Newly Developed Fungal Diet for Artificial Rearing of the Endangered Long-Horned Beetle Callipogon Relictus (Coleoptera: Cerambycidae) Dae-Am YI1,4, Alexander V

Entomological Research 47 (2017) 373–379

RESEARCH PAPER Newly developed fungal diet for artificial rearing of the endangered long-horned beetle Callipogon relictus (Coleoptera: Cerambycidae) Dae-Am YI1,4, Alexander V. KUPRIN2,YongHoonLEE3 and Yeon Jae BAE1

1 Department of Environmental Science and Ecological Engineering, College of Life Sciences and Biotechnology, Korea University, Seoul, South Korea 2 Federal Scientific Center of the East Asia Terrestrial Biodiversity, Russian Academy of Sciences, Vladivostok, Russia 3 Wonju, Gangwon-do, South Korea 4 Research Center of Natural Monument Insects, Yeongwol, Gangwon-do, South Korea

Correspondence Abstract Yeon Jae Bae, Department of Environmental Science and Ecological Engineering, College of In this study, we attempted to develop an artificial diet to effectively rear the Life Sciences and Biotechnology, Korea endangered long-horned beetle Callipogon relictus Semenov to facilitate restoration University, Seoul 02841, Korea. efforts for this species. Fungal mycelia of the white-rot fungus Pleurotus florida Email: [email protected] (Eager) were, for the first time, provided as an artificial diet for the larvae of C. relictus. The experiment started on August 11, 2015 with 53 larvae being Received 10 February 2017; provisioned with the fungal diet under laboratory conditions (25°C, 60 % RH, L:D accepted30March2017. 0:24) without diapause. During the first year, 11 adults (4 males and 7 females, doi: 10.1111/1748-5967.12234 20.8 % of the 53 larvae) emerged between September 10 and October 1, 2016. The body length of the largest male was 106.8 mm, and three of the four males exceeded 100 mm in length. These results show that a fungal diet for rearing C. relictus is useful not only for reducing the larval period to approximately 1/6 of the normal larval period in nature, but also for producing large adults. These results support the notion that fungus may provide nutritional benefits during the larval development of C. relictus, and also indicate that this species is fungivorous.

Key words: Endangered insect restoration, Fungal mycelium diet, Insect rearing, Larval development, Relict long-horned beetle, White-rot fungus Pleurotus.

Introduction in the field is known to be as long as 5 to 6 years (Li et al. 2012). Therefore, reducing the rearing time of the larvae is The endangered long-horned beetle Callipogon relictus considered crucial for effective population restoration. Semenov (Fig. 1), the largest beetle in the Palearctic region, Recently, it has been reported that C. relictus larvae developed is sporadically distributed throughout the Russian Far East, into adults within 6 years in a semi-natural outdoor the northeastern part of China, and the northern part of the environment when provided with sawdust (Li et al. 2012). Korean Peninsula. It is listed in the Red Data Book (Category Two other experiments have shown that the larval growth I) and protected in the Russian Federation and the Republic of stage can be shortened to 42–44 months by terminating Korea. In Korea, it is also designated as a National Natural diapause. The first experiment was performed during the Monument (No. 218) and protected by law. The beetle has period 2009–2013 in Ussuriysk, Russian Federation, by been widely used as an emblematic species in Korea including feeding the larvae naturally decayed Ulmus japonica wood adoption as the symbol of the Entomological Society of under laboratory conditions (25°C, 65 %–75% RH) (Kuprin Korea. et al. 2014). The second experiment was carried out during Special attention has been paid to the restoration of the period 2010–2014 in Yeongwol, Republic of Korea, by C. relictus in Korea and Russia which requires artificial feeding the larvae an artificial diet comprising the sawdust of rearing of the larvae. However, the larval period of C. relictus Quercus mongolica supplemented with additional nutrients

stage. It has also been demonstrated that provision of an artificial fungal diet affects adult size and pupal development time in the bark beetle Dendroctonus ponderosae (Bleiker and Six 2007). Given the findings of these studies, it is very tempting to speculate whether C. relictus, which like many other xylophagous beetles inhabit decayed wood, is also fungivorous. In this study, we tested Lyubarsky’s (1953) assumption that fungal mycelia might be an effective diet for the growth of C. relictus larvae. [Correction added on 12 July 2017, after first online publication: “C. relictusfi” has been changed to “C. relictus”.] For this study, we mainly examined the use of fungal mycelia instead of raw sawdust or sawdust containing additional nutrients as a diet for rearing the larvae of C. relictus.

