Fig. 5

Functional characterization of tick selenoprotein gene knockdowns in R. parkeri-infected (Rp⁺) A. maculatum. A dsRNA-based silencing assay was performed for a SELENOM, b SELENOO, and c SELENOS in Rp⁺ ticks, and the compensatory expression levels of tick antioxidants and selenoproteins were estimated. The dsRNA specific for each selenogene (SELENOM, SELENOO, and SELENOS) was synthesized to include the addition of a T7 RNA polymerase binding site as the flanking sequence in the individual selenogene PCR amplicons from the dsRNA (Additional file 5: Table S1) and the in vitro RNA synthesis (which utilized the HiScribe™ T7 High Yield RNA synthesis kit, New England Biolabs). The dsRNA synthesized for each selenogene, along with irrelevant dsLacZ, were microinjected into 25–30 Rp⁺ ticks or 25–30 R. parkeri-free ticks (Additional file 3: Figure S3). The microinjected ticks were allowed to replete on sheep, and 5–10 ticks were removed from them to study the impact on gene silencing and the impact on Rickettsia parkeri and other symbionts (Figs. 6 and 7) on day 5 post-infestation. In each selenogene-silenced tick tissue (a SELENOM, b SELENOO, and c SELENOS), the transcript levels of a panel of selenogenes (SELENOM, SELENOO, and SELENOS, along with eEFSec, TrxR, SELENOK, SELENON, SELENOT) and redox genes (Cu/Zn-SOD, Mn-SOD, Duox, Catalase, GSHR, Salp25D) were measured. The transcript level for each gene in the control tissues was normalized to 1 for reference and is represented here as a dashed line. Tick GAPDH was used as a reference gene for normalizing the qRT-PCR results. d Oxidative stress in the selenogene-silenced tick midguts and control (dsLacZ) midguts was estimated using a malondialdehyde assay. KD, knockdown