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Fumiam. appl. NemalOl., 1998, 21 (2), 185-198

Survey of entomopathogenic (: Steinernematidae and Heterorhabditidae) in Japan

Mutsuhiro YOSHIDA*, Alexander P. REID**, Bernard R. BRISCOE** and William M. HOMINICK**

* NaLionallnsLiLuLe ofAgro-Environmental Sciences, Kannondai 3-1-1, Tsukuba, lbaraki 305,japan and .'f.* CABllnt.emaLionallnstitute ofParasiLology, 395A HaLfield Road, St. Albans, Heris. AL4 oXV, VK. Accepted for publication 8July 1997.

Surnmary -A survey was conducted for entomopathogenic nematodes in the five climatic regions of Japan: subtropical islands, warm temperate coastal region along the Pacific, central mountainous region, temperate region, and cool temperate region. Over 1400 soil samples from 266 sites were tested for the presence of nematodes using the Galleria baiting technique. One hundred and twenry steinernematids and 29 heterorhabditids were detected in 62 sites and 24 sites, respectively. Eight steinernematid species were recognized: seven undescribed species and Sleinernema kushidai. Each species had a characteristic distribution: three wide-spread common, one coastal, one subtropical, and three mountainous species. In contrast, the hetero­ rhabditids were identified as Heœrorhabditis indica and H. megidis. The former was widely found in the coastal region from the subtropical to the warm temperate zone while the latter was found mainly in the eastern part of the warm temperate coastal region. © Elsevier - ORSTOM

Résumé - Enquête sur les nématodes entomopathogènes (Rhabditida: Steinernematidae et Heterorhabditidae) du Japon - Une enquête a été menée concernant les nématodes entomopathogénes dans les cinq régions climatiques du Japon: les îles subtropicales, la région tempérée chaude des côtes du Pacifique, la région montagneuse australe, la région tempérée et la région tempérée froide. Plus de 1400 échantillons de sol provenant de 266 sites ont été testés pour la présence de nématodes à l'aide de Gallena-piéges. Cent vingt-et-un Steinernematides et 29 Heterorhabditides Ont été détectés dans, respectivement, 62 et 24 sites. Huit espéces de Steinernematides ont été reconnues: sept espéces non décrites et Sœinernema kushidai. Chacune des espéces montre une répartition particuliére : trois sont très répandues, une est côtière, une subtropicale et les trois autres montagnardes. En contraste, les Heterorhabditidae ont ètè identifiès comme Helerorhabidùis indica et H. megidis. La première espèce est largement rèpandue dans la région côtière depuis la zone subtropicale jusqu'à la zone tempérée chaude, tandis que la seconde est surtout rencontrée dans la partie est de la région côtière tempérée. © Elsevier - ORSTOM Keywords: distribution, entomopathogenic nematodes, Helerorhabdilis, Japan, species diversiry, Sleinemema, survey.

Nematodes of the families Steinernematidae and (Mamiya & Ogura, 1989) and Kyushu district Heterorhabditidae have characteristics that make (Kimura & Ishibashi, 1991) revealed the presence of them extremely useful as biological control agents. other steinernematids. Numerous surveys have been conducted world-wide, The present study describes a survey conducted resulting in the isolation of many species and strains; between 1991 and 1994 in five climatic regions, from Australia (Akhurst & Bedding, 1986), North America subtropical to cool temperate regions, mostly in major (Akhurst & Brooks, 1984; Poinar et al., 1987; Mraëek forests and ranging in elevation from near sea level to & Webster, 1993; Rueda et a1., 1993; Nguyen & about 1600 m. This work was aimed at collecting Smart, 1994), Puerto Rico (Roman & Beavers, 1982), indigenous entomopathogenic nematodes adapted to South America (Doucet, 1986; Doucet & Doucet, the various climates for possible use in biological con­ 1990, Nguyen & Smart, 1990; Stock, 1995), Hawaii trol regimens. (Hara et al., 1991), Europe (Mraëek, 1980; Deseo et al., 1984; Husberg et al., 1988; Hominick & Bris­ coe, 1990; Griffin et al., 1991), Africa (Shamseldean Materials and methods & Abd-Elgawad, 1994), India (Poinar et al., 1992), The present survey was conducted from the south Sri .Lanka (Amarasinghe et al., 1994), Asia (Zhang part of Japan in a north-east direction along the et al., 1992; Choo et al., 1995). In Japan, Steinernema Pacifie coast to collect nematodes adapted to dry, hot, kushidai Mamiya, 1988 was first isolated in Shizuoka and warm environments, then from the south to the from a tree nursery where an outbreak and an infesta­ north of the central part (Izu Peninsula - Nagano tion of white grubs had occurred (Kushida et al., Pref.) to collect nematodes adapted to cool or cold 1987). Later surveys conducted in eastern Japan mountainous environments. Moreover, sorne other

