T7 ribomax rnai system

The dsRNA was synthesized using the T7 RiboMAX™ RNAi System (Promega, Madison, WI, USA) according to the manufacturer's recommendations. Oligonucleotide primers containing T7 promoter sequences (in italics at 5′-end) were listed in Supplementary Table 2. A dsRNA targeting luciferase used as a negative control was subjected to the same PCR amplification protocol using luciferase-specific primers (Yu et al., 2013 (link)). These dsRNA were maintained at −80°C until use. Unfed adult ticks (N = 50 females per group, two independent groups) were microinjected with 0.5 μL HSP70 dsRNA (about 1 μg RNA) at the base of the fourth right leg of the ventral surface of ticks. Control ticks were microinjected with unrelated luciferase dsRNA. After microinjection, ticks were maintained in an incubator at 25°C and 95% humidity for 24 h and then allowed to feed on rabbits' ears. Four female ticks per group were collected at 5 days of blood feeding for RNA and protein extraction to characterize gene knockdown by real-time quantitative PCR and Western blot with respect to luciferase control. Remaining ticks were allowed to feed until engorgement, which tick mortality, engorgement rate and engorgement weight was determined in individual engorged female ticks.

Double-stranded RNA (dsRNA) was synthesized using the T7 RiboMAX™ RNAi System (P1700, Promega, Madison, WI, USA) according to manufacturer’s recommendations. Primers containing T7 promoter sequences (Additional file 1: Table S1) were designed to synthesize the dsRNA. The dsRNA to RhEcR, RhUSP, and Luciferase mRNA had about 500 base pairs (bp) each. Because the R. haemaphysaloides genome sequence is not available, the siRNA specificity and potential off-targets were estimated based on similar regions of the Drosophila melanogaster ortholog gene (accession number XP_021710216) and genome sequences using the dsCheck program [38 (link)]. Also, using BLAST, analyses were conducted using publicly available genome sequences of Ixodes persulcatus, Haemaphysalis longicornis, Dermacentor silvarum, Hyalomma asiaticum, R. sanguineus, R. microplus, and I. scapularis to confirm that the dsRNA was specific to the target genes and to reduce the possibility of off-target effects. A dsRNA targeting luciferase was used as a negative control [39 (link)]. The quality of the dsRNA was determined by electrophoresis on 1.0% agarose gel. The dsRNA was stored at −80 °C until use.