Molecular characterisation of Heterorhabditis indica isolates from India, Kenya, Indonesia and Cuba

Nematology, 2000, Vol. 2(5), 477-487 Molecular characterisation of Heterorhabditis indica isolates from India, Kenya, Indonesia and Cuba Colin M. STACK 1, Subbana G. EASWARAMOORTHY 2, Usha K. METHA 2, Martin J. DOWNES 1, Christine T. GRIFFIN 1and Ann M. BURNELL 1,* 1The Institute for Bioengineeringand Agroecology, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland 2SugarcaneBreeding Institute, Indian Council for Agricultural Research, Coimbatore, Tamil Nadu 641 007, India Accepted for publication:20 November 1999 Summary – Isolates of Heterorhabditis were identi ed as H. indica using the following molecular diagnostic features: hybridisation to a H. indica speci c satellite DNA probe; AluI and MboI restrictionpro les of the rDNA ITS PCR product and the AluI pro le of the rDNA IGS PCR product. The Kenyan isolates represent a distinct subgroup of H. indica. These isolates lacked one of the two Hinf I restriction sites which are present in the rDNA ITS product of all the other isolates tested and they also differed from other H. indica isolates in their rDNA IGS HaeIII restriction pro le. The Indian isolates are interfertile. The Kenyan isolates are interfertile but only one Kenyan isolate, Ki3, produced viable progeny when crossed with H. indica LN2. The four Indonesian isolates are interfertile,but only one Indonesian isolate(INA H1) produced viable hybrids when crossed with H. indica LN2. INA H1 was also interfertilewith the Kenyan isolate Ki3. Résumé – Caractérisation moléculaire d’isolats d’Heterorhabditis indica provenant d’Inde, du Kenya, d’Indonésie et de Cuba –Des isolats d’Heterorhabditis ont été identiés comme H. indica par l’utilisation des techniques de caractérisation moléculaire suivantes: hybridation avec une sonde spéci que du DNA satellite de H. indica, produits des pro ls de restriction par PCR de l’ITS du rDNA par AluI et MboI et produit de PCR de l’IGS du rDNA par AluI. Les isolats keniyans constituent un sous-groupe distinct d’H. indica. Un des deux sites de restriction de Hinf I, présent dans les produits de l’ITS du rDNA de tous les autres isolats étudiés, est absent dans ces isolats qui différaient également dans leurs pro ls de restriction de l’IGS du rDNA par HaeIII. Les isolats d’Inde sont interfertiles.Les isolats kenyans sont inter-fertilesmais un seul de ces isolats, Ki3, a produit une descendance viable après croisement avec H. indica LN2. Les quatre isolats indonésiens sont interfertiles,mais un seul d’entre eux (INA H1) a produit des hybrides viables après croisement avec H. indica LN2. INA H1 a été également interfertileavec l’isolat kenyan Ki3. Keywords – crossbreeding,DNA probe, entomopathogenicnematode, molecular diagnostics,rDNA ITS, rDNA IGS, satellite DNA. Entomopathogenic nematodes (EPN) of the families Steinernematidae and Heterorhabditidae, together with their symbioticbacteria Xenorhabdusspp.andPhotorhabdus sp., respectively, are currently being mass produced commerciallyand used to controla variety of soil-dwelling insect pests in Europe, USA, Australia and China(see reviews by Kaya and Gaugler, 1993;Ehlers, 1996).The biological control potentialof EPN has stimulated numerous surveys in an effort to  nd new indigenous isolates and possibly also new species of Heterorhabditis and Steinernema (reviewed by Hominick et al., 1996). Rapid and reliable diagnostic tests are required for species identi- cation in such surveys and DNA  ngerprintingtechniques *Correspondingauthor, e-mail: [email protected] are now becoming more widely used as a  rst screen to determine the species compositionof newly isolated EPN collections.These molecularapproachescan then be supplemented by morphological, morphometric and