Fundam. appl. NemalOl. 1164-5571/98/02/© Elsevier - ORSTOM 185 M. Yoshida et al. regions sampled between 1991 and 1994 were added also by PCR-RFLP analysis of the internai tran­ to the present srudy. scribed spacer region from the ribosomal DNA repeat A total of 1416 soil samples was collected from 276 unit. Specimens for morphological study were pre­ sites, which represented the five climatic regions pared according to Minagawa and Mizukubo (1994). (Fig. 1) in the present study as follows: z) subtropical The extraction of genomic DNA from a single nema­ islands, four islands in Okinawa Pref. (Nansei-shoto tode was conducted according to Joyce el al. (1994c) Isis.), 309 samples from 81 sires; il) warm temperate and Reid el al. (1997). The ITS region was amplified coastal region along the Pacifie coast, the warm region by PCR with the 18S and 26S primer set (Vrain el al., affected by the warm current "Kuroshio", from Cape 1992). Subsequent RFLP analysis was performed as Sata-misaki (south end of Kyushu, Kagoshima Pref.) per Reid el al. (1997). Each isolate was assigned to an to Cape Inubo-zaki (Chiba Pref.), 732 samples from RFLP type based on the restriction fragment patterns 116 sites; iiz) central mountainous region, the cool yielded by Alu 1 and Hinf 1 (Sleinernema spp.) or Alu 1 highland at more than 500m elevation from lzu and Rsa 1 (Helerorhabditis spp.). Seventeen restriction Peninsula (Shizuoka Pref.) to Niigata Pref., 165 sam­ enzymes (Alu l, BstO l, Dde l, EcoR l, Hae III, Hha l, pies from 31 sites; iv) temperare region, Oki Isis. (Shi­ Hind III, Hinfl, Hpa II, Kpn l, Pst l, Pvu Il, Rsa l, Sal mane Pref.) and sorne regions in the eastern and l, Sau3A 1, Sau96 l, Xba 1) were used for the detailed northern part of Japan (Chiba, Ibaraki and Miyagi RFLP analysis of each representative of the RFLP Prefs), 129 samples from 26 sites; v) cool temperate types (Reid et a!., 1997). The detailed RFLP profiles region (Hokkaido Pref.), 44 samples from twelve were compared with the RFLP database of the nema­ sites. The climatic division was derermined based on todes at CABI International Institute of Parasitology, vegetation and elevation, referring to Maekawa (1977) UK. and Nakamura el al. (1996). Samples were collected from November to May before the luxuriant growth Results of weeds and bushes, with the exception of the Positive soil samples with entomopathogenic ne­ Okinawa Is. where samples were taken in June after matodes were obtained from ail sampling regions the rainy season. The number of samples taken from (Fig. 1), and represented 10.0% of the total number each sire was usually five (ranging from three to thirty ofsoil samples (142/1416) or 28.2% of the geographi­ samples/site) with approximately 600-700 g soil col­ cal sires (75/266). Steinernematids were recovered in lected for each sample to a depth of 5-20 cm. 8.5% of the soil samples (12011416), from 23.3% of 3 Approximately 500 cm of soil of each sample was the sites (62/266), and heterorhabditids in 2.0% of the placed in a polythene cup (110 mm diam., 50 mm soil samples (2911416), from 9.0% of the sites (24/ depth) covered with a lid and three ta five final instar 266). The incidence and species diversity of the larvae of were added (Bedding & nematodes in the four main sampling regions were Akhurst 1975). Each cup was incubated at room tem­ obviously different. A much higher prevalence was perature (20-25°C) for 2 weeks. Each container was noted in the central mountainous region (64.5% of checked for larval mortality every 2-3 days from 5 or 6 the sites [20/31]) than in the subtropical islands (8.6% days after setting the Galleria trap up to 14 days. Dead [7/81]), the temperate region (23.1 % [6/26]) and the larvae were placed individually on a polyurethane warm remperate coastal region (33.6% [391116]). sponge (20 mm length, 15 mm width and less than 10 Eighty-two steinernematid isolates from 47 positive mm thickness) in each cell of a twelve-well plate with sires were classified into eight species: seven undes­ water. The plates were kept at room temperature (20­ cribed species and S. kushidai, by means of morpho­ 25°C) and periodically examined for presence of nem­ logy and/or RFLP analysis. The undescribed Slei­ atodes. Cadaver from which infective juveniles began nernema species are herein referred as MY l, MY2, to emerge were individually transferred to a plastic MY3, MY4, MY5, MY6, and MY7, based on RFLP cup (60 mm diam., 35 mm depth) with the poly­ profiles (Fig. 2). The incidence and species diversity urethane sponge and distilled water. In the present of steinernematids increased as follows: subtropical study, an "isolate" represented the nematodes islands (1.2% of the sites [1/81], Steinernema sp. obtained from a single dead Galleria larva and not MY4) < temperate region (23.1 % [6/26], Steiner­ from one soil sample. Infective juveniles isolated in nema spp. MYl, MY3, and unidentified isolates) < this manner were rinsed in distilled water several warm temperate coastal region (27.6% [321116], times in the cup and stored at 5-7°C (Sleinernema spp. Sleinernema spp. MYl, MY2, MY3, S. kushidai, and except S. kushidaz) or 10-12°C (s. kushidai and H ele­ unidentified isolares) < central mountainous region rorhabditis spp.). (64.5% [20/31], Sleinernema spp. MYl, MY3, MY5, Identification of isolates was based on MY6, MY7, S. kushidai, and unidentified isolates) morphometrics of infective juveniles and morphology (Fig. 3). Twenty-eight heterorhabditid isolates from of 1st generation males under light microscopy and 23 positive sites were identified as H. indica Poinar,

186 Fztrldam. appl. NemalOl. EnlOmOpathogenic nemalOdes in Japan

-=---+--~--45N 146E

Hokkaido 001 Temperate Regio 14üE

--=------jl+---'=-o 42N

Central Mountainous Region

Togo Is. Nishinoshima Is'fhc'Y 36N Oki Isis.

