Discussion

We have developed an experimental toolkit for studies on juvenile liver fluke that is built upon their ability to survive and develop over long periods We have developed an experimental toolkit for studies on juvenile liver fluke that is built upon their ability to survive and develop over long periods in vitro. The toolkit comprises robust reverse genetics and target localization tools complimented by a suite of bioassays that inform gene functions relating to survival, motility, growth/development and drug susceptibility (the availability of resistant and susceptible isolates facilitates studies on parasite-drug interplay). High RNAi penetrance extends across diverse neuronal, muscle, gut and stem cell targets. Here we pose the question, can our in vitro worms inform in vivo biology? Whilst they don’t reach sexual maturity, in vitro worms develop We have developed an experimental toolkit for studies on juvenile liver fluke that is built upon their ability to survive and develop over long periods in vitro. The toolkit comprises robust reverse genetics and target localization tools complimented by a suite of bioassays that inform gene functions relating to survival, motility, growth/development and drug susceptibility (the availability of resistant and susceptible isolates facilitates studies on parasite-drug interplay). High RNAi penetrance extends across diverse neuronal, muscle, gut and stem cell targets. Here we pose the question, can our in vitro worms inform in vivo biology? Whilst they don’t reach sexual maturity, in vitro worms develop ex vivo adult-like motility, morphology and excretory/secretory profiles. Also, during development in vitro worms display changes in drug sensitivity similar to those reported for in vivo studies. Worms grow more slowly in vitro than in vivo, a difference that appears to be driven by ~4-fold higher stem cell proliferation in vivo. Indeed, fluke drug susceptibility appears to diminish with increasing stem cell proliferation. Additionally, we find that the transcriptomes of 3-week old in vivo and in vitro juveniles show that ~11% of gene transcripts are differentially expressed, and many of those showing differential expression have proposed functions relating to growth/development. Further, only three of 42 miRNAs showed differential expression between in vitro and in vivo juveniles, with all three involved in developmental regulation. These data support our hypothesis that in vitro maintained juvenile fluke provide a valid model system for the study of parasite biology.

This work reported here has been supported by NC3Rs (NC/N001486/1) and the Department for the Economy Northern Ireland