Journal of Insect Biotechnology and Sericology 71, 61-63 (2002)

Infection of the Silkworm, , with militaris

Ruiying Chen* and Masatoshi Ichida

Faculty of TextileScience, Kyoto Institute of Technology, Saga-ippongi 1, Ukyou-ku,Kyoto 616-8354, Japan

(Received April 3, 2001; Accepted December 29, 2001)

To develop techniques to produce stromata of Cordyceps militaris (L.) Link on a large scale, the silkworm, Bombyx mori, was infected and the growth of stroma was investigated. The 5th instar larvae and the pupae of the silkworm reared in a sterilized indoor environment on an artificial diet and were inoculated with C. militaris by spraying, dipping, or by hypodermic injection. The rate of infection differed among the three methods; the highest being in the hypodermic injection. The rate of infection was also higher in the pupae than in the larvae. The formation and growth of the stromata were better in the silkworm pupae. A conidial suspension was injected at different sites on the pupal body. Infection rates, however, did not differ significantly among the sites. The formation and growth of the stromata were not affected by the injection site either. Key words: Cordyceps militaris, silkworm, Bombyx mori, infection, artificial inoculation, stromata

suspension was adjusted to 10' conidia/ml. This suspension INTRODUCTION was used in the inoculation experiments. Cordyceps militaris (L.) Link. is a parasitic hosted by many species of lepidopteran pupae. It belongs Rearing of host silkworm to the subdivision Ascomycotina whose stroma is The silkworm, Bombyx mori, was reared in a sterilized baculiform (Shimizu, 1997). Recently, many researches indoor environment on an artificial diet (Chen et al., 1992). have been focused on the medicinal properties of the The eggs were disinfected with a 3% formaldehyde for 15 metabolites of C. militaris (Furuya et al., 1983; Ikumoto et min and then hatched in sterilized equipment. The larvae al., 1991). Furthermore, tumor-inhibiting and immune- were kept at 29°C from the first to third instar, and at 25°C enhancing effects of these metabolites have been confirmed from the fourth instar till maturation. After pupation, the (Liu et al., 1992; Jiang et al., 1999). At present, there is an pupae to be used as hosts were removed from the cocoons. increasing demand for C. militaris materials for analyses of Some 5th instar larvae of day 2 were also used as larval its medicinal properties. However, wild C. militaris is rare hosts. and difficult to collect. In this study, in order to develop techniques for the production of C. militaris stromata on a Artificial inoculation large scale, the infection of Bombyx mori with C. militaris Three methods were used to inoculate the larvae and and the growth of stroma were investigated. pupae with the conidial suspension; (1) spraying method in which 3 ml of the conidial suspension per 20 insects was sprayed onto the surface of the host silkworm once a day, MATERIALS AND METHODS for two days; (2) dipping method in which the whole insect Strains body was dipped in the conidial suspension for 20 sec, The stroma of Cordyceps sp. grown on the pupae of once a day, for two days; and (3) hypodermic injection in Lepidoptera moth (unidentified) were collected in the which 0.1 ml of the conidial suspension was directly summer of 1996 at Siping Jilin, China. The fungus was injected into the silkworm body with a syringe. identified as C. militaris by microscopic examination of the The inoculated larvae and pupae were reared at 25°C and stroma and ascospore. The isolate was obtained from the 90% RH, in darkness, and at 23-25 °C and 85-90% RH, ascospore. The fungus was cultured on a slant culture respectively. Also, twenty larvae and pupae were used for medium containing peptone (10 g/l), yeast extract (10 g/l), each treatment. Furthermore, to compare the infection rate glucose (30 g/1) and agar (1.5%) at 25°C for 10 days. When at different injection sites, the conidial suspension was the conidia matured, they were taken out of the tube and injected into the head, thorax and abdomen of the male and suspended in sterilized water. The concentration of conidial female pupae. One hundred pupae were used for each injection. When the infected larvae and pupae became *To whom correspondence should be addressed . sclerosed with C. militaris, they were placed in a culture Fax: +81-75-881-8418 Tel: +81-75-861-0714. glass jar (commercial mayonnaise jar, 65 mm in diam, 130 Email: [email protected]. mm in height) at 20°C and 90% RH, and at a photoperiod 62 Chen and Ichida of 12L-12D (200-300 lux) to develop the stromata. larvae became sclerosed by C. militaris. Two days after the incubation in the culture jar, white hyphae emerged from the intersegmental membrane of the larvae, and four days RESULTS AND DISCUSSION after the incubation, the mycelia covered the surface of the Inoculation of the 5th instar larvae of silkworm body (Fig. I B). At that time, the structure of the original The results of inoculation of the 5th instar larvae are tissue inside the larval body was destroyed beyond shown in Table 1. Fourteen days after the inoculation, out recognition (Fig. 1C). After 3 months of incubation, the of 20 larvae, one individual treated by spraying, 4 treated stromata were 1-2 cm long. Most of the stromata on the by dipping and 12 treated by injection were sclerosed by larvae had no perithecia (Fig. 113), however a little had the C. militaris infection. The infected larvae lost their formed immature perithecia. appetite, became inactive, and developed dark brown specks (Fig. IA). These specks were thought to be the Inoculation of silkworm pupae result of the formation of melanin under the cuticle, which As shown in Table 2, 10 days after the inoculation, one was a response against fungus. Five days after injection individual inoculated by spraying, two by dipping and 19 and 7 days after spraying and dipping, the bodies of the by injection were sclerosed. When the pupae were infected with C.militaris, dark brown specks could be seen on their Table 1. Infection of the 5th instar larvae of the silkworm with body (Fig. 2A1). Five days after injection and 9 days after C. militaris 14 days after inoculation' spraying and dipping, the infected pupae gradually became sclerosed (Fig. 2A2). Three days after the incubation in the culture jar, white hyphae were seen to emerge from the intersegmental membrane and the spiracles (Fig. 2B 1). Five days after the incubation, hyphae could be seen spreading inside the pupal body (Fig. 2B2). Ten days after the incubation, the primordia of stroma, colored orange, emerged from the dead pupae (Fig. 2C). Lastly, approxima- 'Twenty larvae were used for each inoculation . tely 40 days after the incubation, the stromata matured and grew longer than 6 cm in length (Fig. 2D).