0 , jr ~ 3IN '0;)

31N . Cape ~kinnwn Snta-misaki 6N Q Is. ---- .... Yonaguni Is. .Q'" ~~ • spp. - / ~~ • Steinernema spp. -; "* l'h;g~ki b. ,i"'~ À Heterorhabditis spp. & Steinernema spp. [_r .. ---24N X'l v No Entomopathogenic Nematcxies Iriomote Is. ~~ 123E Fig. 1. Localities and regions where samplings for heterorhabditids and steinernematids were conducted. FIgures indicated latitude (N) and longitude (E). Each symbol often represents more than one site.

Karunakar & David, 1992 and H. megidis Poinar, Jak­ of the Ohio isolate (original strain) of the three RFLP son & Klein, 1987. The RFLP profiles of the Japanese types of H. megidis (Ohio, North West European isolates of H. megidis showed the same pattern as that [NWE], and Irish types; Hominick, el al., 1996).

VoL 21, n° 2 - 1998 187 M. Yoshida et al.

h 7 ~ ~ III Il 12 IJ I~ 1" Ih 17 I~ \l Il 7 S 1) 10 11 12 IJ 1~ 15 Ih 17 )S \1

Sleille,."e/l1(1 'p. ,\IY 1 \'","el'lll'Il/II sp. .\ 1Y1

~ ~ .\ ~ (, 7 S 'J '1) 11 12 1. \ 1-lI.' 1(1 17 1S \ 1 (, ... S II 1(1 11 1 1J l-l 1 1" 17 1S \ 1

S/I';III'I'II('II/(I '1'. .\IY3 \/l';III'rl/1'1I1II 'p. \1' ~

h 7 X ~ III Il 12 1.1 I~ 15 1.. 17 IX \1 .\ .1 ::. Il "1 S I) lU Il I~ 1.\ 1.... 1:: Ih 17 IS \1

,\JY~ .\'/eill<'I'/I1'(1/a "p. \/<';'11'/'111'11/11 'p. \ 1Yh

h 7 X ') III 11 12 1.1 1~ 1" 1h 17 I,~ \ 1 '1 111 Il 12 IJ I~ 1" Ih 17 IX \1

.\'/"'"('/'1"'(1111 sp. ,\1),7 S/I';"I'I'(lI'II/(I klllhidai

Fig. 2. Restrielion Fragment Length Polymorphism profiles of se'ven undeseribed Steinernema sp. and S. kushidai. Ethidium bro­ mide slained 1.5 % agarose gel. Lane 18: PCR amplified fragment of hu.ernal Transeribed Spaeer region from rDNA with 18s and 26s primer sel. Lanes 1- 17: restrielion digestion pallern (1: Alu l, 2: BstO l, 3: Dde l, 4: EcoR l, 5: Hae III, 6: Hha l, 7: Hind III, 8: HinfI, 9: Hpa II, 10: Kpn 1,11: Pst 1,12: Pvu II, 13: Rsa 1,14: SalI, 15: Sau3A 1,16: Sau96 l, 17: Xba 1). Lanef Positive control (A 11.1 1 restriction digest of ITS regionfrom S. feltiae site 76 strain UK). Lane M: DNA size marker (from top to bOlwm: 2, 1.2, 0.8, 0.4, 0.2, 0.1 kbp). Eaeh isolales of Steinernema sp. from the following loeality (see Table 1), MY1: Sala; MY2: A LSumi; MY3: Omaezaki; MY4: Yonaguni Is.; MY5: Enzan; MY6: MaLSumoto; MY7: Omaehi; S. kushidai: Toyo.

188 Fundam. appl. Nernawl. E11l0mopathogenic nematodes in Japan

However, another European study (Smits et a!.) 1991; mountainous region (6.5% [2/31], H. megidis) < the Dix et al., 1992; Joyce el al., 1994a) b) c) suggesred subtropical islands (7.4% [6/81], H. indica) < the that the Irish group was not conspecific with the warm temperate coastal region along the Pacifie NWE/Ohio groups. Accordingly, in the present study, (13.8% [16/116], both species and an unidentified the authors treated the three groups showing closely isolare) (Fig. 4). related RFLP pattern of ITS (Reid, 1994) as the H. A total of 39 isolates (38 steinernematids and one megidis species complex. The incidence and species heterorhabditid) could not be identified, because they diversity of heterorhabditids increased as follows: the did not provide living infective juveniles from infected temperate region (0% of the sites [0/26]) < the central wax moth larvae or were lost during culture storage

/~ 41N ffi1 \- J ~ 14?E

146E

140E •• ~O'---++---,,---'>~-42N -~--'=---~- 36N .~ ./J 31N 'OrY

26N "

---.1.=----.31N ~0 2:1N • Steinernema sp. MYI + Steinernema sp. MY5 ... Steinernema sp. MY2 *Steinernetna sp. MY6 lb • Steinernema sp. MY3 T Steinernema sp. MY7 1) occurrence in same site • Steinernema sp. MY4 * Steinernema kushidai 2) strain Hamakita (after Mamiya, 1988)

Fig. 3. Distribution of Sreinernema spp. from a survey ofJapan. Latitude (N) and longitude (E) are indicated. Each symbol often represents more than one site.

