When Reticulitermes flavipes workers encounter spores of Metarhizium adhering to a nestmate’s cuticle, they respond with brief bouts of rapid, whole-body shaking, a behavior the researchers call longitudinal oscillatory movement. This alarm display functions as a call for help, soliciting allogrooming from nearby colony members. Workers use their mouthparts to strip conidia from each other’s bodies, and the ingested spores are effectively destroyed, aided by salivary antimicrobial compounds including beta-1,3-glucanases and a small, cysteine-rich antifungal peptide called termicin. The critical open question has been which molecular signature, or pathogen-associated molecular pattern, actually triggers this rapid hygienic response. Ergosterol, an essential component of fungal membranes, was a leading candidate, particularly after earlier work showed that applying ergosterol to the cuticle of the Formosan subterranean termite Coptotermes formosanus elicited increased allogrooming.

To test this hypothesis rigorously, the team collected thirteen clonally propagated Metarhizium isolates from soils near active termite foraging sites across five US states, representing three species: Metarhizium robertsii, M. brunneum and M. guizhouense. Species and strains were confirmed by sequencing the translation elongation factor 1-alpha gene along with ITS and intergenic spacer regions. The researchers then quantified ergosterol in each isolate’s conidia using a saponification and spectrophotometric method originally developed for measuring ergosterol in Candida, calculating sterol content as a percentage of wet spore weight and correcting for the late pathway intermediate 24(28)-dehydroergosterol, which absorbs at a different wavelength.

The behavioral experiments produced a striking and initially counterintuitive result. In preference assays, termites given a choice between filter paper halves treated with ergosterol, cholesterol or solvent alone aggregated on the sterol-treated sides, but only ergosterol significantly elevated alarm shaking and allogrooming. Filter papers treated with methanol extracts of ergosterol from conidia also triggered alarm responses that correlated positively with the ergosterol content of the source isolates. Yet when termites were challenged with intact, living conidia, the intensity of the alarm response was not positively correlated with ergosterol content at all. Instead, the relationship trended in the opposite direction, suggesting that while ergosterol can serve as a recognition cue on its own, it is not the initial signal that alerts termites to dangerous contamination on a nestmate’s body.

Even more revealing was the correlation between ergosterol and pathogenicity. Across the thirteen isolates, spore ergosterol content was significantly and positively associated with the hazard ratio of death in Cox regression survival analyses, meaning that isolates richer in membrane ergosterol were more lethal to termites, not less. Adhesion assays ruled out the possibility that the most alarming isolate simply deposited more spores on the cuticle, and thin-layer chromatography showed no peroxidation or amino acid acylation of the sterol that might have altered its detectability. The picture that emerged was of a fungus in which high ergosterol somehow confers a survival advantage during the critical early hours after contact with a termite host.

The key to that advantage appears to lie in termicin, the termite’s principal antifungal weapon. When the researchers incubated conidia from each isolate in a five micromolar solution of recombinant R. flavipes termicin, the peptide’s fungicidal activity varied dramatically across isolates and correlated strongly with both virulence and ergosterol content. Termicin was most effective against the most lethal isolates, those with the highest ergosterol levels, and least effective against the least alarming, low-ergosterol strains. In vivo confirmation followed: pretreating conidia of the high-ergosterol isolate MD 002 with termicin significantly improved termite survival over fourteen days, while the same treatment offered no protection against the low-ergosterol isolate MD 353. Because termicin is an amphipathic peptide believed to disrupt fungal membranes, membrane ergosterol content plausibly determines how vulnerable a spore is to this assault.

Together, these findings sketch a mechanistic model of coupled, counterbalancing traits. Fungal cell wall integrity is known to be influenced by membrane ergosterol, and studies of antifungal drugs that block ergosterol synthesis show that reducing membrane sterol is compensated by reinforcing the cell wall. The Towson team proposes that Metarhizium isolates with lower membrane ergosterol develop cell walls richer in whatever unidentified pathogen-associated molecular pattern termites actually detect, making those spores easier to recognize and triggering vigorous allogrooming. Conversely, isolates with high ergosterol are harder for termicin to kill but present a weaker initial alarm signal. The fungus thus faces a genuine trade-off between a shield that protects against the peptide weapon and a signal that betrays it to the colony’s hygiene patrol, and the termites appear to have adaptively exploited exactly this vulnerability.

The evolutionary dimension of the story is equally compelling. Previous work documented a recent selective sweep for an advantageous termicin variant in sympatric populations of R. flavipes and R. virginicus in Maryland, and the two species respond differently to M. brunneum and M. robertsii, hinting at co-adaptation to local fungal lineages. In the new study, genetic distances between Metarhizium isolates correlated significantly with differences in the alarm responses their conidia elicited, even after controlling for collection latitude, indicating that the true recognition cue has diverged along with fungal phylogeny. Ergosterol content, by contrast, showed no correlation with genetic distance, suggesting it is a more plastic trait shaped by environmental conditions. Consistent with that idea, prior research on Argentine ants found that serial laboratory exposure to small ant groups drove Metarhizium to reduce its ergosterol and evade social immunity, a shift that would simultaneously increase the wall-based signal termites detect.

