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Genetic Diversity in Insect-Parasitic Nematodes (Rhabditida: Heterorhabditidae)
JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.
72, 185–189 (1998)
IN984778
Genetic Diversity in Insect-Parasitic Nematodes (Rhabditida: Heterorhabditidae) Ghazala Hashmi1 and Randy Gaugler Department of Entomology, Rutgers University, New Brunswick, New Jersey 08903 Received December 19, 1996; accepted March 25, 1998
Little is known about the genetic structure of various species and populations of entomopathogenic nematodes. We determined genetic variability within and among isolates of seven Heterorhabditis species using random amplified polymorphic DNA (RAPD) markers. We used 10 random primers which were previously identified as useful to quantify genetic variability among these species. Mean percentage similarity among the individuals of conspecific species was 96.25%, whereas, the mean value among different isolates for three species was 83.8%. Mean percentage similarity among different species was 31.3%. The banding patterns produced by RAPDs positively correlated with described morphological classification; however, H. hawaiiensis could not be separated from H. indicus, or H. marelatus from H. hepialius. RAPD profiles placed an unidentified isolate (IS5) with H. indicus. r 1998 Academic Press Key Words: insect parasitic nematode; Heterorhabditis species; genetic variability; RAPD markers.
INTRODUCTION
At present little is known about the genetic structure of entomopathogenic nematodes. The knowledge of within species diversity is fundamental to understanding genetic structure, which in turn can be used in population genetic studies as well as for identification. Various field populations have been collected throughout the world and each has been designated as an ‘‘isolate’’ or ‘‘strain.’’ No knowledge about the genetic structure of these isolates is available. Molecular markers generated with various methods can be used efficiently to determine the genetic variability within and among populations (Curran and Webster, 1989). Protein and DNA markers have been implemented in entomopathogenic nematode research. Most of these 1 To whom correspondence should be addressed at Department of Entomology, Rutgers University, New Brunswick, NJ 08903. Fax: (732) 932-7229. E-mail: [email protected].
studies have been centered on the search of molecular markers for species identification (for review see Curran, 1990; Hashmi et al., 1997). High levels of genetic variability among Heterorhabditis isolates have been reported using both isozyme and DNA markers (Akhurst et al., 1987; Curran and Webster, 1989; also review by Curran, 1990; Gardner et al., 1994; Joyce et al., 1994; Liu and Berry, 1996; Hashmi et al., 1996; Smith et al., 1991). The genus Heterorhabditis comprises nine species: H. argentinensis Stock, H. bacteriophora Poinar, H. brevicaudis Liu, H. hawaiiensis Gardner, Stock, and Kaya, H. indicus Poinar, Karunakar, and Hastings, H. megidis Poinar, Jackson, and Klein, H. zealandica Poinar, H. marelatus Liu and Berry, and H. hepialius Stock, Strong, and Gardner. Entomopathogenic nematode species have traditionally been characterized using a combination of morphological traits and cross breeding (Akhurst and Bedding, 1978; Dix et al., 1991; Poinar, 1986, 1990). Because of the hermaphrodite nature of Heterorhabditis species, unique morphological characters are difficult to observe (Curran and Webster, 1989; Curran, 1990; Poinar, 1990). In spite of low morphological diversity, considerable genetic diversity exists in this genus (Akhurst, 1987; Curran and Webster, 1989; Curran, 1990; Hashmi et al., 1996; Smith et al., 1991). Random amplified polymorphic DNA (RAPD) markers (Williams et al., 1990; Welsh and McClelland, 1990) have been used for the study of genetic diversity of Heterorhabditis and Steinernema species (Hashmi et al., 1996). These techniques also were used for the description of new species (Gardner et al., 1994; Liu and Berry, 1996; Stock et al., 1997). Hashmi et al. (1996) reported different levels of polymorphism (percentage similarity: 24–96%) among Heterorhabditis populations collected from different parts of the world. Therefore, the population genetic structure may differ among different Heterorhabditis species. In the present study we investigated within and among genetic diversity of various isolates belonging to seven Heterorhabditis species.
