Jpn. J. Environ. Entomol. Zool. 28（2）：71－77 （2017） 環動昆 第 28 巻 第 2 号：71－77 （2017） Original Article

The first record of Antennopsis gallica Buchli and Heim, an ectoparasitic fungus on the speratus (Kolbe) in Japan

Ikhsan Guswenrivo1) 2) , Hiroki Sato3), Izumi Fujimoto1), Tsuyoshi Yoshimura1)

1) Lab. Innovative Humano-habitability, Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan 2) Research Center for Biomaterials, Indonesian Institute of Sciences, Jl. Raya Bogor KM.46, Cibinong, Bogor, West Java 16911, Indonesia 3) Department of Forest Entomology, Forestry and Forest Product Research Institute, Matsu-no-sato 1, Tsukuba, Ibaraki 305-8687, Japan

（Received: June 8, 2017；Accepted: July 3, 2017）

Abstract The ectoparasitic fungus Antennopsis gallica Buchli and Heim was found from three colonies of Reticulitermes speratus (Kolbe) collected from Uji, Kyoto Prefecture. This is the first record in Japan of this species. The infection rate was 17.8–25.0% in workers and 0–10% in soldiers. On average, 7.0–7.7 thalli were found per individual. The fungus grew on any surface of the and showed no preference for particular body parts.

Keywords : Ectoparasitic fungi, Antennopsis gallica, Reticulitermes speratus

Introduction behavioral deficiencies, but also cause disease syndromes and even death through slow-acting mechanisms (Weir and As social , termites live in the soil where various Blackwell, 2005). In Japan, more than one hundred species of microorganisms such as fungi, bacteria and viruses are present. ectoparasitic fungi have been reported, mainly from High population density, nest environments and social behavior beetles; until 2001, none of these fungi had yet been reported are the variables that promote the infection of termites by associated with termites (Sugiyama, 1973; Lee and Sugiyama, microorganisms (Sands, 1969; Blackwell and Rossi, 1986; 1986; Terada, 1996; 1998; 2000; 2001). The first report about Rosengaus et al., 2003; Hughes et al., 2008). The presence of ectoparasitic fungi on termites in Japan was made by Hojo et al. microorganisms such as fungi within termite colony may affect (2001 and 2002). They found three types of Termitaria spp. on the termite life. Both symbiotic and pathogenic fungi live with the body surface of Hodotermopsis japonica Holmgren, termites. Termite–fungus symbiosis has been a target of many Reticulitermes speratus (Kolbe), and Coptotermes formosanus termite researchers (Hyodo et al., 2000; Matsuura et al., 2009; Shiraki from 5 localities in Japan. Rosengaus et al., 2011). Pathogenic fungi, particularly In all, 22 species from 9 genera of ectoparasite fungi have endoparasitic fungi, have been evaluated for their possibility as been reported on termites (Blackwell and Rossi, 1986), and biocontrol agents (Culliney and Grace, 2000; Chouvenc et al., one of the most termite-specific genus is Antennopsis. In 1952 2011). Buchli and Heim described Antennopsis gallica on An ectoparasite is a parasite that lives on the outer body Reticulitermes lucifungus Rossi in southern France, proposed surface or in the skin of , and numerous fungal the genus Antennopsis, and placed it in a new order, ectoparasites have been reported. They grow on the host skin Gloeohaustoriales, of the class Hyphomycetes (Buchli, 1951). and may not only impede the host’s typical activity and induce Presently the genus Antennopsis contains three species:

Corresponding author: ikhsan [email protected] - 71 - Guswenrivo et al.

