第32巻 第2号 日本線虫学会誌 20G2年12月 Life history of Rhabdontolaimus psacotheae (Diplogasterida: Diplogasteroididae) and its habitat segregation from Bursaphelenchus conicaudatus (Aphelenchida: Aphelenchoididae) Natsumi Kanzaki' and Kazuyoshi Futail1,2 The life history of Rhabdontolaimus psacotheae was examined and compared with that of a species of mycetophagous nematode Bursaphelenchus conicaudatus, which shares its vector and habitat with R. psacotheae . The third stage dauer juveniles of R. psacotheae enter thereproductive tracts of maleand female yellow-spotted longicom beetles, Psacothea hi- laris, and were transmitted from one Moraceae tree to another. The dauer juveniles of R. psacotheae enter a trees through the oviposition marks of their vector beetles, molt into adults then feed on bacteria proliferaing around the beetle tunnels made in the tree. Although, R. psacotheae was found to be sympatric to B. conicaudatus, R. psacotheae was segregated from B. conicaudatus because of the differences in the food resources in trees and in the or- gan of the vector beetle where they infest. Jpn. J. Nematol. 32 (2), 60-68 (2002) Keywords: bionomics, ecology, life cycle, Rhabdontolaimus, vector system. Two species of nematodes, Rhabdontolaimus psacotheae and Bursaphelenchus conicaudatus have been isolated from the yellow-spotted longicorn beetle, Psacothea hilaris, and its host trees of family Moraceae (Kanzaki et al., 2000; 2002). The life histories of B. conicaudatus and P. hilaris in western Japan are as follows (Kanzaki and Fu- tai, 2001). In early summer, adult beetles of P hilaris infested with dispersal fourth-stage juveniles of B. conicaudatus emerge and feed on the leaves of trees in the family Moraceae for maturation. After feed- ing, females mate and oviposit on their host trees. Nematodes invade their host trees through vector's ovi- position wounds simultaneously with their vector's eggs. Then the nematodes feed on fungi, which ar growning around the beetle's oviposition wounds, until beetle larvae hatch. Beetle larvae hatch about a week after oviposition and make a tunnel into the xylem of the host trees. Nematodes feed on fungi propagating on the surface of the beetles' tunnels, and build up and keep their population until the next early summer. Thus, the nematode, B. conicaudatus, depends its dispersal on their vector beetles, P hi- laris (Kanzaki and Futai, 2001). As for the life history of R. psacotheae, information obtained so far is limited; R. psacotheae is bac- teriophagous species and the dauer juveniles were found in the reproductive tructs of male and female yellow-spotted longicorn beetles (Kanzaki et al, 2002). The present study gives further information on the life history of R. psacotheae in relation to its vector beetle and host tree. The life history of R. psa- cotheae is compared with that of sympatric nematode, B. conicaudatus (Kanzaki and Futai, 2001). 1Laboratory of Environmental Mycoscience, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606- 8502, Japan. 2Corresponding author, e-mail: [email protected] ―60― Vol.32 No.2 Japanese Journal of Nematology December, 2002 MATERIALS AND METHODS The beetle organ harboring nematode, R. psacotheae and the number of the nematodes infested Adults of P. hilaris were collected from a fig tree on the Yoshida campus of Kyoto University, Kyoto (Yoshida), and from a field of mulberry, Morus bombysis at the Kyoto Institute of Technology, Kyoto (KIT), between 12 June and 11 September 1998. Ten female and 15 male P hilaris were collected at Yoshida, while 55 females and 68 males were collected at a field of KIT. To examine vector preference of the nematode, 20 adults of mulberry borer, Apriona japonica and nine adults of tiger longicorn beetle, Xylotrechus chinensis were also collected from the field of KIT, because these two beetle species and yellow-spotted longicorn beetle share mulberry trees as their host. The each individual beetle was deter- mined its fresh weight then ground with ca. 40 ml tap water. The nematodes were extracted from each ground body of beetle using the Baermann funnel technique, and counted. The dauer juveniles of R. psa- cotheae were extracted together with the dispersal fourth stage juveniles of B. conicaudatus, so the num- bers of nematodes were counted separately. The Kolmogorov-Smirnov test was used to examine the nor- mality of the data, and Mann-Whitney's U-test was used to determine the significance of differences in mean numbers of nematodes between two sample sites. The sympatry of R. psacotheae and B. conicau- datus within beetle individuals was examined by regression analysis. Transmission of R. psacotheae from vector beetles to Moraceae trees Fifteen females and 10 males of P. hilaris served for this experiment. Adults of P hilaris were col- lected at the field of KIT on 23 July 1998. Each beetle was placed in a plastic cup, containing