Life History of Rhabdontolaimus Psacotheae (Diplogasterida

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 hilaris, 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 organ 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 oviposition 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 hilaris (Kanzaki and Futai, 2001). As for the life history of R. psacotheae, information obtained so far is limited; R. psacotheae is bacteriophagous 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. psacotheae 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]

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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. psacotheae were extracted together with the dispersal fourth stage juveniles of B. conicaudatus, so the numbers of nematodes were counted separately. The Kolmogorov-Smirnov test was used to examine the normality 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. conicaudatus 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 collected 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 determine 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 experimental 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

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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 beetles (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 between 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 number 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 significant 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' transmission 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 ovipositing 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.

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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. hilaris was strongly suggested in two Moraceae species (p= 0.01)(Fig. 5).

B C

Fig. 1. Reproductive tructs of Psacothea hilaris containing the dispersal third stage (dauer) juvenile of Rhabdonto- laimus psacotheae. A: The ovipositor of female beetle infested with dauer juveniles of nematodes, B, C: geni- taria of male beetle infested with the nematodes. Arrow: nematodes

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A B A

Fig. 3. Sympatry of Rhabdontolaimus psacotheae and Bursaphelenchus conicaudatus in the body of each beetle. A: Yoshida; B: KIT. Numbers of B. conicau- Fig. 2. Dispersal third stage (dauer) juvenile of datus and R. psacotheae isolated from each beetle Rhabdontolaimus psacotheae. Juveniles were plotted horizontal and vertical axis, respec- stick to each other by their oily film. B: tively. Correlation between the numbers of B. coni- Dauer juvenile of R. psacotheae with de- caudatus and those of R. psacotheae was examined generated digestive system. by linear regression analysis.

Table 2. Proportion of Rhabdontolaimus psacotheae recovered from yellow-spotted longicorn beetles, mulberry twigs, and mulberry leaves and frass.

Values are means given in %. Means within each column followed by different letter are significantly different at the 5 % level (one-factor ANOVA, Tukey-Kramer's multiple comparison test).

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Table 3. Numbers of Rhabdontolaimus psacotheae extracted from two host trees, Ficus carica and Morus bombysis.

1% of samples from which nematodes were recovered (numbers of total samples) . 2mean ± sd (number of nematodes/g of dry wood) . A

Fig. 4. The relationship between the transmission rate of Rhabdontolaimus psacotheae from a vector body to a mulberry twig and that of Bursaphe- lenchus conicaudatus. Ratio of Bur- saphelenchus conicaudatus and R. psacotheae transmitted to twigs from Fig. 5. Sympatry of Rhabdontolaimus each female beetle ovipositted were psacotheae and Bursaphe- plotted horizontal and vertical axis, lenchus conicaudatus in host respectively. Correlation between the tree. A: fig tree at Yoshida; B: ratio of B. conicaudatus and that of mulberry trees at KIT. Numbers R. psacotheae was examined by lin- of B. conicaudatus and R. psa- ear regression analysis. cotheae isolated from each wood sample were plotted horizontal and vertical axis, respectively. Correlation between the numbers of B. conicaudatus and those of R. psacotheae was examined by linear regression analysis.

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DISCUSSION

Similar to our previous findings, the dauer (dispersal) juveniles of R. psacotheae were found only in the reproductive tracts of male and female beetles (Kanzaki et al., 2002). This suggests that R. psacotheae prefer vector's reproductive organ as the special place to stay. The dauer juvenile of R. psacotheae was not found in A. japonica and X. cinencis, and no adult or propagative juvenile nematodes were found in the body of P hilaris . The vector selection and vector utilization of R. psacotheae were similar to those of sympatric species, B. conicaudatus, i.e., these two species of nematodes select P. hilaris as their primary vector and enter their vector body in their dauer (dispersal) stage, which has no direct nutritional relationship with their vector (Kanzaki and Futai, 2001). The dauer juveniles of R. psacotheae enter mulberry trees through the oviposition marks of P. hilaris. This study could not clarify whether or not the dauer juveniles kept in the body of male beetles enter the trees. Horizontal transmission of R. psacotheae might occur among beetles during the mating be- havior, because the dauer juveniles of R. psacotheae infest the reproductive tracts of both the sexes of the beetles. Within 48 hours after their invasion of tree, R. psacotheae become fourth stage juveniles or adults, or remained as dauer juveniles. As the case of many bacteriophagous or mycetophagous nematodes (Giblin and Kaya, 1983; Giblin et al., 1984; Giblin-Davis et al., 1993; Griffith, 1987; Poinar, 1983; Rid- dle and Albert, 1997), the dauer juveniles of R. psacotheae are suspected to be the dispersal third stage juveniles. The average numbers of nematodes isolated from each vector beetle were 77.3 (Yoshida) and 9.9 (KIT). The numbers, however, could be underestimated, because the dauer juveniles of R. psacotheae are covered with oily film, and could be somewhat interrupted at the extraction by the Baermann funnel. The timing of R. psacotheae invasion of Moraceae trees and their distribution in the trees are similar to those of B. conicaudatus (Kanzaki and Futai, 2001), i.e., P. hilaris and these two species of nematodes are sympatric in the trees. R. psacotheae invade Moraceae trees simultaneously with B. conicaudatus . R. psacotheae is, however, segregated from B. conicaudatus as to food resources in their host tree, i. e., R. psacotheae is bacteriophagous, while, B. conicaudatus is fungivorus species (Kanzaki et al., 2000; 2002). Thus, the direct competition for food resources would not occur between these two species of nematodes. Within beetle body, these two nematode species were allopatric; R. psacotheae infests vector's reproductive tract and B. conicaudatus infests vector's trachea (Kanzaki and Futai, 2001). The numbers of R. psacotheae carried by each vector were not correlated with those of B. conicaudatus on the same beetles. The average numbers of R. psacotheae carried by a beetle (77.3 in Yoshida and 9.9 in KIT) were apparently lesser than that of B. conicaudatus (268 in Yoshida and 80 in KIT) (Kanzaki and Futai, 2001). The difference in the average number of nematodes carried by a beetle could partly be at- tributed to the difficulty in extraction for R. psacotheae by Baermann funnel technique. Another possible reason could be the difference is departure velocity from vector beetles. The departure ratio within 48 hours of R. psacotheae clearly differed from that of B. conicaudatus . The ninety three per cent of R. psacotheae was released from beetles during the 48 hours of the experimental period, while, only 27% of B. conicaudatus were released during the same period (Kanzaki and Futai, 2001). Thus, R. psacotheae might be released more easily and rapidly than B. conicaudatus . We used the field population of the beetles for this study, so the large portion of R. psacotheae harbored in the beetles might have already been released at the capture of the beetles.

