Spatiotemporal expression patterns and RNA interference efficiency of key diapause genes in Lymantria dispar—Investigating the heritability of RNA interference effects for pest biocontrol

The spongy moth (Lymantria dispar Linnaeus), a major quarantine pest, relies on diapause as a key survival strategy. This study examined the temporal and spatial expression of four diapause-associated genes, LdGCLC, LdGLUD1_2, LdIDH1, and LdIDH2. Using fluorescence in situ hybridization and qPCR, their expression was analyzed across developmental stages and tissues. Additionally, RNA interference (RNAi) via microinjection and immersion was employed to systematically silence these genes. The results revealed that (1) all four genes were expressed in the brain; (2) dsRNA microinjection reduced target gene expression in larvae, with FISH showing a decrease in fluorescence intensity; (3) immersing eggs in dsRNA solution significantly lowered target gene expression, diapause hormone and ecdysone levels, along with a notable reduction in hatchability; (4) pre-immersion eggs in 4.4 M HCl at room temperature for 5 min before bacterial immersion significantly enhanced RNAi efficiency; and (5) maternal dsRNA microinjection decreased target gene expression, diapause hormone and ecdysone levels in offspring eggs, confirming transgenerational RNAi. These findings underscore RNAi's potential for heritable gene silencing in pest control.