crossbreeding techniques to conrm the identication of putative new species(reviewed by Hominick et al., 1997). AlthoughSteinernema and Heterorhabditisshare many similarities in their mode of life and morphology, these similarities result from convergent evolution and are not indicative of a close phylogenetic relationship between the two families (Poinar, 1993; Sudhaus, 1993;Blaxter et al., 1998). Steinernema also appears to be more species rich than is Heterorhabditis. Twenty two Steinernema c ®Koninklijke Brill NV, Leiden, 2000 477 C.M. Stack et al. species are recognised by Hominick et al. (1997). Adams et al. (1998) carried out a phylogenetic analysis based on rDNA internal transcribed spacer 1 DNA sequences of nine described species and one putative species of Heterorhabditis and they suggested that three pairs of sister taxa may be conspeci c, thereby delimiting six species of Heterorhabditis. The study of Adams et al. (1998)did not includeH. brevicaudis(Liu, 1994). Current biogeographicdata suggests that two species of Heterorhabditis,H. indica and H. bacteriophora, have a global distribution. H. indica occurs widely in the tropics and subtropics, having been isolated in southern India (Poinar et al., 1992); Sri Lanka (Amarasinghe et al., 1994); peninsular Malaysia (Mason et al., 1996); Indonesia (Grif n et al., 1999b); the Caribbean region (Arteaga Hernandez & MráÏ cek, 1984; Joyce et al., 1994a; Grenier et al., 1996a; Constant et al., 1998); Egypt (Grenier et al., 1996a) and in subtropical and warm temperate zones in Japan (Yoshida et al., 1998). Curran and Driver (1994) presented data for HaeIII restriction digests of the rDNA intergenic spacer region of a range of tropical isolates of Heterorhabditis from north Australia, Egypt, the Caribbean region, Florida, USA and Hawaii. They distinguished between two restriction pro les designated D1 and D1a. This distinctionbetweenthe D1 and D1a pro les was based on a size polymorphismof the large HaeIII restriction fragment from the rDNA IGS fragment. H. bacteriophora occurs in regions of continental and Mediterranean climate in both the northern and southern hemispheres (reviewed by Hominick et al., 1996). To date, H. megidis has been recorded only from the northern hemisphere (Poinar et al., 1987; Smits et al., 1991; Miduturi et al., 1996; Menti et al., 1997; Yoshida et al., 1998; Grif n et al., 1999a), where it typically has a more northerly and more restricted distribution than H. bacteriophora, although H. megidis can be locally common in coastal regions of North West Europe (Grif-  n et al., 1999a). The remaining described species of Heterorhabditis appear to have a more restricted distribution: H. zealandica has been isolated in New Zealand and Tasmania (Wouts, 1979; Poinar, 1990; Curran & Driver, 1994); H. marelatus has been isolated so far only in Oregon and California, USA (Liu & Berry, 1996; Stock, 1997); H. brevicaudis has been isolated in south east China (Liu, 1994) and H. argentinensis in Argentina (Stock, 1993), although it is possible that H. argentinensis and H. bacteriophora may be conspeci c (Adams et al., 1998). Heterorhabditis species distributions can also be inuenced by altitude(Constant et al., 1998), soil type (Kaya, 1990; Grif n et al., 1994) and vegetation cover (Strong et al., 1996). We report here the isolation of new isolates of H. indica from India and Kenya. We have utilised a range of moleculardiagnostictechniquesin conjunctionwith crossbreeding in the identication of these new H. indica isolates and of isolates of H. indica previously isolated from India, Indonesiaand Cuba. Our data indicatethat the HaeIII rDNA IGS restriction pro le designated D1a by Curran and Driver(1994)also occurs in the H. indica typespecies LN2 and that the Kenyan isolates represent a distinct