Abstract
BackgroundFunctional 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).ResultsThe 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.ConclusionSDNP 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.
Highlights
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
The RNA interference (RNAi) effect on Setaria digitata novel protein (SDNP) transcript level was assessed two dpi, and 14 dpi of Small interfering RNA (siRNA) using Quantitative real-time polymerase chain reaction (qPCR) and the knockdown of the gene was determined by comparing the transcript levels of the worms derived from non-injected, buffer-injected and green fluorescent protein (GFP) siRNA-injected mosquitoes
A significant suppression of the target gene expression was observed in this study, and the transcript abundance of the SDNP was reduced by 93% and 87.4 % in the worms from SDNP siRNA-injected mosquitoes, compared to the non-injected at 2 dpi and 14 dpi, respectively, compared to worms from non-injected mosquitoes (Fig. 2a, b)
Summary
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. Nematodes are the most abundant metazoans, capable of inhabiting both terrestrial and aquatic habitats This facilitates their ability to cause both human and animal health problems, as well as to impair crop production worldwide. Plant-parasitic nematodes destroy approximately 12.3% of the annual crop yield, and animal parasitic nematodes kill millions of livestock animals. This contributes to a loss of billions of US dollars annually and necessitates the allocation of additional billions of US dollars for anthelminthic drugs for livestock [4, 5]. In this context, screening of the nematode genome will enable us to gain a comprehensive knowledge of the molecular mechanisms associated with the development of the nematode, which may facilitate the control of parasitic nematodes [9, 10]
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