Conserved core RNAi machinery in trematode-vectoring freshwater snails indicates gene silencing potential in the absence of classical systemic and amplification effectors

Abstract RNA interference (RNAi) is a widely exploited reverse-genetics tool with potential uses for disease control. Successful RNAi has been reported in trematode-vectoring freshwater snails, but the composition of RNAi effector-encoding gene complements, a key driver for RNAi efficiency, remains unstudied in these species. Using bioinformatics and comparative genomics, we searched for orthologues of 115 RNAi effector sequences in genomes of 4 snail vectors: Biomphalaria glabrata, B. pfeifferi, Bulinus truncatus and Lymnaea stagnalis . Gene expression patterns of selected RNAi effectors were then examined across developmental stages and tissues of the model B. glabrata snail. At least 74 RNAi-related proteins were conserved across all 4 species, including core components known to be essential for gene silencing. Classical systemic RNAi-deficient genes that facilitate systemic RNAi in other systems were absent, suggesting that alternative pathways may compensate for double-stranded RNA uptake and transport. Core effectors of secondary RNAi amplification and heritable RNAi were not detected. Expressions of Dicer-1, Argonaute-2 and the exonuclease Eri-1 did not vary significantly with snail size. A putative RNAi-inhibiting Staufen orthologue showed elevated expression in the ovotestis, while another putative cholesterol-interacting gene was overexpressed in the trunk tissue and may partly contribute to RNAi import. Altogether, our results present the most comprehensive overview of RNAi pathway effectors in major intermediate snail hosts for trematodes. The findings underscore the likely broad potential for RNAi use in these snails as an experimental tool and an alternative control method to CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based approaches.