Fluid absorption in the isolated midgut of adult female yellow fever mosquitoes (Aedes aegypti).

The transepithelial voltage (Vte) and the volume of isolated posterior midguts of adult female yellow fever mosquitoes (Aedes aegypti) were monitored. In all experiments, the initial Vte after filling the midgut was lumen negative, but subsequently became lumen positive at a rate of approximately 1 mV min(-1). Simultaneously, the midgut volume decreased, indicating spontaneous fluid absorption. When the midguts were filled and bathed with mosquito saline, the average rate of fluid absorption was 36.5±3.0 nl min(-1) (N=4, ±s.e.m.). In the presence of theophylline (10 mmol l(-1)), Vte reached significantly higher lumen-positive values, but the rate of fluid absorption was not affected (N=6). In the presence of NaCN (5 mmol l(-1)), Vte remained close to 0 mV (N=4) and fluid absorption was reduced (14.4±1.3 nl min(-1), N=3, ±s.e.m.). When midguts were filled with buffered NaCl (154 mmol l(-1) plus 1 mmol l(-1) HEPES) and bathed in mosquito saline with theophylline, fluid absorption was augmented (50.0±5.8 nl min(-1), N=12, ±s.e.m.). Concanamycin A (10 µmol l(-1)), ouabain (1 mmol l(-1)), and acetazolamide (1 mmol l(-1)) affected Vte in different ways, but all reduced fluid absorption by 60-70% of the value before addition of the drugs.

Signaling by intracellular Ca2+ and H+ in larval mosquito (Aedes aegypti) midgut epithelium in response to serosal serotonin and lumen pH.

The midgut of larval mosquitoes (Aedes aegypti) mediates a cycle of alkali secretion in the anterior segment (AMG) followed by partial reacidification in the posterior segment (PMG); both processes are serotonin-dependent. Here we report that intracellular Ca(2+)(Ca(i)(2+)) as indicated by Fura-2 fluorescence, is elevated in both tissues in response to serotonin, but the time courses differ characteristically in the two gut segments, and Ca(2+)-free solution abolishes the serotonin response in AMG, but not in PMG, whereas Thapsigargin, an inhibitor of endoplasmic Ca(2+) transport, abolished responsiveness to 5-HT in PMG. These results suggest the origins for the Ca(2+) signal differ between the two tissues. Quantitative real-time RT-PCR revealed expression of 5 putative 5-HT receptor types in AMG, including 5-HT(2)-like receptors which would be expected to initiate a Ca(2+) signal. None of these receptors were highly expressed in PMG. Cyclic AMP (cAMP) is a secretagogue for both tissues, but H89, an inhibitor of Protein Kinase A (PKA), is also a secretagogue, suggesting that the stimulatory effect of cAMP involves a non-PKA pathway. Cytochalasins B and D block the effect of 5-HT in AMG, suggesting a vesicle-fusion mechanism of activation of the basal V-ATPase in this tissue. Finally, in PMG, elevation of luminal pH increases (Ca(i)(2+)) and decreases intracellular pH as measured by BCECF fluorescence. These responses suggest that the rate of acid secretion by PMG might be responsive to local demand for luminal reacidification as well as to serosal serotonin.

Functionalization of micro- and nano-apertures with chromate-selective solvent polymeric membrane.

A new miniaturization approach to create micro- and nanoscale ion selective electrodes (ISEs) was demonstrated and the concept tested with an environmentally relevant chromate-selective membrane consisting of 7.7:62.2:31.1 wt % Aliquat336:2-NPOE:PVC. Apertures of 100 nM and 30 microM dimensions were made using MEMS fabrication techniques and functionalized through a macroscale application of solvent polymeric membrane. Performance studies for the microscale ISE showed a response slope of -58.6+/-5.6 mV decade(-1) and limit of detection (LOD) of 2.1 x 10(-5)+/-1.1 x 10(-5) M, versus -65.2+/-4.2 mV decade(-1) and 1.8 x 10(-5)+/-6 x 10(-6) M for the nanoscale ISE. This was consistent with control studies with carefully conditioned coated wire electrodes, which demonstrated a response slope of -61.7+/-2.4 mV decade(-1) and a LOD of 3.0 x 10(-6)+/-1 x 10(-6) M. Response times for the best micro- and nanoscale ISEs were in the 10-20 s timeframe. Electrical resistance measurements were in the GOmega range for the microscale ISEs and nanoscale ISEs. Appropriate ISE geometry was confirmed through AFM measurements and calculations based on electrical properties for micro- and nanoscale apertures. These micro- and nanoscale ISEs are expected to have significant impact in the field of microscale analytical processes.

