The use of cell and larval assays to identify target genes for RNA interference mediate control of the Australian sheep blowfly (Lucilia cuprina)

Flystrike is primarily caused by Lucilia cuprina, which is a major health and welfare issue for the sheep in wool industries. Current chemical-based controls can have limited effectiveness due to emerging resistance in the parasite. RNA interference (RNAi), which uses double-stranded RNA (dsRNA) as a trigger molecule, has been successfully investigated for the development of innovative pest control strategies. Although RNAi offers great potential, the efficient identification, selection of target genes and delivery of dsRNA represent challenges to be overcome for the successful application of RNAi for control of L. cuprina. In this study, the application of RNAi was explored using a novel L. cuprina embryonic cell line as well as in vitro larval bioassays. A primary L. cuprina (blowfly) embryo cell line (BFEC) was established and confirmed as being derived from L. cuprina eggs by PCR amplicon sequencing. The BFECs were successfully transfected with plasmids and messenger RNA (mRNA) expressing fluorescent reporter proteins and dsRNA using lipid-based transfection reagents. The transfection of dsRNA into BEFC in this study demonstrated RNAi-mediated knockdown of target gene expression. Three of the dsRNAs identified in this study, resulted in significant reductions in target gene expression in BFEC and loss of biological fitness in a L. cuprina larvae feeding bioassay. This study confirms that the novel BFEC cell line can be used to improve the efficacy of dsRNA-mediated screening to accelerate the identification of potential target genes in the development of RNAi mediated control approaches for L. cuprina. These results contribute to the development of sustainable control strategies for this important agricultural pest.