subgroup within the D1a group of H. indica. Materials and methods NEMATODE ISOLATES H. indica LN2, the H. indica type species (Poinar et al., 1992), is maintained at the Sugarcane Breeding Institute (SBI), Coimbatore, India. It was originally isolated from soil samples collected at Ramanathapuram 20 km north of Coimbatore, Tamil Nadu. Coimbatore (at 11°N latitude and 77°E longitude),is 120 km from the Arabian sea, 270 km from the Indian ocean and 310 km from the Bay of Bengal.The soil samples(soil type, red loam)were collected from fallow land (the previous crop was monsoon sorghum) and were baited in the laboratory with top borer (Scirpophaga excerptalis, Pyralidae: Lepidoptera) larvae. LN2B — in situ baiting with S. excerptalis larvae was carried out in a  eld at Somayanur,Coimbatore,from which groundnuts had been harvested. The soil type was red loam. The S. excerptalis larvae were placed individually in small plastic lids covered with brass wire mesh. The insect traps were left in the soil for four days and were then returned to the laboratory at SBI and checked for nematode infection. LN4 — this isolate was obtained from an infected white grub (Holotrichia serrata, Scarabaeidae: Coleoptera)larva collected from a sugarcane  eld at Thirupattur,25 km northof Coimbatore.The soiltypewas heavy clay. In collaboration with personnel from the Kenya Agricultural Research Institute(KARI), 21 soil samples, each of about 500 g, were collected from  ve locations in coastal Kenya and two locations inland, in August 1994. One sample was taken near Tambia village, four from the WAU/KARI centre at Mtwapa, one from coral-based soil at an elevated (8 m) coastal site near Mombasa, 13 from vegetation fringing a beach at Kanamai, one from far inland in the south-west near Lolgorien,and one from near 478 Nematology Isolates of Heterorhabditisindica the Sand river in the same area as the last. The samples were  own to NUI Maynooth,where each sample was divided and each half was baited with  ve late instar Galleria mellonella larvae and incubated at 28°C. Heterorhabditis isolates were recovered only at Kanamai, in a vegetation belt extending 15 m inland from the beach edge. Bioluminescentcadavers(indicatingthe presence of Heterorhabditis) were recovered from two of the 13 samples taken from there. Four isolates were collected in a survey of  ve Indonesian islands by Grif n et al. (1999b). Extracted DNA samples from four isolates displaying the D1a IGS restriction pro le (FLGS10, JAM23, JAM79, ST09, ES10)were obtainedfrom Dr Felice Driver(CSIRO, Canberra, Australia). The source and geographicorigin of the other Heterorhabditisspecies and isolates included in this study are listed in Table 1. MOLECULAR CHARACTERISATION DNA was isolated according to Smits et al.(1991). The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of the rDNA cistron were ampli ed by means of the polymerase chain reaction (PCR) as described by Joyce et al. (1994a, b). Ampli cation products were digested with restriction endonucleasesfollowing the manufacturer’s instructions using 5-12 ml PCR product in a 15 ml reaction volume. The entire digest was loaded on a 2% agarose gel and electrophoresed in 1´TBE at 5 V/cm for 3.5 h. Restriction fragments were visualised by ethidium bromidestaining. The dot blot procedure utilised the H. indica speciesspeci c satelliteDNA probe used by Grenier et al.