Gene expression in Rhipicephalus (Boophilus) microplus: Identification of tick antigens and investigation of the tick RNA interference pathway

The cattle tick Rhipicephalus microplus is a hematophagous ectoparasite of major veterinary importance both globally and in Australia. Australian beef cattle producers in the tropical and subtropical regions of the continent suffer estimated annual losses of AU$175m. The development of a novel tick vaccine has therefore been identified as a high priority research goal by all stakeholders of the  Australian beef industry (Playford 2005). A novel bioinformatics methodology for the identification of potential vaccine targets in the R. microplus expressed sequence tags sequences was developed and applied resulting in the identification of 68 potential tick vaccine candidates. Furthermore, the applicability of RNA interference (RNAi) for tick gene functional studies was assessed. This study comprehensively characterized the major components of the RNAi pathway in both R. microplus and Ixodes scapularis, which resulted in the identification of an I. scapularis homologue of Dicer protein, as well as the identification of Argonaute-1 and -2 homologues in both I. scapularis and R. microplus. Most notably, homologues of a C. elegans RNA-dependent RNA polymerase were
found in both R. microplus and I. scapularis, a protein involved in the amplification of the RNAi signal and previously not identified in any other arthropod species. A comparative RNAi study targeting 10 highly conserved R. microplus homologues of Drosophila genes with known RNAi phenotypes was conducted. The R. microplus homologue of D. melanogaster Ubiquitin-63E showed the strongest phenotypic changes in both cell culture and in adult female ticks injected with dsRNA. This Ubiquitin-63E-silencing effect in fully engorged females was subsequently investigated by microarray analysis. The contribution of potential off-target effects to phenotypic changes was
assessed by developing an in silico analysis and combining the prediction results with the microarray data. A total of 224 unique transcripts were detected as differentiallyexpressed. The off-target analysis revealed that 11 off-targets predicted by the in silico
analysis were present in the list of differentially expressed genes. The research presented in this thesis has contributed to the development of novel approaches for reverse vaccinology in ectoparasites and it has furthermore improved the understanding
of the mechanisms of RNAi and the delivery of double-stranded RNA in ticks.