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The midgut of the female mosquito Aedes aegypti was studied immunohistologically with antisera to various regulatory peptides. Endocrine cells immunoreactive with antisera to perisulfakinin, RFamide, bovine pancreatic polypeptide, urotensin 1, locustatachykinin 2 and allatostatins A1 and B2 were found in the midgut. Perisulfakinin, RFamide and bovine pancreatic polypeptide all react with the same, about 500 endocrine cells, which were evenly distributed throughout the posterior midgut, with the exception of its most frontal and caudal regions. In addition, these antisera recognized three to five neurons in each ingluvial ganglion and their axons, which ran longitudinally over the anterior midgut, as well as axons innervating the pyloric sphincter. The latter axons appear to be derived from neurons located in the abdominal ganglia. Antisera to two different allatostatins recognized about 70 endocrine cells in the most caudal area of the posterior midgut and axons in the anterior midgut whose cell bodies were probably located in either the brain or the frontal ganglion. Antiserum to locustatachykinin 2 recognized endocrine cells present in the anterior midgut and the most frontal part of the posterior midgut, as well as about 50 cells in the most caudal region of the posterior midgut. Urotensin 1 immunoreactivity was found in endocrine cells in the same region as the perisulfakinin-immunoreactive cells, but no urotensin-immunoreactive axons were found in the midgut. Double labeling experiments showed that the urotensin and perisulfakinin immunoreactivities were located in different cells. Such experiments also showed that the locustatachykinin and allatostatin immunoreactivities in the most caudal area of the posterior midgut were present in different cells. No immunoreactivity was found in the mosquito midgut when using antisera to corazonin, allatropin or leucokinin IV. Since these peptides have either been isolated from, or can reasonably be expected to be present in mosquitoes, it was concluded that these peptides are not present in the mosquito midgut.

The effects of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and bumetanide (both 10(-4) M) on transepithelial Na+, K+, Cl-, and fluid secretion and on tubule electrophysiology were studied in isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti. Peritubular DBcAMP significantly increased Na+, Cl-, and fluid secretion but decreased K+ secretion. In DBcAMP-stimulated tubules, bumetanide caused Na+, Cl-, and fluid secretion to return to pre-cAMP control rates and K+ secretion to decrease further. Peritubular bumetanide significantly increased Na+ secretion and decreased K+ secretion so that Cl- and fluid secretion did not change. In bumetanide-treated tubules, the secretagogue effects of DBcAMP are blocked. In isolated Malpighian tubules perfused with symmetrical Ringer solution, DBcAMP significantly hyperpolarized the transepithelial voltage (VT) and depolarized the basolateral membrane voltage (Vbl) with no effect on apical membrane voltage (Va). Total transepithelial resistance (RT) and the fractional resistance of the basolateral membrane (fRbl) significantly decreased. Bumetanide also hyperpolarized VT and depolarized Vbl, however without significantly affecting RT and fRbl. Together these results suggest that, in addition to stimulating electroconductive transport, DBcAMP also activates a nonconductive bumetanide-sensitive transport system in Aedes Malpighian tubules.

Evidence is presented for hormone-controlled adenosine 3',5'-cyclic monophosphate (cAMP)-mediated NaCl diuresis in Malpighian tubules of the blood-feeding yellow-fever mosquito Aedes aegypti. Studies in isolated Malpighian tubules reveal that cAMP added to the peritubular bath selectively stimulates NaCl secretion and not KCl secretion by increasing the Na conductance of the basolateral membrane of primary cells. These effects are duplicated by forskolin and theophylline in parallel with increased intracellular concentrations of endogenous cAMP. Two natriuretic peptides that we have isolated by high-pressure liquid chromatography (HPLC) methods from mosquito heads also increase NaCl and fluid secretion in isolated Malpighian tubules together with increased intracellular levels of cAMP. These results are consistent with a mechanism of NaCl diuresis in which the natriuretic peptides and cAMP are respectively the primary and secondary messengers that couple the ingestion of a blood meal to the excretion of the unwanted salt and water fraction of the meal. This hypothesis is supported by in vivo studies that reveal elevated intracellular cAMP levels in Malpighian tubules at the time of maximum NaCl diuresis.

A natriuretic factor that triggers diuresis in isolated Malpighian tubules of the mosquito was isolated from the head of the yellow-fever mosquito Aedes aegypti by passing a saline extract of mosquito heads through low-pressure and then high-pressure liquid chromatography (HPLC) columns. Three fractions with biologic activity eluted during a reverse-phase HPLC linear acetonitrile gradient run. Fraction I depolarized the transepithelial voltage (Vt) of isolated perfused Malpighian tubules but did not not stimulate fluid secretion in the Ramsay assay (J. A. Ramsay, J. Exp. Biol. 31: 104-113, 1954). Fraction II depolarized and fraction III hyperpolarized Vt, and both stimulated fluid secretion three- to fourfold. Even though the effects of fractions II and III on Vt differed, both stimulated fluid secretion by increasing the rate of NaCl secretion without affecting K secretion. The selective stimulation of active secretory Na transport by fraction III is mimicked by cyclic AMP (cAMP), suggesting the second messenger role of cAMP in the effects of fraction III. Because fraction III stimulates a NaCl-rich, as opposed to KCl-rich, fluid, the term mosquito natriuretic factor is proposed for this active fraction.

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.

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