Serotonin-induced high intracellular pH aids in alkali secretion in the anterior midgut of larval yellow fever mosquito Aedes aegypti L.

The anterior midgut of the larval yellow fever mosquito Aedes aegypti generates a luminal pH in excess of 10 in vivo and similar values are attained by isolated and perfused anterior midgut segments after stimulation with submicromolar serotonin. In the present study we investigated the mechanisms of strong luminal alkalinization using the intracellular fluorescent indicator BCECF-AM. Following stimulation with serotonin, we observed that intracellular pH (pH(i)) of the anterior midgut increased from a mean of 6.89 to a mean of 7.62, whereas pH(i) of the posterior midgut did not change in response to serotonin. Moreover, a further increase of pH(i) to 8.58 occurred when the pH of the luminal perfusate was raised to an in vivo-like value of 10.0. Luminal Zn(2+) (10 micromol l(-1)), an inhibitor of conductive proton pathways, did not inhibit the increase in pH(i), the transepithelial voltage, or the capacity of the isolated tissue to alkalinize the lumen. Finally, the transapical voltage did not significantly respond to luminal pH changes induced either by perfusion with pH 10 or by stopping the luminal perfusion with unbuffered solution which results in spontaneous luminal alkalinization. Together, these results seem to rule out the involvement of conductive pathways for proton absorption across the apical membrane and suggest that a serotonin-induced alkaline pH(i) plays an important role in the generation of an alkaline lumen.

The anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): effects of amino acids, dicarboxylic acids, and glucose on the transepithelial voltage and strong luminal alkalinization.

Isolated anterior midguts of larval Aedes aegypti were bathed in aerated mosquito saline containing serotonin (0.2 micromol L(-1)) and perfused with NaCl (100 mmol L(-1)). The lumen negative transepithelial voltage (V(te)) was measured and luminal alkalinization was determined through the color change of luminal m-cresol purple from yellow to purple after luminal perfusion stops. Addition of 10 mmol L(-1) amino acids (arginine, glutamine, histidine or proline) or dicarboxylic acids (malate or succinate) to the luminal perfusate resulted in more negative V(te) values, whereas addition of glucose was without effect. In the presence of TRIS chloride as luminal perfusate, addition of nutrients did not change V(te). These results are consistent with Na(+)-dependent absorption of amino acids and dicarboxylic acids. Effects of serotonin withdrawal indicated that nutrient absorption is stimulated by this hormone. Strong luminal alkalinization was observed with mosquito saline containing serotonin on the hemolymph-side and 100 mmol L(-1) NaCl in the lumen, indicating that alkalinization does not depend on luminal nutrients. Omission of glucose or dicarboxylic acids from the hemolymph-side solution had no effect on luminal alkalinization, whereas omission of amino acids significantly decelerated it. Re-addition of amino acids restored alkalinization, suggesting the involvement of amino acid metabolism in luminal alkalinization.

Revisiting the cellular mechanisms of strong luminal alkalinization in the anterior midgut of larval mosquitoes.

Here we critically review two recent hypotheses about the mechanism of strong alkalinization by the anterior midgut of mosquito larvae and our tests of these hypotheses. We present experimental evidence against the major components of transport models proposed in these hypotheses. Measurements of the transapical and transbasal proton electrochemical gradients provide an indication of driving forces faced by and generated by the transport mechanisms of the tissue. These measurements confirmed that basal V-ATPase energizes alkalinization. Serotonin stimulates the V-ATPase, as indicated by the ensuing increase in proton-motive force across the basal membrane. Moreover, the neurohormone resulted in a surprisingly large increase in the intracellular pH. The results of inhibitor studies indicate that, contrary to previous proposals, carbonic anhydrase is apparently not involved in supplying acid-base equivalents to the respective transporters. Furthermore, any apical processes proposed to be involved in alkali secretion or acid absorption must be Cl(-) independent and insensitive to DIDS, amiloride, Zn(2+) and ouabain. These results argue against the involvement of putative apical Cl(-)/HCO (-)(3) exchangers, apical H(+) channels, apical cation/proton exchangers and the importance of the apical Na(+)/K(+) pump. The studies analyzed here thus provide both a limitation and direction for further studies of the mechanism of strong alkalinization in this system.