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0022-2011/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
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HASHMI AND GAUGLER MATERIALS AND METHODS
Nematode Culture The nematode species and isolates tested included: H. bacteriophora (HP88 and E1), H. indicus (EMS-13, Coimbatore), H. hawaiiensis (MG-13), H. megidis (HSH2 and HO1), H. zealandica, H. marelatus (OH10), and H. hepialius (Bodega Bay). Nematode species H. indicus Coimbatore, H. hawaiiensis MG-13, and H. zealandica were obtained from biosys (Columbia, MD). An Egyptian isolate (EMS-13) of H. indicus was provided by M. Shamseldean (Cairo University), IS5 by I. Glazer (Volcani, Israel), H. hepialius by P. Stock (University of California), and DNA of H. marelatus by J. Liu (Oregon State University). Each isolate was passaged through Galleria mellonella, and emerging infective juveniles were collected (Poinar, 1979). The infective juveniles were surface-sterilized and inoculated on lipid agar media (Dunphy and Webster, 1989), seeded with their symbiotic bacteria, Photorhabdus luminescens. All stages of nematodes were collected by centrifugation (Sulston and Hodgkin, 1988) and stored frozen at 220°C until used for DNA extraction. DNA Extraction and RAPD Analysis DNA extraction of nematodes was performed according to the method of Sulston and Hodgkin (1988). The PCR conditions and reagent concentrations were as described previously (Hashmi et al., 1996). Based on the previous study with 80 random primers (Hashmi et al., 1996) 10 primers were chosen. Those primers (OP-A02, OP-A13, OP-A18, OP-C06, OP-C11, OP-H04, OP-H08, OP-H18, OP-H19, OP-S07) were obtained from Operon Technologies, Inc. (Alameda, CA). The amplification products were electrophoresed in 1% w/v, agarose gel in 13 TBE buffer at 1 V/cm for 12–16 h and visualized by staining with ethidium bromide. Reproducibility of DNA profiles was determined by replicating all RAPD reactions at least three times. Reactions were replicated with DNA extracted from different batches of nematodes at different times. DNA Extraction and RAPD Analysis of Single Nematode DNA from single infective stage juvenile was extracted as described for Caenorhabditis elegans by Williams et al. (1992) with the following modifications. Ten nematodes of each species were picked randomly under sterile conditions and individually placed in a drop (15 µl) of nematode lysis buffer on a Petri plate, cut with a syringe, transferred in an Eppendorf tube, and processed. The DNA of each nematode was divided into three aliquots and used in PCR with three different primers (OP-A13, OP-C06, and OP-H19). The PCR conditions were similar to those described for bulk DNA.
Data Analysis Two independent DNA preparations of each species were used except for H. marelatus. Each experiment was replicated at least three times. Genomic DNA was amplified with each of the 10 primers. For single nematode analysis, 10 infective stage juveniles of each species were hand-picked with a needle and DNA was isolated and analyzed as described above. All polymorphic bands were scored for the presence versus absence of a specific amplification product. DNA fragments at low intensities were scored as present only when they were reproducible in repeated experiments. Banding patterns of DNA generated by each primer were analyzed in a pair-wise comparison using the method described by Nei and Li (1979). Percentage similarity was calculated for shared DNA fragments per primer for each species as F 5 2CXY/(TX 1 TY). Where F is the similarity for primer, CXY is the number of common bands produced by both species and TX and TY are the bands generated by isolates X and Y, respectively. Average linkage method (Sneath and Sokal, 1973) was used for cluster analysis on the values of 12F, using the unweighted pair-group method algorithm (UPGMA) and dendrograms were plotted (SAS Institute, 1992). RESULTS
Representative banding patterns generated with individual nematodes of two Heterorhabditis species are shown (Fig. 1). Primer-specific bands generated with individual nematode of each genotype were similar. Some minor differences were observed in band intensities due to differences in DNA concentration of individual nematodes. Although isolates of Heterorhabditis spp. used in this study were from different geographical regions, they showed little variability in the band patterns (Table 1). On average the similarity among isolates of H. bacteriophora, H. indicus, and H. megidis was 83.8%. Overall mean within species similarity for these three Heterorhabditis spp. was 89.75%. High within population similarity (mean 96.2%) was observed with randomly selected individual nematodes, whereas, interspecific similarities based on RAPD markers were 31.3%. The Heterorhabditis species tested were easily distinguished by RAPD analysis (Fig. 2). Similarities between seven Heterorhabditis species ranged from 11.1 to 95% (Table 2) when analyzed for shared DNA fragments. The recently described species H. marelatus (Liu and Berry, 1996) showed a high similarity (95%) with H. hepialius, followed by H. megidis (35.7%), H. bacteriophora (29.6%), H. zealandica (19.0%), and H. indicus (16.6%). H. indicus shared a high similarity with H. hawaiiensis (92.0%). Low levels of similarities were observed between H. indicus and other species
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FIG. 1. RAPD-PCR profiles of two Heterorhabditis species (H. bacteriophora and H. indicus) produced with primer OP-A13 using individual nematodes. (A) DNA banding pattern of DNA isolated from all stages of nematodes (lane 1), DNA isolated from the same population and amplified under similar conditions used for individual nematodes (lane 2), and individual nematode (lanes 3–6) of H. bacteriophora. M, molecular weight marker l DNA cut with HindIII. (B) DNA banding patterns of five individual nematodes (lanes 1–5) of H. indicus. M, molecular weight marker l DNA cut with HindIII and fX174 DNA/HaeIII.
