Persistent parental RNAi in the beetle Tribolium castaneum involves maternal transmission of long double-stranded RNA

Abstract

Parental RNA interference (pRNAi) is a powerful and widely used method for gene-specific knockdown. Yet in insects its efficacy varies between species, and how the systemic RNAi response is transmitted from mother to offspring remains elusive. Using the flour beetle Tribolium castaneum, we report an RT-qPCR strategy to unmask the presence of double-stranded RNA (dsRNA) distinct from endogenous mRNA. We find that the injected dsRNA is directly transmitted into the egg and persists throughout embryogenesis. Despite this depletion of dsRNA from the mother, we show that strong pRNAi can persist for months before waning at strain-specific rates. In seeking the receptor proteins for cellular uptake of long dsRNA into the egg, we lastly present a phylogenomics profiling approach to ascertain macroevolutionary distributions of candidate proteins. We demonstrate a visualization strategy based on taxonomically hierarchical assessment of orthology clustering data to rapidly assess gene age and copy number changes, refined by several lines of sequence-based evidence. We use this approach to document repeated losses of SID-1-like channel proteins in the arthropods, including wholesale loss in the Heteroptera (true bugs), which are nonetheless highly sensitive to pRNAi. Overall, we elucidate practical considerations for insect pRNAi against a backdrop of outstanding questions on the molecular mechanism of dsRNA transmission to achieve long-term, systemic knockdown.