The septate junction protein Tetraspanin 2A is critical to the structure and function of Malpighian tubules in Drosophila melanogaster.

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.

The septate junction protein Mesh is required for epithelial morphogenesis, ion transport, and paracellular permeability in the Drosophila Malpighian tubule.

Septate junctions (SJs) are occluding cell-cell junctions that have roles in paracellular permeability and barrier function in the epithelia of invertebrates. Arthropods have two types of SJs, pleated SJs and smooth SJs (sSJs). In Drosophila melanogaster, sSJs are found in the midgut and Malpighian tubules, but the functions of sSJs and their protein components in the tubule epithelium are unknown. Here we examined the role of the previously identified integral sSJ component, Mesh, in the Malpighian tubule. We genetically manipulated mesh specifically in the principal cells of the tubule at different life stages. Tubules of flies with developmental mesh knockdown revealed defects in epithelial architecture, sSJ molecular and structural organization, and lack of urine production in basal and kinin-stimulated conditions, resulting in edema and early adult lethality. Knockdown of mesh during adulthood did not disrupt tubule epithelial and sSJ integrity but decreased the transepithelial potential, diminished transepithelial fluid and ion transport, and decreased paracellular permeability to 4-kDa dextran. Drosophila kinin decreased transepithelial potential and increased chloride permeability, and it stimulated fluid secretion in both control and adult mesh knockdown tubules but had no effect on 4-kDa dextran flux. Together, these data indicate roles for Mesh in the developmental maturation of the Drosophila Malpighian tubule and in ion and macromolecular transport in the adult tubule.

Voltages and resistances of the anterior Malpighian tubule of Drosophila melanogaster.

The small size of Malpighian tubules in the fruit fly Drosophila melanogaster has discouraged measurements of the transepithelial electrical resistance. The present study introduces two methods for measuring the transepithelial resistance in isolated D. melanogaster Malpighian tubules using conventional microelectrodes and PClamp hardware and software. The first method uses three microelectrodes to measure the specific transepithelial resistance normalized to tubule length or luminal surface area for comparison with resistances of other epithelia. The second method uses only two microelectrodes to measure the relative resistance for comparing before and after effects in a single Malpighian tubule. Knowledge of the specific transepithelial resistance allows the first electrical model of electrolyte secretion by the main segment of the anterior Malpighian tubule of D. melanogaster The electrical model is remarkably similar to that of the distal Malpighian tubule of Aedes aegypti when tubules of Drosophila and Aedes are studied in vitro under the same experimental conditions. Thus, despite 189 millions of years of evolution separating these two genera, the electrophysiological properties of their Malpighian tubules remains remarkably conserved.

The plasticity of extracellular fluid homeostasis in insects.

In chemistry, the ratio of all dissolved solutes to the solution's volume yields the osmotic concentration. The present Review uses this chemical perspective to examine how insects deal with challenges to extracellular fluid (ECF) volume, solute content and osmotic concentration (pressure). Solute/volume plots of the ECF (hemolymph) reveal that insects tolerate large changes in all three of these ECF variables. Challenges beyond those tolerances may be 'corrected' or 'compensated'. While a correction simply reverses the challenge, compensation accommodates the challenge with changes in the other two variables. Most insects osmoregulate by keeping ECF volume and osmotic concentration within a wide range of tolerance. Other insects osmoconform, allowing the ECF osmotic concentration to match the ambient osmotic concentration. Aphids are unique in handling solute and volume loads largely outside the ECF, in the lumen of the gut. This strategy may be related to the apparent absence of Malpighian tubules in aphids. Other insects can suspend ECF homeostasis altogether in order to survive extreme temperatures. Thus, ECF homeostasis in insects is highly dynamic and plastic, which may partly explain why insects remain the most successful class of animals in terms of both species number and biomass.

Targeting renal epithelial channels for the control of insect vectors.

