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 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.

The smORF-containing genemille-pattesis required for moulting andTrypanosoma cruzimetacyclogenesis in the Chagas disease vectorRhodnius prolixus

“…At least two technical limitations must be reported for the analysis and interpretation of Rp- mlpt RNAi results. First, the injection of a dsRNA in the haemolymph of insects leads to a systemic knock-down of gene expression [41,42], thus, it is not clear where Rp- mlpt encoded peptides are produced and act in R. prolixus wild-type nymphs. Second, the small size of smORF-encoded peptides precludes the development of antibodies and their applications in western-blots and immunostainings as well their detection via common proteomic techniques.…”

Disruption of transmission of plant pathogens in the insect order Hemiptera using recent advances in RNA interference biotechnology

“…pRNAi is an RNAi response where the phenotype of the progeny of the treated organism is observed after gene knockdown. In T. castaneum , a strong pRNAi was observed to persist for months before waning at strain‐specific rates (Figures 1 and 2) (Horn et al, 2021).…”

RNAi-based pest control: Production, application and the fate of dsRNA

“…dsRNA cellular uptake is an indispensable step to generate RNAi responses and two pathways have been identified to play roles in internalizing dsRNA from gut lumen: 1) Systemic RNA Interference Deficient protein 1 (SID1)-mediated uptake pathway and 2) clathrin-dependent endocytic pathway. Regarding the SID1-mediated dsRNA uptake, SID1 orthologues (also known as Sil) have been identified in many insects except Diptera ( Horn et al, 2022 ). A. mellifera administrated with dsRNA showed a significant increase in AmSid1 expression level ( Aronstein et al, 2006 ) and knockdown of Sil led to a decreased RNAi efficiency in insects such as L. decemlineata , N. lugens and D. virgifera virgifera , suggesting the functional role of SID1 in dsRNA internalization ( Xu et al, 2013 ; Miyata et al, 2014 ; Cappelle et al, 2016 ).…”