Functional analysis of a novel parasitic nematode-specific protein of Setaria digitata larvae in Culex quinquefasciatus by siRNA mediated RNA interference

Abstract

BACKGROUND: Functional analysis of animal parasitic nematode genes is often quite challenging due to the unavailability of standardised in vitro culture conditions and lack of adequate tools to manipulate these genes. Therefore, this study was undertaken to investigate the suitability of Culex quinquefasciatus, as an in vivo culture platform for Setaria digitata larvae and RNA interference (RNAi), as a post-transcriptional gene silencing tool to study the roles of a vital gene that encodes a novel parasitic nematode-specific protein (SDNP). RESULTS: The red colour fluorescence detected following RNAi injection to the thorax of C. quinquefasciatus indicated the uptake of dsRNA by S. digitata larvae. The reduction of SDNP transcripts in siRNA treated larvae compared to non-treated larvae, as determined by qPCR, indicated that the siRNA pathway is operational in S. digitata larvae. The observation of motility reductions and deformities during the development indicated the association of SDNP in larvae locomotion and development processes, respectively. The irregularities in the migration of larvae in mosquitoes and elevated survival rates of mosquitoes compared to their untreated counterparts indicated reduced parasitism of S. digitata larvae in mosquitoes upon targeted downregulation of SDNP by siRNA treatment. CONCLUSION: SDNP plays vital roles in muscle contraction, locomotion, development processes, larval development and parasitism of S. digitata. Its ubiquitous presence in parasitic nematodes and its absence in their hosts provide a tantalising prospect of the possibility of targeting SDNP for future development of anthelmintic drugs. The susceptibility of the larval stages of S. digitata for RNAi in Culex quinquefasciatus was also demonstrated for the first time in this study.