RNA interference dynamics in juvenile Fasciola hepatica are altered during in vitro growth and development

Abstract

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 .

Characteristics of environmental RNAi in potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Psylloidea: Triozidae)

“…Second, the extent of RNAi persistence in adult PoP was determined for the minimal effective concentration resulting in optimal gene silencing, determined as 100 ng/μL. Finally, to evaluate the possible effects of psyllid developmental stage on PoP RNAi responsiveness ( Araujo et al, 2006 ; Terenius et al, 2011 ; Ulrich et al, 2015 ; Vogel et al, 2019 ; McCusker et al, 2020 ), RNAi penetrance was evaluated for PoP third instar nymphs, immature or “teneral” adults, and mature or “reproductive stage” adults.…”

Neoblast-like Stem Cells ofFasciola hepatica

“…Italian strain F. hepatica (Ridgeway Research, UK) were excysted as described by McVeigh et al and McCusker et al [83,84]. A full excystment protocol is available at http://dx.doi.org/10.17504/protocols.io.14egn212qg5d/v1.…”

“…Following excystment, NEJs/juveniles were soaked in 100 ng/µL dsRNA (diluted in RPMI) for 24 hours twice a week for the duration of the experiment to ensure knockdown [84]. Worms were imaged each week to monitor growth using ImageJ (as described in [91]), and a subset of worms exposed to EdU to monitor proliferation, as described previously.…”

The Differences in the Susceptibility Patterns to Triclabendazole Sulfoxide in Field Isolates of Fasciola hepatica Are Associated with Geographic, Seasonal, and Morphometric Variations

“…TCBZ-susceptible and -resistant F. hepatica strains collected from livestock have been characterized and maintained in the laboratory for decades [22]. These laboratory adapted F. hepatica isolates are used as models for in vitro studies of drug susceptibility [23][24][25][26][27]. Even among the limited number of laboratory parasite clones, significant differences in susceptibility profiles to anthelminthic drugs including TCBZ have been documented.…”