Figure 1 A Callipogon relictus female laying eggs on Ulmus japonica infected by the fungus Pleurotus citrinopileatus in Ussuriysk, Russia. Materials and methods under laboratory conditions at constant temperature (20°C, Insects 65 %–75% RH) (Yi 2014). In the former study, the larval growth period was successfully reduced to 42 months, We imported two pairs of live C. relictus collected in North whereas in the latter it was reduced to 44 months (Fig. 2). Korea (Cheonma-san, Pyeonganbuk-do, N. Korea, coll. July – Although there have been numerous previous studies on the 28 August 4, 2015) via China on August 10, 2015. A customs use of artificial diets for long-horned beetles (Harley and clearance to South Korea was approved (#15408195) by the Wilson 1968; Sugiyama 1969; Gardiner 1970; Yamane 1973, Incheon International Airport Regional Office of Animal and 1974; Akutsu et al. 1980; Murakoshi and Aono 1981; Kevin Plant Quarantine Agency (APQA) under the permission of and Robinson 1982; Dubois et al. 2002), little is known about the Ministry of Environment of Korea. utilizing fungal mycelia as a diet for any species of long-horned The adults were divided into pairs, and induced to mate in beetles (Kukor and Martin 1986). In the case of stag beetles two plastic cages (63 cm in length, 44 cm in width, and (Lucanidae) that consume dead or decayed wood during their 23 cm in height) on August 11, 2015. Each of five pieces larval growth period, it is commonly known to insect breeders of decayed wood of Mongolian oak Quercus mongolica – – and collectors that larvae tend to be of a larger size at sites (8 9cmindiameterand12 13 cm in length) to be used for where white-rot fungi are observed on the base of trees. egg-laying were prepared and soaked in water for 2 h. The Most lucanid beetles are well known to be fungivorous wood pieces were then sterilized in a microwave oven for (Kojima 1996; Tanahashi et al. 2009; Tanahashi and Kubota 30 min. After observing several mating activities, the males 2013), and some species of Anobiidae, such as the death- were removed from the cages and isolated. The females were – watch beetle Xestobium rufovillosum are highly associated left in the cages at room temperature (22 25°C) until post- with fungus (Campbell 1941) for the duration of the larval oviposition and death. Commercial sugar gelatins were provided as the adult’s food and were frequently replanished. On September 11, after a month of rearing, the wood pieces were split in order to collect the grown larvae. Fifty-three 1st instar larvae were taken from one cage, but no larvae were found in the other cage.

Artificial fungal diet The fungal diet used for rearing larvae was newly separated and cultivated out of mycelia of the white-rot fungus Pleurotus florida (Eager). We used commercial potato dextrose agar powder (PDA: 213400-BD; Difco, Becton Dickinson, Franklin Lakes, NJ, USA) as the medium for sub-culturing Figure 2 Comparison of the larval development period of Callipogon P. florida. PDA agar powder was mixed in a 1000 mL relictus with that observed in previous studies. graduated cylinder, at a ratio of 19.5 mg of powder in

