Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubriformis

RNA interference in parasitic nematodes of animals: a reality check?

“…The ease with which RNAi can be used in C. elegans, together with the fact that the RNAi mechanism itself is conserved in a large number of organisms [4][5][6], has almost automatically led to the assumption that RNAi could be applied to related parasitic nematodes. However, recent evidence indicates that the application of RNAi to parasitic nematodes is not as straightforward as was expected [7,8] and efficacy has been extremely variable [9][10][11]. This variability is particularly intriguing.…”

“…Following the soaking of H. contortus larvae in fluorescently labelled dsRNA, uptake was found to be relatively weak in third-stage larvae (L3) compared with L2 and L4 stages [10], which might reflect the lack of functioning mouthparts in L3s [19]. Electroporation has been used to introduce dsRNA into several trichostrongylid nematodes of sheep and cattle [9][10][11]. This seems to be the most effective means of delivery [9] but it remains unclear at which anatomical site the dsRNA penetrates the worm and to what extent it spreads in the tissues.…”

“…Electroporation has been used to introduce dsRNA into several trichostrongylid nematodes of sheep and cattle [9][10][11]. This seems to be the most effective means of delivery [9] but it remains unclear at which anatomical site the dsRNA penetrates the worm and to what extent it spreads in the tissues. In addition, this approach requires careful optimization because pulses of current of the wrong length or intensity cause cell damage or rupture [20].…”

“…To date, the life stages tested for parasitic nematodes range from newly hatched L1 of Trichostrongylus colubriformis [9] to adult Nippostrongylus brasiliensis [22]. Clearly, it would be much easier if all experiments could be carried out using the free-living larval stages.…”

“…A possibility is that standard dsRNA preparations, which are used at relatively high concentrations, contain contaminating partially degraded dsRNA, antisense RNA or siRNAs at a concentration that is sufficient to induce RNAi downstream of the interaction between RDE-4 and DICER, two key components of the RNAI pathway [13,23]. It is noteworthy that siRNA is more efficiently delivered in T. colubriformis than is the longer dsRNA [9], although the converse is true in C. elegans [26].…”