Distribution Pattern of Termitomyces Types Symbiotic with the Title Fungus-Growing Termite Odontotermes Formosanus on Okinawa Island( Text 全文 )
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Distribution pattern of Termitomyces types symbiotic with the Title fungus-growing termite Odontotermes formosanus on Okinawa Island( Text_全文 ) Author(s) Hojo, Masaru Citation Entomological Science, 22(4): 398-403 Issue Date 2019-10-09 URL http://hdl.handle.net/20.500.12000/47726 Rights 1 Distribution pattern of Termitomyces types symbiotic with the fungus-growing termite 2 Odontotermes formosanus on Okinawa Island 3 4 Masaru Hojo 5 University of the Ryukyus, Senbaru 1, Nishihara, Okinawa 903-0213, Japan 6 7 Correspondence: Masaru Hojo, University of the Ryukyus, Senbaru 1, Nishihara, Okinawa 8 903-0213, Japan. 9 E-mail: [email protected] 10 11 Abstract 12 Fungus-growing termites (subfamily Macrotermitinae) cultivate the symbiotic basidiomycete 13 fungus Termitomyces in their fungus comb to digest cellulosic materials and to supply nitrogen- 14 rich fungal diet. In Japan, the fungus-growing termite Odontotermes formosanus is found on 15 the Yaeyama Islands and Okinawa Island, Okinawa Prefecture. O. formosanus is thought to 16 have been recently and artificially introduced to Okinawa Island as its distribution is 17 discontinuous and restricted to small areas. Previous DNA analyses revealed that two types of 18 Termitomyces, namely Termitomyces sp. Type A and Termitomyces sp. Type B, whose fruiting 19 bodies correspond to Termitomyces microcarpus-like pseudorhiza-lacking small mushroom and 20 Termitomyces intermedius, respectively, are cultivated by O. formosanus on the Yaeyama 21 Islands. However, information about the Termitomyces types cultivated by O. formosanus on 22 Okinawa Island is limited. To define the fungal types cultivated by O. formosanus on Okinawa 23 Island, I developed a diagnostic PCR method using primer sets specific to the nuclear ribosomal 24 DNA sequences consisting of the internal transcribed spacers (ITS1 and ITS2) and 5.8S rDNA 25 of Termitomyces not using fungal mycelium, but using the termite gut metagenome including 26 fungal DNA as a template. The results demonstrated that the same two Termitomyces types from 27 Iriomote Islands are cultivated by O. formosanus in Okinawa Island. The distribution pattern 28 of Termitomyces types on Okinawa Island showed that Termitomyces sp. Type A is limited to 29 the mountainous side of Sueyoshi Park, despite Termitomyces sp. Type B being widely 30 distributed in the area in which O. formosanus is found. This finding implies that O. formosanus 31 on Okinawa Island was recently introduced from Iriomote Islands to Sueyoshi Park. 32 Key words: Blattodea, fungus farming, mutualism, social insect, Termitidae 33 34 Many termites harbor symbiotic microorganisms inside their bodies, such as in the hindgut, to 35 aid the digestion of primarily cellulosic materials (Bignell 2000). By contrast, fungus-growing 36 termites (subfamily Macrotermitinae), a derived termite group, digest cellulosic materials with 37 the aid of basidiomycete fungi Termitomyces (Agaricomycetes, Agaricales, Lyophyllaceae), 38 located outside of their bodies, along with gut bacteria (Sands 1969; Wood & Thomas 1989; 39 Rouland-Lefèvre et al. 2006; da Costa et al. 2019). In these termites, worker termites, which 40 are the sterile helper caste in the termite society, construct fungus combs in their nest using their 41 particular feces evacuated by young workers, which contain incompletely digested plant 42 materials (Badertscher et al. 1983). The workers manage the fungus combs and the mass of 43 white fungus mycelium, called nodule, on the fungus combs (Sieber & Leuthold 1981; Gerber 44 et al. 1988). Then, the termites consume the nodules and old fungus combs in which the 45 cellulosic component has been degraded by the Termitomyces mycelium (Badertscher et al. 46 1983; Darlington 1994; Bignell 2000; Rouland-Lefèvre 2000). Finally, termites decompose 47 oligosaccharides and fungal biomass with the aid of their gut bacterial enzymes (da Costa et al. 48 2019). Termitomyces also contributes to provide nitrogen-rich fungal diet to the termites (Hyodo 49 et al. 2003; Sapountzis et al. 2016). This ectosymbiosis creates an obligate mutualistic 50 relationship; that is, it is impossible for one species to survive without the other (Darlington 51 1994). 52 Till date, the fruiting bodies (mushrooms) of approximately 30 species of Termitomyces 53 have been described worldwide based on the morphology of the basidiocarp and basidiospore 54 (Frøslev et al. 2003; Kirk et al. 2008). Termitomyces are primarily divided into the following 55 two subgenera based on the presence or absence of pseudorhiza, a plant root-like structure 56 connected to the fungus comb (Frøslev et al. 2003; Wei et al. 2009; Tibuhwa 2012): 57 Praetermitomyces, the member of which has a very small fruiting body and lacks pseudorhiza, 58 and Eu-termitomyces, members of which have large fruiting bodies and possess pseudorhiza. 