500 mL water. To aid solubilization, the solution was Table 1 The total period of larval development and body size of male transferred to a 1000 mL conical flask and mixed with a adults fed with fungal diet magnetic stirrer on a heating plate. When completely solubilized, the solution was divided into 20 mL portions, Emerged Laval period Length Weight which were poured into 50 mL round-bottomed glass test No. date (2016) (day) Sex (mm) (g) tubes (27.3 mm in inner diameter), which were capped with 1Sep.10358♂ 101.4 14.59 silicon stoppers. The glass test tubes were then autoclaved at 2Sep.15363♂ 90.2 13.09 2 121°C for 20 min at a vapor pressure of 1.2 kg/cm ,and 3Sep.16364♂ 106.8 16.90 placed in a tilted position until solidified. 4Sep.21369♂ 100.8 15.84 For production of the sawdust medium, we used crushed Mean (±SE) 363.5 99.80 15.105 Mongolian oak wood to prepare sawdust, which was (±2.3) (±3.47) (±0.821) maintained at a 50 %–60 % moisture content. One thousand- gram amounts of the moistened sawdust were placed into polypropylene bottles (1400 mL). The surface of the sawdust Table 2 The total period of larval development and body size of female was then tamped, and a hole of 1 cm diameter was made in the adults fed with fungal diet middle, from the surface to the bottom of the bottle. The bottles were closed with a sterilized cotton cap. The sawdust Emerged Laval period Length Weight No. date (2016) (day) Sex (mm) (g) medium was autoclaved at 121°C for 2 h, at a vapor pressure 2 of 1.2 kg/cm . The sterilized sawdust was then cooled to 25°C, 1Sep.21369♀ 84.4 13.06 and moved to an aseptic room. PDA medium was then placed 2Sep.25373♀ 80.1 12.03 in the bottles of sawdust and cultivated. The temperature of the 3Sep.27375♀ 78.9 11.13 cultivation room was set at 25°C, and the humidity was 4Sep.28376♀ 79.2 12.30 maintained under 70 %. The growth period was 20 days and 5Sep.28377♀ 90.3 18.75 the medium was cultivated in the dark. 6Oct.01380♀ 80.8 13.30 7Oct.01380♀ 84.3 13.23 Mean (±SE) 375.7 82.57 13.40 Rearing (±1.5) (±1.54) (±0.94) We collected 53 larvae at the 1st instar stage from the wood in which eggs had been laid. The larvae were immediately measured (Mitutoyo digital calipers; Mitutoyo, Kawasaki, the pre-pupation period (Fig. 3), although there was no Japan) and weighed (Shimadzu EL200 electronic balance; decrease in head capsule size (Fig. 4). The relationship Shimadzu, Kyoto, Japan). The larvae were individually placed between head capsule size and body length was proportional into wide mouthed (17.4 mm in diameter) glass vials (Fig. 5). On December 1, 2016, the survival rate of the (26.6 mm in diameter, 102 mm in length) filled with cultivated remaining 42 larvae was 100 %. sawdust medium, and reared in a growth chamber (Sanyo The earliest male emerged 11 days before the earliest MLR-351H; Sanyo, Osaka, Japan). The larvae were allowed female. After the emergence of four males, seven females to grow for 2 months at 25°C and 65 % RH, and a photoperiod of L:D 0:24. After 2 months, the larvae were transferred from the glass vials to the polypropylene bottles (1,400 mL) that had been filled with the sawdust cultivated PDA medium. At bi-monthly intervals, all the larvae were observed, measured, and transferred to a new bottle of sawdust.

Results Of the 53 larvae reared, 11 adults (20.8 %) emerged in September 2016 after approximately 1 year (Tables 1 and 2). It is noteworthy that during the larval development, there was a marked increase in growth over the first 6 months, particularly with regard to the larval weight (Fig. 2). It was also observed that growth rates in terms of length and weights Figure 3 Growth rate of Callipogon relictus in terms of the body length peaked at the 10th month and then gradually decreased during of larvae.

