Diversity of RNAi efficiency-related nuclease in lepidopteran insects

RNA interference (RNAi) is a conserved gene-silencing mechanism; however, its efficiency varies among insect taxa and is particularly low in lepidopterans. A Lepidoptera-specific nuclease, RNAi efficiency-related nuclease (REase), has recently been identified as an RNAi suppressor that degrades double-stranded RNA (dsRNA) upon exposure. To investigate the diversity and potential functions of the REase genes, a phylogenetic analysis was conducted across lepidopteran species and a gene expression analysis of Spodoptera exigua was performed. Three lepidopteran-specific REase groups (REase1-3) and one conserved group related to Asteroids were identified, with all previously reported REase genes classified as REase1. REase3 likely originated from an ancestral duplication of an Asteroid, from which REase1 and REase2 subsequently diverged. REase1 and REase2 were broadly distributed across Lepidoptera, although absent in some lineages. Notably, REase1 exhibited extensive copy number variation among species, whereas REase2 and REase3 were mostly in single-copy form. Expression analysis showed that both REase1 and REase2 were strongly upregulated following dsRNA injection, along with core RNAi components such as Dicer2 and Argonaute2. This dsRNA responsiveness, combined with the accelerated evolution of the REases, suggests their involvement in an evolutionary arms race involving fast-evolving immune challenges, such as viruses. Furthermore, the rapid evolution and frequent gene loss of REase paralogs, which are hallmarks of functionally redundant genes, suggest that REase2 acts as an RNAi suppressor, similar to REase1. These findings highlight the evolutionary diversity and possible functional redundancy of the REases and underscore their potential importance in modulating RNAi efficiency in Lepidoptera.