Antennopsis gallica Heim and Buchli, A. grassei Buchli and A. measured. Five hundred workers and twenty soldiers were gayi Buchli. Each species of Antennopsis is specific to a chilled by a refrigerator and examined for infection by different termite species. Buchli (1960b) discovered A. grassei ectoparasitic fungus by observing them individually under a on Neotermes amplus Sjost, Neotermes desneuxi Sjost and dissecting microscope (S8APO, Leica, Wetzlar, Germany). Glyptotermes longiceps Paulian from Madagascar. Antennopsis gayi was found on Coptotermes elisae Desneux and C. Morphological identification obiratus Hill from New Guinea (Buchli, 1966). Antennopsis The infected termites were separated and kept at 4oC prior to grassei and A. gayi occurred on termites in Madagascar, South identification. Mounting samples were prepared for America, Africa and Asia (Blackwell and Rossi, 1986). morphological identification. An infected body part of R. Antennopsis gallica was found on Kalotermes flavicolis speratus was dissected using an entomological pin (5ST Fabricius, R. virginicus Banks and R. flavipes (Kollar) in Italy, 11252-00, Dumont, Heidelberg, Germany) and mounted with USA, Canada, and Brazil (Buchli, 1960b; Gouger and lactophenol solution. The fungus was observed using a light Kimbrough, 1969; Blackwell, 1980; Blackwell and Rossi, microscope (BX51, Olympus, Tokyo) and a digital microscope 1986; Myles et al., 1998; Blackwell and Kimbrough, 1978). (VHX-5000, Keyence, Osaka, Japan) for the identification Arai et al. (2009) presented the Antenopsis sp. from process. Reticulitermes speratus colonies in Japan, but the identification of the species was not done yet. Infection rate and fungal distribution on the body surface In the present study, we sought to find Antennopsis species Infection rates of 500 workers and 20 soldiers were counted associated with Reticulitermes speratus (Kolbe), which is a by counting the numbers of thalli per individual termite, and termite widely distributed in Japan and constitutes a major pest recording the infected body parts. there. Results and Discussion Material and Methods Morphological identification Sampling and preparation for observation The ectoparasite fungus was found from all the 3 colonies of Termites were collected from Uji City, Kyoto Prefecture, R. speratus. It showed three major thallus parts: namely, a Japan (34°53'11.5" N 135°49'3.1" E) on September 5, 2015. holdfast (H), conidiophores (C) and conidial head (Ch) (Fig. Three colonies of termite were collected from dead tree 1). The holdfast (H) has an ellipse shape 21.49 μm in length branches on the forest ground, and were wrapped with paper and 8.90 μm in width on average. As the part that attaches to and kept inside plastic bags, then brought back to the the termite cuticle (Tc), it consists of four cells with an average laboratory. The weather conditions during sampling were cell size of 5.17 μm. One or two conidiophores (C) were

Fig 1. Antennopsis gallica Heim and Buchli. (a) A thallus growing on the leg (red circle) of a Reticulitermes speratus

worker. (b) A thallus from the holdfast with an immature conidial head. (c) Two thalli from the holdfast, with one thallus releasing spores (Sp). (d) Different focal planes of figure (c). Termite cuticle (Tc), holdfast (H), conidiophore (C), and conidial head (Ch).

- 72 - The first record of Antennopsis gallica commonly observed from the two-center holdfast cells, and The distribution of A. gallica covers a wide geographical they were 104.12 μm in length and 5.10 μm in width. They range with different termite species as hosts: Antenopsis comprised 18–27 cells with strong constriction at the base. The gallica was found on R. lucifungus santonensis later conidial head (Ch) was 23.18 μm in length by 8.92 μm width synonymized with R. flavipes ; Austin et al., 2005 in France on average, comprising 3–10 cells, and showing an oval shape (Heim, 1951), on R. virginicus and R. flavipes in Florida at maturity. Spherical spores ca. 3–4 μm in diameter were (Gouger and Kimbrough, 1969), Georgia (Blackwell, 1980), released from the conidial head. These morphological Wisconsin and Michigan (Blackwell and Rossi, 1986), on characteristics clearly suggested that the fungus was A. gallica, Reticulitermes sp. in Lousiana (Blackwell and Rossi, 1986), on although only two spores were observed. Kalotermes flavicollis in Yugoslavia (Buchli, 1969), on R. The morphology of A. gallica in the present study was lucifungus in Italy (Rossi and Cesari Rossi, 1977) and on R. similar to that first described by Buchli on the fungus obtained flavipes in Canada (Myles et al., 1998). The new record of A. from Reticulitermes lucifungus in France (Buchli, 1952). He gallica in Japan with a different host termite, R. speratus, described it as an extraordinary fungus with 3 major body further expands the known geographical range of the fungus. A structures, namely conidial head, conidiophores and holdfast country-wide survey is required to gain more information on (Buchli, 1960a). From his description, A. gallica has a its distribution in Japan. Also, in order to clarify the precise 23-μm-long ellipsoid holdfast and a 100- to 280-μm-long morphological development including infection process and conidiophore on average. In 1969, Gouger and Kimbrough lifecycle of A. gallica, artificial cultivation of the fungus is reported the existence of A. gallica on R. virginicus and R. indispensable. flavipes in and around Gainesville, Florida, USA. They measured the size of the holdfast (24–27 μm in length, 8–9 μm Infection rate width, and 6–6.5 μm height), the length of conidiophores The infection rate of A. gallica in 3 colonies of R. speratus (100–500 μm) and illustrated the fungus development stage in from Uji City was stable in 500 workers (17.8–25.0%) (Table detail. 1). For soldiers, a 0–10% infection rate (0–2 individuals) was A specimen of A. gallica was deposited at the National observed for all 3 colonies (Table 1). Myles et al. (1998) and Museum of Nature and Science, Japan with registration Buchli (1952) reported that nymphoids and soldiers were the number TNS-F-54006. castes of R. flavipes most heavily infected by A. gallica, but

Table 1 The infection rate of Antennopsis gallica in workers and soldiers of 3 colonies of Reticulitermes speratus.

