The research team, working with strains held in the BIOTEC culture collection in Thailand, isolated C. blackwelliae from a lepidopteran pupa collected in Khao Yai National Park and two C. javanica strains from lepidopteran larvae found in Nam Nao National Park and at Wang Takrai Waterfalls. Phylogenetic analysis of ITS and tef gene sequences confirmed the identity of the C. blackwelliae strain, placing it squarely with the type species described in earlier taxonomic work. These species belong to a genus that has undergone extensive reclassification in recent years, with molecular data splitting what was once a broad catch-all group into some 23 accepted genera within the family Cordycipitaceae.

To measure how dangerous each strain was to insects, the researchers ran virulence assays against the beet armyworm, Spodoptera exigua, a well-established pest model. Spores were harvested from cultures, adjusted to a concentration of 10^8 spores per milliliter, and injected into larvae, with 30 insects per strain divided across three independent replicates. The team tracked two distinct measures of mortality over seven days: total mortality, counting every dead insect, and mycelium-associated mortality, counting only those corpses that showed visible fungal outgrowth. This distinction matters because entomopathogens must balance rapid killing against the need to keep the host alive long enough to accumulate nutrients for fungal development and sporulation.

The results revealed clearly different infection strategies. One C. javanica strain, BCC 82944, was the most virulent, achieving 100 percent total mortality by day three, while the second strain, BCC 79245, averaged 80 percent total mortality. Both C. javanica strains produced visible mycelial outgrowth on only about 20 to 40 percent of cadavers. C. blackwelliae showed intermediate virulence, killing roughly 90 percent of insects by day three, but it never produced visible external mycelia even after a full week. The authors interpret heavy mycelial outgrowth as a sign of moderate toxin production combined with tissue-degrading enzymatic activity, whereas its absence points to rapid, systemic killing driven by metabolites acting at the cellular level.

To see what chemistry lay behind these differences, the team cultured both species in yeast malt liquid medium until glucose was depleted, then analyzed crude extracts by ultrahigh-performance liquid chromatography coupled with diode array detection and ion mobility tandem mass spectrometry. Computational prediction of compound classes from the mass spectra, using the CANOPUS tool, showed that the two species occupy distinct chemical territory. Alkaloid-like and fatty-acid-like features dominated the C. blackwelliae extracts, while amino acids and peptides, including the cyclodepsipeptides known as beauveriolides, were the major products of C. javanica. Beauveriolides I, Jb and L, confirmed against authentic standards, were entirely absent from C. blackwelliae.

Feature-based molecular networking, however, told a subtler story. Although C. blackwelliae did not make the known beauveriolides, it produced distinct analogs that clustered within the same molecular family, suggesting that beauveriolide biosynthesis is conserved across both species but with different amino acid building blocks. Manual inspection supported by the ModiFinder software pointed to tyrosine-containing analogs in C. blackwelliae, with two features whose neutral masses of 579.3588 and 607.3499 daltons likely correspond to alanine being swapped for tyrosine in beauveriolides I and L. C. blackwelliae also produced beauvericins, compounds not detected in C. javanica, hinting at a broader chemical arsenal that intriguingly mirrors its different virulence profile.

Scaling up cultivation allowed the researchers to isolate and characterize individual compounds. From C. javanica grown in GG1 medium, they purified seven beauveriolide congeners, three of which had never been described before. One of these, named beauveriolide Q, resisted conventional analysis because it would not dissolve in deuterated solvents and would not form crystals large enough for X-ray diffraction. The team turned instead to 3D electron diffraction, a technique that works with micron-scale crystals, collecting data on a transmission electron microscope at liquid nitrogen temperature. Because the data workflow can introduce pattern flipping that corrupts the assignment of absolute stereochemistry, the researchers pre-calibrated the entire pipeline using a five-amino-acid peptide of known configuration, then solved and refined the structure, establishing beauveriolide Q as a cyclodepsipeptide containing tryptophan, alanine and leucine residues around a 3-hydroxy-4-methyldecanoyl unit.