(1996a). GenomicDNA (100 ng)samples were denaturedby adding 1MNaOH and 200 mMEDTA pH 8.2 to each sample to give a  nal concentration of 0.4 MNaOH, 10 mM Table 1. Source and geographic origin of the Heterorhabditis species and isolates included in this study. Species Isolate Geographic Origin Source H. indica LN2 Coimbatore, India Easwaramoorthy1 H. indica LN2B Coimbatore, India This study H. indica LN4 Coimbatore, India This study H. indica Ki3 Kanami, Kenya This study H. indica K4A Kanami, Kenya This study H. indica INA H1 West Java, Indonesia Grif n et al. (1999b) H. indica INA H9 Ambon, Indonesia Grif n et al. (1999b) H. indica INA H17 Seram, Indonesia Grif n et al. (1999b) H. indica INA H23 Moluccas, Indonesia Grif n et al. (1999b) H. indica P2M Artemisia, Cuba Mracek2 H. indica D1 Darwin, Australia Bedding3 H. indica FLGS10 Florida, USA Curran & Driver3 H. indica JAM23 Jamaica Curran & Driver H. indica JAM79 Jamaica Curran & Driver H. indica ST09 Virgin Islands Curran & Driver H. indica ES10 Egypt Curran & Driver H. bacteriophora HP88 Utah, USA Akhurst3 H. zealandica NZH3 New Zealand Bedding3 H. marelatus OH-10 Oregon, USA Liu4 H. hepialus Bodega Bay California, USA Stock5 H. megidis HL81 Leeuwarden, The Netherlands Westerman6 H. ‘Irish type’ K122 Wexford, Ireland Grif n et al. (1994). 1Sugar Cane Breeding Institute,Coimbatore, India; 2Institute of Entomology, Czech Academy of Sciences, Ceske Budejovice, Czech Republic; 3CSIRO, Canberra, Australia; 4Oregon State University, OR, USA; 5University of California, Davis, CA, USA; 6Van Hall Instituut, Leeuwarden, The Netherlands. Vol. 2(5), 2000 479 C.M. Stack et al. EDTA and the samples were then boiled for 10 min in a water bath. The samples were then transferred by vacuum suction onto a positively charged nylon membrane (Amersham Life Sciences Ltd., Amersham, Buckinghamshire, UK) in a slot blot apparatus (Schleicher & Schuell, D37582 Dassel, Germany) as per manufacturer’s instructions. The DNA was then  xed onto the nylon membrane by UV cross-linking using Stratagene’s Stratalinker (Stratagene, La Jolla, CA 92037, USA). The recombinant pUC plasmids Hi12 and HP88s9 which contained respectively the H. indica and H. bacteriophora satellite DNA monomers were obtained from Dr Pierre Abad, INRA, Antibes, France and ampli ed using PCR. The PCR primers used to amplify the H. indica monomer from the Hi12 plasmid were 5¢-CTGAAGCACTTGGGACAGAGC-3¢and 5¢-CTCCTCGTTGAGGACGGGAGT3¢(Abadonet al., 1998;Grenier,pers. comm.).The H. bacteriophora monomer was ampli ed from the HP88s9 plasmid using the following PCR primers 5¢-AGCTATGCCAGAATGATCGCC-3¢and 5¢-AGATTCTCTGTACGATGAGTA-3¢(Grenier et al., 1996b; Grenier, pers. comm.). DNA was ampli ed using the following conditions: one cycle of 94°C for 5 min was followed by 35 cycles of denaturation at 94°C for 0.5 min annealing at 52°C for 1 min and extension at 72°C for 2 min, with a  nal cycle of extension at 72°C for 5 min. The probes were labelled using the ECL direct nucleic acid detection system (Amersham) following the manufacturer’s instructions. Hybridisations were conducted at 42°C overnight. After hybridisation, the  lters were washed  rst with 0.5 ´SSC for 40 min at 42°C and then with 0.1´SSC for 5 min at room temperature.After posthybridisation washes,  lters were exposed to Hyper imECL (Amersham) following the manufacturer’s instructions. The membrane was then stripped and reprobed using an 18S rDNA probe as a loading control. The 18S probe was obtained by PCR from H. indica LN2 genomic DNA using the primers: 18SR2B 5¢-TACAAAGGGCAGGGACGTATT-3¢and 18S1.2 5¢-GGCGATCAGATACCGCCCTAGTT-3¢(T.O. Powers, pers. comm.). CROSS-BREEDING TESTS Cross-breedingstudieswere carried outas describedby Dix et al. (1992) with the following controls being set up for each cross: virginity test — 20 virgin females were placed on a lipid agar plate that had been inoculated and pre-incubatedwith the primary form of the LN2 bacteria; self-cross — ten virgin females and ten males of the same isolate were placed on lipid agar plates containing the bacterial symbiont. The result of any cross between different isolates was taken as valid only if there were no progenyin the virginitytest and there