Isolation, Culture and Characterization of Hirsutella sinensis Mycelium from Caterpillar Fungus Fruiting Body

Abstract

The caterpillar fungus Ophiocordyceps sinensis (previously called Cordyceps sinensis) has been used for centuries in Asia as a tonic to improve health and longevity. Recent studies show that O. sinensis produces a wide range of biological effects on cells, laboratory animals and humans, including anti-fatigue, anti-infection, anti-inflammatory, antioxidant, and anti-tumor activities. In view of the rarity of O. sinensis fruiting bodies in nature, cultivation of its anamorph mycelium represents a useful alternative for large-scale production. However, O. sinensis fruiting bodies harvested in nature harbor several fungal contaminants, a phenomenon that led to the isolation and characterization of a large number of incorrect mycelium strains. We report here the isolation of a mycelium from a fruiting body of O. sinensis and we identify the isolate as O. sinensis' anamorph (also called Hirsutella sinensis) based on multi-locus sequence typing of several fungal genes (ITS, nrSSU, nrLSU, RPB1, RPB2, MCM7, β-tubulin, TEF-1α, and ATP6). The main characteristics of the isolated mycelium, including its optimal growth at low temperature (16°C) and its biochemical composition, are similar to that of O. sinensis fruiting bodies, indicating that the mycelium strain characterized here may be used as a substitute for the rare and expensive O. sinensis fruiting bodies found in nature.

Fig 1. Culture of H. sinensis mycelium derived from O. sinensis fruiting body.

(A) O. sinensis fruiting body or stroma (top) protruding from the shell of a caterpillar insect (bottom) was obtained in the Naqu prefecture in Tibet. HSM strain CGB 999335 was isolated from a similar fruiting body. (B) Colony of HSM strain CGB 999335 cultured for 28 days at 18°C on PDA agar. (C) CGB 999335 mycelium observed under optical microscopy. (D) Sterile FM1 liquid medium used to culture CGB 999335 mycelium in the present study (left tube; containing 1.2% of yeast extract as a source of nitrogen) and sterile liquid 1.2% (w/v) soybean broth commonly used in other laboratories (tube on the right). Notice the pellet of undissolved powder in the tube on the right. See the Methods section for more details. (E) Dark-field optical microscopy image of CGB 999335 mycelium cultured in FM1 medium. (F) CGB 999335 mycelium cultured in soybean broth seen in D (tube on the right). Undissolved, brown material can be seen among mycelial cells.

Fig 2. 5.8S-ITS rDNA phylogenetic tree of Ophiocordyceps species.

The evolutionary relationship of Ophiocordyceps 5.8S-ITS rDNA genes was determined using the neighbor-joining method [32]. Evolutionary distances were assessed using the maximum composite likelihood (MCL) method. The bootstrap consensus tree, which represents the evolutionary relationship of the analyzed taxa, was inferred from 500 replicates as before [27]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches. Evolutionary distances (i.e., number of base substitutions per site) were computed using the maximum composite likelihood method [24].

Fig 3. Multi-locus sequence typing-based phylogenetic analysis of Ophiocordycipitaceae, Bionectriaceae, Hypocreaceae and Nectriaceae for a five-gene dataset.

The evolutionary relationship of a five-gene dataset (nrSSU, nrLSU, RPB1, RPB2, EF-1a) was determined using the maximum likelihood method based on the Tamura-Nei model [25]. Initial tree(s) for the heuristic search were obtained using the neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach. Shown here is the bootstrap consensus tree inferred from 500 bootstrap replicates. The percentage of replicate tree that clustered with associated taxa is indicated.

Fig 4. Effect of temperature on the culture of H. sinensis mycelium.

CGB 999335 mycelium was cultured in liquid FM1 medium with mixing for eight days at the temperature indicated. Mycelium cells were obtained by centrifugation, followed by drying and measurement of biomass weight.

Fig 5. Culture of H. sinensis mycelium at various pH.

CGB 999335 mycelium was cultured in liquid FM1 medium at 16°C with mixing. Prior to culture, the pH of the culture medium was adjusted to the indicated value by adding 1 M HCl or NaOH. After five days of culture, mycelium cells were obtained by centrifugation, followed by drying and measurement of biomass weight.

Fig 6. Culture of H. sinensis mycelium with time.

CGB 999335 mycelium was cultured in liquid FM1 medium with mixing at 16°C for the time indicated. Mycelium cells were obtained by centrifugation, followed by drying and measurement of biomass weight.

Fig 7. HPLC chromatograms of H. sinensis mycelium and natural Ophiocordyceps specimens.

HPLC chromatogram of (A) O. sinensis fruiting body and insect; (B) O. sinensis fruiting body; and (C) CGB 999335 mycelium. HPLC was performed on a reverse-phase column as described in Methods. Peaks were identified based on the use of pure standard compounds processed under the same conditions. Nucleosides and nitrogenous bases were monitored using a UV detector. The results shown are representative of experiments performed in triplicate. Peak intensities are given in Table 2.