Three small molecules were identified in high throughput screens that 1) block renal inward rectifier potassium (Kir) channels of Aedes aegypti expressed in HEK cells and Xenopus oocytes, 2) inhibit the secretion of KCl but not NaCl in isolated Malpighian tubules, and after injection into the hemolymph, 3) inhibit KCl excretion in vivo, and 4) render mosquitoes flightless or dead within 24h. Some mosquitoes had swollen abdomens at death consistent with renal failure. VU625, the most potent and promising small molecule for development as mosquitocide, inhibits AeKir1-mediated currents with an IC50 less than 100 nM. It is highly selective for AeKir1 over mammalian Kir channels, and it affects only 3 of 68 mammalian membrane proteins. These results document 1) renal failure as a new mode-of-action for mosquitocide development, 2) renal Kir channels as molecular target for inducing renal failure, and 3) the promise of the discovery and development of new species-specific insecticides.

Localization and role of inward rectifier K(+) channels in Malpighian tubules of the yellow fever mosquito Aedes aegypti.

Malpighian tubules of adult female yellow fever mosquitoes Aedes aegypti express three inward rectifier K(+) (Kir) channel subunits: AeKir1, AeKir2B and AeKir3. Here we 1) elucidate the cellular and membrane localization of these three channels in the Malpighian tubules, and 2) characterize the effects of small molecule inhibitors of AeKir1 and AeKir2B channels (VU compounds) on the transepithelial secretion of fluid and electrolytes and the electrophysiology of isolated Malpighian tubules. Using subunit-specific antibodies, we found that AeKir1 and AeKir2B localize exclusively to the basolateral membranes of stellate cells and principal cells, respectively; AeKir3 localizes within intracellular compartments of both principal and stellate cells. In isolated tubules bathed in a Ringer solution containing 34 mM K(+), the peritubular application of VU590 (10 µM), a selective inhibitor of AeKir1, inhibited transepithelial fluid secretion 120 min later. The inhibition brings rates of transepithelial KCl and fluid secretion to 54% of the control without a change in transepithelial NaCl secretion. VU590 had no effect on the basolateral membrane voltage (Vbl) of principal cells, but it significantly reduced the cell input conductance (gin) to values 63% of the control within ∼90 min. In contrast, the peritubular application of VU625 (10 µM), an inhibitor of both AeKir1 and AeKir2B, started to inhibit transepithelial fluid secretion as early as 60 min later. At 120 min after treatment, VU625 was more efficacious than VU590, inhibiting transepithelial KCl and fluid secretion to ∼35% of the control without a change in transepithelial NaCl secretion. Moreover, VU625 caused the Vbl and gin of principal cells to respectively drop to values 62% and 56% of the control values within only ∼30 min. Comparing the effects of VU590 with those of VU625 allowed us to estimate that AeKir1 and AeKir2B respectively contribute to 46% and 20% of the transepithelial K(+) secretion when the tubules are bathed in a Ringer solution containing 34 mM K(+). Thus, we uncover an important role of AeKir1 and stellate cells in transepithelial K(+) transport under conditions of peritubular K(+) challenge. The physiological role of AeKir3 in intracellular membranes of both stellate and principal cells remains to be determined.

The excretion of NaCl and KCl loads in mosquitoes. 1. Control data.

The handling of Na(+) and K(+) loads was investigated in isolated Malpighian tubules and in whole mosquitoes of Aedes aegypti. Isolated Malpighian tubules bathed in Na(+)-rich Ringer solution secreted Na(+)-rich fluid, and tubules bathed in K(+)-rich Ringer solution secreted K(+)-rich fluid. Upon Na(+) loading the hemolymph, the mosquito removed 77% the injected Na(+) within the next 30 min. The rapid onset and magnitude of this diuresis and the excretion of more Na(+) than can be accounted for by tubular secretion in vitro is consistent with the release of the calcitonin-like diuretic hormone in the mosquito to remove the Na(+) load from the hemolymph. Downstream, K(+) was reabsorbed with water in the hindgut, which concentrated Na(+) in excreted urine hyperosmotic to the hemolymph. Upon K(+) loading the hemolymph, the mosquito took 2 h to remove 100% of the injected K(+) from the hemolymph. The excretion of K(+)-rich isosmotic urine was limited to clearing the injected K(+) from the hemolymph with a minimum of Cl(-) and water. As a result, 43.3% of the injected Cl(-) and 48.1% of the injected water were conserved. The cation retained in the hemolymph with Cl(-) was probably N-methyl-d-glucamine, which replaced Na(+) in the hemolymph injection of the K(+) load. Since the tubular secretion of K(+) accounts for the removal of the K(+) load from the hemolymph, the reabsorption of K(+), Na(+), Cl(-), and water must be inhibited in the hindgut. The agents mediating this inhibition are unknown.

