Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

Sebastian Kurscheid, Ala E. Lew-Tabor, Manuel Rodriguez Valle, Anthea G. Bruyeres, Vivienne J. Doogan, Ulrike G. Munderloh, Felix D. Guerrero, Roberto A. Barrero, Matthew I. Bellgard

Research output: Contribution to journalArticlepeer-review

Abstract

Background: The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype. Results: We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying. Conclusion: We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.

Original languageEnglish (US)
Article number26
JournalBMC Molecular Biology
Volume10
DOIs
StatePublished - Mar 26 2009

Bibliographical note

Funding Information:
The authors acknowledge Dr Bing Zhang for his assistance with culture qRT-PCR analysis and Ms Catherine Minchin for maintenance of the BME26 cell lines and for undertaking the culture knockdown experiments. The authors also wish to acknowledge the expertise and diligence provided by Mr Daniel Jarrett in the preparation of molecules for the RNAi diagram (Figure 5) and Dr Leo Salividar (USDA) for assistance with identifying Gen-Bank Accession numbers for all relevant R. microplus consensus and clone sequences. We would like to thank Dr Wayne Jorgensen and Prof Rudi Appels for a critical review of the manuscript. This research was funded by the Cooperative Research Centre for Beef Genetic Technologies, Armi-dale, NSW, Australia.

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