Silencing of a putative immunophilin gene in the cattle tick Rhipicephalus (Boophilus) microplus increases the infection rate of Babesia bovis in larval progeny

Abstract

BackgroundThe cattle tick Rhipicephalus (Boophilus) microplus is involved in the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. Interactions between ticks and protozoa are poorly understood and the investigation of tick genes that affect tick fitness and protozoan infection can set the stage for dissecting the molecular interactions between the two species.ResultsIn this study, RNA interference was used to silence R. microplus genes that had been previously shown to be up-regulated in response to B. bovis infection. The silencing of a putative immunophilin gene (Imnp) in female ticks fed on a calf acutely infected with B. bovis decreased the hatching rate and survival of larval progeny. Interestingly, Imnp was up-regulated significantly in ovaries of R. microplus in response to B. bovis infection and its silencing in female ticks significantly increased the infection rate of the protozoan in larval progeny. The results also showed that the silencing of a putative Kunitz-type serine protease inhibitor (Spi) gene and a putative lipocalin (Lpc) gene decreased the fitness of R. microplus females, but had no significant effect on the infection rate of B. bovis in larval progeny.ConclusionThe silencing of the Imnp, Spi or Lpc genes decreased the fitness of R. microplus females fed on a calf during acute B. bovis infection. The Imnp gene data suggest that this putative immunophilin gene is involved in the defense system of R. microplus against B. bovis and may play a role in controlling the protozoan infection in tick ovaries and larval progeny.

Tick Passage Results in Enhanced Attenuation of Babesia bovis

“…Parasites were quantified from these samples using the msa-1 qPCR assay as described above. A positive control containing 10 ng of T2Bo_vir gDNA and gDNA negative controls from naïve-larva, calf, and tick tissues were included (4,15). Genomic DNA was also extracted from 100 l of individual tick hemolymph using the DNeasy blood and tissue DNA kit as per the manufacturer's recommendation (Qiagen, Valencia, CA).…”

“…Genomic DNA was also extracted from 100 l of whole blood using a DNeasy blood and tissue DNA kit as per the manufacturer's recommendation (Qiagen, Valencia, CA). Quantitative PCR targeting the merozoite surface antigen 1 gene (msa-1) was performed as previously described (4,15). A positive control containing 10 ng of T2Bo gDNA and a negative control consisting of naive tissue or blood DNA were included.…”

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control

“…(B.) microplus had negative impact on the reproductive performance of the tick and also increased the infection rate of B. bovis in larval progeny [41], while knockdown of TROSPA, serum amyloid A, and calreticulin reduced the infection level of B. bigemina in Rhipicephalus ticks [126]. Silencing a Kunitz-type serine protease inhibitor from D. variabilis increased the rickettsial infection in the midgut [146], whereas in R.…”

“…(B.) microplus, silencing a Kunitz-type serine protease inhibitor, Spi, tended to increase the infection rate of B. bovis in larval progeny [41], but silencing of another Kunitz-type protease inhibitor 5 (KTPI) did not have any effect on Babesia infection [126].…”

“…Injection has been accomplished using a 33-36-gauge needle attached to a Hamilton syringe particularly in large tick species, such as Amblyomma americanum [37], Dermacentor variabilis, and D. marginatus [38], while microinjection using a microcapillary drawn to a fine point needle and inserted to a micromanipulator has been commonly employed in smaller tick species, including Ixodes [39], and Haemaphysalis [26] ticks. Different injection sites include the lower right quadrant of the ventral surface of the exoskeleton [40], the groove between the basis capituli and the scutum [37], the ventral torso of the idiosoma, away from the anal opening [39], and the coxal membrane in the fourth coxae [26,41]. In some reports, dsRNA was injected through the spiracle [29,42,43] and anal pore; the latter inducing midgut-specific silencing of the target gene [44].…”