Functional dissection of conserved RNAi pathways in antiviral immunity of several hemipteran insects

The small silencing RNA pathways are crucial post-transcriptional gene silencing mechanisms involved in antiviral defense, gene regulation, and transposon suppression. In insects, the three main pathways are the microRNA (miRNA), Piwi-interacting RNA (piRNA), and small interfering RNA (siRNA) pathways. While these pathways are well-studied in model insects like fruit flies and mosquitoes (Order: Diptera), their functions in non-model insects remain largely unexplored. In this study, we conducted a comprehensive functional screening of core RNAi-related genes in a hemipteran insect, the small brown planthopper (Laodelphax striatellus). Our results demonstrated that genes in the miRNA pathway (AGO1 and DCR1) and the piRNA pathway (Piwi and AGO3) are essential for the survival, development, and fecundity of L. striatellus, but they are not actively involved in the antiviral response. Unexpectedly, knocking down the key siRNA pathway gene DCR2 did not produce a detectable change in viral accumulation or the abundance of virus-derived small interfering RNAs. This finding suggests that substantial depletion of DCR2 does not detectably impair the measured antiviral siRNA responses in L. striatellus under our experimental conditions. A similar lack of detectable antiviral phenotypes following DCR2 knockdown was observed in three other representative hemipteran insects from different families/genera (Sogatella furcifera, Nilaparvata lugens, and Bemisia tabaci). Additionally, CRISPR/Cas9-mediated knockout of DCR2 resulted in embryonic lethality in homozygous mutant strains of L. striatellus, while heterozygous mutants showed significantly prolonged developmental duration. Functional RNAi-of-RNAi assays further demonstrated that DCR2 pre-knockdown did not detectably reduce silencing of a subsequently introduced dsRNA reporter in L. striatellus, and DCR2-heterozygous mutants with reduced DCR2 expression showed no change in viral accumulation. These results reveal an essential developmental role for DCR2 and indicate that partial DCR2 depletion does not measurably compromise antiviral immunity under the tested conditions. Collectively, our findings broaden our understanding of the organization and biological functions of small silencing RNA pathways in hemipteran insects.