Submitted to: Journal of Insect Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/22/2010
Publication Date: 8/15/2010
Publication URL: http://doi:10.1016/j.jinsphys.2010.04/017
Citation: Harvey, W.R., Okech, B.A., Linser, P.J., Becnel, J.J., Ahearn, G.A., Sterling, K.M. 2010. H+ V-ATPase-Energized Transporters in Brush Border Membrane Vesicles from Whole Larvae of Aedes Aegypti. Journal of Insect Physiology. 56:1377-1389. Interpretive Summary: There is a critical need for the development of new larvicides for control of mosquitoes that vector diseases to man and animals but are safe for non-target organisms and have minimal environmental impacts. The ability to study and discover new target sites in the larval midgut is difficult due to their small size. In this collaborative research project between ARS and University of Florida researchers, a method has been developed to isolate large quantities of midgut components (brush border membranes) from larvae of the yellow fever mosquito Aedes aegypti. This technology can be utilized to conduct detailed studies on midgut transporters and receptors that can be targeted for the development of novel larvicides.
Technical Abstract: Brush Border Membrane vesicles (BBMVs) from Whole larvae of Aedes aegypti (AeBBMVWs ) contain an H+ V-ATPase (V), a Na+/H+ antiporter, NHA1 (A) and a Na+-coupled, nutrient amino acid transporter, NAT8 (N), VAN for short. All V-ATPase subunits are present in the Ae. aegypti genome and in the vesicles. AgNAT8 was cloned from An. gambiae, localized in BBMs and characterized in Xenopus laevis oocytes. AgNHA1 was cloned and localized in BBMs but characterization in oocytes was compromised by an endogenous cation conductance. AeBBMVWs complement Xenopus oocytes for characterizing membrane proteins, can be energized by voltage from the V-ATPase and are in their natural lipid environment. BBMVs from caterpillars were used in radio-labeled solute uptake experiments but ~10,000 mosquito larvae are needed to equal 10 caterpillars. By contrast, functional AeBBMVWs can be prepared from 10,000 whole larvae in four hours. Na+-coupled 3Hphenylalanine uptake mediated by AeNAT8 in AeBBMVs can be compared to the Phe-induced inward Na+ currents mediated by AgNAT8 in oocytes. Western blots and light micrographs of samples taken during AeBBMVW isolation are labeled with antibodies against all of the VAN components. The use of AeBBMVWs to study coupling between electrogenic V-ATPases and the electrophoretic transporters is discussed.