Bilayer lipid membrane (BLM) based ion selective electrodes at the meso-, micro-, and nano-scales.

This paper presents a novel method for making micron-sized apertures with tapered sidewalls and nano-sized apertures. Their use in bilayer lipid membrane-based ion selective electrode design is demonstrated and compared to mesoscale bilayers and traditional PVC ion selective electrodes. Micron-sized apertures are fabricated in SU-8 photoresist films and vary in diameter from 10 to 40 microm. The tapered edges in SU-8 films are desired to enhance bilayer lipid membrane (BLM) formation and are fabricated by UV-light overexposure. Nano-apertures are made in boron diffused silicon film. The membranes are used as septa to separate two potassium chloride solutions of different concentrations. Lecithin BLMs are assembled on the apertures by ejecting lipid solution. Potassium ionophore, dibenzo-18-crown-6, is incorporated into BLMs by dissolving it in the lipid solution before membrane assembly. Voltage changes with increasing potassium ion concentrations are recorded with an A/D converter. Various ionophore concentrations in BLMs are investigated. At least a 1% concentration is needed for consistent slopes. Electrode response curves are linear over the 10(-6) to 0.1M range with a sub-Nernstian slope of 20mV per Log concentration change. This system shows high selectivity to potassium ions over potential interfering sodium ions. BLMs on the three different aperture sizes at the meso-, micro-, and nano-scales all show similar linear ranges and limits of detection (LODs) as PVC ion selective membranes.

Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na+/K+-ATPase.

Recently, Na(+)/K(+)-ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na(+)/K(+)-ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na(+)/K(+)-ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na(+)/H(+)-exchanger. Altogether the results of the present investigation indicate that apical Na(+)/K(+)-ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.

Alkalinization in the isolated and perfused anterior midgut of the larval mosquito, Aedes aegypti.

In the present study, isolated midguts of larval Aedes aegypti L. (Diptera: Culicidae) were mounted on perfusion pipettes and bathed in high buffer mosquito saline. With low buffer perfusion saline, containing m-cresol purple, transepithelial voltage was monitored and luminal alkalinization became visible through color changes of m-cresol purple after perfusion stop. Lumen negative voltage and alkalinization depended on metabolic energy and were stimulated in the presence of serotonin (0.2 micromol l(-1)). In some experiments a pH microelectrode in the lumen recorded pH values up to 10 within minutes after perfusion stop. The V-ATPase inhibitor concanamycin (50 micromol l(-1)) on the hemolymph side almost abolished V(te) and inhibited luminal alkalinization. The carbonic anhydrase inhibitor, methazolamide (50 micromol l(-1)), on either the luminal or hemolymph-side, or the inhibitor of anion transport, DIDS (1 mmol l(-1)) on the luminal side, had no effect on V(te) or alkalinization. Cl(-) substitution in the lumen or on both sides of the tissue affected V(te), but the color change of m-cresol purple was unchanged from control conditions. Hemolymph-side Na(+) substitution or addition of the Na(+)/H(+) exchange inhibitor, amiloride (200 micromol l(-1)), reduced V(te) and luminal alkalinization. Luminal amiloride (200 micromol l(-1)) was without effects on V(te) or alkalinization. High K(+) (60 mmol l(-1)) in the lumen reduced V(te) without affecting alkalinization. These results indicate that strong luminal alkalinization in isolated and perfused anterior midgut of larval A. aegypti depends on basolateral V-ATPase, but is apparently independent of carbonic anhydrase, apical Cl(-)/HCO(3)(-) exchange or apical K(+)/2H(+) antiport.

