protein has an extremely short carboxy terminus. RT-PCR analysis shows that both clones are expressed in the mosquito Malpighian tubules at the larval and pupal stages, in addition to the adult stage before and after blood-feeding. Expression of both clones was also detected in adult mosquito ovaries, midguts, and hindguts.

Na(+)/H(+) exchange in mosquito Malpighian tubules.

Fluid secretion and intracellular pH were measured in isolated mosquito Malpighian tubules to determine the presence of Na(+)/H(+) exchange. Rates of fluid secretion by individual Malpighian tubules in vitro were inhibited by 78% of control in the presence of 100 microM 5-(N-ethyl-n-isopropyl)-amiloride (EIPA), a specific inhibitor of Na(+)/H(+) exchange. Steady-state intracellular pH was measured microfluorometrically by using 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in individual Malpighian tubules. Bathing the Malpighian tubules in 0 mM extracellular Na(+) or in the presence of 100 microM EIPA reduced the steady-state intracellular pH by 0.5 pH units. Stimulation of the Na(+)/H(+) exchanger by using the NH(4)Cl pulse technique resulted in a rate of recovery from the NH(4)Cl-induced acute acid load of 8.7 +/- 1.0 x 10(-3) pH/s. The rates of recovery of intracellular pH after the acute acid load in the absence of extracellular Na(+) or in the presence of 100 microM EIPA were 0.7 +/- 0.6 and -0.3 +/- 0.3 x 10(-3) pH/s, respectively. These results indicate that mosquito Malpighian tubules possess a Na(+)/H(+) exchanger.

Isolation of the V-ATPase A and c subunit cDNAs from mosquito midgut and malpighian tubules

Using conserved amino acid sequences for the design of oligonucleotide primers, we isolated cDNA clones for two subunits of the V-ATPase from the midgut and Malpighian tubules of Aedes aegypti larvae. The 3.1 kb cDNA of the A subunit of the peripheral catalytic V1 sector codes for a protein of 68.6 kDa. The protein contains conserved motifs, including an ATP/GTP binding site, found in all other A subunits. Southern analysis using the A subunit as a probe suggests the presence of only a single copy of gene in the Aedes aegypti. The 0.85 kb cDNA of the c subunit of the membrane H+ conducting V0 sector codes for a protein of kDa. This protein has four transmembrane domains and contains a conserved glutamic acid that serves as the binding site for dicyclohexylcarbodiimide. Southern analysis using the c subunit as a probe suggests the presence of more than one related gene in the genome of Aedes aegypti. Pileup analysis of various A and c subunits shows that these subunits fall into distinct clusters, including one in which all arthropod proteins are clustered.

Leucokinin increases paracellular permeability in insect Malpighian tubules

There are two major transport pathways across epithelia: a transcellular pathway through cells and a paracellular pathway between cells. Previous electrophysiological studies in mosquito Malpighian tubules suggested that the neuropeptide leucokinin-VIII (LK-VIII) increases the chloride permeability of the paracellular pathway. To test the effect of LK-VIII on the paracellular pathway further, we measured transepithelial permeabilities of inulin and sucrose in isolated Malpighian tubules from the mosquito Aedes aegypti. Cell membranes are impermeable to inulin and sucrose, leaving the paracellular pathway as the only route for their transepithelial permeation. LK-VIII (10(-6) mol l-1) significantly increased transepithelial permeability to both inulin (by 73.8 %) and sucrose (by 32.4 %) in parallel with a significant increase in rates of transepithelial fluid secretion (by 75­90 %). Cyclic adenosine monophosphate (cyclic AMP, 10(-4) mol l-1), which is known to stimulate transcellular transport, also increased rates of transepithelial fluid secretion (by 57­59 %), but it did so without increasing the permeability to sucrose and inulin. Thus, LK-VIII increases the permeability of the paracellular pathway whereas cyclic AMP does not.

