Differential expression of microRNAs and tRNA fragments mediate the adaptation of the liver fluke Fasciola gigantica to its intermediate snail and definitive mammalian hosts

Highlights

• Fifty-six microRNAs (miRNAs) from 33 conserved families and four Fasciola-specific miRNAs were identified.

• miRNAs are mainly involved in the regulation of metabolism, biosynthesis, growth and developmental processes.

• The expression profiles of 5′ halves derived by tRNA^-Lys and tRNA^-Gly were mainly related to the buffalo-infecting stages.

• This study provided an overview of the dynamic changes of small RNAs that occur during the lifecycle stages of F. gigantica.

• This global analysis of F. gigantica lifecycle stages revealed new roles of miRNAs and tRFs in parasite development.

Abstract

The tropical liver fluke Fasciola gigantica affects livestock and humans in many Asian countries, large parts of Africa, and parts of Europe. Despite the public health and economic impacts of F. gigantica, understanding of F. gigantica biology and how the complex lifecycle of this liver fluke is transcriptionally regulated remain unknown. Here, we tested the hypothesis that the regulatory small non-coding RNAs (sncRNAs), microRNAs (miRNAs) and tRNA-derived fragments (tRFs) play roles in the adaptation of F. gigantica to its intermediate and definitive hosts. We sequenced sncRNAs of eight lifecycle stages of F. gigantica. In total, 56 miRNAs from 33 conserved families and four Fasciola-specific miRNAs were identified. Expression analysis of miRNAs suggested clear stage-related patterns. By leveraging the existing transcriptomic data, we predicted a miRNA-based regulation of metabolism, transport, growth and developmental processes. Also, by comparing miRNA complement of F. gigantica with that of Fasciola hepatica, we detected a high level of conservation and identified differences in some miRNAs, which can be used to distinguish the two species. Moreover, we found that tRFs at each lifecycle stage were predominantly derived by tRNA^-Lys and tRNA^-Gly at 5′ half sites, but relatively high expression was related to the buffalo-infecting stages. Taken together, we provided a comprehensive overview of the dynamic transcriptional changes of small RNAs that occur during the developmental stages of F. gigantica. This global analysis of F. gigantica lifecycle stages revealed new roles of miRNAs and tRFs in parasite development and will facilitate future research into understanding of fasciolosis pathobiology.

Introduction

With an estimate of more than 15,000 species, trematodes (Platyhelminthes, Trematoda) include the vast majority of all known flatworm species, and some of them are involved in opportunistic infections of human and livestock (Furst et al., 2012, Torgerson et al., 2015). The genus Fasciola (Fasciola gigantica and Fasciola hepatica) represents an important group of liver flukes with notable economic and public health relevance (Mas-Coma, 2005). Previous studies showed that fascioliasis can affect between 2.4 and 17 million people, and 180 million are at risk of infection (Toet et al., 2014, Cwiklinski et al., 2016, Mas-Coma et al., 2018). The actual numbers of infections in humans and animals are likely underestimated due to the lack of comprehensive or coordinated investigations, and limited availability of diagnostic tools in some developing countries (Toet et al., 2014, Harrington et al., 2017). Fascioliasis is recognised as a neglected tropical disease by the World Health Organization due to the significant impacts on public health and socioeconomic losses (https://www.who.int/foodborne_trematode_infections/fascioliasis/en/).

