FIG. 4.

Knockdown of longicin by RNAi facilitates transmission of Babesia parasite through the vector ticks. Unfed adult female ticks were injected with longicin dsRNA (ds longicin) into the hemocoel through the fourth coxae by use of fine-point glass needles. Control ticks were injected with PBS alone. Ticks were collected from the ear of a Babesia gibsoni-infected dog on day 6 after attachment. (A) Microinjection-treated ticks at day 3 on the ear of a dog. Injection of longicin dsRNA inhibited endogenous longicin expression (green) in the midgut. Figures are representative of three independent RNAi experiments. (B) RT-PCR analysis (day 3). Note the reduced expression of longicin mRNA in longicin dsRNA-injected ticks. (C) Immunoblot analysis of endogenous longicin expression (day 3). Results showed the absence of longicin expression, indicating that the effective knockdown of longicin mRNA was achieved by dsRNA injection. (D) Images of the tick midgut. B. gibsoni parasites were visualized by use of mouse anti-B. gibsoni antibody (green). The two right panels highlight the lumen and epithelium from the merge section of the midgut. (E) Ticks on day 6. The body weights of ticks with silencing longicin were significantly lower than those of control ticks at day 6. Suppression of endogenous longicin expression in longicin dsRNA-treated ticks was seen up to engorgement. Preoviposition, oviposition, and egg periods of ticks treated with dsRNA were similar to those of PBS control ticks. Smaller engorged ticks silenced with longicin subsequently transmitted larger numbers of Babesia sp. parasites than did PBS control ticks. One scale, 1 mm. (F) Prevalence and intensity of B. gibsoni infection. The numbers of invaded parasites were evaluated by P18 genes on the B. gibsoni genome DNA by use of a real-time quantitative PCR. (G) Representative image of the migrating Babesia parasite in the tick ovary. (H) Intensity of B. gibsoni infection. Data represent the means ± SEM for three experiments with five ticks. Quantitative results demonstrated that repression of longicin enhanced the B. gibsoni transmission in the vector tick. The error bars indicate SEM for three independent experiments with three ticks.