Functional analysis of a Midgut Proteolytic Gene in Tsetse Fly (Glossina pallidipes) by RNA Interference

Abstract

Trypanosomiasis a devastating disease for human and animals in sub-Saharan Africa is transmitted via a tsetse fly (Genus: Glossina) bite. In absence of vaccine, effective and affordable drugs, the disease control relies heavily on vector control strategies because trypanosomes require an obligatory passage through the vector. Understanding the factors involved in tsetse-trypanosome interactions such as the midgut proteolytic genes that are known to be potential barriers to the initial trypanosome establishment stands to enhance vector control. However, very few in vivo studies have been undertaken to analyze such genes. The study describes functional analysis of a midgut Glossina proteolytic gene (GPL) by RNA interference to establish its influence on tsetse fly feeding success and survival. The coding region of the gene was amplified by RT-PCR and sequenced. The gene classified as a serine protease belonging to the trypsin family, with> 90% identity to the previously reported GPL genes from G. austeni and G. f fuscipes. Sequence prediction showed it has a pi of 4.99, molecular weight of 29.47 KD and a hydrophobic leader peptide (16aa) indicating it is secretory in nature. When compared to other serine protease of Diptera origin, it exhibited close phylogenetic relatedness to those of other haematophagous insects. A reverse genetic approach to study the isolated gene function was limited by the short lived nature of the achieved knockdown as confirmed by semi quantitative RT-PCR. The highest transcript reduction (60%) lasted for two days, feeding success in this group was not significantly impaired (p<0.05) but, it improved with time as the transcripts level recovered. Also, the observed mortality was low and survival of the fly was normal when compared to the uninjected control group. The short lived GPL knockdown suggests it is crucial to tsetse fly physiology and may contribute to bloodmeal digestion. The study results through targeted transcript knockdown together with GPL dual role during trypanosome establishment could provide a foundation for the development of a genetic approach to control trypanosomiasis based on this gene.