Endogenous RNA virus-Derived elements exhibit functional divergence in modulating development and virus susceptibility in Bombyx mori.

Non-retroviral endogenous viral elements (nrEVEs) are increasingly recognized as potential regulators of host biology, but their functional roles in Lepidoptera remain largely unexplored. Here, we identified seven nrEVEs derived from four RNA virus families in Bombyx mori. All seven nrEVEs were transcriptionally active and exhibited distinct developmental and tissue-specific expression patterns. Their small-RNA profiling revealed that these elements could generate small interfering RNA (siRNA)-like RNAs, primary PIWI-interacting RNA (piRNA)-like RNAs, or ping-pong-associated piRNAs. Functional analyses of the Totiviridae-derived BmToEVE and the Chuviridae-derived BmChuEVE3 revealed markedly divergent physiological roles. Depletion of either nrEVE delayed larval development and reduced body weight, while differentially affecting reproductive and cocoon traits. Yeast two-hybrid screening and transcriptomic analyses further implicated both nrEVEs with cuticle-associated and reproductive processes. Importantly, BmChuEVE3 knockdown broadly suppressed metabolic and immune responses, accompanied by increased B.mori nucleopolyhedrovirus (BmNPV) and cypovirus (BmCPV). By contrast, BmToEVE silencing markedly reduced BmNPV accumulation and enhanced host defense-related transcriptional responses. Its effect on BmCPV was comparatively modest and emerged only at the later infection stage. Overall, our findings reveal functional diversification of silkworm nrEVEs and highlight the contribution of virus-derived sequences to developmental regulation and antiviral physiology in a lepidopteran host.