RNAi-mediated knockdown of daf-12 in the model parasitic nematode Strongyloides ratti

Abstract

The gene daf-12 has long shown to be involved in the dauer pathway in Caenorhabditis elegans (C. elegans). Due to the similarities of the dauer larvae of C. elegans and infective larvae of certain parasitic nematodes such as Strongyloides spp., this gene has also been suspected to be involved in the development of infective larvae. Previous research has shown that the application of dafachronic acid, the steroid hormone ligand of DAF-12 in C. elegans, affects the development of infective larvae and metabolism in Strongyloides. However, a lack of tools for either forward or reverse genetics within Strongyloides has limited studies of gene function within these important parasites. After determining whether Strongyloides had the requisite proteins for RNAi, we developed and report here the first successful RNAi by soaking protocol for Strongyloides ratti (S. ratti) and use this protocol to study the functions of daf-12 within S. ratti. Suppression of daf-12 in S. ratti severely impairs the formation of infective larvae of the direct cycle and redirects development towards the non-infective (non-dauer) free-living life cycle. Further, daf-12(RNAi) S. ratti produce slightly but significantly fewer offspring and these offspring are developmentally delayed or incapable of completing their development to infective larvae (L3i). Whilst the successful daf-12(RNAi) L3i are still able to infect a new host, the resulting infection is less productive and shorter lived. Further, daf-12 knockdown affects metabolism in S. ratti resulting in a shift from aerobic towards anaerobic fat metabolism. Finally, daf-12(RNAi) S. ratti have reduced tolerance of temperature stress.

Author summary

Strongyloides ratti is a model parasitic nematode of interest for its use in understanding basic biology and the development of novel helminth therapies. However a lack of genetic tools has stymied progress, although CRISPR/Cas9 has recently been reported. After determining whether RNAi might work in S. ratti by profiling the RNAi pathway proteins, we developed a successful RNAi protocol, which was used to study the gene daf-12. In Caenorhabditis elegans, daf-12 is involved in various developmental and metabolic processes, including the formation of long-living dauer larvae which are considered to be similar to the infective larvae of Strongyloides. Based on the external application of dafachronic acid (the ligand of DAF-12 in C. elegans) and gene expression studies, it was proposed that daf-12 has conserved functions in Strongyloides. Using our RNAi method, we provide the first proof of successful gene knockdown within S. ratti and demonstrate that daf-12 in S. ratti is involved in the same processes as C. elegans. This supports that daf-12 functions are conserved in distantly related nematodes and that daf-12 is an important target for the development of novel antihelminthics.