a mulberry twig and a leaf. The cups were kept at room temperature, in the darkness for 48 hours. Afterwards, the nematodes were extracted from each beetle, the twigs and leaves, and from beetle's frass, and then counted. Also the number of beetle's eggs laid on the twigs was counted. The number of the nematodes remained in each beetle's body, those invaded into twigs, and those fallen over leaves and frass were counted separately.The Kolmogorov-Smirnov test was used to examine the normality of the data, and the one-way analysis of variance (ANOVA) and Tukey-Kramer's multiple comparison test were used to de- termine the significance of differences in the transmission ratio (proportion of nematodes recovered from twigs, see Table 2) among three groups of beetles; females which had laid their eggs during the experi- mental period, those which had not, and males. The simultaneousness of invasion of mulberry tree by R. psacotheae and by B. conicaudatus was examined by regression analysis. Distribution and stage of R. psacotheae in host trees of P. hilaris The fig tree at Yoshida from which beetles had been obtained was cut down on 10 September, 1998, and 18 wood samples were taken, i.e. three from healthy part of the trunk, the other three from decayed part of the trunk, and twelve from branches. Half the branch samples had beetle oviposition marks and the other half had no marks. Three mulberry trees at the field of KIT were cut down on 21 December 1999. From the trees, 39 wood samples were obtained; 30 samples from trunks with beetle tunnels, and nine samples from those without beetle tunnels. Among the former 30 samples, nine, eight, and 13 samples were obtained from sapwood, heartwood, and surface tissue of beetle's tunnel, respectively. Among the latter nine samples, four were from sapwood, and five were from heartwood. The sympatry of R. psacotheae and B. conicaudatus within host trees was examined by regression ―61― 第32巻 第2号 日本線虫学会誌 2002年12月 analysis. RESULTS The beetle organ harboring nematode, R. psacotheae and the number of the nematodes infested The dauer juveniles of R. psacotheae were found in the reproductive tracts of male and female bee- tles (Fig. 1) but were not found in the testis or ovary. The dauer juveniles were easily distinguished from those of B. conicaudatus, because the juveniles of R. psacotheae were covered with oily film (lipophilic) and were smaller than those of B. conicaudatus (Fig. 2). Ninety six percent of the beetles obtained at Yoshida were infested with R. psacotheae, and 77 nematodes in average carried by a beetle. While 58% of the beetles at KIT were infested with the nematode, and an average number of nematodes was 10. Thus, there was significant difference (p= 0. 05) in the numbers of nematodes carried by a beetle be- tween these two sampling sites (Table 1). Body weights of beetles showed marked variance within each of the two populations (Table 1). However, there were not any relationships (p= 0. 05) between the num- ber of nematodes carried by a beetle and the body weight of respective beetles. No nematodes were extracted from A. japonica or from X. chinensis (Table 1). There was no sig- nificant correlation between the number of R. psacotheae carried by a beetle and that of B. conicaudatus on the same beetle (Fig. 3). Transmission of R. psacotheae from vector beetle to Moraceae tree Ninety three per cent of R. psacotheae, which had been carried, moved from ovipositing female beetles into twigs of Moraceae tree, while no or small numbers of nematodes migrated into leaves and frass. Almost all the nematodes carried by males or non-ovipositing females remained in vector body (Table 2). There is no relationship between the numbers of eggs laid by a beetle and nematodes' trans- mission rates. The R. psacotheae found in mulberry twigs, leaves or beetle frass were dauer juveniles, fourth stage propagative juveniles or adults. There was significant correlation between the transmission ratio of R. psacotheae from a oviposit- ing female beetle to a mulberry twig and that of B. conicaudatus to the same twig (p= 0. 01) (Fig. 4). Table 1. Numbers of Rhabdontolaimus psacotheae obtained from beetles. 1 Mean •} sd (range) followed by different letters are significantly different at p < 0. 05 (Mann- Whitney's U-test). 2Mean •} sd. 3Kyoto Institute of Technology. ―62― Vol.32 No.2 Japanese Journal of Nematology December, 2002 Distribution and stage of R. psacotheae in host trees Rhabdontolaimus psacotheae was found in three decayed trunk samples from the fig tree and in all the branch and twig samples with oviposition marks, but was not detected in sound trunk samples or twigs without oviposition marks (Table 3). In the mulberry tree samples nematodes were detected mainly from trunk samples with beetles' tunnel (Table 3). The sympatry of R. psacotheae and B. conicaudatus in the host trees of P.