Journal of Insect Biotechnology and Sericology 83, 77-81 (2014)

CO2 anesthesia enhances infection rate of militaris (: Clavicipitaceae) on pupae of the silkworm,

Shinichi Abe1, 3, Kei-ichiro Yamamoto1,＊ , Ying An2, Jun Saeki3, Takashi Itagaki3 , Hisayoshi Kofujita2 and Koichi Suzuki4,＊＊

1 United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550, Japan 2 Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan 3 Nichihara Research & Development Laboratories, Inc., Tsuwano-cho 699-5207, Shimane, Japan 4 Office of Research Exchange, Iwate University, Morioka 020-8550, Japan (Received August 1, 2014; Accepted October 15, 2014; Accepted in revised form November 25, 2014)

An entomogenous , Cordyceps militaris, grows on larvae and pupae of the silkworm, Bombyx mori. To overcome problems in the establishment of fungal infection caused by advancing pupal-adult development, we

tested the effects of carbon dioxide (CO2) anesthesia. Pupae inoculated with C. militaris were held in CO2 anes- thesia for 2 days. Almost all pupae exposed to CO2 were infected with C. militaris, but the rate of infection of chilled or untreated pupae decreased with increasing stage of pupal-adult development at inoculation. The re-

sults indicate that exposure of silkworm pupae to CO2 enhances infection rate. This treatment will support the mass production of C. militaris for pharmacological uses. Key words: Cordyceps militaris, Bombyx mori pupae, carbon dioxide anesthesia, infection rate

Shrestha et al., 2012). INTRODUCTION Although much effort has been applied to scaling up Cordyceps, Ophiocordyceps and spp. (Luangsa-ard the rate of infection with C. militaris, advancing pupal- et al, 2005; Sung et al., 2007) are entomogenous fungi tra- adult development before injection inoculation reduces ditionally used as treatments for cancer, metabolic diseases infection rate and hinders the mass production of C. mili- such as diabetes, cardiovascular diseases, and neural dis- taris. Here, we tested whether the use of carbon dioxide orders, albeit without good scientific evidence (Paterson, (CO2) anesthesia could enhance infection rate. CO2 is rou- 2008; Zhou et al., 2009; Das et al., 2010; Tsushima et al., tinely used as an insect anesthetic in many laboratories 2010; Yue et al., 2012; Patel and Ingalhalli, 2013). Wild C. (Nilson et al., 2006). This is the first report to demon- militaris grows extremely slowly in a limited natural strate its use to enhance infection rate, although it has range, and the stroma (fruiting body) is very small. Sub- been tested in the artificial culture of Isaria japonica (= merged culture under optimized conditions (Shih et al., Pacelomyces tenuipes) (Inatomi et al., 2000). 2007) offers greater mycelial production with less chance of contamination, but constituent compounds tend to MATERIALS AND METHODS change. For example, some biopharmaceutical constituents of Ophinocordyceps sinensis differed between natural and Host silkworms cultured samples (Li et al., 2004). The trasnscriptional Silkworms (B. mori, Shurei × Shogetsu) were reared on profiles of genes in C. militaris stromata grown on silk- an artificial diet (Nihon Nosan Kogyo Co. Ltd., Japan) worm (Bombyx mori) larvae differed from those produced until the 4th instar at Ehime Sanshu Co. Ltd. (Ehime in culture (Xiong et al., 2010). Thus, the formation and Prefecture, Japan), and 5th instar larvae were grown on growth of stromata of C. militaris in silkworm pupae have mulberry leaves at Nichihara Research & Development been studied and the rate of infection by injection inocu- Laboratories Co. Ltd. (Shimane Prefecture, Japan). lation is higher within a limited age of pupal development than in the larvae (Chen and Matsubara, 2002; Chen and Preparation of Cordyceps militaris inoculum Ichida, 2002; Sato and Shimazu, 2002; Hong et al., 2010; Cultures of C. militaris (NBRC No. 100741) were pur-  chased from the National Institute of Technology and * Present address: Nishizawa & Associates, Intellectual Property: Evaluation Biological Resource Center (Chiba Prefecture, Patents, Kudan Horie Bldg. 6F. Kudan-kita, 4-3-14, Chiyoda-ku, Japan). Dried pupae (50 g) with 400 mL of distilled water Tokyo 102-0073, Japan were boiled for 60 min. The extract of silkworm pupae **To whom correspondence should be addressed. (200 mL) was mixed with agar (20 g), and the mixture Fax & Tel: +81-19-621-6917. was poured into 10 ml of test tubes and autoclaved at Email: [email protected] 121°C for 40 min. Each test tube was inoculated with the 78 Abe et al. culture of C. militaris and the inoculated media were cul- tured at 25°C for 35 days. Hyphal bodies of C. militaris were collected and suspended in water (8 × 104 cells/mL). Then 50 μL of the suspension was injected into the hae- mocoel of each pupa by the methods of Sato and Shimazu (2002) and Chen and Ichida (2002).

