Variability in mountain pine beetle (Coleoptera: Curculionidae) gene expression in response to increasing concentrations of species-specific double-stranded RNA.

Sensitivity to double-stranded RNA (dsRNA)-induced gene silencing has been observed in multiple coleopterans. Initial work with mountain pine beetle, Dendroctonus ponderosae Hopkins, determined that specific ubiquitously expressed essential genes can cause mortality when silenced by carefully designed dsRNAs. However, current dsRNA production methods are not economically feasible for production at scale. Therefore, identifying the minimum efficacious dose is a critical step to deployment. To investigate the relationship between dsRNA dose and subsequent gene expression, adult mountain pine beetles were fed either a target or control dsRNA across a gradient of concentrations, including 2.5, 1.0, 0.25, and 0.025 µg/µl, and relative gene expression of the target gene was computed between dsRNA treatments of the same concentration. Differences in relative gene expression were detected along the dsRNA concentration gradient. Interestingly, higher doses of dsRNA caused significant overexpression of the target gene, heat shock protein 70 kDa, while the more miniscule doses showed no statistically significant upregulation. Overexpression of target mRNA levels in the presence of exogenous dsRNA has been demonstrated in multiple other coleopterans, but the mechanism is not yet clear. The interplay between dsRNA dose, target sequence, and other factors will likely vary by target organism, and as such, rigorous optimization assays will be necessary to understand the unique factors necessary for consistent gene silencing.