Distribution pattern of types symbiotic with the Title -growing 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 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 (subfamily ) 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 -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: , fungus farming, mutualism, social ,

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 (, , ),

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, 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 leaves, on Iriomote Island in 2017 (Table

111 S1). The DNAs from the guts of workers were extracted using PowerSoil DNA Isolation Kit

112 (Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. The nuclear rDNA including

113 ITS regions were amplified by PCR using PrimeSTAR HS DNA Polymerase (Takara Bio, Shiga,

114 Japan) with the aforementioned primer sets. The reaction was performed under the following

115 conditions: 35 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 5 s, and extension

116 at 72°C for 1 min. The PCR products were electrophoresed in a 2% agarose gel including

117 Midori Green Advance DNA Stain (Nippon Genetics, Tokyo, Japan), and the UV-

118 transilluminated gels were photographed using the ChemiDoc Touch Imaging System (Bio-Rad,

119 Hercules, CA, USA). Agarose gel electrophoresis of PCR products using a termite gut

120 metagenome sample as a template revealed the presence of one clear band in each well (Fig. 1).

121 The bands were of two sizes: one approximately 670 bp and the other approximately 550 bp.

122 This result was obtained using DNA from the guts of major workers, and I was able to obtain

123 the same results using DNA from the whole bodies of major workers, minor workers, and

124 soldiers and the guts of alates of both sexes. To confirm the Termitomyces types of each band,

125 the DNA sequences of the amplified products were also determined. The purified PCR products

126 were used as templates for direct sequencing reactions, which were performed by the dideoxy-

127 nucleotide cycle sequencing procedure using a BigDye Terminator v3.1 Cycle Sequencing kit

128 (Applied Biosystems, Foster City, CA, USA). Data collection was performed using an

129 automatic DNA sequencer, ABI PRISM 3130xl Sequencer (Applied Biosystems). The

130 determined Termitomyces rDNA including ITS sequences from termite guts were deposited in

131 the DDBJ, EMBL, and Embank databases (accession nos. LC421530–LC421538, LC459621–

132 LC459623 (Table 1 and S1)). From the ITS sequences, the samples of the longer bands were

133 very similar to those of Termitomyces sp. Type A registered by Katoh et al. (2002) (accession 134 nos. AB051879, AB051880, AB051882, AB051883 and AB051886) (Fig. S2), whereas the

135 samples of the shorter bands were almost identical to those of Termitomyces sp. Type B

136 (accession nos. AB051881, AB051884, AB051885, AB051887 and AB051890) (Fig. S3).

137 These results concluded that the longer band originated from Termitomyces sp. Type A and the

138 shorter band originated from Termitomyces sp. Type B.

139 To investigate the distribution pattern of Termitomyces types cultivated by O. formosanus

140 on Okinawa Island, O. formosanus were collected from 81 colonies at 25 sites around Naha and

141 Urasoe cities in 2015 and 2017 (Table S2). Because it is difficult to define the range of an O.

142 formosanus colony, I considered that the two colonies are different if the distance between the

143 two collection points was more than 30 m. The GPS coordinates of the collection points were

144 recorded by Oregon 550TC (Garmin, Switzerland). Figure 2 shows the collection sites of

145 termites and ratio of Termitomyces types at Naha and Urasoe cities in Okinawa Island. The

146 diagnostic PCR results indicated that the habitat of Termitomyces sp. Type B expanded

147 throughout the whole habitat of O. formosanus on Okinawa Island. However, the habitat of

148 Termitomyces sp. Type A was restricted at site no. 11, which is a mountainous area of Sueyoshi

149 Park (Fig. 2). The distribution of Termitomyces sp. Type A at this site expanded throughout at

150 the site no. 11. A previous study suggested that only Termitomyces sp. Type A is distributed in

151 Okinawa Island (Katoh et al. 2002). They analyzed only one fungus comb owing to the

152 sampling problem and found that the colony possessed Termitomyces sp. Type A. My findings

153 indicate that O. formosanus on Okinawa Island cultivate Termitomyces sp. Type A and

154 Termitomyces sp. Type B, the same as on Iriomote Island. It is most likely that O. formosanus

155 were introduced to site no. 11 from Iriomote Island because Termitomyces sp. Type A is limited

156 at this site.

