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

Submitted to: BioMed Central (BMC) Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 4, 2009
Publication Date: March 26, 2009
Citation: Kurscheid, S., Lew-Tabor, A.E., Rodriguez Valle, M., Bruyeres, A.G., Doogan, V.J., Munderloh, U.G., Guerrero, F., Barrero, R.A., Bellard, M.I. 2009. Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila. BioMed Central (BMC) Molecular Biology. 10:Article 26.

Interpretive Summary: 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 the Ixodes scapularis tick genome sequencing project and extensive R. microplus expressed gene sequences, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks. RNAi is a powerful technique for assessing gene function and will be used to help identify candidate antigens to develop an effective anti-tick vaccine. We screened 13,643 R. microplus ESTs and I. scapularis genome reads and were especially interested in finding tick gene sequences with sequence similarity to well characterized genes from Drosophila melanogaster that have significant effects on viability. Our analysis identified 30 RNAi proteins in R. microplus including a putative tick Dicer, RISC associated (Ago-2, TudorSN, FMRp), RNA dependent RNA polymerase (EGO-1) and 20 homologues implicated in double-stranded RNA uptake and processing. We then selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi screens for viability in both BME26 R. microplus embryonic cells and female ticks in vivo. In the cell line assays, only genes associated with proteasomes had an effect on cell viability. In vivo RNAi showed that 9 genes had significant effects on viability either by causing lethality or impairing egg laying. 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.

Technical Abstract: 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.