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Generally, the nematodes of the genus Rhabdontolaimus are considered to be in close relationships with coleopteran insects (Andrassy, 1984; Gurando, 1979; Kanzaki et al., 2002; Massey, 1974; Ruhm, 1956), however, the detailed bionomics of these nematodes in their relation to its vector insects have scarcely been studied so far, and more information are needed to be accumulated.

ACKNOWLEDGEMENT

We thank Dr. M. Ichida, Kyoto Institute of Technology, and Dr. N. Maehara, Forest and Forestry Product Research Institute, for providing beetle samples.

LITERATURE CITED

Andrassy, I.(1984) Klasse Nematoda Gustav Fischer Verlag, Stuttgart, 509 pp. Giblin, R. M. and Kaya, H. K.(1983) Bursaphelenchus seani n. sp.(Aphelenchoididae), a phoretic associate of bees in the genus Halictus (Hymenoptera: Halictidae). Revue de Nematologie 7, 177-189. Giblin, R. M., Swan, L. and Kaya, H. K.(1984) Bursaphelenchus kevini n. sp.(Aphelenchida: Aphelenchoidi- dae), as associate of Anthophora bomboides stanfordiana Cockwell, 1904 (Hymenoptera: Anthophoridae). Revue de Nematologie 6, 39-50. Giblin-Davis, R. M., Mundo-Ocampo, M., Baldwin, J. G., Norden, B. B. and Batra, S. W. T.(1993) Description of Bursaphelenchus abruptus n. sp.(Nemata: Aphelenchoididae), an associate of a digger bee. Journal of Nematology 25, 161-172. Griffith, R.(1987) Red ring disease of coconut palm. Plant Disease 71, 193-196. Gurando, E. V.(1979) Nematodes of the lesser pine shoot beetle Blastophagus minor. Vestnik Zoologii 0,28-33. Kanzaki, N., Tsuda, K. and Futai, K.(2000) Description of Bursaphelenchus conicaudatus n. sp.(Nematoda: Aphelenchoididae), isolated from the yellow-spotted longicorn beetle, Psacothea hilaris (Coleoptera: Cerambycidae) and fig trees, Ficus carica. Nematology 2, 165-168. Kanzaki, N. and Futai, K.(2001) Life history of Bursaphelenchus conicaudatus (Nematoda: Aphelenchoididae) in relation to the yellow-spotted longicorn beetle, Psacothea hilaris (Coleoptera: Cerambycidae). Nematol- ogy 3, 477-484. Kanzaki, N., Minagawa, N. and Futai, K.(2002) Description of Rhabdontolaimus psacotheae n. sp.(Diplogaste- rida: Diplogasteroididae), isolated from the yellow-spotted longicom beetle, Psacothea hilaris (Coleoptera: Cerambycidae) and fig trees, Ficus carica. Japanese Journal of Nematology 32, 7-12. Massey, C. L.(1974) Biology and taxonomy of nematodes of bark beetles in the United States. USDA Agricul- ture Handbook No. 446, 233 pp. Poinar, G. O.(1983) The Natural History of Nematodes. Prentice-Hall, New Jersey, 323pp. Riddle, D. L. and Albert, P. S.(1997) Genetic and environmental regulation of dauer larva development. In: C. ELEGANS II.(Riddle, D. L. Blumenthal, T. Meyer, B. J. and Priess, J. R., eds.) Cold Spring Harbor Labo- ratory Press, New York, pp. 739-768. Ruhm, W.(1956) Die Nematoden der Ipiden. Gustav Fischer verlag, Jena, 437pp.

Received September 30, 2002

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和文摘要

Rhabdontolaimus psacotheaeの生活史と .Bursaphelenchus conicaudatus

との棲み分け

神崎菜摘・二井一禎

Rhabdontolaimus psacotheaeの生活史を調査した。Rhabdontolaimus psacotheaeはクワ科植物の材部に生息し、その耐久型(分散型第三期)幼虫は媒介者であるキボシカミキリの生殖器に侵入し、寄主樹木間を伝搬される。耐久型幼虫は、キボシカミキリの産卵痕を経由して寄主樹木に侵入し、材内で脱皮後、キボシカミキリ幼虫の坑道周辺で細菌類を食餌源として増殖していた。この種のこのような生活史を生息域、媒介昆虫を同じくする菌食性線虫、Bursmhelenchus conicaudatuSのそれと比較した。これら2種は、樹木材内では同所的に生息していたが、その食餌源に関して棲み分けが見られた。また、媒介者虫体内ではその侵入部位に関して棲み分けが見られた。

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