Development of an in vitro model system for studying bacterially expressed dsRNA-mediated knockdown in Neoparamoeba genus

Paula Lima, Natasha Botwright, James Harris, Mathew Cook

    Research output: Contribution to journalArticle

    4 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)447-455
    Number of pages9
    JournalMarine Biotechnology
    Volume16
    Issue number4
    DOIs
    Publication statusPublished - Aug 2014

    Keywords

    • Amoeba
    • Aquaculture
    • RNA interference
    • Salmon
    • Soaking

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