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1. Exploiting somatic piRNAs in Bemisia tabac i enables novel gene silencing through RNA feeding

   https://doi.org/10.26508/lsa.202000731  

   Mondal, Mosharrof; Brown, Judith K; Flynt, Alex (August 2020, Life Science Alliance)

   null (Ed.)

   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. 

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2. Mechanisms, Applications, and Challenges of Insect RNA Interference

   https://doi.org/10.1146/annurev-ento-011019-025224  

   Zhu, Kun Yan; Palli, Subba Reddy (January 2020, Annual Review of Entomology)

   The RNA interference (RNAi) triggered by short/small interfering RNA (siRNA) was discovered in nematodes and found to function in most living organisms. RNAi has been widely used as a research tool to study gene functions and has shown great potential for the development of novel pest management strategies. RNAi is highly efficient and systemic in coleopterans but highly variable or inefficient in many other insects. Differences in double-stranded RNA (dsRNA) degradation, cellular uptake, inter- and intracellular transports, processing of dsRNA to siRNA, and RNA-induced silencing complex formation influence RNAi efficiency. The basic dsRNA delivery methods include microinjection, feeding, and soaking. To improve dsRNA delivery, various new technologies, including cationic liposome–assisted, nanoparticle-enabled, symbiont-mediated, and plant-mediated deliveries, have been developed. Major challenges to widespread use of RNAi in insect pest management include variable RNAi efficiency among insects, lack of reliable dsRNA delivery methods, off-target and nontarget effects, and potential development of resistance in insect populations. 

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3. A Tale of Two Lobsters—Transcriptomic Analysis Reveals a Potential Gap in the RNA Interference Pathway in the Tropical Rock Lobster Panulirus ornatus

   https://doi.org/10.3390/ijms231911752  

   Banks, Thomas M.; Wang, Tianfang; Fitzgibbon, Quinn P.; Smith, Gregory G.; Ventura, Tomer (October 2022, International Journal of Molecular Sciences)

   RNA interference (RNAi) has been widely utilised in many invertebrate models since its discovery, and in a majority of instances presents as a highly efficient and potent gene silencing mechanism. This is emphasized in crustaceans with almost all taxa having the capacity to trigger effective silencing, with a notable exception in the spiny lobsters where repeated attempts at dsRNA induced RNAi have demonstrated extremely ineffective gene knockdown. A comparison of the core RNAi machinery in transcriptomic data from spiny lobsters (Panulirus ornatus) and the closely related slipper lobsters (Thenus australiensis, where silencing is highly effective) revealed that both lobsters possess all proteins involved in the small interfering and microRNA pathways, and that there was little difference at both the sequence and domain architecture level. Comparing the expression of these genes however demonstrated that T. australiensis had significantly higher expression in the transcripts encoding proteins which directly interact with dsRNA when compared to P. ornatus, validated via qPCR. These results suggest that low expression of the core RNAi genes may be hindering the silencing response in P. ornatus, and suggest that it may be critical to enhance the expression of these genes to induce efficient silencing in spiny lobsters. 

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4. Target-specific requirements for RNA interference can arise through restricted RNA amplification despite the lack of specialized pathways

   https://doi.org/10.7554/eLife.97487.3  

   Knudsen-Palmer, Daphne R; Raman, Pravrutha; Ettefa, Farida; De_Ravin, Laura; Jose, Antony M (August 2024, eLife)

   Since double-stranded RNA (dsRNA) is effective for silencing a wide variety of genes, all genes are typically considered equivalent targets for such RNA interference (RNAi). Yet, loss of some regulators of RNAi in the nematodeCaenorhabditis eleganscan selectively impair the silencing of some genes. Here, we show that such selective requirements can be explained by an intersecting network of regulators acting on genes with differences in their RNA metabolism. In this network, the Maelstrom domain-containing protein RDE-10, the intrinsically disordered protein MUT-16, and the Argonaute protein NRDE-3 work together so that any two are required for silencing one somatic gene, but each is singly required for silencing another somatic gene, where only the requirement for NRDE-3 can be overcome by enhanced dsRNA processing. Quantitative models and their exploratory simulations led us to find that (1) changingcis-regulatory elements of the target gene can reduce the dependence on NRDE-3, (2) animals can recover from silencing in non-dividing cells, and (3) cleavage and tailing of mRNAs with UG dinucleotides, which makes them templates for amplifying small RNAs, are enriched within ‘pUG zones’ matching the dsRNA. Similar crosstalk between pathways and restricted amplification could result in apparently selective silencing by endogenous RNAs. 

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5. Implications of Endogenous Small Regulatory RNAs Survey in Mollusks on Gene Silencing Approaches

   https://doi.org/10.1007/s10126-025-10519-9  

   Von_Eiff, Cory; Schueng_Zancanela, Beatriz; Gima, Megan; Quito, Kevin; Kotikalapudi, Manitejus; Valdivia, Sergio; Santos-Ortega, Yulica; Flynt, Alex_Sutton (October 2025, Marine Biotechnology)

   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. 

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