Summary
To date, there has been considerable focus on the identification of biological targets in nematodes against which control agents can be developed. RNA interference allows determination of whether a specific gene is essential for development or survival and has potential as a technique for the high-throughput screening of genes for deleterious effects. RNAi as a technique has been applied to parasitic nematodes with variable success. This project aimed to characterise the pathway for RNAi in the parasitic nematode Haemonchus contortus as compared to the pathway already defined by studies in Caenorhabditis elegans.
A continual bioinformatics approach was utilised over the entire project as new H. contortus sequence was released into the public domain. Next-Generation Roche 454-Titanium sequencing of xL3 and adult nematodes was incorporated into the project as an extra deliverable and provided a great deal of very high quality data for H. contortus. All available H. contortus sequence (both in-house and public) was collected and compiled into a searchable database. This database was then queried using a variety of parameters in an attempt to increase the sequence information available for orthologues of the molecules involved in the RNAi mechanism. Considerable sequence information was obtained for ten of these genes (sid-1, dcr-1, drh-1, eri-1, pir-1, rsd-3, rrf-1, rde-1, rrf-3 and ego-1). Sequence information was not obtained for two genes (rsd-6 and rde-4), that do not appear to be present. However, sequence was identified for a possible family of argonaute proteins; these may fulfil the functional role of the missing C. elegans orthologues. A small amount of sequence was identified for the elusive sid-2, this gene appears to be rather divergent and has been extremely difficult to find. Primers were designed for seven of these genes (sid-1, dcr-1, eri-1, pir-1, rsd-3, rde-1, and ego-1), each representing a different processing branch in the RNAi mechanism. These primers were designed to amplify a small conserved coding region for each gene and were used to generate a stage-specific expression profile and localisation profile by quantitative PCR. All genes were expressed during the parasite life-cycle with variations in expression profiles. This provides evidence that the RNAi mechanism is functional in H. contortus, although it does differ from C. elegans. It should be noted that the compiled sequence library provides a strategic resource that will benefit discovery efforts in parasitic nematodes and may enable identification of new drug/vaccine targets in future projects.
Our RNAi assay that was originally developed in Project AHW.031 was significantly improved early in Project WP166. This improved RNAi assay was utilised in this project for a detailed functional characterisation of six genes, Nov0374, Nov0491, S3_0055, S1_452, ben-1 and ubq-1. These genes are expressed in all life cycle stages as determined by quantitative PCR. No RNAi phenotype was observed for Nov0374 and only slight mortality at L1 for ubq-1. A more dramatic developmental arrest was observed for ben-1 and S3_0055 with approximately 20% sick L1s at day 4 and 20% sick L1s and 20% sick L2s at day 8. Nov0491 had increased mortality at L1 and L2, while S1-452 had developmental arrest at L2 with 40% at day 8. In situ hybridisation experiments were undertaken to determine the localisation profiles for these genes. The differential gene silencing patterns did not correlate with the localisation of available mRNA transcripts for each gene. Alternatively, the variability of gene silencing by RNAi in H. contortus may be due to variable rates of protein turnover for the selected targets. Further studies would be required to investigate this possibility.
