Comparative and Functional Analysis of Tsetse Fly Aquaporins

Abstract

Tsetse flies (Glossina sp) are the vector of African trypanosomes, the causative agents of sleeping sickness in man and Nagana in cattle. The insect is strictly hematophagous, taking in significantly high quantities of water during bloodmeals that present a considerable osmotic challenge. This necessitates machinery for eliminating excess water. One potential candidate is a group of membrane channel proteins called aquaporins (AQPs) that are responsible for the movement of water across cell membranes. In other insect species, aquaporins have been shown to facilitate physiological processes including desiccation and freeze tolerance. Gmmdripa aquaporin has been shown to play an important role in diuresis and larval development in Glossina morsitans morsitans. This study sought to determine presence, tissue distribution and function of gmmdripa homologs proteins in three tsetse fly species. Using bioinformatics approach, three putative aquaporin genes named GfmAQP1, GffAQP1 and GlAQP1 from Glossina fuscipes martini, Glossina fuscipes fuscipes and Glossina longipennis respectively, were identified. Multiple sequence alignment and phylogenetic analysis using Neighbour-Joining method showed that these AQPs had high homology to Glossina morsitans morsitans integral protein A (DripA) and belonged to the water-specific Drosophila integral protein (DRIP) group of aquaporins. Further amino acids multiple alignment revealed that the AQPs had two asparagine-proline-alanine (NPA) motifs and six transmembrane domains that are evolutionarily conserved in most AQPs. Semi-quantitative RT-PCR showed higher levels of AQP expression in midgut as compared to proboscis, legs and head. In addition, the genes are expressed during all life stages with higher levels in newly emerged teneral flies as compared to larvae, pupae and adult. Functional analysis using RNA interference of Gfm AQP1 in G. f. martini showed decreased rate of diuresis after a blood meal but no significant effect on the fly survival (p<0.655). However, differences on the feeding success between the test and uninjected control groups were significant (p<0.0113), indicating the negative effects of injury inflicted by the needle during injections. These findings indicate an important role of AQP1 in regulation of water in tsetse.