Vol. 21, na 2 - 1998 189 M. Yoshidq et al. manipulation. In 1993, five steinernematid isolates sites, in the eastern part of the warm temperate (obtained from five of ten samples) were recovered coastal region. Steinernema sp. MY3 was isolated from from Mihama (Wakayama Pref.). They yielded no thirteen sites in Shikoku and Honshu, from coastal to infective juveniles and several 1st generation adults mountain regions. Steinernema sp. MY4 was isolated obtained from sorne dead Galleria larvae indicated only from one inland site in Yonaguru Is. in the sub­ unfavourable conditions for propagation of steiner­ tropical region. Steinernema spp. MY5, MY6 and nematids. These isolates couId clearly be separated MY? were isolated from one, one, and two sites, from ail other Japanese isolates by the spicule shape. respectively, in the central mountainous region. In the following year, the same site was sampled and S. kushidai was isolated from five sites in the warm the isolates obtained on this occasion (three positive temperate coastal region and one site in the central samples/ten samples) showed the same spicule shape mountainous region. H. indica was isolated from four­ and the same RFLPs pattern as those of Steinernema teen sites in the subtropical to warm temperate coastal sp. MY1. Accordingly, the first isolates from Mihama regions. H. megidis was isolated from seven sites in the were not included in this grouping. eastern part of the warm temperate coastal region and two sites in the central mountainous region. The Environmental characters and geographical features steinernematid distribution was very broad, ranging of the sites with nematades are indicated in Table 1. from beach sites ta inland sites and from subtropical The geographical distribution of steinernematids and ta cool temperate regions, while most of the heter­ heterorhabditids identified to species level is summa­ orhabditids occurred in the coastal area from the rized in Figs 3 and 4. Sceinernema sp. MY1 was iso­ warm region (Fig. 1). lated from 28 sites in Kyushu, Honshu, Hokkaido, and Oki Isis, from coastal to mountainous regions. The vertical distribution of the steinernematids Sceinernema sp. MY2 was isolated from three coastal ranged from beaches ta mountains about 1400 m in

• Heterorhabditis indica • Heterorhabditis megidis

o 36N --""--

26N Cl

~ Jj~ 24N 123E

Fig. 4. Distribution of Heterorhabditis spp. from a survey ofJapan. Latitude (N) and longitude (E) are indicated. Each symbol often represems more than one site.