Similar to other digenetic trematodes, F. gigantica has a complex lifecycle. The individual stages are very similar to those found in the sister species F. hepatica and only differ in the intermediate mollusc host species (Andrews, 1999; Howell, A., 2011. Snail-borne diseases in bovids at high and low altitude in Eastern Uganda: Integrated parasitological and malacological mapping. MSc dissertation, Liverpool School of Tropical Medicine, UK). Briefly, the egg, a weather-resistant/free-living stage, passes in faeces from the mammalian host into the environment, and then develops into a miracidium, a free-living stage, that actively seeks a snail as an intermediate host. Within the snail, the miracidium undergoes further development into a sporocyst, redia and cercaria (Phalee et al., 2015, Zhang et al., 2019b). A large number of cercariae emerge from snails and encyst on aquatic plants as metacercariae. The metacercaria, an another weather-resistant/free-living stage, on vegetation has the capability to remain viable for up to 6 months (Howell, 2011, MSc dissertation, cited earlier; Cwiklinski et al., 2016). Upon ingestion of vegetation or water contaminated with metacercariae by mammalian definitive hosts, the parasite emerges from their cyst in the intestine, as a newly excysted juvenile (NEJ) before crossing the intestinal wall into the peritoneal cavity. The developing juvenile fluke migrates through the liver parenchyma (~6–8 weeks), and then enters the gallbladder and/or bile ducts. Within the mammalian hosts, the parasite survives by feeding on bile and blood to maintain their metabolism and growth until formation of the adult stage and the production and release of eggs with the host’s faeces. Although the developmental stages and their roles in the lifecycle of F. gigantica are already known, the molecular and gene regulatory processes that control their adaptation to their host are still poorly defined.

MicroRNAs (miRNAs), are a large class of endogenously expressed single-stranded non-coding RNAs, which function as post-transcriptional gene regulators by complementary sequence paring of the seed region of the miRNA with the 3′ untranslated sequences (3′ UTRs) of protein coding mRNAs (Bartel, 2018). This mechanism of post-transcriptional regulation is highly conserved in animals and miRNAs are involved in many cellular and organismal processes such as development (Gebert and MacRae, 2018). The miRNA complement of several trematode groups, including a few free-living and parasitic stages, have been described using samples from pooled adults (see Cai et al., 2016). However, due to the small size of intra-snail larval stages and the developmental differences of intra-mammallian juvenile stages of flatworms, the analysis of miRNA contribution to the various developmental stages from egg to adult of any flatworm has remained challenging. Compared with miRNAs, tRNA-derived fragments (tRFs) and the tRNA half are a new class of small non-coding RNAs (sncRNAs) derived from mature tRNA precursors. They may play similar functional roles in post-transcriptional control of genes (Kanai, 2015, Cristodero and Polacek, 2017), and currently they have been reported to be involved in parasite development, and leading to potential pathogenesis (Fontenla et al., 2015, Nowacki et al., 2015, Fricker et al., 2019).

Fasciola spp. have been extensively studied from individual samples/developmental stages such as NEJ, adult and the parasites’ extracellular vesicles (EVs) with a focus on F. hepatica (Xu et al., 2012, Fontenla et al., 2015, Fromm et al., 2015b, Fromm et al., 2017, Ovchinnikov et al., 2020). An early study on F. gigantica identified the partial miRNA complement from the adult stage (Xu et al., 2012), and a recent study also demonstrated the presence of F. gigantica-derived miRNAs in the serum of infected mammalian hosts (Guo and Guo, 2019). However, due to the absence of a reference genome of F. gigantica, the miRNA complement likely remained incomplete, which in turn makes mechanistic analysis of miRNA functions difficult to perform. Fortunately, with the recent availability of a genome (Pandey et al., 2020) and transcriptome (Young et al., 2011, Zhang et al., 2017, Zhang et al., 2019b) of F. gigantica, and the reannotated miRNA complement in F. hepatica (Fromm et al., 2015b, Fromm et al., 2017), it is now possible to fill this gap.

In the present study, we performed small RNA sequencing of eight lifecycle stages of F. gigantica including egg, miracidium, redia, cercaria, metacercaria, two juvenile stages, and adult, respectively. Out of the detected 56 conserved miRNAs, 46 miRNAs were not previously described. We found that miRNA complement of F. gigantica is similar to F. hepatica, and identified miRNA genes with single nucleotide (nt) differences which interestingly enable differentiation of the two liver fluke species (F. gigantica and F. hepatica). Additionally, we detected expression differences of particular miRNAs across the lifecycle stages, and by using miRNA target prediciton and previously published RNA-seq data from the same stages, we found that respective target genes behave similarly. Futhermore, we examined the presence of tRFs in the obtained small RNA sequencing data.