Culekinin depolarizing peptide: a mosquito leucokinin-like peptide that influences insect Malpighian tubule ion transport

A peptide termed culekinin depolarizing peptide (CDP) was isolated from approximately 1.2 million mosquitos (94% Culex salinarius). The peptide was isolated on the basis of a rapid myotropic assay that utilized a hindgut preparation from Leucophaea maderae and a transepithelial voltage assay that used mosquito Malpighian tubules from Aedes aegypti. A 15% trifluoroacetic acid extraction from the mosquitos, two solid phase extraction steps, and six HPLC steps resulted in the isolation of 9.7 nmol of CDP. This value corresponds to approximately 8 fmol/mosquito. Edman degradation indicated the following sequence for CDP: Asn-Pro-Phe-His-Ser-Trp-Gly-NH 2. The sequence was confirmed as the suspected C-terminal amide form of the peptide, since native and synthetic CDP had identical chemical and biological properties. CDP is a member of the leucokinin family of neuropeptides. The leucokinins have been found in three other insect species ( Leucophaea maderae, Acheta domesticus and Locusta migratoria) where these peptides were isolated by their myotropic properties alone. CDP shares a C-terminal sequence homology (i.e., Phe-X-Ser-Trp-Gly-NH 2) with the rest of the leucokinins. CDP corresponds to the strongest tubule depolarizing activity in the C. salinarius extract. These findings agree with previous structure-activity studies that suggest that mosquitos would contain a leucokinin-like factor that had Phe-His-Ser-Trp-Gly-NH 2 as the C-terminal pentapeptide. This is the first leucokinin isolated from blood feeding or holometabolous insects.

Arachidonic acid and prostaglandin E 2 in malpighian tubules of female yellow fever mosquitoes

The fatty acid compositions of Malpighian tubules from adult females of the mosquito Aedes aegypti were determined for total lipids, phospholipids, triacylglycerols and three phospholipid fractions, namely phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol/ phosphatidylserine (PI/PS). The prostaglandin precursor arachidonic acid (20:4n-6) occurred in total lipids and phospholipids, but not triacylglycerols. Within phospholipids, nearly all of the 20:4n-6 was detected in PC, with only traces in PE, and none was detected in PI/PS. Isolated Malpighian tubules incorporated exogenous radioactive 20:4n-6 into tissue phospholipids and diacylglycerols, with most of the radioactivity recovered in diacyglycerol. These data indicate selective incorporation of 20:4n-6 into tissue lipids. PGE 2 was detected in Malpighian tubule whole mounts by immunohistochemical staining. These findings support the idea that prostaglandins are physiologically active in mosquito Malpighian tubules.

Leucokinins, a new family of ion transport stimulators and inhibitors in insect Malpighian tubules

Leucokinins are octapeptides isolated from heads of the cockroach Leucophaea maderae . In the cockroach they increase motility of the isolated hindgut. Surprisingly, synthetic leucokinins have biological activity in a different insect and in a different tissue. In isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti , leucokinins depolarize the transepithelial voltage. This effect on voltage is dependent on extracellular Cl. One leucokinin, LK-8, the effects of which were studied further in isolated Malpighian tubules, was found to inhibit transepithelial fluid secretion at low concentrations (10 −11 M threshold), and to stimulate fluid secretion at high concentrations (3.5 × 10 −9 M threshold). Together, the depolarizing effects on voltage and the stimulation of fluid secretion suggest that leucokinins increase the Cl permeability of the tubule wall thereby increasing the availability of Cl for secretion with Na, K and water. Structure-function comparisons of the seven leucokinins studied suggest that the active region of the octapeptide is segregated to the C-terminal pentapeptide. In view of the known effects of leucokinins on hindgut motility in the cockroach, our finding of effects in mosquito Malpighian tubules suggests that leucokinins may be widely distributed in insects where they may have diverse functions in a variety of organs.

Intracellular pH.

Intracellular pH.