59 Only the species Termitomyces microcarpus has belonged to Praetermitomyces. 60 In Japan, the fungus-growing termite Odontotermes formosanus is distributed on the 61 Yaeyama Islands and Okinawa Island, Okinawa Prefecture (Ikehara 1966; Yasuda et al. 2000). 62 It is also distributed in Southeast Asia, Taiwan, and southern mainland China (Ahmad 1965; 63 Cheng et al. 2007; Chiu et al. 2010). Okinawa Island is the most eastern habitat of O. 64 formosanus. However, as this termite is discontinuously distributed in a small area around Shuri 65 district, it is believed to have been artificially introduced to Okinawa Island. In addition to host 66 termite information, three nomina of Termitomyces fruiting bodies have been identified via 67 morphological description (Otani 1979; Otani & Shimizu 1981; Otani 1982; Takahashi & 68 Taneyama 2016). They all possess pseudorhiza, and are identified as Termitomyces eurrhizus 69 (Berk.) R. Heim, Termitomyces clypeatus R. Heim, and Termitomyces intermedius Har. Takah. 70 & Taneyama, respectively. Recently, Hojo and Shigenobu (2019) were the first to describe 71 pseudorhiza-lacking fruiting body of Termitomyces in Japan. This T. microcarpus-like fruiting 72 body and the previously identified three nomina of Termitomyces fruiting bodies are largely 73 different considering the size of the pileus (Hojo and Shigenobu, 2019). Apart from basidiocarp 74 morphology-based classification, DNA analysis of host termite and fungal mycelium cultured 75 on the fungus comb of O. formosanus indicate that Japanese O. formosanus, which is the only 76 host termite of Termitomyces in Japan, cultivate two types of Termitomyces, which have been 77 termed Termitomyces sp. Type A and Termitomyces sp. Type B (Katoh et al. 2002). Their survey 78 indicated that Termitomyces sp. Type A is distributed in Iriomote, Ishigaki, and Okinawa islands 79 while Termitomyces sp. Type B is restricted to Iriomote Island. DNA analysis revealed that the 80 fruiting body of Termitomyces sp. Type A is T. microcarpus-like pseudorhiza-lacking small 81 mushroom (Hojo & Shigenobu 2019), whereas that of Termitomyces sp. Type B is T. 82 intermedius (Takahashi & Taneyama 2016). 83 To hypothesize the migration process of O. formosanus to Okinawa Island, the 84 identification of the Termitomyces types that are cultivated is also indispensable. However, 85 information about the Termitomyces types cultivated by O. formosanus on Okinawa Island is 86 limited. Species classification of Termitomyces by molecular analysis of nuclear ribosomal 87 DNA (rDNA) sequences consisting of the internal transcribed spacers (ITS1 and ITS2) is very 88 useful (Siddiquee et al. 2015). However, it is difficult to obtain fungal DNA from fungus combs 89 because O. formosanus do not construct conspicuous mounds above ground; rather, they 90 construct fungus combs underground with no signs on the soil above. Furthermore, the time 91 frame for collecting fungal DNA from the fruiting body of Termitomyces is very short because 92 the fruiting body of this genus is seasonal and highly perishable. By contrast, it is easy to collect 93 host termites because the workers of O. formosanus make conspicuous foraging tunnels on the 94 ground and trees around their habitat during all seasons. Therefore, I developed a diagnostic 95 PCR method to distinguish the two types of Japanese Termitomyces using primer sets specific 96 to the rDNA including ITS regions of Termitomyces and the termite gut metagenome including 97 fungal DNA as a template. 98 To develop a diagnostic PCR method for distinguishing the two types of Termitomyces 99 ingested by termites, the primers were designed with consideration of the following points: 1) 100 the size of the amplified product should be very different between Termitomyces sp. Type A and 101 Termitomyces sp. Type B, and this difference can be detected through agarose gel 102 electrophoresis of PCR products; 2) as there are various fungi growing on the fungus comb, 103 such as Xylaria (Sands 1969; Moriya et al. 2005; Rogers et al. 2005; Ju & Hsieh 2007; Okane 104 & Nakagiri 2007; Visser et al. 2009), fungi other than Termitomyces should not be amplified; 105 and 3) all Termitomyces species registered in the database can be amplified. The primer 106 sequences for rDNA including ITS regions were 5′-CTGCGGAAGGATCATTATTGAA-3′ 107 (forward) and 5′-CCTGATTTGAGGTCAAATGGTC-3′ (reverse). Figure S1 shows sequence 108 alignments of the primer regions of Japanese Termitomyces types and other fungi associated 109 with fungus-growing termite nests. The workers of the host termite O. formosanus were 110 collected from foraging sites, such as dead trees or