were progenyin the self-cross. At least ten second generation virgin females were used for each cross. Results RESTRICTION PROFILES OF THE RDNA INTERNAL TRANSCRIBER SPACER (ITS) REGION All of the tropical isolates of Heterorhabditis yielded aca 1 kb fragment upon PCR ampli cation with the ITS primers. These ampli cation products were digested with the diagnostic restriction endonucleases used by Joyce et al. (1994a) for species diagnosis in Heterorhabditis (viz. AluI, Hinf I and MboI). When digested with AluI (Fig. 1) the Indian isolates LN2B and LN4 shared the same restriction pro le as the H. indica LN2 type species, as did the Indonesian isolates, the P2M isolate from Cuba, and the Kenyan isolates. When digestedwith MboI, the Kenyan isolates shared the same restriction pro le Fig. 1. AluI restrictiondigests of the PCR amplication products of the rDNA internal transcribed spacer region of Heterorhabditis isolates, separated on a 2% agarose gel and stained with ethidium bromide. M: 1 kb marker; 1: H. indica LN2; 2: P2M; 3: LN2B; 4: LN4; 5: INA H23; 6: INA H9; 7: INA H17; 8: INA H1; 9: Ki3; 10: K4A; 11: Heterorhabditis‘Irish type’ K122; 12: H. megidis HL81. 480 Nematology Isolates of Heterorhabditisindica as H. indica LN2 and P2M (Fig. 2). The Indonesian isolates also displayed the same MboI restriction pro le as H. indica LN2 (data not shown). When digested with Hinf I (Fig. 3) the Indian, Indonesian and Cuban isolates but not the Kenyan isolates,possessed the same restriction pro le as H. indica LN2. The Kenyan isolates have only a single Hinf I restriction site in the ITS rDNA fragment, yielding two restriction fragments of ca 620 and 450 bp, unlikeall the otherH. indica isolatestested,which possess two HinfI sites in this region, yielding three restriction fragments of ca 450, 360 and 240 bp. Fig. 2. MboI restriction digests of the PCR amplication products of the rDNA internal transcribed spacer region of Heterorhabditisisolates, separated on a 2% agarose gel and stained with ethidium bromide. M: 1 kb marker; 1: H. indica LN2; 2: P2M; 3: Ki3; 4: K4A; 5: H. bacteriophora HP88. RESTRICTION PROFILES OF THE RDNA INTERGENIC SPACER (IGS) REGION The PCR ampli cation products for the IGS rDNA region of the tropical isolates varied in size from 1.5 kb to 1.7 kb. The ampli ed IGS rDNA fragments were restricted with the endonucleases AluI and HaeIII. A diagnostic AluI restriction pattern was obtained for H. indica LN2 and this was shared by all the Indian, Indonesian, Cuban and Kenyan isolates (Fig. 4) and the D1 and D1a type isolates(Fig. 6). When HaeIII was used to digest the IGS rDNA region, H. indica LN2 displayed a distinctive ve fragment pro le of 740, 280, 190, 160 and 120 bp (Fig. 5) and, with the exception of the Kenyan isolates, four of these fragments were shared by the other tropical isolates. The Kenyan isolates Ki3 (lane 9) and K4A (lane 10) had a distinct restriction pro le yielding HaeIII fragments of 700, 280 (a doublet), 185 and 120 bp. The largest H. indica HaeIII fragmentwas highlypolymorphic in size between the tropical isolates, ranging in size from 700 to 850 bp, with some isolateshavingfragments of intermediatesize ca 740 bp.Size polymorphismin this fragment was the basis on which the types D1 and D1a were recognised by Curran and Driver (1994). The HaeIII pro-  les of the other three speciesof Heterorhabditisincluded in Fig. 5 were all distinctly different from that of H. indica. As can be seen from Fig. 6, the HaeIII rDNA IGS restriction pro le designated D1a by Curran and Driver (1994)(lanes 4-7) is similar to that of H. indica LN2, but the HaeIII pattern of the Kenyan isolates (Fig. 6, lane 3; Fig. 5, lanes 9,10) is unique. Fig. 3. HinfI restriction digests of the PCR amplication products of the rDNA internal transcribed spacer