(16.1% with H. megidis and 16.6% with H. zealandica and H. marelatus). DNA profiles generated with an Israeli isolate (IS5) of Heterorhabditis showed a similarity of 89% to H. indicus (data not shown) and it is considered an isolate of this species. H. bacteriophora showed highest similarity with H. zealandica (63%), whereas the similarity with all other species was less than 30%. Four species groups can be identified using cluster analysis (Fig. 3) as follows: (1) H. bacteriophora and H.
TABLE 1 Percentage Similarity within and among Isolates of Heterorhabditis Species Based on Shared DNA Fragments Nematode H. bacteriophora H. hepialius H. hawaiiensis H. indicus H. megidis H. zealandica Average H. bacteriophora H. indicus H. megidis Average Average H. b vs H. z H. b vs H. m
Percent similarity
Nematode
Percent similarity
Within isolates 95.3 98.9 97.2 98.0 93.8 94.3 96.25 6 3.5 Among isolates 83.3 85.2 82.9 83.8 6 1.5 Within species a 89.75 6 3.0 Among species b 63 24
H. b vs H. mar H. b vs H. hep H. b vs H. i H. b vs H. h H. z vs H. m H. z vs H. mar H. z vs H. hep H. z vs H. i H. z vs H. h H. m vs H. mar H. m vs H. hep H. m vs H. i H. m. vs H. h H. mar vs H. hep H. mar vs H. i H. mar vs H. h H. i vs H. h Average
29.6 29.6 22.2 22.2 11.1 19.0 19.0 16.6 16.6 35.7 35.7 16.1 15.0 95.0 16.6 16.6 92.0 31.3 6 5.1
a Within species average of H. bacteriophora, H. indicus and H. megidis. b H. bacteriophora (H.b), H. hepialius (H. hep), H. hawaiiensis (H. h), H. indicus (H. i), H. megidis (H. m), H. marelatus (H. mar), H. zealandica (H. z).
zealandica; (2) H. megidis; (3) H. indicus and H. hawaiiensis, and (4) H. marelatus and H. hepialius. DISCUSSION
Entomopathogenic nematodes show great potential in biological control of insect pests (Gaugler, 1988; Poinar, 1986). Although research has been conducted on the biocontrol abilities of these nematodes (Gaugler, 1988; Nickle et al., 1988; Bedding, 1990), knowledge is scarce about the genetic structure of natural populations. The genetic differentiation among species is the result of the interaction of random chance, migration, and natural selection. Thus, various field isolates are often different from one another at different degrees. DNA and isozyme markers have shown a wide range of similarity among isolates of Heterorhabditis spp. (Akhurst, 1987; Curran and Webster, 1989; Hashmi et al., 1996). These studies suggest the presence of many variants among species collected from different geographical areas. The question is how can we classify the different variants found among these species? Variants
FIG. 2. Comparison of RAPD-PCR banding pattern of six Heterorhabditis species using primer OP-H19. M, molecular weight markers lDNA cut with HindIII and fx174 DNA/HaeIII; 1, H. bacteriophora; 2, H. megidis; 3, H. marelatus; 4, H. hawaiiensis; 5, H. zealandica; 6, H. indicus.
