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 µmol 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 µmol l-1) on the hemolymph side almost abolished Vte and inhibited luminal alkalinization. The carbonic anhydrase inhibitor, methazolamide (50 µmol 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 Vte or alkalinization. Cl- substitution in the lumen or on both sides of the tissue affected Vte, 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 µmol l-1), reduced Vte and luminal alkalinization. Luminal amiloride (200 µmol l-1) was without effects on Vte or alkalinization. High K+ (60 mmol l-1) in the lumen reduced Vte 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-/HCO3- exchange or apical K+/2H+ antiport.

SUMMARY The present investigation studied the influence of a number of neuropeptides on semi-open preparations of the isolated and perfused anterior stomach of larval Aedes aegypti. Effects of peptides were observed on the lumen negative transepithelial voltage (Vte) that is present with serotonin in the bath; this voltage most likely reflects active HCO3– secretion involved in alkalization of the larval anterior stomach. The five different A. aegypti allatostatins(allatostatin A 1–5) all affected Vte in almost identical ways, causing a 10–15% reduction of the voltage at 10–7 mol l–1. A. aegyptineuropeptide F and proctolin reduced Vte at submicromolar concentrations. At 10–6 mol l–1,neuropeptide F reduced Vte by 30% and proctolin reduced Vte by 50%. In contrast, A. aegypti allatotropin, A. aegypti head peptides I and III and A. aegypti short neuropeptide F were without effect on Vte. During the investigation it was observed that the peristaltic contractions of the preparations caused a dynamic component of Vte. Peristaltic contractions and the correlated voltage fluctuations depended on the presence of serotonin. Peristaltic activity and Vtedeflections were progressively inhibited by A. aegypti head peptides I and III by A. aegypti short neuropeptide F and by A. aegypti neuropeptide F when the peptide concentrations were increased from 10–8 to 10–6 mol l–1. These observations show that physiological concentrations of some of the tested neuropeptides affect two processes that require coordination: ion transport and motility of the larval anterior stomach.

SUMMARYThe lumen-negative transepithelial voltage (Vte) of the isolated and perfused anterior stomach of mosquito larvae (Aedes aegypti) was studied with a `semi-open' preparation in which one end of the gut was ligated onto a perfusion pipette and the other end remained open to the bath. All experiments were performed with serotonin-stimulated preparations. Vte was abolished after addition of 2.5 mmol l-1 dinitrophenol and depended on the presence of Cl-. Na+ substitution experiments showed that a major part of Vte depended on the presence of this cation in the hemolymph side of the epithelium. Addition of 10 μmol l-1concanamycin (78±6% inhibition) or 2.5 mmol l-1 ouabain(15±2% inhibition) to the bath partially inhibited Vte. DPC (0.5 mmol l-1) or DIDS (0.1 mmol l-1) reduced Vte when applied to the hemolymph side of the epithelium (to 49±8% or 78±3% of the control,respectively). When present on both sides of the epithelium, these inhibitors caused further Vte reductions (to 23±4% or 35±4% of the control, respectively). Hemolymph-side furosemide (0.1 mmol l-1) or BaCl2 (5 mmol l-1) reduced Vte by 13±3% or 23±4% of the control,respectively. When applied to the hemolymph side of the epithelium, amiloride(0.2 mmol l-1) significantly decreased Vte by 35±6% of the control, whereas the drug caused no further effect when it was subsequently also applied to the luminal side of the epithelium. The above results are the basis for an extended model for the cellular mechanisms of NaHCO3 secretion/HCl absorption involved in alkalization of the anterior stomach of mosquito larvae.

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 microns. The tapered edges in SU-8 films are desired to enhance bilayer lipid membrane (BLM) formation and are fabricated by UV-light overexposure. Nanoapertures 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 0.1 M range with a sub-Nernstian slope of 20 mV 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.

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.Abbreviation:S-Iserotonin-like immunoreactivityRF-IFMRFamide-like immunoreactivitySCP-Ismall cardioactive peptide b-like immunoreactivity