region of Heterorhabditis isolates, separated on a 2% agarose gel and stained with ethidium bromide. M: 1 kb marker; 1: LN2; 2: P2M; 3: LN2B; 4: LN4; 5: INA H23; 6: INA H9; 7: INA H17; 8: INA H1; 9: Ki3; 10: K4A; 11: Heterorhabditis‘Irish type’ K122; 12: H. megidis HL81. Vol. 2(5), 2000 481 C.M. Stack et al. Fig. 4. AluI restriction digests of the PCR ampli cation products of the rDNA intergenic spacer region of Heterorhabditis isolates, separated on a 2% agarose gel and stained with ethidium bromide. M: 1kb size marker; 1: LN2; 2: P2M; 3: LN2B; 4: LN4; 5: INA H23; 6: INA H9; 7: INA H17; 8: INA H1; 9: Ki3; 10: K4A; 11: Heterorhabditis ‘Irish type’ K122; 12: H. megidis HL81; 13: H. bacteriophoraHP88. Fig. 5. HaeIII restriction digests of the PCR amplication products of the rDNA intergenic spacer region of Heterorhabditis isolates, separated on a 2% agarose gel and stained with ethidium bromide. M: 1kb size marker; 1: LN2; 2: P2M; 3: LN2B; 4: LN4; 5: INA H23; 6: INA H9; 7: INA H9; 8: INA H1; 9: Ki3; 10: K4A; 11: Heterorhabditis‘Irish type’ K122; 12: H. megidis HL81; 13: H. bacteriophora HP88. USE OF H.INDICA SPECIES SPECIFIC SATELLITE DNA PROBE When Southern blots of total genomic DNA of the tropical isolates were probed with the H. indica speciesspeci c satellite DNA probe described by Abadon et al. (1998), the probe hybridised only with the isolates classi ed from PCR analysis as being H. indica, including the Kenyan isolates Ki3 and K4A (Fig. 7A). No hybridisation was detected by this probe to any of the other species included as controls on the Southern blot. This blot was then reprobed with the H. bacteriophoraspeci c satellite DNA probe described by Grenier et al. (1996), and this probe hybridised only with the HP88 isolate of H. bacteriophora included on the Southern blot (data not shown). 482 Nematology Isolates of Heterorhabditisindica Fig. 6. HaeIII and AluI restriction digests of the PCR amplication products of the rDNA intergenic spacer region of Heterorhabditis isolates, including isolates designated type D1a by Curran and Driver (1994). M: 1kb size marker; 1: H. indica LN2; 2: D1; 3: K4A; 4: ES10; 5: JAM79; 6: ST09; 7: FLGS10; M: 1 kb marker; 9: H. indica LN2; 10: D1; 11: ES10; 12: JAM79; 13: ST09; 14: FLGS10. Lanes 1 to 7 contain HaeIII digests and lanes 9 to 14 contain AluI digests. The digests were separated on a 2% agarose gel and stained with ethidium bromide. Table 2. The results of cross-breeding experiments of six Heterorhabditis isolates from India, Indonesia and Kenya. Female Male H. indica LN 4 INA H1 INA H23 Ki3 K4A LN2 India India Indonesia Indonesia Kenya Kenya H. indica + + + 0 + 0 LN2 LN 4 + + 0 0 + + INA H1 + + + + + 0 INA H23 0 0 + + 0 0 Ki3 + + + 0 + + K4A 0 0 0 0 + + + cross resulted in fertile progeny; 0 no progeny detected. CROSS BREEDING ANALYSIS Selected cross-breedingresults are presented in Table 2. All the Indian isolates are interfertile. The Kenyan isolates, which differed from H. indica in their HinfI ITS restriction pro le and their HaeIII IGS restriction pro le, are interfertile amongst each other but only one Kenyan isolate, Ki3, produced viable progeny when crossed with the H. indica LN2 type species. The Indonesian isolates are interfertile, but surprisingly, only one of the Indonesian isolates tested (INA H1) produced viable hybrids when crossed with H. indica LN2. The INA H1 isolate was also interfertile with the Kenyan isolate Ki3. Discussion The results presented here con rm and extend previous studies which show that a combination of molecular diagnostictools can be reliably used for species identi cation in Heterorhabditis. Isolates of Heterorhabditis from India, Kenya, Indonesia and Cuba hybridised to the H. indica speci c satellite DNA probe (Grenier et al., 1996; Abadon et al., 1998). H. indica