Colony Observed termite number Infected termite (%)

number Workers Soldiers Workers Soldiers

C1 500 20 93 (18.6) 0

C2 500 20 125 (25.0) 2 (10)

C3 500 20 89 (17.8) 2 (10)

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Table 2 The number of Antennopsis gallica thalli recorded from the body

of workers of Reticulitermes speratus.

Colony Number of thalli per worker Average

Number 1–5 6–10 11–15 16–20 ≥21

C1 46 24 18 3 2 7.7

C2 53 21 37 10 4 7.0

C3 59 23 5 2 0 7.1

they did not show any comparable data on infection rates. Termites were collected in September, when the monthly Their results conflict with our observation with R. speratus that average temperature was 22.9oC with 70% relative humidity the workers had a higher infection rate than that of soldiers (RH). Generally, fungi can grow in a wide range of (Table 1). Our results might be explained by the high activity temperatures and RH, but their growth is affected by of workers compared to soldiers. Workers are the major caste environmental conditions. The observation of A. gallica on R. in termite colonies, and they do most of the duties in the speratus showed a lower infection rate in comparison with colony such as foraging, nursing and grooming. When they those of most records of ectoparasitic fungi on termites. Buchli forage outside the nest, they have more opportunities to come (1952) reported that he took his sample during winter and into contact with fungi and bring them back to the nest. found higher infection rates both per individual and per colony. Workers of R. speratus also engage in grooming activities, and The relatively low infection rates in the present study may they groom and clean themselves and nestmates including have a relation with the season. Further seasonal studies are soldiers (Wilson, 1971). This social behavior facilitates the needed to determine the effect of season on infection rate. transmission of ectoparasitic fungi even though grooming also Due to difficulties in observing A. gallica in a living and has the benefit in termites of conferring resistance and is an moving termite, host insects were examined after being killed. antifungal defense strategy (Boucias et al., 1996; Shimizu and Therefore, the observation of effects of A. gallica on termite Yamaji 2003; Yanagawa and Shimizu, 2005 and 2007). Further behavior such as movement, feeding activity, and grooming study of R. speratus colonies from other parts of Japan is was not available. In the present investigation, the visual required to discuss the caste-dependent infection trends. observation of infected termites showed no differences of body size and color from those of uninfected individuals. Myles et al. Distribution of the fungus on termite body surface (1998) also observed that there was no difference in size and Our observation showed that the thalli number on R. color of infected R. flavipes termites compared to speratus varied from 3 up to 50 per individual termite worker infection-free individuals. (Table 2). The thalli numbers were classified into 5 groups: Thalli of A. gallica did not restrict themselves to specific 1–5, 6–10, 11–15, 16–20 and ≥21 thalli/worker. The number of body sections of R. speratus. The fungal thalli of A. gallica thalli per termite showed a similar pattern in the 3 colonies. were found on every body part including head capsule, The majority of infected workers had 1–5 thalli, followed by antennae, mandible, thorax, legs, and abdomen. Of the 307 6–10 and/or 11–15; workers with 21 or more thalli showed the infected workers from 3 colonies of R. speratus, the legs lowest rate in all the colonies. The number of thalli on a single showed the highest thallus number followed by antennae, infected termite has a reported range of 1–35 (Gouger and abdomen, head capsule, mandible and thorax (Table 3). Kimbrough, 1969; Blackwell, 1980; Blackwell and Kimbrough, According to the report by Myles et al. on R. flavipes in 1978; Kimbrough and Gouger, 1970). Another report Canada (1998), A. gallica were found on many parts of the mentioned that each individual termite can be infested with up body, with the abdomen being the most infected area, followed to 150 thalli (Blackwell and Kimbrough, 1976). The highest by the thorax and the head. These results strongly suggest that number of fungal thalli was counted by Myles et al. (1998) on A. gallica does not show a preference for the particular body R. flavipes: 479 thalli on one infected termite. part of termites on which to grow.

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Table 3 Distribution of the fungus Antennopsis gallicaJ. in body parts of 307 workers of Reticulitermes speratus.

