For over a decade RNA interference (RNAi) has been an important molecular tool for functional genomics studies in parasitic flatworms. Despite this, our understanding of RNAi dynamics in many flatworm parasites, such as the temperate liver fluke (Fasciola hepatica), remains rudimentary. The ability to maintain developing juvenile fluke in vitro provides the opportunity to perform functional studies during development of the key pathogenic life stage. Here, we investigate the RNAi competence of developing juvenile liver fluke. Firstly, all life stages examined possess, and express, core candidate RNAi effectors encouraging the hypothesis that all life stages of F. hepatica are RNAi competent. RNAi effector analyses supported growing evidence that parasitic flatworms have evolved a separate clade of RNAi effectors with unknown function. Secondly, we assessed the impact of growth/development during in vitro culture on RNAi in F. hepatica juveniles and found that during the first week post-excystment liver fluke juveniles exhibit quantitatively lower RNAi mediated transcript knockdown when maintained in growth inducing media. This did not appear to occur in older in vitro juveniles, suggesting that rapidly shifting transcript dynamics over the first week following excystment alters RNAi efficacy after a single 24 h exposure to double stranded (ds)RNA. Finally, RNAi efficiency was found to be improved through use of a repeated dsRNA exposure methodology that has facilitated silencing of genes in a range of tissues, thereby increasing the utility of RNAi as a functional genomics tool in F. hepatica.

十多年来，RNA 干扰 (RNAi) 一直是寄生扁虫功能基因组学研究的重要分子工具。尽管如此，我们对许多扁虫寄生虫（如温带肝吸虫（肝片吸虫））的 RNAi 动力学的理解仍处于初级阶段。在体外维持发育中的幼吸虫的能力为在关键致病生命阶段的发育过程中进行功能研究提供了机会。在这里，我们调查了发育幼年肝吸虫的 RNAi 能力。首先，所检查的所有生命阶段都拥有并表达核心候选 RNAi 效应子，支持以下假设：肝镰刀菌的所有生命阶段有 RNAi 能力。RNAi 效应器分析支持了越来越多的证据，即寄生扁虫已经进化出一个独立的 RNAi 效应器分支，其功能未知。其次，我们在评估生长/发展的影响，在体外对RNAi的文化肝片吸虫少年，发现在第一周后脱包囊肝吸虫青少年表现出定量较低的RNAi介导的转录击倒时维持在生长诱导培养基。这在较老的体外似乎没有发生幼鱼，表明在单次暴露于双链 (ds) RNA 24 小时后，在脱囊后第一周内快速变化的转录动力学改变了 RNAi 的功效。最后，发现通过使用重复的 dsRNA 暴露方法提高了 RNAi 的效率，该方法促进了一系列组织中基因的沉默，从而增加了 RNAi 作为功能基因组学工具在F. livera 中的效用。