speci c DNA restriction pro les were also obtained for all the tropical isolates of Heterorhabditis investigated here when the ITS rDNA region was restricted using the diagnostic restriction enzymes AluI and MboI (Joyce et al., 1994a). The utility of restriction digests of the rDNA ITS region in species diagnosis of EPN has been con rmed in several studies (Joyce et al., 1994a,b; Miduturiet al., 1996; Hominick et al., 1997; Yoshida et al., 1998; Grif n et al., 1999a;Pamjav et al., 1999). Restriction of the rDNA IGS region with AluI also yieldeda H. indica speci c restriction pro le for all the tropical isolates tested in this study. The PCR ampli cation product which we obtained for the H. indica rDNA IGS varied in size from 1.2 to 1.6 kb. Hominick et al. (1997) also observed length heterogeneity in the rDNA IGS region among geographic isolates of single Steinernema spp. and they suggested that this heterogeneity may make restriction pro les of this DNA fragment unreliable for species identication. The length heterogeneity which we detected in the H. indica IGS PCR ampli cation product did not affect the AluI restriction pro le of the product and a clear-cut seven restriction fragment pattern was common to all the H. indica isolates Vol. 2(5), 2000 483 C.M. Stack et al. Fig. 7. A: Slot blot analysis of genomic DNA (100 ng) of the Heterorhabditisisolates using a H. indica specic satellite DNA probe; B: The membrane used for Fig. 7A was stripped and reprobed using an rDNA 18S probe as a loading control. A1: H. indica LN2; A2: D1; A3: P2M; A4: LN2B; A5: LN4; A6: INA H23; B1: INA H9; B2: INA H17; B3: INA H1; B4: Ki3; B5: K4A; B6: H. bacteriophoraHP88; C1: H. zealandicaNZH3; C2: H. marelatus OH-10; C3: H. hepialus Bodega Bay; C4: Heterorhabditis‘Irish type’ K122. tested. When the IGS region was digested with HaeIII, H. indica LN2 displayed a distinctive  ve fragment pro-  le. All of the H. indica isolates, with the exception of the Kenyan isolates, also displayed a  ve fragment pro-  le. Four of these fragments were conserved among the isolates, but the largest fragment (of ca 740 in H. indica LN2) was polymorphicamong the H. indica isolates. This fragment, which seems to be associated with the length polymorphism in the H. indica IGS region, is the basis of the D1/D1a polymorphism observed by Curran and Driver (1994). In most species studied to date, the rDNA IGS contains tandem arrays of subrepeats (Gerbi, 1985; Williams et al., 1990; Vahidi & Honda, 1991; Novak et al., 1993; Linares et al., 1994; Crease, 1995). This results in the length of the IGS region being variable both between and within species. The length heterogeneity of the H. indica IGS PCR product did not affect the AluI restriction pro le of this fragment, which suggests that AluI has a restriction site within a tandem repetitive unit in the rDNA IGS of H. indica. In contrast, HaeIII appears not to cut within this tandem repeat region with the result that one of the HaeIII restrictionfragments is polymorphic,its length presumably depending on the number of AluI repeat units which it contains. The HaeIII rDNA IGS restriction pro le of the D1a isolate is similar to that of H. indica LN2, but the HaeIII pattern of the Kenyan isolates is unique. The large HaeIII fragment of the Kenyan isolate K4A is the same size as that of D1a, thus the Kenyan isolates appear to represent a distinct subgroup within D1a. Tropical isolates from Darwin (North Australia), Puerto Rico, Hawaii, the Virgin Islands and Egypt were found by Curran and Driver (1994) to possess the D1 pro le, while the D1a pro le was described by these authors from isolates collected in Puerto Rico, Jamaica, Florida, the Virgin Islands and Egypt. Comparison of the D1 and D1a HaeIII and AluI rDNA IGS restriction pro les with those of