BIOLOGY OF A TYLENCHID PARASITIC ON THE JAPANESE SAWYER,

BY

N. OGURA and H. KOSAKA Laboratory of Nematology, Forestry and Forest Product Research Institute, P.O. Box 16, Tsubuka Norin Kenkyu Danchi, Ibaraki 305, Japan

A nematode which parasitizes the reproductive organs of the Japanese pine sawyer, Monochamus alternatus, has both a parasitic cycle and a mycetophagous free-living cycle. Entomoparasitic adult females lay eggs and hatched juveniles develop to 4th stage juveniles within the ovary and testis of M. alternatus adults. The 4th stage juveniles leave M. alternatus adults during defecation and during egg deposition. They moult to free-living adult males or infective adult females in the presence of M. alternatus larvae. The juveniles also moult to mycetophagous free-living adult males or females and propagate on the mycelia of an as yet unidentified fungus. The life history of the nematode is similar to that of Deladenusspp. but the morphology of the entomoparasitic female adult is similar to that of Contortylenchusspp. Keywords: Entomogenous nematode, Allantonematidae, Neotylenchidae, fungus, , reproductive organ.

The Japanese pine sawyer, Monochamus alternatus Hope, transmits the pine wood nematode, xylophilus (Steiner and Buhrer), which induces pine wilt disease (Mamiya, 1986). Endo- and ectoparasites of M. alternatus have been sought for use as biological control agents against the . A parasitic nematode found in the reproductive organs of M. alternatus adults captured in central Japan in 1969 (N. Enda, pers. comm.) may be a candidate as a biological control agent. The present study was conducted to describe the biology of the nematode. From our results, it is clear that the life history of this nematode is quite similar to that of Deladenus spp. but that the behaviour and morphology of the entomoparasitic adult females are more similar to that of Contortylenchus spp.

MATERIALS AND METHODS

M. alternatus. Larvae and prepupae of M. alternatus were collected in central Japan during January, 1989. These were placed individually in glass tubes (1.6 cm diam. x 7 cm long). Prepupae to be used for the infection tests and adults were grown under an artificial rearing system (Kosaka & Ogura, 1990) at 25 ± 1 °C under 16 L:8 D photoperiod. Nematode. Juvenile were isolated from the ovaries and testes of M. alternatus adults using a Baermann funnel procedure. Approximately 10,000 juveniles were washed 3 times with 5 ml sterilized distilled water (DW). 456

Next they were soaked in 5 ml of a 0.1 % benzethonium chloride solution for 15 min and then washed 3 times with 5 ml DW. FiftY!l1 of the juvenile suspen- sion, containing approximately 2000 juveniles, were pipetted on to agar or PDA in culture bottles (3 x 4 cm base x 2 cm height). The nematodes which grew on the mycelia of the fungus in the culture bottle were washed out of the bottles with 5 ml DW. A 100 yl aliquot of the nematode suspension containing approximately 2000 nematodes and several pieces of mycelium was pipetted on to PDA in another culture bottle and incubated at 25 + 1 ° C . The nematode culture was then subcultured at 30 days intervals. Fungus. When nematode juveniles recovered from M. alternatus were inoculated on PDA, several species of fungi grew on the PDA. One of these fungi which grew in association with a single dead juvenile supported develop- ment and propagation of the nematodes. Small pieces of agar containing the fungus (ca. 5 x 5 x 5 mm) were transplanted to PDA in culture bottles and an homogeneous fungus line was obtained after several successive transplanta- tions ; this was used for nematode culture. The fungus was also cultured axenically in liquid culture of 0.2% yeast extract (Difco, U.S.A.) and 2% dextrose. Agar and PDA. A 2 To agar suspension was autoclaved at 120°C for 15 min then mixed with the same volume of an antibiotic solution (400 jig tetracyclin, 400 yg streptomycin, and 400 units penicillin per ml) which had been passed through 0.2 ym pore-size filter. Five ml of the agar-antibiotic solution were poured into sterilized culture bottles (final concentration: 1 % agar, antibiotics 200 yg or units per ml). The bottles were closed with silicon plugs to allow ven- tilation. To make PDA with antibiotics, 40 g potato were boiled in 100 ml DW for 15 min and particulate materials were removed by paper filtration. The potato filtrate was combined with 4 9lo dextrose and 2 % agar and autoclaved. The antibiotic solution described above was added and 5 ml of the mixture were poured into each culture bottle. Crossing experiment. A small cluster of mycelia in yeast extract dextrose solu- tion was inoculated on to the agar pad in the culture bottle. A juvenile of the mycetophagous free-living nematode was released on the mycelia. After 10 days an adult male of the mycetophagous free-living cycle was released to half of the culture bottle where an adult female appeared. After 20 days the cultures were examined to determine if progeny had been produced. Inoculation. Prepupae of M. alternatus that had been chilled to release them from prepupal diapause were used for an infection test. A prepupa was placed on an agar pad in a culture bottle and 0.5 ml of a nematode suspension (5000 of 4th stage juveniles taken from M. alternatus) was pippetted on to the plate. Other prepupae or pupae were placed in culture bottles containing PDA on which 2000 mycetophagous nematodes had been cultured for 30 days. This 30 day culture yielded approximately 10,000-20,000 nematodes in each culture bottle. The prepupae and pupae were held at 25 ± 1 °C for 10 days in the culture bottles before transfer to the artificial rearing system.