Validation of a feeding strategy to deliver bacterially expressed dsRNA to marine amoeba from the genusNeoparamoeba

Abstract

RNA interference (RNAi) mediated by double stranded RNA (dsRNA) has emerged as one of the most promising techniques to study gene function of non-model protozoan parasites. We have previously demonstrated that bacterially expressed dsRNA delivered by immersion elicited successful knockdown in Neoparamoeba pemaquidensis, the non-infective species closely related to the causative agent of salmonid amoebic gill disease (AGD). However, considering that amoebae naturally feeds on microorganisms, direct ingestion of bacteria designed to express dsRNA could allow rapid and low-cost analysis of gene function on a large-scale. Therefore, the main objective of this study was to investigate whether ingestion of bacteria-expressing dsRNAs would also induce suppression of N. pemaquidensis β-actin and EF1α. Despite effective bacterial uptake, no significant variation in EF1α relative copy number was triggered by dsRNA ingestion. β-actin, on the other hand, presented similar silencing efficiency to what was observed in our previous soaking study. However, the observed RNAi response was delayed by at least 72 h. The present work provides evidence that delivery of bacterially expressed dsRNA through feeding can be successfully achieved in N. pemaquidensis, albeit not as efficiently as by soaking. Therefore, further investigation is required to develop more efficient and specific RNAi delivery systems in Neoparamoeba species. To our knowledge, this is the first time that RNAi-mediated knockdown through ingestion was attempted to manipulate gene function of a marine amoeba.