Ergosterol may also play a subtler secondary role in the hygiene cascade. The authors speculate that when termicin disrupts the membranes of high-ergosterol spores, released sterol could then act as a secondary elicitor, amplifying aggregation and allogrooming precisely when the initial wall-based alarm is weak. Residual ergosterol has been detected on the cuticles of C. formosanus workers an hour after Metarhizium exposure, lending plausibility to this compensation mechanism. Candidate molecules for the primary cue include cell wall beta-glucans, although inhibiting termite beta-1,3-glucanases did not reduce allogrooming in earlier experiments, leaving the identity of the true trigger unresolved. What is clear is that termite survival rests on a finely tuned balance between a behavioral response that is energetically costly and a peptide weapon whose effectiveness varies with the biochemistry of each fungal strain it meets.

Beyond its elegance as an evolutionary case study, the work carries practical implications. Metarhizium species are widely deployed as biological control agents against termites and other soil pests, and understanding why some isolates fail, namely because they trigger strong social immunity while resisting termicin, could guide the selection or engineering of strains that slip past termite defenses. Conversely, the finding that ergosterol content can shift plastically under selection pressure suggests that pathogens can rapidly adapt to dense, patchily distributed social insect populations. For now, the termites hold their ground through a two-layered strategy: an unidentified cell wall signal that sounds the alarm, and a membrane-targeting peptide that strikes hardest at the strains most adept at staying quiet. The arms race beneath the forest floor, it turns out, is negotiated one sterol molecule at a time.

Subject of Research: The role of ergosterol in termite social immunity and fungal pathogen detection against Metarhizium infections

Article Title: The influence of ergosterol on termites’ defenses against Metarhizium infections

Article References: Rosenbloom, J. H., Luitel, B., & Bulmer, M. S. (2026). The influence of ergosterol on termites’ defenses against Metarhizium infections. The Science of Nature, 113(6), Article 130. https://doi.org/10.1007/s00114-026-02183-3

Image Credits: AI Generated

DOI: 10.1007/s00114-026-02183-3

Keywords: ergosterol, termites, Metarhizium, social immunity, allogrooming, termicin, antifungal peptide, pathogen-associated molecular pattern, Reticulitermes flavipes, entomopathogenic fungus, fungal cell membrane, host-pathogen coevolution

The research began in the field. Specimens were collected from Gool in District Ramban, at altitudes of roughly 2,500 to 3,500 meters above sea level, during the monsoon months between June and August. The club-shaped, orange stromata the researchers found were closely associated with insect larvae and pupae, whose bodies the fungus had colonized and mummified with its mycelium. Back at the Plant Pathology, Mycology and Microbiology Laboratory in Srinagar, the team documented the fungus’s macroscopic anatomy in detail, noting stromata measuring 3.8 to 6.2 centimeters in length, semi-immersed ovoid perithecia, and the darker fertile heads that signal maturity.

Identification did not rest on appearance alone. Microscopic examination revealed subcylindrical conidiophores, slender flask-shaped phialides, and variably shaped conidia, along with eight-spored cylindrical asci containing filiform ascospores. The researchers then turned to molecular tools, extracting DNA by the CTAB method and amplifying the internal transcribed spacer region of the ribosomal DNA with the universal primers ITS-1F and ITS-4R. Sequencing showed 99.78 percent identity with a reference strain of C. militaris, and the sequence was deposited in GenBank under accession number PQ810010. A maximum-likelihood phylogenetic analysis of 18 ITS sequences placed the Kashmir isolate firmly within the C. militaris species complex, supported by a bootstrap value of 100 percent. The specimen itself was preserved as voucher number 9337-KASH in the herbarium at the University of Kashmir.

With identity confirmed, the researchers set out to solve a practical problem: the fungus grows slowly. On ordinary Sabouraud’s dextrose agar, C. militaris needed roughly 27 to 28 days to cover a plate, and the full cultivation cycle to mature stromata in prior work stretched to two or three months. For a fungus whose value lies in compounds such as cordycepin, adenosine, gamma-aminobutyric acid, ergothioneine, lovastatin, carotenoids and a suite of minerals, time is money. The team compared four solid media, Sabouraud’s dextrose agar, potato dextrose agar, corn meal agar and Richard’s synthetic agar, measuring mycelial diameter every three days over three weeks of incubation at 25 degrees Celsius.