190 Fundam. appl. NemaLOI. EnlOmOpalhogenic nemalOdes in Japan elevation. In the case of the survey at Enzan (Yama­ species sympatrically occurred in fourteen sampling nashi Pref.) in the central mountainous region, 30 soil sites and steinernematids and heterorhabditids were samples were taken from six sites (five samples/site) isolated from the same soil samples in six of these from rwo mountains (two sites about 1000 m and fourteen sites (Table 2). However rwo or more conge­ 1400 m in elevation from one mountain and two sites neric species were never isolated from the same soil 1000 m to 1200 m, one site about 1400 m, and one sample. H. megidis and the steinernematids were sire about 1600 m in elevation from a nearby moun­ found sympatrically in eight of nine sites with tain). Sreinernematids were isolated from five of the H. megidis, while steinernematids were found in only six sites (1000-1400 m in elevation). No steinernema­ three of fourteen sites with H. indica. The rwo hetero­ tids were isolated from the site at about 1600 m. rhabditid species were always isolated allopatrically. In Steinernema spp. MY5 (one isolate at about 1000 m) the case of the adjacent occurrence of the rwo hete­ and MY7 (one iso1ate at about 1400 m) were isolated rorhabditid species, the two sampling sites in Omae­ from the first mountain and Steinernema spp. MYI zaki (Shizuoka) were separated by less than 5 km and (five isolates at about 1000-1400 m) and MY3 (one the site where H. indica occurred· was closer to the isolate at about 1000-1200 m) from the second Pacific coast than the site where H. megidis occurred. mountain. There was sorne frozen soil under the sur­ face in the two sites at 1400 and 1600 m in the second mountain, when the sampling was conducted (April). Discussion Steinernema sp. MY1 was isolated from the soil sur­ rounded by frozen soil in the former site. Steinernema Although most of the soil samples were taken from sp. MY1 was found from the coast close to sea level to woodlands, the area surveyed covered a wide range of mountains about 1400 m in elevation. Steinernema sp. climates ranging from subtropical to cool temperate MY2 was isolated only near sea level. Steinernema sp. regions and forests are the dominant habitat in Japan. MY3 was found from the coast close to sea level to AIso, vertical distribution of nematodes was studied mountains about 1000-1200 m in elevation. Steiner­ from sea level to high altitudes (up to 1600 m). nema sp. MY4 was isolated at about 100 m. Steiner­ Accordingly, the present results are an indication of nema sp. MY5 was isolated at about 1000 m, Steiner­ the natural distribution of steinemematids and hete­ nema sp. MY6 was isolated at about 700-800 m. Stei­ rorhabditids in Japan, corresponding to the climatic nernema sp. MY7 was isolated at about 800-1400 m. and/or geographical variation. Steinernematids were S. kushidai was found from the coast nearly at sea level more common than heterorhabditids, as is the case in to a coastal hill at less than 100 m and in one moun­ most surveys except for North Carolina (Akhurst & tain site at about 700-800 m (Table 1). The vertical Brooks, 1984), New Jersey (S tuart & Gaugler, 1994), distribution of heterorhabditids ranged from beaches Hawaii (Hara et al., 1991), and Egypt (Shamseldean to low mountains about 200 m in elevation, with the & Abd-Elgawad, 1994), in which heterorhabditids exception of two isolates (H. megidis) from mountains were more common. However, in the subtropical about 700-800 m in elevation (Table 1). To date, region (Nansei-show Isis.), heterorhabditids were S. kushidai and H. megidis have n.ot been isolated from more prevalent than steinernematids, although the the inland part berween Shizuoka Pref. and Nagano overall nematode prevalence was much lower. Hetero­ Pref. (Figs 3, 4). rhabditids occurred mostly in the coastal area, while steinernematids were found in only one inland site. Steinernema spp. MY4, MY5, MY6, and MY7, S. The prevalence of the two families was similar to the kushidai, and H. megidis were isolated only from Hawaiian survey (6.8% positive sites for enromopa­ woodlands. Steinernema spp. MY1, MY2, and MY3 thogenic nematodes, 6.3% positive for heterorhab­ and H. indica were isolated from both grasslands and ditids, and 0.6% positive for steinernematids; Hara woodlands (Table 1). Steinernema spp. MY 1 and et al., 1991). In the Hawaiian survey, 95.5% (21/22) MY3 were isolated from vegetation ranging from of isolates were collected from beach sites and one iso­ grasslands close to woodlands to well-developed for­ late at 61 m elevation (Hara et al., 1991). Conversely, ests (climax vegetation). Steinernema sp. M Y2 and H. in this survey only one isolate was collected from a indica were isolated from vegetation ranging from beach site, while six were recovered from coastal for­ grasslands far from woodlands to well-developed for­ ests or a grassland away from the beach (Table 1). ests. Steinernema spp. MY5 and MY6 were isolated This difference may be due to the different species from sparse forests (secondary forests). S. kushidai involved (H. hawaiiensis Gardner, Stock & Kaya, 1994 and H. megidis were isolated from sparse to well­ from Hawaiian Isis., H. indica from Nansei-shoto developed forests. Steinernema spp. MY4 and MY7 Isis.) and their host insects, as Akhurst and Bedding were isolated from well-developed forests. (1986) mentioned. It should be also considered that Although in most of the positive sampling sites only differences in geological origin (Hawaiian Isis. are one nematode species was recovered at a time, sorne oceanic islands, Nansei-shoto isls. are continental

Vol. 21, n° 2 - 1998 191 M. Yoshida et al.

Table 1. Habitai charaClerislics of positive sampling sites ofSteinernema and Heterorhabditis species

Species Locality Climatic Configuraùon Elevation Distance Habitat2) Vegetation of the site region1) (m) from sea(m)

Steinernerna species kushidai Kochi Toyo WTC Coast <10 <10 W:Roadside Conilignosa (CrYPlOmeria) Kochi Tosa-shimizu WTC Coastal Mountain 100 <30 W:Roadside Laurilignosa Wakayama Hidaka WTC Coastal Mountain 50-100 <100 W:Slope Laun"lignosa Wakayama Kushimoto WTC Coast 70-80 <50 W:Beside park Launlignosa Aichi Atswni WTC Coast (Tip of <10 <100 W Pine, Evergreen Headland) Nagano Matsumoto CM Mountain 700-800 Inland W:Beside park Deciduilignosa (Oak) MYI Kagoshima Sata WTC Coast 5-10 100-200 W:Road-side Laurilignosa Kagoshima Sata WTC Coastal Mountain <100 100-200 W Evergreen CrYPlOmeria Shimane Nishinoshima T Hill 200 Inland W Conilignosa (Pine) Shimane Saigo T Coast 100-200 <100 W+G Pine, Evergreen, (Tip of Cape) Glass Shimane Fuse T Mountain 400-500 Inland W Decidul1ignosa Shimane Fuse T Mountain 300 Inland W:Beside pass Deciduilignosa Wakayama Kushimoto WTC Coast 70-80 200 W Laurilignosa Wakayama Kushimoto WTC Coast 70-80 50-100 W:Beside park Laurilignosa Wakayama Kushimoto WTC Coast 70-80 <20 W+G:Park Pine, Evergreen, (Tip of Cape) Turf Wakayama Mihama WTC Coastal Mounrain 200-300 500 W+G:Open- Pine, Broad- land(Park) leaved, Grass Shizuoka Fujinomiya CM Mountain 500-600 Inland W:River-side Deciduilignosa Shizuoka Atami CM Mountain 534 Inland W:Forest Park Decidul1ignosa Shizuoka Syuzenji CM Mountain 500 Inland W:Forest Park Deciduilignosa, Bamboo grass Shizuoka Toi \VfC Coast 40-50 <50 W Laurilignosa, (Tip of Cape) Bamboo grass Shizuoka Nishi-Izu WTC Coastal Hill 10-20 500-600 W Laurilignosa Shizuoka Minami-Izu WTC Coastal Hill 200 500-600 W Laurilignosa Shizuoka Minami-lzu WTC Coast 50-60 <100 W:Beside park Laurilignosa Yamanashi Enzan CM Mountain 1100-1200 Inland W Deciduilignosa Yamanashi Enzan CM Mountain 1400 Inland W Deciduilignosa, Bamboo grass Nagano Matsumoto CM Mountain 700-800 Inland W:Beside park Deciduilignosa (Oak) Nagano Omachi CM Mountain 800-900 Inland W+G Grass,Oak Chiba Amatsu- WTC Coastal Mountain 100-200 50-100 W Laurilignosa Kominato Chiba Wada WTC Coast (Beach) <5 <100 W:Sheiter belt Pine To be cominued neXI page