Excretion of NaCl and KCl loads in mosquitoes. 2. Effects of the small molecule Kir channel modulator VU573 and its inactive analog VU342.

The effect of two small molecules VU342 and VU573 on renal functions in the yellow fever mosquito Aedes aegypti was investigated in vitro and in vivo. In isolated Malpighian tubules, VU342 (10 µM) had no effect on the transepithelial secretion of Na(+), K(+), Cl(-), and water. In contrast, 10 µM VU573 first stimulated and then inhibited the transepithelial secretion of fluid when the tubules were bathed in Na(+)-rich or K(+)-rich Ringer solution. The early stimulation was blocked by bumetanide, suggesting the transient stimulation of Na-K-2Cl cotransport, and the late inhibition of fluid secretion was consistent with the known block of AeKir1, an Aedes inward rectifier K(+) channel, by VU573. VU342 and VU573 at a hemolymph concentration of about 11 µM had no effect on the diuresis triggered by hemolymph Na(+) or K(+) loads. VU342 at a hemolymph concentration of 420 µM had no effect on the diuresis elicited by hemolymph Na(+) or K(+) loads. In contrast, the same concentration of VU573 significantly diminished the Na(+) diuresis by inhibiting the urinary excretion of Na(+), Cl(-), and water. In K(+)-loaded mosquitoes, 420 µM VU573 significantly diminished the K(+) diuresis by inhibiting the urinary excretion of K(+), Na(+), Cl(-), and water. We conclude that 1) the effects of VU573 observed in isolated Malpighian tubules are overwhelmed in vivo by the diuresis triggered with the coinjection of Na(+) and K(+) loads, and 2) at a hemolymph concentration of 420 µM VU573 affects Kir channels systemically, including those that might be involved in the release of diuretic hormones.

Roles of PKC and phospho-adducin in transepithelial fluid secretion by Malpighian tubules of the yellow fever mosquito.

The diuretic hormone aedeskinin-III is known to increase the paracellular Cl- conductance in Malpighian (renal) tubules of the mosquito Aedes aegypti via a G protein-coupled receptor. The increase serves the blood-meal-initiated diuresis and is associated with elevated levels of Ca2+ and phosphorylated adducin in the cytosol of tubule. In the present study we have cloned adducin in Aedes Malpighian tubules and investigated its physiological roles. Immunolabeling experiments are consistent with the association of adducin with the cortical cytoskeleton, especially near the apical brush border of the tubule. An antibody against phosphorylated adducin revealed the transient phosphorylation of adducin 2 min after stimulating tubules with aedeskinin-III. The PKC inhibitor bisindolylmaleimide-I blocked the phosphorylation of adducin as well as the electrophysiological and diuretic effects of aedeskinin-III. Bisindolylmaleimide-I also inhibited fluid secretion in control tubules. Phorbol 12-myristate 13-acetate increased phosphorylated adducin levels in Malpighian tubules, but it inhibited fluid secretion. Thus, the phosphorylation of adducin by PKC alone is insufficient to trigger diuretic rates of fluid secretion; elevated levels of intracellular Ca2+ may also be required. The above results suggest that the phosphorylation of adducin, which is known to destabilize the cytoskeleton, may (1) facilitate the traffic of transporters into the apical brush border supporting diuretic rates of cation secretion and (2) destabilize proteins in the septate junction thereby enabling paracellular anion (Cl-) secretion at diuretic rates. Moreover, PKC and the phosphorylation of adducin play a central role in control and diuretic tubules, consistent with the dynamic behavior of both transcellular and paracellular transport pathways.