Figure 4 Growth rate of Callipogon relictus in terms of the head capsule width of larvae. Figure 6 The largest male of Callipogon relictus, measuring 106.8 mm emerged on September 16, 2016, approximately 1 year after commencing the rearing of larvae on an artificial fungal diet.

had emerged by October 1, 2016. Thus, under the experimental conditions used in this study, the period of larval growth was approximately 1/6 of that occurring in the field, where the larval stage of C. relictus takes 5 to 6 years to complete development (Li et al. 2012). It is also less than approximately 1/3 of the larval growth periods observed in other studies in which larvae had been reared in decayed wood or in sawdust with nutrient supplimentation (Fig. 7). Previous studies have indicated that at room temperature the average period from oviposition to egg hatching in C. relictus is 12 (±2 SE) days, and that the pupal period is 25 (±5 SE) days (Yi 2014). It is thus estimated that the duration of the larval period recorded in this study was 333–355 days, if we assume that eggs were deposited immediately after mating had been Figure 5 Relationship between head capsule width and body length of Callipogon relictus larvae. observed on August 11, 2015. This indicates that we could markedly shorten the larval period to just a single year under laboratory conditions by feeding the larvae with a fungal diet. emerged (Tables 1 and 2). The sex ratio of females to males It is also worth noting that the death rate of larvae reared on was thus 6.4:3.6. fungal diet is markedly lower than that observed in The largest adult male measured 106.8 mm in body length (Fig. 6), and three of the four males exceeded 100 mm in length (Table 1). The average size of the seven females was 82.57 ± 1.54 mm (Table 2).

Discussion In this study we aimed to shorten the larval period and to enlarge the adult size of C. relictus by rearing the larvae of this species on an artificial diet of fungal mycelia for the first time. The results indicate that C. relictus is primarily fungivorous, like many other wood-boring saprophagous Coleoptera that spend their larval period inside dead or decaying wood feeding on fungal mycelia. Of the 11 adults that emerged, Figure 7 Comparison of larval periods of Callipogon relictus reared on the first emerged on September 10, 2016, and all 11 adults various diets.

Table 3 The size data of C. relictus grown in nature and collected during the previous century in Russia

Female (I) Female (I-cont.) Male

Coll. date Body length (mm) Stored Coll. sate Body length (mm) Stored Coll. date Body length (mm) Stored

1898.12 81.56† ZIN 2002.07 74.54 IBSS 1902. 74.24 ZIN 1898.12 64.48 ZIN 2007.07 79.90 IBSS 1910.07 71.63 ZIN 1899.12 67.89 ZIN 2008.07 64.24 IBSS 1910.08 85.14 ZIN 1908.07 75.89 ZIN 2008.07 67.78 IBSS 1926.08 84.10 ZIN 1910.07 72.00 ZIN 2008.07 68.10 UNR 1926.08 77.99 ZIN 1910.07 76.58 ZIN 2008.07 69.43 UNR 1926.08 68.94 ZIN 1910.07 66.87 ZIN 2008.07 89.41 UNR 1926.08 30.80 ZIN 1910.07 64.44 ZIN 2009.08 60.79 UNR 1930.08 72.69 ZIN 1910.07 74.36 ZIN 2009.07 68.55 UNR 1934.08 85.15 IBSS 1910.07 90.12 ZIN 2009.08 71.08 UNR 1935.07 96.72 ZMMU 1911.06 66.01 ZMMU 2009.08 69.32 UNR 1968.10 88.81 ZIN 1911.06 68.88 ZIN 2009.08 65.87 UNR 1972.08 89.15 ZIN 1911.07 55.56 ZIN 2010.08 69.50 UNR 1973.08 93.05 ZMMU 1911.07 54.78 ZIN 2010.08 69.39 UNR 1971–73 88.91 ZIN 1911.07 81.12 ZIN 2011.08 78.80 UNR 1972–73 90.30 ZIN 1911.07 59.63 ZIN 2011.08 80.10 UNR 1979.07 92.78 ZMMU 1912.06 78.36 ZIN 2012.08 67.40 UNR 1982.07 98.99 ZMMU 1912.07 74.14 ZIN 2013.07 80.20 IBSS 1983.08 88.01 ZMMU 1912.08 54.78 ZIN 2013.07 69.23 UNR 1986.08 92.09 ZIN 1912.08 74.53 ZIN 2013.07 74.56 UNR 2013.07 97.70 UNR 1916.07 87.98 ZIN 2013.07 77.89 UNR 2013.07 95.01 UNR 1926.08 77.99 ZIN 2013.07 76.80 UNR 1926.08 77.50 ZIN 2013.07 78.90 UNR 1926.08 58.97 ZIN 2013.07 88.90 UNR 1926.08 67.40 ZIN 2013.07 78.99 UNR 1926.08 67.99 ZIN 2013.07 86.70 UNR 1926.08 70.99 ZIN 2014.07 80.50 UNR 1926.08 67.59 ZIN 2014.07 73.81 UNR 1929.05 77.71 ZIN 2014.07 88.05 UNR 1937.08 68.90 IASE 2016.08 84.99 UNR 1937.08 76.58 IASE 2016.08 77.90 ZMMU 1968.08 70.50 IASE No label 79.12 ZMMU 1971.06 65.65 IASE No label 69.42 ZMMU 1971.08 62.21 IASE No label 69.23 ZMMU 1971–73 77.20 IASE No label 85.69 ZIN 1972–73 78.63 IASE No label 79.25 IASE 1978.08 61.82 IBSS No label 79.10 IBSS 1979.08 73.12 IBSS No label 78.99 IASE 1985.08 81.10 ZMMU No label 79.96 IASE 1985.08 68.85 ZMMU 1993.08 85.14 IASE Mean (±SE) 75.70 (±1.16) Mean (±SE) 83.91 (±3.28)