192 Fundam. appl. NemalOl. Eneomopachogenic nemalOdes in Japan

Table 1. (cane.)

Species Locality Climatic Configuration of Elevation Distance Habitat2) Vegetation region 1) the site (m) from sea(m)

Chiba Kyonan wrc Coastal Mountain 200 1.1-1.2k W Laurilignosa Chiba Iioka wrc CoastaJ Hill 100-200 700-800 W Laurilignosa Miyagi Sendai T Plain 40 Jnland W:Coniferous CrYPlOmeria plantation Hokkaido Yubari CT Mounrain 500-600 Jnland W Deciduilignosa, Bamboo grass Hokkaido Obihiro CT Plain 40 Jnland W Deciduilignosa, Bamboo grass MY2 Aichi Atsumi wrc Coast <10 <50 W Pine, Evergreen (Tip of Headland) Chiba Shirahama wrc Coast (Beach) <5 10-50 G:Road-side Bush, Grass Chiba Wada wrc Coast (Beach) <5 10-50 W:Shelter belt Pine MY3 Kochi Muroto wrc Coastal Mountain 200

Vol. 21, nO 2 - 1998 193 M. Yashida et al.

Table 1. (cam.)

Species Localiry Climatic Configuration Elevation Distance Habitat2). Vegetation of the site region 1) (m) from sea(m)

Hewrorhabditis species indica Okinawa Ishigaki Is. SI Coast 30 300-400 G:Beside cropland Grass (Arundo donax) Okinawa Ishigaki Is. SI Coast (Beach) 1-2 10-20 W:Park Laurilignosa Okinawa Iriomote Is. SI Plain 20

194 Fundam. appl. NemalOl. EnlOrnopalhogenic nernalOdes in Japan

Table 2. Sympam'c occurrence of sl.einememalids and helerorhabdùids in sorne sùes.

Locality Site No. Sleinernema spp. Helerorhabdùis spp. Total

kushidai MYI MY2 MY3 MY6 sp.? indica megidis

Kagoshima Sata 2 1/11 1/ Il 2111 Wakayama Kushimoto 3 1/10 1/10 2/10 Kushimoto 4 2/10 2110 3110 1) Aichi Atsumi 1 1/30 1/30 3/30 1/30 6/30 Shizuoka Omaezaki 1 2/11 1/11 3/11 Omaezaki 2 1/12 1112 2112 Atami 1 3/7 1/7 4/7 Toi 1 3/5 1/5 3/52) Nishi-Izu 1 1/2 112 1/2 2/2 3) Minami-lzu 1 1/5 115 2/5 Minami-Izu 2 2/10 1110 211 02) Nagano Matsumoto 1 1/5 1/5 1/5 1/5 3/52) Matsumoto 2 1/5 1/5 2/5 Chiba Iioka 3 1/7 2/7 2/72) Each value indicates the no. positive samples / no. samples from a site. sp. ?: unidentified isolate 1) Sleinernema sp. MYI and Helerorhabdilis indica from a single sample. 2) Sleinernema sp. MYI and H. rnegidis from a single sample. 3) Sleinernerna sp.? and H. rnegidis from a single sample.

