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Abstract We highlight the recent 5 years of research that contributed to our understanding of the mechanisms of RNA interference (RNAi) in insects. Since its first discovery, RNAi has contributed enormously as a reverse genetic tool for functional genomic studies. RNAi is also being used in therapeutics, as well as agricultural crop and livestock production and protection. Yet, for the wider application of RNAi, improvement of its potency and delivery technologies is needed. A mechanistic understanding of every step of RNAi, from cellular uptake of RNAi trigger molecules to targeted mRNA degradation, is key for developing an efficient strategy to improve RNAi technology. Insects provide an excellent model for studying the mechanism of RNAi due to species‐specific variations in RNAi efficiency. This allows us to perform comparative studies in insect species with different RNAi sensitivity. Understanding the mechanisms of RNAi in different insects can lead to the development of better strategies to improve RNAi and its application to manage agriculturally and medically important insects. 

Abstract RNA interference (RNAi) is a promising technology for the development of next‐generation insect pest control products. Though RNAi is efficient and systemic in coleopteran insects, it is inefficient and variable in lepidopteron insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW),Spodoptera frugiperdaby conjugating double‐stranded RNA (dsRNA) with biodegradable chitosan (Chi). dsRNA conjugated with chitosan was protected from degradation by endonucleases present in Sf9 cell‐conditioned medium, hemolymph, and midgut lumen contents collected from the FAW larvae. Chi–dsRNA complexes showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing chitosan formulated dsRNA in Sf9 cells and the tissues induced a significant knockdown of endogenous genes. Chi–dsIAP fed to FAW larvae induced knockdown ofiapgene, growth retardation, and mortality. Processing of dsRNA into small interfering RNA was detected with chitosan‐conjugated³²P‐UTP‐labeled ds green fluorescent protein in Sf9 cells and FAW larval tissues. Overall, these data suggest that dsRNA conjugated with chitosan helps dsRNA escape from the endosomes and improves RNAi efficiency in FAW cells and tissues. 

Abstract RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW),Spodoptera frugiperdacells by formulating dsRNA with Cellfectin II (CFII) transfection reagent. The CFII formulated dsRNA was protected from degradation by endonucleases present in Sf9 cells conditioned medium, hemolymph and midgut lumen contents collected from the FAW larvae. Lipid formulated dsRNA also showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing Sf9 cells and tissues to CFII formulated dsRNA caused a significant knockdown of endogenous genes. CFII formulated dsIAP fed to FAW larvae induced knockdown ofiapgene, growth retardation and mortality. Processing of dsRNA into siRNA was detected in Sf9 cells andSpodoptera frugiperdalarvae treated with CFII conjugated³²P‐UTP labeled dsGFP. Overall, the present study concluded that delivering dsRNA formulated with CFII transfection reagent helps dsRNA escapes from the endosomal accumulation and improved RNAi efficiency in the FAW cells and tissues. 

Abstract Apoptosis has been widely studied from mammals to insects. Inhibitor of apoptosis (IAP) protein is a negative regulator of apoptosis. Recent studies suggest thatiapgenes could be excellent targets for RNA interference (RNAi)‐mediated control of insect pests. However, not much is known aboutiapgenes in one of the well‐known insect model species,Tribolium castaneum. The orthologues of fiveiapgenes were identified inT. castaneumby searching its genome at NCBI (https://www.ncbi.nlm.nih.gov/) and UniProt (https://www.uniprot.org/) databases usingDrosophila melanogasterandAedes aegyptiIAP protein sequences as queries. RNAi assays were performed inT. castaneumcell line (TcA) and larvae. The knockdown ofiap1gene induced a distinct apoptotic phenotype in TcA cells and induced 91% mortality inT. castaneumlarvae. Whereas, knockdown ofiap5resulted in a decrease in cell proliferation in TcA cells and developmental defects inT. castaneumlarvae which led to 100% mortality. Knockdown of the other threeiapgenes identified did not cause a significant effect on cells or insects. These data increase our understanding ofiapgenes in insects and provide opportunities for developingiap1andiap5as targets for RNAi‐based insect pest control. 

More than 100 RNA chemical modifications to cellular RNA have been identified.N⁶-methyladenosine (m⁶A) is the most prevalent modification of mRNA. RNA modifications have recently attracted significant attention due to their critical role in regulating mRNA processing and metabolism. tRNA and rRNA rank among the most heavily modified RNAs, and their modifications are essential for maintaining their structure and function. With our advanced understanding of RNA modifications, increasing evidence suggests RNA modifications are important in regulating various aspects of insect life. In this review, we will summarize recent studies investigating the impact of RNA modifications in insects, particularly highlighting the role of m⁶A in insect development, reproduction, and adaptation to the environment.