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

Abstract

Background: The 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.

Results: In 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.

Conclusion: The 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.

Figure 1

Parasitemia of B. bovis during acute infection and timeline indicating the time-point for double stranded RNA injection, collection of ticks to assess gene silencing, and feeding period. The B. bovis parasitemia was determined by quantitative real-time PCR and presented as log₁₀ parasites per ml of peripheral blood.

Figure 2

Transcript level of the imunophilin (Imnp) (A), serine protease inhibitor (Spi) (B) and lipocalin (Lpc) (C) genes in ovaries and guts of six partially engorged R. microplus females injected with dsRNA (grey bar) or buffer (black bar) and fed on a calf acutely infected with B. bovis. Imnp was silenced 92.9% (± 3.1%) and 93.6% (± 2.8%) in ovaries and guts, respectively. Spi was silenced 93.4% (± 7.0%) and 85.3% (± 26.4%) in ovaries and guts, respectively. Lpc was silenced 81.5% (± 9.6%) and 86.8% (± 12.5%) in ovaries and guts, respectively. The data represent mean values of relative expression of the genes of interest. Asterisk (*) indicates difference between control and dsRNA-injected groups as determined by t-test (P < 0.05).

Figure 3

Cumulative percentage of engorgement of R. microplus females injected with dsRNA to silence the immunophilin (Imnp) (black lozenge), serine protease inhibitor (Spi) (black square) or lipocalin (Lpc) (black triangle) genes and control females (white circle). The graphic shows the effect of gene silencing on engorgement and survival of female ticks throughout the feeding period. Asterisk (*) indicates difference between the control and the Spi dsRNA-injected groups as determined by Chi-squared test (P < 0.01).

Figure 4

Infection rate of B. bovis in larval progeny from gene silenced R. microplus females. Imnp - a putative immunophilin gene; Spi - a putative Kunitz-type serine protease inhibitor; Lpc - a putative lipocalin gene. The percentages of larval progeny infected with B. bovis were compared by Chi-squared test and P values for each experimental group are shown.

Figure 5

Transcript level of the imunophilin (Imnp) (A), serine protease inhibitor (Spi) (B) and lipocalin (Lpc) (C) genes in ovaries and guts of six partially engorged R. microplus females fed on either an uninfected calf (black bar) or a B. bovis-infected calf (grey bar). The data represent mean values of relative expression of the genes of interest. Asterisk (*) indicates difference between dsRNA-injected and control groups as determined by t-test (P < 0.05).