The isolated anterior stomach of larval mosquitoes (Aedes aegypti): voltage-clamp measurements with a tubular epithelium.

The anterior stomach of larval Aedes aegypti was isolated and perfused via two pipettes. For transepithelial voltage (V(te)) measurement, the inflow pipette and the bath were connected via agar bridges to calomel electrodes. For voltage-clamping, the lumen of the tissue contained an Ag/AgCl wire held by the outflow pipette, and the preparation was placed in a bath within a spiral of Ag/AgCl wire. After equilibrating the tissue in mosquito saline on both sides, a V(te) of -8+/-1 mV was measured (+/-S.E.M., N=32). Current-voltage curves (+/-100 mV) demonstrated ohmic behaviour of the epithelium. Short-circuiting resulted in a current (I(sc)) of 103+/-16 microA cm(-2) and a mean transepithelial conductance (G(te)) of 11.8+/-1.3 mS cm(-2) (+/-S.E.M., N=32). A Yonath-Civan plot of G(te) of individual preparations over the corresponding I(sc) resulted in a straight line (r(2)=0.8422), indicating that the difference in I(sc) of individual preparations is mainly based on different transcellular conductances (G(c)). This analysis allowed to estimate the mean leak conductance (G(l) approximately 3.9 mS cm(-2)) and the mean transcellular electromotive force (E(c) approximately 13 mV). After administering 0.2 micromol L(-1) serotonin, I(sc) and G(te) significantly increased, to 457+/-49 microA cm(-2) and to 21.3+/-2.3 mS cm(-2) (+/-S.E.M., N=31, P<0.05), respectively. The Yonath-Civan plot after serotonin resulted again in a straight line (r(2)=0.8219), indicating a mean G(l) of about 1 mS cm(-2) and a mean E(c) of about 22 mV. Dinitrophenol (2.5 mmol L(-1)) almost abolished I(sc) and significantly reduced G(te) (N=6). Concanamycin A (100 micromol L(-1)) reduced I(sc) by more than 90% without significantly affecting G(te).

Comparison of immunoreactivity to serotonin, FMRFamide and SCPb in the gut and visceral nervous system of larvae, pupae and adults of the yellow fever mosquito Aedes aegypti.

In all life stages, the gut of the mosquito is innervated by a small number (typically 4) of central neurons immunoreactive to serotonin (SI). The serotonergic system appears to pass through metamorphosis largely intact, despite extensive remodeling of the gut. Axons immunoreactive to antibodies raised against molluscan FMRFamide (RF-I) constitute peptidergic innervation that anatomically parallels the serotonergic system. In the larva, two clusters of 3 neurons project to the anterior regions of the gut, whereas in the pupa and adult, typically two large RF-I neurons located next to the esophagus send several processes posteriorly. In adults, these neurons branch throughout the diverticula and anterior stomach. In pupae, but not in larvae or adults, the gut RF-l system coexpresses reactivity to antibodies raised against a member of another peptide family, molluscan small cardioactive peptide b (SCP-I). SCP-I immunoreactivity is localized independently of RF-l immunoreactivity in the ganglia of all stages and in neurons that project along the gut of the adult. We did not find any colocalization of S-I and the peptide markers. Distinct populations of enteroendocrine cells populate different regions of the gut at different life stages. Changes in staining pattern suggest that these cells are replaced at metamorphosis along with the other gut cells during the extensive remodeling of the tract. Distributed in the gut epithelium are subpopulations that express either RF-I or SCP-I; a small fraction of these cells bind antibodies to both peptides. The stomachs of adult females are larger than those of males, and the numbers of SCP-I and RF-I enteroendocrine cells are proportionately greater in females. In all the life stages, the junctions between different regions of the gut are the focus of regulatory input. The larval cardiac valve possesses a ring of cells, the necklace cells, which appear to receive extensive synaptic inputs from both the serotonergic system and the peptidergic system. Another focus of control is the pyloric valve, which is encircled by axon-like processes. The immunoreactive pattern of this region differs across life stages, expressing SCP-I in larvae, S-I in pupae, and both SCP-I and RF-I in adults.