Figure 8.

Silencing of ap-1 in nymphs by RNAi reduces iafgp expression and survival of ticks at cold temperatures. (A) QRT-PCR results showing ap-1 transcripts in uninfected or A. phagocytophilum-infected ticks incubated at 10 ± 1 °C for 8 hrs. Gel shift assays with biotinylated iafgp TATA (B) probe (DS704943, 18338–18387 bp) or probe containing AP-1 binding (C) site (DS704943, 18144–18193 bp) and nuclear proteins (2.5 µg) prepared from uninfected (UI) or A. phagocytophilum-infected (I) nymphs incubated at 10 ± 1 °C for 8 hrs is shown. Dotted arrow indicates free probe and solid arrow indicates shift. NE indicates nuclear extracts and + or − indicates presence or absence, respectively. QRT-PCR analysis showing reduced ap-1 (D) or iafgp (E) mRNA levels in ap-1-dsRNA–injected uninfected nymphal ticks compared with the mock-treated controls. (F) Percentage survival of mock- or ap-1-dsRNA–injected nymphal ticks at the LT₅₀ time point is shown. (G) Mobility (in cm) by mock- or ap-1-dsRNA–injected ticks at LT₅₀ time point (−20 °C, 25 min) is shown. In panel A,D,E and G each circle represents one individual tick. Whereas, in panels F each circle represents one experiment performed with 10 ticks/group. Statistical analysis was performed using Student’s t test and P value less than 0.05 was considered significant in panels A and D–G.