†Holotype IASE, Institute of Animal Systematics and Ecology, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia. IBSS, Institute of Biology and Soil Science, Far East Branch of the Russian Academy of Sciences, Vladivostok, Russia. UNR, Ussuri Nature Reserve, Far East Branch of the Russian Academy of Sciences, Ussuriysk, Russia. ZIN, Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia ZMMU, Zoological Museum of M.V. Lomonosov State University, Moscow, Russia.

Entomological Research 47 (2017) 373–379 377 © 2017 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd D.-A. Yi et al. experiments using raw sawdust under semi-field conditions Acknowledgments (Li et al. 2012), and also lower than that when using sawdust The authors express their sincere gratitude to Dr. Sci. B.A. with nutrient supplimentation in the laboratory (Yi 2014). Korotyaev (Zoological Institute of the Russian Academy of During the rearing period in this study for more than 14 months Sciences, St. Petersburg, Russia), Dr. A.A. Gusakov by December 1, 2016, no larvae were found dead at (Zoological Museum of the Moscow State University, bi-monthly observations. Moscow, Russia), and Prof. Dr. Sci. A.S. Lelej (Institute of The emergence order of the different sexes recorded in Biology and Soil Science, Far East Branch of the Russian the present study is consistent with the observation that Academy of Sciences, Vladivostok, Russia) for providing males appear 13–20 days earlier than females in a natural the opportunity to study materials on C. relictus. The present environment (Lyubarsky 1953): the earliest male emerged study was conducted as a part of collaborative project between 11 days before the earliest female. Although the number the Research Center of Natural Monument Insects, Yeongwol, of adults that emerged in the present study was small, Korea, and Ussuri Nature Reserve, Far East Branch of the the sex ratio (female:male 6.4:3.6) is somewhat similar to Russian Academy of Science with the support of both that recorded based on a collection established over the Yeongwol County and the Cultural Heritage Administration, course of more than a century in Russia (female:male Korea. The work of A.V. Kuprin was funded by Russian 7.9:2.1) in terms of female dominant phenomenon Fund for Basic Research under the research project No. (Table 3). With respect to body size, rearing larvae on fungal mycelia 16-34-00323. proved highly effective, producing adult males exceeding 100 mm in length and females with lengths exceeding 80 mm following a larval development period of References approximately 1 year. On the basis of our exhaustive survey Akutsu K, Honda K, Arai S (1980) Mass rearing method of the udo of the entire specimen data for C. relictus collected since longicorn beetle, Acalolepta luxuriosa Bates (Coleoptera: 1898 (Semenov 1899), which are currently stored at five Cerambycidae) on an artificial diet. Japanese Journal of Applied scientific institutions located in the Russian Federation Entomology and Zoology 24:119–121. 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