H. indica LN2, indicates that the isolates designated D1 and D1a by Curran and Driver (1994) belong to H. indica. An important step towards achieving an effective nematode bacterium complex for pest control is to seek naturally occurring endemic EPN isolates, as such isolates are likely to possess physiological traits that are adapted to local climatic and ecological conditions. The Kenyan isolates described in this study clearly belong to a distinct sub-group of H. indica and it is probable that these isolates also share a distinctive phenotype. The two Kenyan isolates are interfertile but only one of these isolates(Ki3) produced viable progeny when crossed with the H. indica LN2 type species. All of the Indonesian Heterorhabditis isolates were indistinguishable from H. indica in the diagnostic molecular tests and they were interfertile with each other, but only one of the Indonesian isolates (INA H1) was interfertile with H. indica LN2 and INA H1 was also interfertile with the Ki3 isolate from Kenya. These results suggest that, although H. indica has a global distribution in tropical and subtropical regions of the world, gene ow within the species may be quite restricted. We havepreviouslyobservedreproductiveincompatibilitybe484 Nematology Isolates of Heterorhabditisindica tween isolates of H. bacteriophora from Europe (Grif n et al., 1999a) and from North America (Dix et al., unpubl.). We have not determined whether this reproductive isolation is caused by genetic means or is the result of cytoplasmic factors. Cytoplasmic factors such as endosymbiont bacteria (see reviews by Werren, 1997; Johanowicz & Hoy, 1998) and chromosomal factors such as transposons (reviewed by Kidwell, 1990; Petrov et al., 1995) have been shown to be frequent causes of reproductiveincompatibilityin arthropods. Endosymbiontbacteria have recently been shown to cause reproductiveincompatibility in  larial nematodes (Hoerauf et al., 1999). Since the reproductiveincompatibilityobservedin this study does not yield clear groupsof compatible and incompatiblestrains and in view of the consistencyof the DNA based diagnostic tests, any assignment of biological species within the H. indica group would be injudicious at present. Adams (1988)evaluatesphylogeneticand biologicalspecies concepts in the delimitationof speciesin the H. bacteriophora group. The global distribution of H. indica throughout the tropics and subtropics suggests that H. indica possesses a range of phenotypic characters which give it an advantage in this climatic zone. Such phenotypesmight include adaptationto high temperatures, desiccationtoleranceand good dispersal ability. It is unlikely that the widespread distribution of H. indica in the tropics arises because this species was extant before the continents began breaking up and drifting. The best supported hypothesis of phylogenetic relationshipsamong Heterorhabditisspecies, in a phylogeny derived from the rDNA ITS 1 DNA sequence data, puts H. indica as the most ancient Heterorhabditis lineage (Adams et al., 1998). However, the phylogenetic tree for Heterorhabditis and Steinernema of Reid (1994) shows that the genetic distance between Heterorhabditis species is considerably less than that between Steinernema species and given the small genetic distance detected by Reid (1994) between the most divergent of the Heterorhabditis species in his study (a D value of ca 0.04 as calculated by the method of Nei and Li, 1979), it seems unlikely that H. indica speciated before the Jurassic/Cretaceousbreak up of Pangaea(an estimated 200 million years ago). Acknowledgement We acknowledgethe support of the European Community STD-3 Programme (TS3 CT 940273). References ABADON, M., GRENIER, E., LAUMOND, C. & ABAD, P. (1998). 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