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 response is transmitted from mother to offspring remains elusive. Using the beetle Tribolium castaneum, an RT‐qPCR strategy to distinguish the presence of double‐stranded RNA (dsRNA) from endogenous mRNA is reported. It is found that injected dsRNA is directly transmitted into the egg and persists throughout embryogenesis. Despite this depletion of dsRNA from the mother, it is shown 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, a phylogenomics profiling approach of candidate proteins is also presented. A visualization strategy based on taxonomically hierarchical assessment of orthology clustering data to rapidly assess gene age and copy number changes, refined by sequence‐based evidence, is demonstrated. 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, are thereby documented. Overall, practical considerations for insect pRNAi against a backdrop of outstanding questions on the molecular mechanism of dsRNA transmission for long‐term, systemic knockdown are elucidated.

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.

dsRNA Uptake In article 2100064 by Kristen A. Panfilio and co‐workers, the cuticle exoskeleton of flour beetle larvae reveals normal anatomy (above: head‐to‐tail in blue‐to‐red) and long‐term parental RNAi knockdown (below), here showing a mirror‐image duplication of the abdomen (red termini to yellow center). Strong knockdown can persist for months despite transmission of full‐length double‐stranded RNA (dsRNA) from the mother into the egg, depleting maternal dsRNA levels.