157 As O. formosanus is also distributed in Southeast Asia, Taiwan, and southern mainland

158 China (Ahmad 1965; Cheng et al. 2007; Chiu et al. 2010), to confirm that O. formosanus in 159 Okinawa Island are derived from Iriomote Island, molecular phylogenetic analysis of host

160 termite was conduced. The reference mitochondrial cytochrome oxidase subunit II (COII) gene

161 sequence for O. formosanus were obtained from the National Center for Biotechnology

162 Information (NCBI) nucleotide database. Sequences for Odontotermes hainanensis (accession

163 no. EU253896) and Odontotermes longignathus (accession no. AB051877) were also included

164 as outgroups. Alignment was conducted using the Clustal W program package (Thompson et

165 al., 1994). The phylogenetic relationships were inferred using the maximum likelihood method

166 based on the Hasegawa-Kishino-Yano model (Hasegawa et al. 1985). The analysis involved 18

167 nucleotide sequences. All positions containing gaps and missing data were eliminated. The final

168 dataset included 507 positions. Evolutionary analyses were conducted using MEGA7 (Kumar

169 et al., 2016). Phylogenetic analysis of O. formosanus indicated no differences in COII

170 sequences between the samples from Okinawa Island and the Yaeyama Islands (Iriomote and

171 Ishigaki islands), while the sequences were largely different between Japanese O. formosanus

172 and Chinese or Taiwanese O. formosanus (Fig. S4). This also supports that O. formosanus on

173 Okinawa Island was introduced from Iriomote Island.

174 O. formosanus have been distributed on Okinawa Island for at least 100 years (Nawa 1914).

175 In the last century, several kinds of trees have been introduced to Okinawa Island from the

176 Yaeyama Islands (Ocean Expo. Commemorative Park Management Foundation 1997). For

177 example, Satakentia liukiuensis, a species of palm tree endemic to Iriomote Island and Ishigaki

178 Island (Johnson 1998), is found in Sueyoshi Park (Kuroshima 1974). Sexual spores discharged

179 from the fruiting body of Termitomyces are necessary for the inoculation of fungi in the fungus

180 comb in the nest of the next generation nest of Odontotermes (Johnson et al. 1981; Sieber 1983;

181 Korb & Aanen 2003). The worker caste, which first fledges from the incipient nest, carries the

182 sexual spores of the Termitomyces species into their fungus comb from outside to initiate fungi

183 cultivation. The dispersion of termites is normally limited to the distance that a termite alate 184 can fly (Nutting 1969). However, in the case of fungus-growing termites, symbiosis with

185 Termitomyces will be unsuccessful if sexual spores are absent near their nesting site. Therefore,

186 the distance of O. formosanus dispersion is thought to be connected to the scattering distance

187 of fungal spores and the distance that the termite can fly. At least two O. formosanus nests,

188 including a fungus comb of Termitomyces sp. Type A and Termitomyces sp. Type B with a queen

189 and king, must have also been introduced as a result of planting. The findings of the present

190 study suggest that O. formosanus and Termitomyces on Okinawa Island were introduced in the

191 Sueyoshi Park from the Iriomote Island and that only Termitomyces sp. Type B was dispersed

192 from this site. This may possibly be related to the size of the fruiting body and the number of

193 Termitomyces spores that were dispersed as it was recently reported that the fruiting body of

194 Termitomyces sp. Type A is very small (Hojo & Shigenobu 2019). Future studies on fungal

195 spore dispersion will provide us with more information about fungus-growing termite

196 dispersion.

197

198 ACKNOWLEDGMENTS

199 I thank Prof. Mitsuru Moriguchi (Okinawa University) and Haruhiko Fujii (facility staff of

200 Sueyoshi Park ‘Mori no Ie Minmin’) for a great deal of advice about planting on Okinawa. This

201 work was financially supported by a JSPS KAKENHI (No.15K07798) to M.H. provided by the

202 Ministry of Education, Culture, Sports, Science and Technology, Japan.

203

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317 SUPPORTING INFORMATION

318 Figure S1 Comparison of primer regions for nuclear ribosomal DNA sequences consisting of

319 the internal transcribed spacers and 5.8S rDNA of Termitomyces species and other fungi

320 associated with fungus-growing termite nests. Dots indicate identical nucleotides with primers.

321 Non-identical nucleotides with primers are highlighted in gray boxes.

322 Figure S2 Nucleotide sequence alignment of nuclear ribosomal DNA sequences including ITS

323 regions of Termitomyces sp. Type A.

324 Figure S3 Nucleotide sequence alignment of nuclear ribosomal DNA sequences including ITS

325 regions of Termitomyces sp. Type B.

326 Figure S4 Maximum likelihood analysis of Odontotermes formosanus based on the

327 mitochondrial cytochrome oxidase subunit II (COII) gene sequence. Bootstrap values > 50%

328 are shown above or below the nodes. There were no sequence differences between the three

329 Islands in Japan.

330 Table S1 Collection sites of host termites on Iriomote Island used for the development of

331 diagnostic PCR.

332 Table S2 Collection sites of host termites on Okinawa Island used for diagnostic PCR.

333 334 Figure legends

335 Figure 1 PCR amplification using the termite gut metagenome as a template. The left and the

336 right lanes show the 100 bp DNA ladder marker (M). One band different in size is shown in

337 each sample. Samples are described in Table 1.

338 Figure 2 Collection sites of Odontotermes formosanus on Okinawa Island and the ratio of

339 Termitomyces types symbiotic with O. formosanus.

340