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(+).

Protein kinase A-dependent and -independent activation of the V-ATPase in Malpighian tubules of Aedes aegypti.

Transepithelial ion transport in insect Malpighian tubules is energized by an apical V-ATPase. In hematophagous insects, a blood meal during which the animal ingests huge amounts of salt and water stimulates transepithelial transport processes linked to V-ATPase activation, but how this is accomplished is still unclear. Here we report that membrane-permeant derivatives of cAMP increase the bafilomycin-sensitive ATPase activity in Malpighian tubules of Aedes aegypti twofold and activate ATP-dependent transport processes. In parallel, membrane association of the V(1) subunits C and D increases, consistent with the assembly of the holoenzyme. The protein kinase A inhibitor H-89 abolishes all cAMP-induced effects, consistent with protein kinase A (PKA) being involved in V-ATPase activation. Metabolic inhibition induced by KCN, azide and 2,4-dinitrophenol, respectively, also induces assembly of functional V-ATPases at the membrane without PKA involvement, indicating a phosphorylation-independent activation mechanism.

A dynamic paracellular pathway serves diuresis in mosquito Malpighian tubules.

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 the opening of the paracellular pathway via G protein-coupled receptors and the elevation of intracellular [Ca(2+)], which leads to the reorganization of the cytoskeleton associated with the septate junction (SJ). 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 SJ and in epithelial polarization are largely known for ectodermal epithelia, but the proteins that form and mediate the dynamic functions of the SJ in Malpighian tubules remain to be determined.

Role of an apical K,Cl cotransporter in urine formation by renal tubules of the yellow fever mosquito (Aedes aegypti).

The K,Cl cotransporters (KCCs) of the SLC12 superfamily play critical roles in the regulation of cell volume, concentrations of intracellular Cl(-), and epithelial transport in vertebrate tissues. To date, the role(s) of KCCs in the renal functions of mosquitoes and other insects is less clear. In the present study, we sought molecular and functional evidence for the presence of a KCC in renal (Malpighian) tubules of the mosquito Aedes aegypti. Using RT-PCR on Aedes Malpighian tubules, we identified five alternatively spliced partial cDNAs that encode putative SLC12-like KCCs. The majority transcript is AeKCC1-A(1); its full-length cDNA was cloned. After expression of the AeKCC1-A protein in Xenopus oocytes, the Cl(-)-dependent uptake of (86)Rb(+) is 1) activated by 1 mM N-ethylmaleimide and cell swelling, 2) blocked by 100 µM dihydroindenyloxyalkanoic acid (DIOA), and 3) dependent upon N-glycosylation of AeKCC1-A. In Aedes Malpighian tubules, AeKCC1 immunoreactivity localizes to the apical brush border of principal cells, which are the predominant cell type in the epithelium. In vitro physiological assays of Malpighian tubules show that peritubular DIOA (10 µM): 1) significantly reduces both the control and diuretic rates of transepithelial fluid secretion and 2) has negligible effects on the membrane voltage and input resistance of principal cells. Taken together, the above observations indicate the presence of a KCC in the apical membrane of principal cells where it participates in a major electroneutral transport pathway for the transepithelial secretion of fluid in this highly electrogenic epithelium.

The single kinin receptor signals to separate and independent physiological pathways in Malpighian tubules of the yellow fever mosquito.