Carbon dioxide anesthesia

Generally, commercial, food, or industrial grade CO2 is used for CO2 anesthesia (Newton, 1993; Branscome et al., 2005). As a convenient and effective method (Kamimura et al., 1972; Hayakawa et al., 1986), we used solid CO2

(dry ice, Tairiku Co. Ltd., Japan) in a CO2 gas generator (11 L, 250 mm × 300 mm × 150 mm) connected by four tubes to four anesthesia apparatuses (the capacity of an apparatus is 114 L: 440 mm × 740 mm × 350 mm) (Fig. 1).

CO2 gas generated from dry ice (3 kg/day) was continu- ously supplied to the anesthesia apparatus, and the con- centration was measured with a high-density gas detector (XP-3140, Rex Co. Ltd., Japan). Placing the gas generator Fig. 1. CO2 generator and anesthesia apparatus. For details 80 cm above the anesthesia apparatus allowed a constant see Materials and Methods. supply of gas at >85% concentration. Long tubing al- lowed the CO2 to warm to room temperature. A small Statistical analyses hole in the lid of the apparatus ensured a high CO2 con- Data on CO2 anesthesia were analyzed by ANOVA fol- centration in each apparatus to be ventilated slowly. lowed by Dunnett’s test. Data on chilling anesthesia were analyzed by Student’s t-test. Data on eclosion were ana- Chilling anesthesia lyzed by the Kaplan-Meier method (failure rate plot). Dif- Many laboratories also use ether, chilling, or nitrogen an- ferences at P < 0.05 were considered significant. esthesia (Sillans and Biston, 1979; Branscome et al., 2005). To comapre chilling at 5°C with CO anesthesia, we used 2 RESULTS chilling anesthesia in independent experiment. Effects of carbon dioxide anesthesia on fungal Experimental schedules of carbon dioxide and infection chilling anesthesia The rate of infection of inoculated pupae incubated in

For CO2 anesthesia, pupae were picked out of their co- normal air (control) decreased with increasing dates of in- coons at 8 days after mounting, and those at the eye pig- oculation, which was synchronized to days after eye pig- mentation stage were used. For each experiment, 200 mentation (Fig. 3). In contrast, almost all pupae exposed pupae were randomly divided into five groups (all n = 40, to CO2 anesthesia were infected regardless of dates of in- Fig. 2), which were inoculated at 1, 2, 3, 4, or 5 days af- oculation. Differences in infection rate became significant ter extraction. Controls were continuously incubated at on days 4 (***P < 0.001) and 5 days (****P < 0.0001)

25°C under normal air. After exposure of CO2 anesthesia after inoculation. for 2 days, the pupae in each group were transferred to Of the 600 control pupae, 47.7% eclosed, 12% rotted, normal air as experienced by the controls (Fig. 2). Hard- and the rest (40.3%) were infected with C. militaris. Of ened pupae were considered to be infected and eclosion the 600 CO2-anesthesized pupae, only 1.7% eclosed and was recorded daily for 25 days. The experiment was per- 2.5% rotted, and 95.8% were infected. Alternatively, stro- formed six times. mata were formed from all infected pupae and no signifi- For chilling anesthesia at 5°C , a similar experimental cant difference was observed in producing apparent schedule was followed (Fig. 2). Controls were continuous- stromata between control and CO2-anethesized pupae (data ly incubated at 25°C. Eclosion was additionally recorded not shown). These results show that CO2 anesthesia of pu- daily for 25 days. The experiment was perfomed three pae inoculated with C. militaris is highly effective at en- times. hancing infection rate. CO2 anethesia and Cordyceps militaris from the silkworm pupae 79

Fig. 2. Experimental schedule of CO2 (6 experiments with 200 pupae each) or chilling (3 experiments with 200 pu- pae each) anesthesia treatment.

Effects of chilling anesthesia on fungal infection insect pests (Cheng et al., 2012; Seki and Murai, 2012), it The rates of infection of inoculated pupae incubated at is commonly used to anesthesize many insect species by a 25°C and at 5°C for 2 days both decreased with increas- variety of physiological processes. Its effects on the heart ing dates of inoculation, which was synchronized days af- and the skeletal neuromuscular junctions of Drosophila ter eye pigmentation (Fig.4). These results show that melanogaster larvae are directly mediated by a reduced chilling anesthesia of pupae inoculated with C. militaris is sensitivity to glutamate (Badre et al., 2005). It does not ineffective at enhancing infection rate. kill cockroaches, but movement and food consumption are affected for hours or days after presumed recovery (Branscome et al., 2005). In the codling moth (Cydia po- DISCUSSION monella), high concentrations of CO2 reduce metabolic

Although CO2 anesthesia is effective at controlling some heart rate and prevent the use of ATP (Neven and Hansen, 80 Abe et al.

ACKNOWLEDGEMENTS This study was supported by the JSPS KAKENHI Grant No. 23228001.

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