Transcriptomic and metabolomic insights into the long-term survival strategies of Ophiocordyceps sinensis following host immune perturbation

Abstract Background Ophiocordyceps sinensis is a premier medicinal fungus with a unique slow-killing parasitic strategy, yet the molecular basis of its long-term persistence in host hemocoel remains elusive. This fungus coexists with Thitarodes xiaojinensis larvae for over six months, ultimately leading to host death at the last instar before pupation. To decode the fungal survival strategies masked by host resistance, this study integrated omics approach combining sublethal imidacloprid-induced transcriptomics with longitudinal metabolomic profiling. We investigated the transcriptomic response of O. sinensis within the larval hemocoel under exposure to a sublethal dose of imidacloprid, a stressor utilized here to suppress host immunity via neuro-immune crosstalk. This approach allowed us to infer the core molecular responses of O. sinensis across three critical infection stages (30, 90, and 150 days post-infection, dpi). Results Differential gene expression and functional enrichment analyses revealed that O. sinensis significantly responds to host immune disturbance induced by sublethal dose of imidacloprid. During the early infection stage (30 dpi), Imidacloprid exposure suppressed fungal cell proliferation genes while activating secondary metabolite pathways and immune defense genes. This suggests that during a conventional infection, O. sinensis secondary metabolism was suppressed under the host immunity to maintain a balanced blastospore proliferation. Beyond similar response in fungal secondary metabolites and host immunity, imidacloprid also suppressed protein biosynthesis during the mid-infection stage (90 dpi), indicating that the fungus undergoes metabolism suppression while continuing to maintain balance between blastospore proliferation and host immunogenicity for the conventional infection. During the late stage (150 dpi), genes associated with hydrolytic activity, transport and cell wall degradation were upregulated while fatty acid synthesis genes were downregulated by imidacloprid, suggested that the conventional infection of O. sinensis at this stage exhibits metabolic polarization to shift blastospore to hypha, tightly regulating hydrolysis to balance nutrient acquisition with host viability and increasing fatty acid biosynthesis. Metabolomic analysis showed that the accumulation of chitin precursors in the late stage, including D-glucosamine-6-phosphate, marks the critical blastospore-to-hyphae transition. Furthermore, the accumulation of compounds like chlorogenic acid, L-glutathione oxidized, and hesperidin indicates the dual responses of secreting antimicrobials and deploying antioxidants to protect and sanitize the nutrient base. Conclusions This study provides insights into the dynamic, stage-specific gene expression profiles of O. sinensis that enable its long-term survival in the host hemocoel. Through coordinated regulation of proliferation, metabolism, secondary metabolite production, and immune evasion, the fungus sustains persistent infection and successfully completes its life cycle. These findings advance our understanding of the strategies employed by slow-killing fungal parasites.