Ammonium secretion by Malpighian tubules ofDrosophila melanogaster: application of a novel ammonium-selective microelectrode

Abstract

Summary Ammonia is a toxic nitrogenous waste product of amino acid metabolism that may accumulate to high levels in the medium ingested by larvae of the fruit fly, Drosophila melanogaster. Here we report measurements of haemolymph NH4+ concentration and the secretion of NH4+ by the Malpighian (renal) tubules. Measurement of NH4+ concentrations in secreted droplets is complicated either by the requirement for large sample volumes for enzymatic assays or by the inadequate selectivity of NH4+-selective microelectrodes based on nonactin. We have developed a novel liquid membrane NH4+-selective microelectrode based on a 19-membered crown compound (TD19C6), which has been used previously in ammonium-selective macroelectrodes. In conjunction with an improved technique for correcting for interference of potassium, NH4+-selective microelectrodes based on TD19C6 permit accurate measurement of ammonium concentration in haemolymph samples and nanoliter droplets of fluid secreted by the Malpighian tubules of Drosophila melanogaster. The results indicate that active secretion of ammonium into the Malpighian tubule lumen is sufficient to maintain concentrations of approximately 1 mmol l-1 ammonium in the haemolymph of larvae reared on diets containing 100 mmol l-1 ammonium chloride.

An RNA-Seq Screen of the Drosophila Antenna Identifies a Transporter Necessary for Ammonia Detection

“…The simplest mechanistic explanation for how Amt acts is that it plays a role in clearing ammonia from the sensillar lymph ( Figure S8 ). Drosophila larval haemolymph contains ∼1 mM ammonia [98] , derived from the animal's internal metabolism. Sensillar lymph may also contain ammonia from the fly's metabolism, and in addition is exposed to volatile ammonia from the fly's environment.…”

“…Sensillar lymph may also contain ammonia from the fly's metabolism, and in addition is exposed to volatile ammonia from the fly's environment. Ammonium levels in Drosophila culture vials have been measured at ∼20–30 mM [98] , [99] , primarily from the microbial ammonification of waste products. Although a low concentration of ammonia in sensillar lymph may be inconsequential to the function of most sensilla, it may interfere with the sensitive detection of ammonia by the IR92a-expressing ORN in ac1 sensilla.…”

The taste response to ammonia in Drosophila

“…In olfaction, low concentrations of airborne ammonia enter the sensillum via openings in the sensillar walls, and are then detected by olfactory neurons expressing IR92a. The basal ammonia concentration of the sensillum lymph is unknown, but the concentration in the larval hemolymph is ~1 mM53, and it is possible that the transport uptake function of Amt is needed to keep the ammonia concentration in the lymph of an olfactory sensillum sufficiently low to allow detection of low levels of airborne ammonia. By contrast, the ammonia levels that activate taste neurons (10–100 mM) and those found in a fly culture vial (~30 mM)45 are much higher than 1 mM.…”

Ammonia and urea handling by early life stages of fishes

“…Our understanding of these extra-branchial contributions may be increased through the use of in vitro approaches using isolated guts or nephrons of embryonic and larval fishes. One example of such an approach is the scanning ion-selective electrode technique (SIET) that has been used to measure epithelial NH 4 + fluxes in Malpighian tubules of fruit flies (Browne and O'Donnell, 2013).…”