In me case of the American survey, H. megidis was most prevalent species in Japan, which is similar to the found from grassland (golf course; Poinar el al., dominance of S. feltiae in Ireland (Blackshaw, 1988) 1987), while in the Canadian survey it was found only and Britain (Hominick & Briscoe, 1990). It occurred from orchards (Mracek & Webster, 1993). Other in various temperature regions from me hot environ­ European studies (Smits el al., 1991; Joyce el al., ment of Cape Sata-misaki, at me south end of Kyu­ 1994a; Hominick el al., 1995; Miduturi el al., 1996) shu, to the cold environment of Hokkaido and from recorded the H. megidis species complex from inland me coast ta mountains about 1400 m in elevation. location. Accordingly, it seems mat the occurrence of Sleinernema sp. MY3 was me second most prevalent me H. megidis species complex is restricted neimer by species in the temperate zone and occurred from the me vegetation nor by me configuration at the species coast to mountains about 1000-1100 m in elevation. group level, as mese isolates are considered for the Recently, this species was isolated from the mountain moment to be very closely related or conspecifie. The area of me northern part of Kyushu and Hokkaido different habitat preference among me isolates in (Yoshida unpubl.). The two species had almost the mese various countries is probably due to differences same distribution pattern and were isolated from sim­ in me distribution and/or me species of suitable host ilar environments (Table 1), but mey occurred sepa­ insects, in the subspecies or strains of me nematade, rately from each other except on two sites (Table 2). and in the vegetation differences among mese coun­ S. kushidai occurred widely in me warm temperate tries. coasral region from Kochi to Shizuoka, especially in From the isolation pattern (Fig. 3, Table 1), the coastal forest. The species was first detected from an steinernematid species could be divided into four inland tree nursery (Hamakita City, Shizuoka Pref.), groups: three temperate widespread species (Steiner­ in me warm remperate region (Kushida el al., 1987). nema spp. MYI and MY3, and S. kushidaz); mree It has been considered ta be more adapted to warm mountainous species (Sleinernema spp. MY5, MY6, environments because of their activity (Kushida el al., and MY7); one coastal species (Sleinernema sp. 1987). However, this species was also isolated from MY2); and one subtropical species (Sleinernema sp. me mountain region in a cool climate zone in this sur­ MY4) (Fig. 3, Table 1). Steinernema sp. MY1 was the vey (Table 1).

Vol. 21, n° 2 - 1998 195 M. Yoshida et al.

In contrast wim the former three wide-spread spe­ me RFLP patterns were significantly different bet­ cies, another five uncommon species were found only ween these two species. Cross breeding tests have not in specific regions (Fig. 3, Table 1) and mis may been conducted yet, since Sleinernema sp. MY4 was reflect a greater adaptation to a specific habitat. Stei­ not able to reproduce in either in vivo or in vitro cul­ nernema sp. MY4 is considered to be a subtropical tures (despite the nematode being isolated by using species, found only in laurel forest. A subtropical me Galleria trap method). The RFLP pattern of Stei­ island, Yonaguni Is. is close to Taiwan rather man the nernema sp. MY7 was similar to mat of S. imerme­ ]apanese main land and me species was not isolared dium. However the morphometrics of infective juve­ from any omer subtropical islands in ]apan. Sreiner­ niles were different. nerna sp. MY2 is considered to be a coastal species, The seven steinernematid species other than S. found only in beach-side forest or grassland and sandy kusht'dai are new ta science as indicated by RFLP soil. Natural sandy beaches were weil conserved in the analysis and/or morphologicaJ study. To date, mese mree sites where this species was isolated. Sleinernema steinernematid species are indigenous to ]apan. By spp. MY5, MY6, and MY7 were rare and isolared contrast, ail me heterorhabditid isolates were either only from me forest at high elevation. These species H. indica or H. megidis. H. indica occurs widely in may therefore be considered to prefer cool or coId tropical zones in Egypt (Shamseldean & Abd-Elga­ mountain environment. wad, 1996), India (Poinar el al., 1992), Sri Lanka, Wim respect to the hererorhabditids, it was consi­ normern Australia (Hominick el al., 1996), and Cuba dered mat H. indica was a widespread, coastal species Qoyce el al., 1994a). Therefore, it could be that the mat was not restricted to beach sites and mat H. distribution pattern of H. indica in ]apan was influ­ megidis was a temperate widespread species. Moreover enced by me warm current "Kuroshio" which stans me two species also appear to differ in their vegetation from me Philippine Sea, flows along Taiwan, Nansei­ preference range. H. indica occurred in various vege­ shoto Isis., and me sourhern coast of ]apan and tation: laurel and pine forest, grassland beside crop­ departs from ]apan off me east coast of the Boso land, a turf grass site in a park and a beach having Peninsula (Wadachi, 1987). Ir has been suggested mat only a few grasses and no trees (Table 1), from dry mis warm current affects me distribution of a number open land like a beach to wet, well-developed forest. of insects, especially wood-boring insects (Browne, By contrast, most of the ]apanese isolates of H. 1961; Nobuchi & Makihara, 1987). Browne (1961) megidis occurred only in woodland: laurel, deciduous noted "Currents have also undoubtedly assisred in and pine forest (Table 1). Accordingly, in ]apan, it is conveying elements of me Malaysian fauna to soum­ considered mat me occurrence of H. indica is ern ]apan." Moreover, Konishi (1991) suggested mat restricted by me configuration (coastal site) and mat sorne parasitic wasps of wood-boring insects enlarged me occurrence of H. megidis is restricted by me vege­ meir distribution area by means of driftwood floating tation (forest site). on the ocean current wim their hosts inhabiting the In previous surveys, one isolate of S. carpocapsae woods. (= S. felliae) and four isolates of Sœinernema spp. were The H. megidis species complex occurs widely in reported from the norm part of Honshu (Mamiya & temperate zones in North America (Poinar el al., Ogura, 1989), and two isolates of S. carpocapsae and 1987; Mracek & Webster, 1993) and Europe (Reid, 22 isolates of S. felliae from Kyushu (Kimura & Ishi­ 1994; Hominick el al., 1995). There appears to be bashi, 1991). Although the present survey did not mree c10sely related RFLPs types -Ohio, NWE, and extensively sample mose areas (only Kagoshima, Iba­ Irish Qoyce el al., 1994a; Reid, 1994; Hominick, el raki, and Sendai overlapped wim regions of me previ­ al., 1996)- and the ]apanese isolates were determined ous surveys; Fig. 1), neither S. carpocapsae nor S. to be of me Ohio type. Many similarities between felliae were detected. There were no isolates having eastern Asian biota and easrern North American biota short infective juveniles like S. carpocapsae, other man are also known (Pielou, 1979). Accordingly, it may be s. kushidai. Many isolates examined morphologically mat ]apanese H. megidis is a native species and not an had infective juveniles wim sorne measurements mat introduced species such as the Australian S. felliae overlapped those of S. felliae. However no isolates (Akhurst & Bedding, 1986), because of its occurrence were identified as S. felliae from eimer RFLP patterns inland (Fig. 4) and coexistence with steinernematids or morphological observations. Moreover, a recent re­ (Table 2) in well-developed forest. However, as the sampling conducted in Kyushu (including sorne sites distribution of the heterorhabditids in the coastal where S. carpocapsae or S. felliae had been detected) region of lreland and Britain was suspected to be only found Sleinernema spp. MYI and MY3 (Yoshida affected by transport by sea water (Griffin el al., unpubl.). Sleinernema sp. MY4 was morphologically 1994a), more surveys in eastern Asia are necessary to similar to S. affine, which to date has only been show the distribution pattern of H. megidis and the reported from northem Europe (Poinar, 1990), but origin of the ]apanese isolates.

