By employing a culture-dependent and -independent 16S rRNA and ITS gene high-throughput sequencing analyses, comprehensive information was obtained on the gut bacterial and fungal communities in the ghost moth larvae of three different geographic locations from high-altitude on Tibet plateau and from low-altitude laboratory. Twenty-six culturable bacterial species belonging to 21 genera and 14 fungal species belonging to 12 genera were identified from six populations by culture-dependent method. Carnobacterium maltaromaticum was the most abundant bacterial species from both the wild and laboratory-reared larvae. The most abundant OTUs in the wild ghost moth populations were Carnobacteriaceae, Enterobacteriaceae for bacteria, and Ascomycota and Basidiomycota for fungi. Larval microbial communities of the wild ghost moth from different geographic locations were not significantly different from each other but significant difference in larval microbial community was detected between the wild and laboratory-reared ghost moth. The larval gut of the wild ghost moth was dominated by the culturable Carnobacterium. However, that of the laboratory-reared ghost moth exhibited significantly abundant Wolbachia, Rhizobium, Serratia, Pseudomonas, and Flavobacterium. Furthermore, the larval gut of the wild ghost moth had a significantly higher abundance of Ophiocordyceps but lower abundance of Candida and Aspergillus than that of the laboratory-reared ghost moth.

Insects protect themselves through their immune systems. Entomopathogenic nematodes and their bacterial symbionts are widely used for the biocontrol of economically important pests. Ascarosides are pheromones that regulate nematode behaviors, such as aggregation, avoidance, mating, dispersal, and dauer recovery and formation. However, whether ascarosides influence the immune response of insects remains unexplored. In this study, we co-injected ascarosides and symbiotic Photorhabdus luminescens subsp. kayaii H06 bacteria derived from Heterorhabditis bacteriophora H06 into the last instar larvae of Galleria mellonella. We recorded larval mortality and analyzed the expressions of AMPs, ROS/RNS, and LPSs. Our results revealed a process in which ascarosides, acting as enhancers of the symbiotic bacteria, co-induced G. mellonella immunity by significantly increasing oxidative stress responses and secreting AMPs (gallerimycin, gloverin, and cecropin). This led to a reduction in color intensity and the symbiotic bacteria load, ultimately resulting in delayed host mortality compared to either ascarosides or symbiotic bacteria. These findings demonstrate the cross-kingdom regulation of insects and symbiotic bacteria by nematode pheromones. Furthermore, our results suggest that G. mellonella larvae may employ nematode pheromones secreted by IJs to modulate insect immunity during early infection, particularly in the presence of symbiotic bacteria, for enhancing resistance to invasive bacteria in the hemolymph.

Chinese cordyceps, a highly valued traditional medicine, is formed when the fungal parasite Ophiocordyceps sinensis infects an underground caterpillar (Thitarodes). This interaction progresses slowly, as the larvae have a long developmental period and the fungus requires several months to complete its growth. The medicinal fungus O. sinensis has a complex life cycle that requires successful transformation from blastospores to hyphae for the formation of Chinese cordyceps. Building on our previous identification of diverse microbial communities associated with Thitarodes xiaojinensis larvae, this study investigates the role of host microbiota in enhancing O. sinensis blastospore transformation under in vitro conditions. Cultured supernatant of Fusarium pseudonygamai associated with T. xiaojinensis larvae significantly increased transformation rates by 31.6% after 8 days. Transcriptomic profiling revealed early upregulation of genes involved in energy metabolism, stress response, detoxification, and hyphal morphogenesis (notably CYC1, hmp, gedE, and fahA), supporting the cellular reprogramming required for fungal development. Additionally, mannitol isolated from F. pseudonygamai culture acted as a key promoter of transformation. Further functional assays confirmed that modulation of mannitol biosynthesis genes (M1PDH and MDH) through chemical agonists and inhibitors directly influenced mannitol levels and transformation efficiency. Collectively, these results highlight the pivotal role of microbiota-derived metabolites, particularly mannitol, in regulating O. sinensis transformation, offering potential strategies to improve artificial cultivation of Chinese cordyceps.