Research article

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^1,2 , Ala E Lew-Tabor^{* 1,2,3} , Manuel Rodriguez Valle^1,3 , Anthea G Bruyeres^1,3 , Vivienne J Doogan^1,3 , Ulrike G Munderloh⁴ , Felix D Guerrero^1,5 , Roberto A Barrero² and Matthew I Bellgard^1,2

¹Cooperative Research Centre for Beef Genetic Technologies, Armidale, NSW, Australia

²Centre for Comparative Genomics (CCG), Murdoch University, Perth, Western Australia 6150, Australia

³Department of Primary Industries and Fisheries, Emerging Technologies, Locked Mail Bag No. 4, Moorooka 4105, Queensland, Australia

⁴Department of Entomology, University of Minnesota, St Paul, Minnesota 55108, USA

⁵USDA-ARS, Knipling Bushland US Livestock Insect Research Laboratory, 2700 Fredericksburg Road, Kerrville, TX 78028, USA

author email corresponding author email* Contributed equally

BMC Molecular Biology 2009, 10:26doi:10.1186/1471-2199-10-26

Published:	26 March 2009

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.