Abstract
RNA interference (RNAi) has been extensively used to study gene function in non-model organisms and has the potential to identify parasite target molecules in order to develop alternative treatment strategies. This technology could assist in further development of preventive methods against amoebic gill disease (AGD), the main health problem affecting the Atlantic salmon aquaculture industry in Tasmania (Australia) and now a significant emerging issue in Europe. Using β-actin and EF1-α as candidate genes, we investigated the feasibility of gene knockdown by double-stranded RNA (dsRNA) in Neoparamoeba pemaquidensis, the non-infective strain closely related to the causative agent of AGD, Neoparamoeba perurans. Bacterially expressed dsRNA targeting the selected target genes was administered by soaking (2, 20 and 50 μg/mL) and a time course sampling regime performed. Quantitative real-time PCR analysis showed that candidate genes were successfully downregulated with silencing efficiency and duration both target and dose-dependent. Additionally, β-actin deficient trophozoites unexpectedly transformed into a cyst-like stage, which has not been previously reported in this species. An effective RNAi model system for N. pemaquidensis was validated in the current study. Such findings will greatly facilitate further application of RNAi in the aetiological agent of AGD. To our knowledge, this is the first time that RNAi-mediated technology has been successfully employed in a member of the Neoparamoeba genus.
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Acknowledgments
The authors would like to thank Dr David Cummins for their valuable suggestions and comments on the manuscript. Authors also thank Min Rao (CSIRO Animal, Food and Health Sciences) for kindly providing L4440 plasmid vector and E. coli HT115 strain and Russell McCulloch (CSIRO Animal, Food and Health Sciences) for the assistance with microscopy. This work formed part of a project of the Australian Seafood Cooperative Research Centre (CRC), and received funds from the Australian Government’s CRCs Programme, the Fisheries Research & Development Corporation and other CRC Participants.
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Lima, P.C., Botwright, N.A., Harris, J.O. et al. Development of an In Vitro Model System for Studying Bacterially Expressed dsRNA-Mediated Knockdown in Neoparamoeba genus. Mar Biotechnol 16, 447–455 (2014). https://doi.org/10.1007/s10126-014-9561-4
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DOI: https://doi.org/10.1007/s10126-014-9561-4