In the past, we have used the kinins of the cockroach Leucophaea (the leucokinins) to evaluate the mechanism of diuretic action of kinin peptides in Malpighian tubules of the yellow fever mosquito Aedes aegypti. Now using the kinins of Aedes (the aedeskinins), we have found that in isolated Aedes Malpighian tubules all three aedeskinins (1 microM) significantly 1) increased the rate of fluid secretion (V(S)), 2) hyperpolarized the basolateral membrane voltage (V(bl)), and 3) decreased the input resistance (R(in)) of principal cells, consistent with the known increase in the Cl(-) conductance of the paracellular pathway in Aedes Malpighian tubules. Aedeskinin-III, studied in further detail, significantly increased V(S) with an EC(50) of 1.5 x 10(-8) M. In parallel, the Na(+) concentration in secreted fluid significantly decreased, and the K(+) concentration significantly increased. The concentration of Cl(-) remained unchanged. While the three aedeskinins triggered effects on V(bl), R(in), and V(S), synthetic kinin analogs, which contain modifications of the COOH-terminal amide pentapeptide core sequence critical for biological activity, displayed variable effects. For example, kinin analog 1578 significantly stimulated V(S) but had no effect on V(bl) and R(in), whereas kinin analog 1708 had no effect on V(S) but significantly affected V(bl) and R(in). These observations suggest separate signaling pathways activated by kinins. One triggers the electrophysiological response, and the other triggers fluid secretion. It remains to be determined whether the two signaling pathways emanate from a single kinin receptor via agonist-directed signaling or from a differentially glycosylated receptor. Occasionally, Malpighian tubules did not exhibit a detectable response to natural and synthetic kinins. Hypothetically, the expression of the kinin receptor may depend on developmental, nutritional, and/or reproductive signals.

A SLC4-like anion exchanger from renal tubules of the mosquito (Aedes aegypti): evidence for a novel role of stellate cells in diuretic fluid secretion.

Transepithelial fluid secretion across the renal (Malpighian) tubule epithelium of the mosquito (Aedes aegypti) is energized by the vacuolar-type (V-type) H(+)-ATPase and not the Na(+)-K(+)-ATPase. Located at the apical membrane of principal cells, the V-type H(+)-ATPase translocates protons from the cytoplasm to the tubule lumen. Secreted protons are likely to derive from metabolic H(2)CO(3), which raises questions about the handling of HCO(3)(-) by principal cells. Accordingly, we tested the hypothesis that a Cl/HCO(3) anion exchanger (AE) related to the solute-linked carrier 4 (SLC4) superfamily mediates the extrusion of HCO(3)(-) across the basal membrane of principal cells. We began by cloning from Aedes Malpighian tubules a full-length cDNA encoding an SLC4-like AE, termed AeAE. When expressed heterologously in Xenopus oocytes, AeAE is both N- and O-glycosylated and mediates Na(+)-independent intracellular pH changes that are sensitive to extracellular Cl(-) concentration and to DIDS. In Aedes Malpighian tubules, AeAE is expressed as two distinct forms: one is O-glycosylated, and the other is N-glycosylated. Significantly, AeAE immunoreactivity localizes to the basal regions of stellate cells but not principal cells. Concentrations of DIDS that inhibit AeAE activity in Xenopus oocytes have no effects on the unstimulated rates of fluid secretion mediated by Malpighian tubules as measured by the Ramsay assay. However, in Malpighian tubules stimulated with kinin or calcitonin-like diuretic peptides, DIDS reduces the diuretic rates of fluid secretion to basal levels. In conclusion, Aedes Malpighian tubules express AeAE in the basal region of stellate cells, where this transporter may participate in producing diuretic rates of transepithelial fluid secretion.

The developmental, molecular, and transport biology of Malpighian tubules.

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.

Vacuolar-type proton pumps in insect epithelia.

Active transepithelial cation transport in insects was initially discovered in Malpighian tubules, and was subsequently also found in other epithelia such as salivary glands, labial glands, midgut and sensory sensilla. Today it appears to be established that the cation pump is a two-component system of a H(+)-transporting V-ATPase and a cation/nH(+) antiporter. After tracing the discovery of the V-ATPase as the energizer of K(+)/nH(+) antiport in the larval midgut of the tobacco hornworm Manduca sexta we show that research on the tobacco hornworm V-ATPase delivered important findings that emerged to be of general significance for our knowledge of V-ATPases, which are ubiquitous and highly conserved proton pumps. We then discuss the V-ATPase in Malpighian tubules of the fruitfly Drosophila melanogaster where the potential of post-genomic biology has been impressively illustrated. Finally we review an integrated physiological approach in Malpighian tubules of the yellow fever mosquito Aedes aegypti which shows that the V-ATPase delivers the energy for both transcellular and paracellular ion transport.