Sabouraud’s dextrose agar emerged as the clear winner, producing the maximum mycelial growth of 54.0 millimeters after 21 days, ahead of potato dextrose agar, corn meal agar and Richard’s synthetic agar. But the researchers did not stop there. Knowing that the carbon-to-nitrogen ratio strongly influences mycelial growth in this species, they supplemented the best medium with malt extract, a rich carbon source, and yeast extract, a rich nitrogen source. Malt extract at 6 grams per liter pushed the colony diameter to 70.30 millimeters, but adding more than that brought no further gain. The decisive step came with yeast extract: the optimal combination of 6 grams per liter malt extract and 4 grams per liter yeast extract produced the maximum diameter of 84.60 millimeters after just 17 days of incubation, roughly ten days faster than unsupplemented Sabouraud’s medium.

Faster mycelium is only half the battle; producing the fruiting bodies, or stromata, that contain the medically interesting compounds requires moving the fungus onto solid substrates. Here the team prepared liquid inoculum in two nutrient broths of differing sugar and nitrogen composition, agitating flasks on a shaker at 170 revolutions per minute for up to eight days. Broth I, based on dextrose with peptone and yeast extract plus magnesium sulfate, potassium phosphate and vitamin B1, produced visible growth after seven days, five days sooner than broth II. The liquid mycelial culture also colonized grain substrates faster than mycelium grown on solid media, confirming that liquid spawn is the more efficient route to fruiting.

For the cultivation trials, the researchers chose four locally available and inexpensive grains: brown rice, white rice, corn kernels and wheat. Each 20-gram portion of grain received 45 milliliters of one of the two nutritional broths before sterilization and inoculation. Jars were incubated in darkness at 20 degrees Celsius and 65 to 70 percent relative humidity for the spawn run, then exposed to fluorescent light at 800 to 1,000 lux for twelve hours daily at 85 to 90 percent humidity to trigger primordia formation and stroma development. The logic of using local grains was deliberate: they are cheap, produced by regional farmers, and sustainable, reducing production costs for would-be growers in Jammu and Kashmir.

The results were unambiguous. Brown rice combined with nutritional broth I delivered the fastest performance on every measure: a spawn run of only 14 days, primordia after 11.5 days, and mature stromata after about 25.75 days, for a total cultivation cycle of 51.25 days from inoculation to harvest, the shortest of any combination tested. This substrate pairing also produced the longest stromata, at 78.0 millimeters on average, and the greatest number of stromata per jar, roughly 63 compared with a low of about 28 on wheat grain with broth II. Most strikingly, brown rice with broth I achieved a biological efficiency, the ratio of fresh yield to dry substrate weight, of 72.75 percent, far outperforming corn, white rice and wheat. Wheat grains did produce the thickest stromata, at just over 4 millimeters in diameter, but nowhere near the overall yield of brown rice.

The findings align with a growing international literature. Previous studies have found brown rice superior for C. militaris fruiting, reported maximum stroma yields on whole rice grains at slightly acidic pH, and shown that grain type and vitamin supplementation significantly shape mycelial growth and antioxidant capacity. Other work has optimized liquid culture conditions, identifying ideal glucose, yeast extract and mineral concentrations for biomass production. What distinguishes the Kashmir study is its focus on a wild, locally isolated strain rather than a commercial or imported one. Local strains may carry physiological adaptations to regional climate and substrates, and the authors argue that strain-specific optimization of this kind offers new insight into how cultivation protocols should be tailored to geographic origin rather than assumed universal.

The practical implications reach beyond mycology. C. militaris was approved by China’s Ministry of Public Health in 2009 as the first novel food of its kind, and its bioactive profile is considered comparable to that of the rare and costly caterpillar fungus Ophiocordyceps sinensis, making it a viable substitute in both traditional and modern therapeutic applications. Cordycepin and adenosine, its flagship compounds, show antibacterial, antioxidant, anti-inflammatory and anticancer activities in laboratory studies. By shortening the cultivation cycle, specifying cheap local substrates, and demonstrating conditions, 20 degrees Celsius, moderate humidity and a simple light regimen, that small facilities can maintain, the Kashmir team has laid a foundation for sustainable commercial cultivation that could benefit mushroom growers and entrepreneurs across the Indian Himalayas while easing pressure on wild populations.

Subject of Research: Optimization of nutrient media and grain substrates for the commercial cultivation of the wild medicinal fungus Cordyceps militaris from Jammu and Kashmir.

Article Title: Nutrient media and substrate optimization for commercial cultivation of Cordyceps militaris (L.) Fr., a novel medicinal mushroom from Jammu and Kashmir

Article References: Shrikhandia, P., Lone, S. A., Wani, A. H., & Bhat, M. Y. (2026). Nutrient media and substrate optimization for commercial cultivation of Cordyceps militaris (L.) Fr., a novel medicinal mushroom from Jammu and Kashmir. Discover Biotechnology, 3(1), Article 3. https://doi.org/10.1007/s44340-026-00048-z

Image Credits: AI Generated

DOI: 10.1007/s44340-026-00048-z

Keywords: Cordyceps militaris, medicinal mushroom, cultivation, substrate optimization, brown rice, cordycepin, mycelial growth, biological efficiency, Jammu and Kashmir, entomopathogenic fungus, Sabouraud dextrose agar, stroma formation