Effect of injection site on the infection rate Sclerosis rates at 10 days after injection in the pupae injected with C. militaris at different sites are shown in Table 3. Approximately 50 days after the injection, stromata had grown on all dead pupae and matured perithecia were formed (Fig. 3A, B). The infection rate and stromata growth were not affected by the injection site or pupal sex. The results indicated that the rate of infection by C. militaris in silkworms differed among the three inoculation methods. Rates of infection were much higher through hypodermic injection than by either spraying or dipping. Rates were also higher in the pupae than in the 5th instar larvae.

Table 2. Infection of the silkworm pupae with C. militaris 10 days after inoculation'

Fig. 1. A, Dark brown specks appeared on the infected larvae. B, White mycelia filled the body. C, The inside of a larva killed by C. militaris. D, Immature stromata of C. militaris grew ' Twenty pupae were used for each inoculation on the silkworm larvae. Bars=10 mm. . Infection of Silkworm with Cordyceps militaris 63

Fig. 3. A, Perithecia on the C. militaris stroma of a silkworm pupa on the 50th day after inoculation. Bar=1 mm. B, Stromata of C. militaris matured on sclerosed pupae. Bar=l Omm.

In this study, no difference could be found in the symptoms and mycelial growth between the 5th instar larvae and the pupae of the silkworm infected with C. militaris, however the formation of stroma and perithecia differed. Nutritional requirements of the fungus for the mycelia growth and for the reproductive organ develop- ment have been shown to be different (Kitamoto et al., 1984). It is possible that pupae contains more components Fig. 2. Al, Dark brown specks appeared on the infected required for the formation of C. rnilitaris stroma and pupae. A2, The inside of a pupa killed by C. militaris on the 6th day after hypodermic injection. B1, White mycelia emerged perithecia than larvae. from the intersegmental membrane and the spiracles. B2, The inside of a sclerosed silkworm pupa on the 10th day after hypodermic injection. C, Primordia of stroma colored orange REFERENCES emerged from the silkworm pupae. D, Stromata of C. militaris Chen, R., Mori, H., Sumida, M., Yuan, X., Kitamaru, Y., and grew on the pupae. Bars=10 mm for Al, A2, B2, C, D; 1 mm for B1. Matsubara, F. (1992) All the year round sericulture by an aseptic rearing system of silkworms on an artificial diet. J. Seric. Sci. Jpn. 61, 172-179. Table 3. Sclerosis rates for the silkworm pupae injected with Furuya, T., Hirotani, M., and Matsuzawa, M. (1983) N6-(2- C. militaris at different sites 10 days after injection hydroxyethyl)adenosine,a biologically active compound from cultured mycelia of Cordyceps and species. Phytochemistry22, 2509-2512. Harada, Y., Akiyama,N., Yamamoto,K., and Shirota, Y. (1995) Production of Cordyceps militaris by fruit bodies on artificially inoculated pupae of Mamestra brassicae in the laboratory.Nippon KingakukaiKaiho 36, 67-72. Ikumoto,T., Sasaki, S., Namba, H., Tayama, R., Morimoto,H., Harada et al. (1995) reported that no stromata grew from and Mouri, T. (1991) Physiologicallyactive compounds in the extracts from Tochukaso and cultured mycelia of the pupae of Mamestra brassicae by injection of an Cordycepsand Isaria. YakugakuZasshi 111, 504 -509. ascospore suspension of C. militaris, whereas many Jiang, X., and Sun, Y. (1999) The determination of active sromata grew from pupae infected by dipping. The components in vaious Cordyceps militaris strains. Acta. infection of insects by C. militaris is thought to be related Edulis Fungi 6, 47-50. Kitamoto, Y., and Suzuki, A. (1984) MycologicalMethods (K. to many factors. These factors include the pathogen- Aoshima, K. Tubaki, and K. Miura, eds.), pp.39-47, defense responses of the host, the dose of inoculum Kyouritsusyuppan,Tokyo. injected, and the differences in pathogenicity between the Liu, J., Chen, Z., and Yang,X. (1992) Anticarcinogeniceffect conidia and ascospore. Since the fungus strain, spore and of Cordyceps militaris Link. J. Norman Bethune Univ. Med. host insect used in this study were different from those Sci. 18, 423-425. used by Harada et al. (1995), different results might be Shimizu, D. (1997) Illustrated Vegetable Wasps and Plant Wormsin Colour, pp.70-109,lenohikari Kyoukai, Tokyo. obtained.