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Abstract Mollusks are an abundant group of animals, with many ecologically and economically important members that are phylogenetically distinct from nearly all genetic model organisms. This study provides a clade-wide evaluation of small RNA biogenesis pathways, with emphasis on the eastern oyster, Crassostrea virginica. A more thorough characterization of these molecules supports rationale design of RNA interference (RNAi) approaches for manipulation of mollusk genetics. Like other animal groups, mollusks have conserved microRNAs (miRNAs), with some shared with ecdysozoans and deuterostomes; however, there was no evidence of a dedicated endogenous small-interfering RNA (siRNA) pathway. These findings suggest that alternatives to long double-stranded RNA (dsRNA)-mediated knockdown, specifically short-hairpin RNAs or small duplex RNAs, are likely more appropriate for gene silencing in mollusks. The study also finds abundant Piwi-interacting RNAs (piRNAs) in both soma and gonads with some mollusk-specific aspects. Many invertebrates exhibit somatic piRNAs; however, mollusk piRNAs appear to be restricted to a subset of cells, suggesting that the potential of piRNA-based RNAi is also limited. Further, individual animals also express a unique collection of piRNAs that seem to be only partially determined through inheritance from parents. Together, this work defines the RNAi mechanisms in mollusks and provides insights into the phenotypic diversity seen in this group. 

Abstract Over 20 years ago double‐stranded RNA (dsRNA) was described as the trigger of RNAi interference (RNAi)‐based gene silencing. This paradigm has held since, especially for insect biopesticide technologies where dsRNAs, similar to those described in 1998, are used to inhibit gene expression. In the intervening years, investigation of RNAi pathways has revealed the small RNA effectors of RNAi are diverse and rapidly evolving. The rich biology of insect small RNAs suggests potential to use multiple RNAi modes for manipulating gene expression. By exploiting different RNAi pathways, the menu of options for pest control can be expanded and could lead to better tailored solutions. Fortunately, basic delivery strategies used for dsRNA such as direct application or transgenic expression will translate well between RNAs transiting different RNAi pathways. Importantly, further engineering of RNAi‐based biopesticides may provide an opportunity to address dsRNA insensitivity seen in some pests. Characterization of RNAi pathways unique to target species will be indispensable to this end and may require thinking beyond long dsRNA. © 2020 Society of Chemical Industry 

RNAi is an evolutionarily fluid mechanism with dramatically different activities across animal phyla. One major group where there has been little investigation is annelid worms. Here, the small RNAs of the polychaete developmental model Capitella teleta are profiled across development. As is seen with nearly all animals, nearly 200 microRNAs were found with 58 high-confidence novel species. Greater miRNA diversity was associated with later stages consistent with differentiation of tissues. Outside miRNA, a distinct composition of other small RNA pathways was found. Unlike many invertebrates, an endogenous siRNA pathway was not observed, indicating pathway loss relative to basal planarians. No processively generated siRNA-class RNAs could be found arising from dsRNA precursors. This has a significant impact on RNAi technology development for this group of animals. Unlike the apparent absence of siRNAs, a significant population of piRNAs was observed. For many piRNAs, phasing and ping-pong biogenesis pathways were identified. Interestingly, piRNAs were found to be highly expressed during early development, suggesting a potential role in regulation in metamorphosis. Critically, the configuration of RNAi factors in C. teleta is found in other annelids and mollusks, suggesting that similar biology is likely to be present in the wider clade. This study is the first in providing comprehensive analysis of small RNAs in annelids. 

RNAi promises to reshape pest control by being nontoxic, biodegradable, and species specific. However, due to the plastic nature of RNAi, there is a significant variability in responses. In this study, we investigate small RNA pathways and processing of ingested RNAi trigger molecules in a hemipteran plant pest, the whitefly Bemisia tabaci . Unlike Drosophila , where the paradigm for insect RNAi technology was established, whitefly has abundant somatic piwi-associated RNAs (piRNAs). Long regarded as germline restricted, piRNAs are common in the soma of many invertebrates. We sought to exploit this for a novel gene silencing approach. The main principle of piRNA biogenesis is the recruitment of target RNA fragments into the pathway. As such, we designed synthetic RNAs to possess complementarity to the loci we annotated. Following feeding of these exogenous piRNA triggers knockdown as effective as conventional siRNA-only approaches was observed. These results demonstrate a new approach for RNAi technology that could be applicable to dsRNA-recalcitrant pest species and could be fundamental to realizing insecticidal RNAi against pests.