The miRNome of Fasciola hepatica juveniles endorse the existence of a reduced set of highly divergent miRNAs in parasitic flatworms.

The liver fluke Fasciola hepatica is a foodborne zoonotic parasite affecting livestock worldwide with increasing relevance in human health. The first developmental stage that the host meets after ingestion of the parasite is the newly excysted juvenile (NEJ), that actively transverse the gut wall and migrates to their final location in the liver. The regulation of the early developmental events in NEJs is still poorly understood and a relevant target for control strategies. Here we investigated the putative involvement of small regulatory RNAs in the invasion process. The small RNA population of the NEJ fall into two classes, one represented by miRNAs and a secondary group of larger (32- 33 nucleotides) tRNA derived sequences. We identified more than 30 different miRNAs most of them belonging to ancient miRNAs conserved in protostomes and metazoans, notably with an miR-125b variant as highly predominant. Remarkably, several protostomian and metazoan conserved families were not detected in consonance with previous reports of drastic miRNome reduction in parasitic flatworms. Additionally, a set of 11 novel miRNAs was identified, probably associated with specific gene regulation expression needs in F. hepatica. While sequence conservation in mature miRNA is high across the metazoan tree, we observed that flatworm miRNAs are more divergent suggesting that mutation rates in parasitic flatworms could be high. Finally, the distinctive presence of tRNA derived sequences, mostly 5'' tRNA halves of selected tRNAs in the small RNA population of NEJs could indicate that this parasite uses both miRNA and tRNA fragments for the regulation of gene expression. Overall design: SmallRNA profile of Fasciola hepatica juvenile stage generated by deep sequencing using illumina GAIIx

肝片形吸虫（Fasciola hepatica）是一种食源性人兽共患寄生虫，广泛感染全球家畜，对人类健康的影响日益凸显。宿主经口摄入该寄生虫后，最先接触的发育阶段为新脱囊幼虫（newly excysted juvenile, NEJ），其可主动穿透肠壁并迁移至肝脏内的最终寄生位点。目前对于NEJ早期发育事件的调控机制仍不甚明晰，而这也是寄生虫防控策略的关键靶标。本研究探讨了小型调控RNA在该寄生虫入侵过程中的潜在作用。对NEJ的小型RNA群体进行分析后发现，其可分为两类：一类为microRNA (miRNA)，另一类为长度32~33核苷酸的tRNA来源序列。本研究共鉴定出30余种miRNA，其中多数属于原口动物与后生动物中保守的古老miRNA家族，尤为显著的是miR-125b变体占据了极高的表达丰度。值得关注的是，多个原口动物与后生动物保守的miRNA家族未被检出，这与此前关于寄生扁形动物miRNA组发生大幅缩减的报道一致。此外，本研究还鉴定出11种新型miRNA，其可能与肝片形吸虫特有的基因调控表达需求相关。尽管成熟miRNA的序列保守性在整个后生动物类群中普遍较高，但本研究观察到扁形动物miRNA的序列分化程度更高，这提示寄生扁形动物的突变率可能较高。最后，NEJ小型RNA群体中特有的tRNA来源序列（多为特定tRNA的5'端tRNA半分子）表明，该寄生虫可能同时利用miRNA与tRNA片段来调控基因表达。实验设计概述：本研究通过Illumina GAIIx平台开展深度测序，获取了肝片形吸虫幼虫阶段的小型RNA表达谱。