Location of fungi infection Colony Head Number Antenna Thorax Mandible Legs Abdomen capsule

C1 77 135 39 45 337 83

C2 79 145 45 58 452 96

C3 63 120 31 50 288 75

To clarify the biology of Antennopsis gallica, more data are Invert. Pathol. 35: 101–104. required. The latest report regarding A. gallica was published Blackwell, M. and J.W. Kimbrough (1976) A developmental in Canada as a new biodiversity report (Myles et al., 1998). study of the termite-associated fungus Coreomycetopsis Unfortunately, there are no comparable data regarding Oedipus. Mycologia 68: 551–558. environmental factors and infection rates. Country-wide and Blackwell, M. and J.W. Kimbrough (1978) Hormiscioideus seasonal surveys will be the next targets. In addition, the filamentous gen. et. sp. nov., A termite-infesting fungus artificial cultivation of A. gallica is indispensable to investigate from Brazil. Mycologia 70: 1275–1280. the behavioral effect of A. gallica on termites and to evaluate Blackwell, M. and W. Rossi (1986) Biogeography of fungal the possibility of using this widely distributed ectoparasitic ectoparasites of termites. Mycotaxon 25: 581–601. fungal group as a biocontrol agent. Boucias, D.G., C, Stokes, G. Storey, and J.C. Pendland (1996) The effects of imidacloprid on the termite Reticulitermes Acknowledgement flavipes and its interaction with mycopathogen Beauveria bassiana. Pflanzenschutz-Nachr. Bayer 49: 103–144. We thank Professor Junji Sugiyama and Professor Hiroyuki Buchli, H.H.R. (1952) Antennopsis gallica, a new parasite on Yano of the Research Institute for Sustainable Humanosphere termites. Trans. IXth Int. Congr. Entomol. 1: 519–524. (RISH), Kyoto University for the use of the lab equipment Buchli, H. (1960a) L’effect du champignon parasite during this study. This work was financially supported in part Antennopsis gallica sur les jeunes colonies de termites. C. by a Monbukagaku Sho (MEXT; Ministry of Education, R. Hebd. Séances Acad. Sci. 250: 1320–1231. Culture, Sports, Science, and Technology) Scholarship and a Buchli, H. (1960b) Une nouvelle espéce de champignon Japan Society for the Promotion of Science (JSPS) Kakenhi parasite du genre Antennopsis Heim sur les termite de grant, no. 15H04528. Madagascar. C. R. Hebd. Séances Acad. Sci. 250: 3365–3367. References Buchli, H.H.R. (1966) Notes sur les parasites fongiques des Isoptéres. Rev. Ecol. Biol. Sol. 3: 589–610. Austin, J.W., A.L. Szalanski, R.H. Scheffrahn, M.T. Messenger, Chouvenc, T., N.-Y. Su, and J.K. Grace (2011) Fifty years of S. Dronnet, and A.-G. Bafneress (2005) Genetic evidence attempted biological control of termites : Analysis of a for the synonymy of two Reticulitermes species: failure. Biol. Control 59: 69–82. Reticulitermes flavipes and Reticulitermes santonensis. Culliney, T.W., and J.K. Grace (2000) Prospects for the Ann. Entomol. Soc. Am. 98: 395–401. biological control of subterranean termite (Isoptera: Arai, K., R. Iwata and Y. Abe (2009) Discovery of an ), with special reference to Coptotermes ectoparasitic fungus Antennopsis sp. from Reticulitermes formosanus. Bull. Entomol. Res. 90: 9–21. speratus in Japan (in Japanese). Abstract of the 30th Gouger, R.J. and J.W. Kimbrough (1969) Antennopsis gallica anniversary conference the Society of Urban Pest Heim and Buchli (Hyphomycetes: Gloeohaustoriales), an Management Japan. entomogenous fungus on subterranean termites in Florida. Blackwell, M. (1980) New records of termite-infesting fungi. J. Invert. Pathol. 13: 223–228.

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ヤマトシロアリにおける外部寄生菌 Antennopsis gallica Buchli and Heim の本邦初記録

Ikhsan Guswenrivo・佐藤大樹・藤本いずみ・吉村 剛

京都府宇治市において野外採集したヤマトシロアリ 3 コロニーより外部寄生菌 Antennopsis gallica Buchli and

Heim が発見された. これは本邦における初記録である. 3 コロニーにおける感染率は職蟻が 17.8～25.0%, 兵蟻が

0～10%であった. １個体当たり平均 7.0– 7.7 個の感染が認められた. ヤマトシロアリ体表のどの場所にも感染が

観察され，特に嗜好性の高い部位は観察されなかった.

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