Chemical clues to infection: A pilot study on the differential secondary metabolite production during the life cycle of selected Cordyceps species

<i>Cordyceps</i> species are widespread entomopathogens and promising biocontrol agents that produce diverse secondary metabolites, yet the roles of these molecules during the infection process remain unclear. To interpret how fungal chemistry contributes to host colonization, we compared the metabolomes and virulence traits of two strains of phylogenetically distinct <i>Cordyceps</i> species (<i>C. javanica</i> and <i>C. blackwelliae</i>) and assessed their effects on beet armyworms (<i>fungiSpodoptera exigua</i>). Virulence assays revealed species-dependent pathogenicity, with <i>C. javanica</i> showing the highest virulence. Combining untargeted metabolomics, feature-based molecular networking (FBMN), 3D electron-diffraction crystallography and comprehensive 1D/2D NMR, we gained insights into their metabolomic traits. For instance, <i>C. javanica</i> displayed notable beauveriolide diversity, including three previously undescribed derivatives (<b>1</b>-<b>3</b>), while <i>C. blackwelliae</i> produced mainly diketopiperazines <i>in vitro.</i> The FBMN results revealed putative beauveriolide analogs in the <i>C. blackwelliae</i> extracts, unlike the cadaver analysis, revealing beauvericins in infected corpses. Remarkably, the crude extracts obtained from authentic insect cadavers contained beauveriolides and beauvericins, providing <i>in vivo</i> chemical evidences of their production during infection for the first time. Moreover, bioassays with purified compounds showed that insecticidal activity cannot be attributed across all beauveriolides but depends on amino-acid composition, implying multifunctional roles beyond direct toxicity. Altogether, these results reveal context-dependent metabolic reprogramming and species-specific chemical strategies in entomopathogenic fungi, with implications for microbial ecology, host specificity, and the rational development of fungal biocontrol agents. The results of this study also give rise to the need for more intensified study on the chemical composition of the insect cadavers that are colonized by other entomopathogens.