A second new compound, beauveriolide R, was soluble enough for full nuclear magnetic resonance analysis, which revealed the same tryptophan-alanine-leucine sequence but with a slightly longer fatty acid side chain, explaining its 28-dalton mass difference from beauveriolide Q. A third novel congener, beauveriolide S, could not be crystallized, so the team relied on detailed tandem mass spectrometry fragmentation patterns, showing it to be a variant in which phenylalanine is replaced by alanine. Meanwhile, C. blackwelliae yielded five known compounds: three diketopiperazines and two beta-carboline derivatives. Notably, an independent report of the crystal structure of beauveriolide I appeared while this study was under way, and comparing the two structures showed that despite different aromatic substituents, the macrocyclic core adopts nearly identical conformations, suggesting the ring is relatively rigid.

Bioassays then probed what these molecules actually do. Antimicrobial testing showed only weak activity: beauveriolides S and I inhibited Staphylococcus aureus at a minimum inhibitory concentration of 66.6 micrograms per milliliter, beauveriolide I also inhibited Bacillus subtilis at the same concentration, and beauveriolide M weakly inhibited the fungus Mucor hiemalis. Against human cancer cell lines, beauveriolide I was potent, with half-maximal inhibitory concentrations of 0.9 micromolar against lung carcinoma cells, 1.2 micromolar against epidermoid carcinoma cells and 3.5 micromolar against prostate carcinoma cells, while showing no apparent toxicity toward mouse fibroblasts. None of the C. blackwelliae compounds showed antimicrobial or cytotoxic effects. When purified beauveriolides I and M were injected into beet armyworm larvae, beauveriolide I killed a third of the insects over the first five days, rising to 67 percent total mortality by day seven, whereas beauveriolide M was not insecticidal at all and performed worse than the negative controls. A positive control, the mycotoxin cytochalasin D, killed 83 percent of larvae within two days.

The most consequential finding came from the original insect cadavers preserved as herbarium material. Organic extraction of tissue from the interface between the insect body and the emerging fungal growth revealed beauveriolides in C. javanica cadavers and beauvericins in C. blackwelliae cadavers, providing what the authors describe as the first direct evidence that these metabolites are produced in vivo during natural host colonization. The contrast is telling: C. blackwelliae made diketopiperazines in culture but switched to beauvericins inside the host, indicating species-specific metabolic programs that reconfigure themselves during infection. The work also carries a note for traditional medicine, since the fungal stromata sold alongside insect remains in Asian markets, such as those of Ophiocordyceps sinensis, are known to contain metabolite profiles very different from laboratory cultures, and the carcass chemistry has now been examined directly for the first time. The authors caution that whether these patterns hold across the genus remains an open question, and that future work with broader taxonomic sampling, genomic data and spatially resolved metabolite mapping will be needed to link biosynthetic regulation to ecological function and to guide the development of safer, more effective biocontrol agents.

Subject of Research: Secondary metabolite production and virulence in the entomopathogenic fungi Cordyceps javanica and Cordyceps blackwelliae

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

Article References: Charria-Girón, E., Toshe, R., Khonsanit, A., Kobmoo, N., Kwanthong, P., Gorelik, T. E., Luangsa-ard, J. J., Ebada, S. S., & Stadler, M. (2025). Chemical clues to infection: A pilot study on the differential secondary metabolite production during the life cycle of selected Cordyceps species. IMA Fungus, 16, Article e172651. https://doi.org/10.3897/imafungus.16.172651

Image Credits: AI Generated

DOI: 10.3897/imafungus.16.172651

Keywords: Cordyceps, entomopathogenic fungi, beauveriolides, secondary metabolites, metabolomics, virulence, biocontrol, cyclodepsipeptides, beauvericins, 3D electron diffraction, beet armyworm, traditional Chinese medicine