Determination of pH in Regions of the Midguts of Acaridid Mites

The pH of the guts of mites strongly affects their digestive processes. This study was carried out to determine the pH in the guts of 12 species of stored product and house dust mites. Eighteen pH indicators were chosen and offered to the mites in the feeding biotest. Based on the color changes of the indicators, the gut contents of acaridid mites were determined to be within a pH range of 4 to neutral. The gut contents showed a gradient in pH from the anterior to the posterior part. The anterior midgut (ventriculus and caeca) of most species had a pH ranging from 4.5 to 5, or slightly more alkaline for most of the species, while the middle midgut (intercolon/colon) had a pH of 5 to 6. Finally, the pH of the posterior midgut (postcolon) was between 5.5 and 7. Except for Dermatophagoides spp., no remarkable differences in the pH of the gut were observed among the tested species. Dermatophagoides spp. had a more acidic anterior midgut (a pH of 4 to 5) and colon (a pH of 5) with postcolon (a pH of below 6). The results characterizing in vivo conditions in the mite gut offer useful information to study the activity of mite digestive enzymes including their inhibitors and gut microflora

Neurotransmitter Transporter-Like: A Male Germline-specific SLC6 Transporter Required for Drosophila Spermiogenesis

The SLC6 class of membrane transporters, known primarily as neurotransmitter transporters, is increasingly appreciated for its roles in nutritional uptake of amino acids and other developmentally specific functions. A Drosophila SLC6 gene, Neurotransmitter transporter-like (Ntl), is expressed only in the male germline. Mobilization of a transposon inserted near the 3′ end of the Ntl coding region yields male-sterile mutants defining a single complementation group. Germline transformation with Ntl cDNAs under control of male germline-specific control elements restores Ntl/Ntl homozygotes to normal fertility, indicating that Ntl is required only in the germ cells. In mutant males, sperm morphogenesis appears normal, with elongated, individualized and coiled spermiogenic cysts accumulating at the base of the testes. However, no sperm are transferred to the seminal vesicle. The level of polyglycylation of Ntl mutant sperm tubulin appears to be significantly lower than that of wild type controls. Glycine transporters are the most closely related SLC6 transporters to Ntl, suggesting that Ntl functions as a glycine transporter in developing sperm, where augmentation of the cytosolic pool of glycine may be required for the polyglycylation of the massive amounts of tubulin in the fly's giant sperm. The male-sterile phenotype of Ntl mutants may provide a powerful genetic system for studying the function of an SLC6 transporter family in a model organism

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

The video (3:35 minutes at four times accelerated speed) shows a typical experiment with an isolated and perfused anterior midgut of larval A. aegypti. Luminal solution: low buffer mosquito saline with m-cresol purple (0.04%). Hemolymph-side solution: high buffer mosquito saline with serotonin (0.2 μmol l−1). The posterior part of the preparation is tied with a human hair onto a perfusion pipette (left) and a glass rod inserted into the lumen (right) maintains the tissue in the focus of the microscope. Luminal perfusion was stopped at the beginning of the video. The color change of m-cresol purple that reflects alkalinization (from yellow to purple) begins at the anterior end of the midgut (right) and continues towards more posterior regions of the anterior midgut (left). The peristaltic muscular activity can also be easily observed (cf. Onken et al. 2004b). At 2:40 minutes, the perfusion pump is started again and the purple, alkaline solution is flushed from the lumen of the preparation.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.Financial support for this work was provided by the National Science Foundation (IBN 0091208 to D.F.Moffett and S.B. Moffett) and by the National Institutes of Health (1R01AI063463-01A2 to D.F. Moffett, S.B. Moffett and H. Onken)

The SLC6 transporters: perspectives on structure, functions, regulation, and models for transporter dysfunction

The human SLC6 family is composed of approximately 20 structurally related symporters (co-transporters) that use the transmembrane electrochemical gradient to actively import their substrates into cells. Approximately half of the substrates of these transporters are amino acids, with others transporting biogenic amines and/or closely related compounds, such as nutrients and compatible osmolytes. In this short review, five leaders in the field discuss a number of currently important research themes that involve SLC6 transporters, highlighting the integrative role they play across a wide spectrum of different functions. The first essay, by Gary Rudnick, describes the molecular mechanism of their coupled transport which is being progressively better understood based on new crystal structures, functional studies, and modeling. Next, the question of multiple levels of transporter regulation is discussed by Reinhard Krämer, in the context of osmoregulation and stress response by the related bacterial betaine transporter BetP. The role of selected members of the human SLC6 family that function as nutrient amino acid transporters is then reviewed by François Verrey. He discusses how some of these transporters mediate the active uptake of (essential) amino acids into epithelial cells of the gut and the kidney tubule to support systemic amino acid requirements, whereas others are expressed in specific cells to support their specialized metabolism and/or growth. The most extensively studied members of the human SLC6 family are neurotransmitter reuptake transporters, many of which are important drug targets for the treatment of neuropsychiatric disorders. Randy Blakely discusses the role of posttranscriptional modifications of these proteins in regulating transporter subcellular localization and activity state. Finally, Dennis Murphy reviews how natural gene variants and mouse genetic models display consistent behavioral alterations that relate to altered extracellular neurotransmitter levels