Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference

Abstract

The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.

Expression profiling, gene silencing and transcriptional networking of metzincin metalloproteases in the cattle tick, Rhipicephalus (Boophilus) microplus

“…Similar degrees of silencing have been reported previously in R. microplus (Nijhof et al, 2007). Further, real-time PCR studies and data analysis revealed that significant non-specific silencing took place between As51 and AsC: injection of AsC dsRNA, in Group D, not only significantly reduced the normalized levels of AsC, but also resulted in a significant reduction of As51 (Table 3).…”

“…Injection of R. microplus females with dsRNA was performed as previously described in Nijhof et al (2007). Freshly molted female ticks were injected with 0.5 μl dsRNA solution…”

Molecular and biological characterization of the Amblyomma americanum organic anion transporter polypeptide

“…RNAi gene silencing was carried out according to previously published methods (Nijhof et al, 2007;Decrem et al, 2008;Hatta et al, 2007). Using the cloned AamOatp plasmid DNA as template and PCR primers (forward, 5ЈTAA TAC GAC TCA CTA TAG GGG GAC CCG GCA AGC ACT TGC TAG TT3Ј; reverse, 5ЈTAA TAC GAC TCA CTA TAG GGT GTT CTT GGA GAC CGC GTC CTC C3Ј) with added T7 promoter sequence (in bold), were used to amplify the template for AamOatp double-stranded RNA (dsRNA).…”

Advances in the genomics of ticks and tick-borne pathogens

“…Suppression can be persistent and act transstadially. For example, injection of double-stranded RNA into engorged R. (Boophilus) microplus females led to the downregulation of mRNA levels in eggs and subsequently in hatched larvae for several weeks [25]. RNAi has been used to study gene function and the role of tick genes in pathogen transmission, and as a screening tool for the identification of potential tick antigens for vaccine development [26,27].…”