196 FUr/dam. appl. Nemawl. Enlomopalhogenic nemalOdes in Japan

This survey indicates that the ]apanese environmenr tion of biological species in the genus Heterorhabditis has a rich enromopathogenic nematode fauna, much (Nematoda: Heterorhabditidae) by breeding second gen­ of it specific to ]apan, and that each nematode has a eration amphimictic adults. Parasitology, 104: 509-518. characteristic distribution pattern. Especially, nine of DOUCET, M. M. A. DE (1986). A new species of Neoaplec­ ten species were recorded from mountains. This may !ana Steiner, 1929 (Nematoda: Steinernematidae) from result from the geographic complexity and biological Cord6ba, Argentina. Revue NémalOl., 9: 317-323. diversity of the mountain environmenr that provides DOUCET, M. M. A. DE & DOUCET, M. E. (1990). Sleiner­ numerous and various insects as potential hosts. As nema ritteri n. sp. (Nematoda: Steinernematidae) with a most of the land in ]apan is mounrainous, it might be key to the species of the genus. NemalOlogica, 36: 257-265. speculated that most of the ]apanese entomopatho­ GARD!'ŒR, S. L., STOCK, S. P. & KAYA, H. K (1994). A genic nematodes were more adapted and/or special­ new species of Helerorhabditis from the Hawaiian islands. J Parasit., 80: 100-106. ized to a mountain environmenr. It is expected that GRIFFIN, C. T, FINNEGAN, M. M. & DOWNES, M .J. other species or strains of steinernematids and hetero­ (l994a). Environmental talerance and the dispersal of rhabditids will be isolated from other environments, Heterorhabditis: survival and infectivity of European Hete­ as many potenrial habitats remain ta be explored. rorhabditis following prolonged immersion in seawater. Acknowledgtnents Fundam. appl. NemalOl., 17: 415-421. We thank N. Minagawa, NIAES (Present address: GRIFFlN, C. T, JOYCE, S. A., DIX, l, BURNELL, A. M. National Agricultural Research Center), K Hirara, NIAES & DO\XTNS, M. J. (1994b). Characterization of the (Present address: Yokohama Plant Protection Station), entomopathogenic nematode Heterorhabditis (Nematoda: T Mizukubo, Kyushu Agricultural Experimental Station, Heterorhabditidae) from Ireland and Britain by molecu­ and H. Tanabe, S. Yamanaka, and T Takeuchi, SDS Bio­ lar and cross-breeding techniques, and the occurrence tech, for providing soil samples. We are indebted ta of the genus in these island. Fundam. appl. NemalOl., 17: M. Araki, NIAES for critical reading of the manuscript. 245-253. This work was supported in part by a Grant-in-Aid for Sci­ GRJFFlN, C. T., MOORE, J. F. & DOWNES, M. J. (1991). entific Research, ministry of Education, Culture and Sci­ Occurrence of insect-parasitic nematodes (Steinernema­ ence, Japan (No. 02506001). tidae, Heterorhabditidae) in the Republic of Ireland. NemalOlogica, 37: 92-100. References HA RA, A. H., GAUGLER, R, KAYA, H. K, & LEBECK, AKHURST, R J. & BEDDING, R. A. (1986). Narural occur­ L. M. (1991). 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198 Fundam. appl. NemalOl.