Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi

We report improved and effective in vitro RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, Brugia malayi. The cellular disorganization observed in B. malayi embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their C. elegans orthologs. Targeting the B. malayi cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in C. elegans. Cellular phenotypes induced by our in vitro RNAi procedure can be observed by immunofluorescence in as little as one week.We observed cytological defects following RNAi targeting all seven B. malayi transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism C. elegans. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.Filarial nematodes cause debilitating pathologies in tropical areas with > 1 billion people at risk. Current anthelmintic drugs predominantly target larval stages only, and a developing resistance has been indicated [1-3]. Since almost all filarial species that cause disease in humans rely on the bacterial endosymbiont Wolbachia for proper embryogenesis, development and viability, these symbionts have become a major drug target for novel anti-filarial strategies [4,5]. It is crucial to characterize the molecular and cellular mechanisms underlying Wolbachia transmission, since their segregation patterns in the embryo determine the localization in adult hypodermal chords [6]. Such knowledge could lead to the molecu