Cellular mechanism of bicarbonate regulation and excretion in an insect inhabiting extremes of alkalinity

The saltwater mosquito larva, Aedes dorsal is, is one of the only organisms capable of inhabiting hypersaline lakes composed almost entirely of high concentrations of NaHC03 and Na2C03 salts. Under laboratory conditions larvae survived and developed normally in saline media with pH values up to 10.5, HC0₃⁻ concentrations up to 250 mM, or C0₃²- concentrations up to 100 mM. Despite ingestion of these alkaline media at rates equivalent to 130% of larval body weight per day, these insects regulated hemolymph pH (7.55-7.70) and HCO₃⁻ concentrations (8.0 - 18.5 mM) within narrow physiological limits. Micropuncture and microcannulation studies on the rectal salt gland demonstrated that this organ was an important site of pH and HC0₃⁻ regulation. Microcannulated salt glands secreted a strongly hyperosmotic fluid containing 402 mM HCO₃⁻ and 41 mM C0₃²⁻ at a rate of 38 nl-h⁻¹. Lumen-to-bath HC0₃⁻ and C0₃²⁻ gradients of 21:1 and 241:1, respectively, were generated by the salt gland epithelium against a transepithelial potential of -25 mV (lumen negative) demonstrating clearly the active nature of HCO₃⁻ secretion. To study the mechanisms of HCO₃⁻ transport, an in vitro microperfused rectal salt gland preparation was developed. Net total CO₂ transport (J[sup=JCo₂; sub=net]) as measured by microcalorimetry in perfused salt glands was unaffected by bilateral Na⁺ or K⁺ and serosal Cl⁻ substitutions, or by serosal addition of 1.0 mM ouabain, 2.0 mM amiloride or 0.5 mM SITS. Removal of luminal Cl⁻ inhibited (J[sup=JCo₂; sub=net] by 80%, while serosal addition of 1.0 mM acetazolamide or 0.5 mM DIDS inhibited by (J[sup=JCo₂; sub=net] 80% and 40%, respectively. Perfusion of the anterior and posterior rectal segments demonstrated clearly that the anterior rectum was the site of CO₂ secretion in the microperfused salt gland. Net Cl⁻ reabsorption in the anterior segment was measured by electron microprobe analysis and was equivalent to the rate of CO₂ secretion. In addition, Cl⁻ reabsorption in the anterior segment was completely inhibited by bilaterally replacing C0₂ and HC0₃⁻ with a phosphate or HEPES buffered saline. These data provide strong quantitative evidence for the presence of a 1:1 Cl⁻/HCO₃⁻ exchange mechanism located in the anterior rectal salt gland segment. Microcannulation studies on the individual salt gland segments demonstrated that both rectal segments are capable of secreting a hyperosmotic fluid containing Na⁻, Cl⁻ and HCO₃⁻. Based on these results and the results of studies in which the effects of serosal ion substitutions on salt gland fluid secretion were examined, it has been suggested tentatively that both segments secrete a NaCl-rich fluid and that fluid secretion is driven by coupled NaCl transport. It is further suggested that once this fluid enters the salt gland lumen its composition is modified by ion exchange and reabsorptive processes which are dependent upon the ionic regulatory needs of the animal. In larvae inhabiting low Cl⁻, NaHCO₃-CO₃ lakes, this modification involves a 1:1 exchange of luminal Cl⁻ for serosal HCO₃⁻. The cellular mechanisms of anterior salt gland HCO₃⁻ and Cl⁻ transport were examined using ion and voltage-selective microelectrodes in conjunction with a microperfused anterior segment preparation which allowed complete changes in serosal and mucosal saline composition to be made in