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can be used to describe the limits and distribution of a local population. Various names used to describe variants of entomopathogenic nematode species include isolate, strain, or line. Knowledge of the patterns of genetic difference within all the relevant species is essential for their use as efficient biological control agents. Our study attempted to quantify the variations among Heterorhabditis species. Several DNA preparations of two isolates each of H. bacteriophora, H. megidis, and H. indicus were used to determine similarity among isolates within the same species. Mean percentage similarity of 83.8% was observed among these isolates. An unidentified isolate IS5 was similar (89%) to H. indicus, indicating that more than 80% similarity defined the same genotype among Heterorhabditis spp. Percentage similarity determined by the size of the DNA fragments generated with RAPD also indicated high similarity (mean 96.25%) within isolates. These results are not surprising with Heterorhabditis species considering their life cycle, where the first generation is hermaphroditic and thus individuals in a population can be closely related. These data suggest that heterorhabditids are locally inbred populations with genetic difference between isolates from different geographical regions. Results of the genetic diversity study showed high similarity (63.0%) of shared DNA fragments between H. bacteriophora and H. zealandica, whereas similarity with all other species was less than 30%. H. hawaiiensis has been reported close to H. indicus in morphological characters (Gardner et al., 1994). These two species also showed high similarity (92%) at the DNA level. RAPD profiles generated with several primers produced similar banding patterns for these two species. Because of the high similarities we consider that H. hawiiensis is conspecific with H. indicus. These findings are in agreement with other molecular studies where these species have shown high similarities (Power et al., 1995). H. marelatus and H. hepialius also showed high similarities (92%). Differences if observed were only in band intensities with many markers and may represent the same species. Comparative studies both at morphological and molecular levels are needed to
TABLE 2 Genetic Similarities for Shared DNA Fragments among Heterorhabditis Species Identified with RAPD Markers Species
1
2
3
4
5
6
7
H. bacteriophora H. zealandica H. megidis H. marelatus H. hepialius H. indicus H. hawaiiensis
100 63.0 24.0 29.6 29.6 22.2 22.2
100 11.1 19.0 19.0 16.6 16.6
100 35.7 35.7 16.1 15.0
100 95.0 16.6 16.6
100 16.6 16.6
100 92.0
100
FIG. 3. Unweight pair-group method algorithm (UPGMA) dendrogram showing estimated average genetic distance between Heterorhabditis species. Average linkage cluster analysis was performed on the values of 12F as described under Materials and Methods. Hb, H. bacteriophora; Hz, H. zealandica; Hm, H. megidis, Hmer, H. meralatus; Hhep, H. hepialus; Hi, H. indicus; Hha, H. hawaiiensis.
resolve the status of these species. The high similarities among these two species groups can be attributed to specific environment and habitat preference. Although heterorhabditids have been isolated from different geographical regions (Poinar, 1990; De Doucet and Gabarra, 1994; Smith et al., 1991; Li and Wang, 1989), they are frequently distributed in similar habitats (Hara et al., 1991; Griffin et al., 1994; Amarasinghe et al., 1994; Poinar, 1993). Habitat specificity has been suggested for steinernematids collected from the United Kingdom and The Netherlands (Hominick et al., 1995). In our studies, two isolates of H. indicus and one of H. hawaiiensis, collected from different geographical areas with similar habitats (Gardner et al., 1994; Poinar et al., 1992; M. Shamseldean, personal communication), showed high similarities. This explanation seems quite plausible since H. hepialius and H. marelatus also were isolated from coastal areas with similar environmental conditions (Stock et al., 1996; Liu and Berry, 1996). Although more comparative data are needed on the genetic structure of different populations of the same species, our data suggest that the presence of a population in an area is closely linked with genotype-environmental interactions. These populations can be adapted to specific hosts and environments; therefore similar genotypes are present in a particular habitat. Determination of biological and genetic variability within and among species is important in order to develop an efficient biological control program. An understanding of population level and species level variability will be important for designing strategies for monitoring the release of new isolates, with subsequent recovery and positive identification of released isolates. The present study is the first report of the quantification of genetic diversity among Heterorhabditis species that will be useful in understanding the genetic structure of these nematodes.
GENETIC DIVERSITY IN NEMATODES ACKNOWLEDGMENTS We thank P. Grewal, P. Stock, M. Shamseldean, and J. Liu for providing nematode samples, M. Wilson for reviewing the manuscript, and Dan Collins for technical assistance. New Jersey Agriculture Experiment Station Publication No. D-08256-11-96. REFERENCES Akhurst, R. J. 1987. Use of starch gel electrophoresis in the taxonomy of the genus Heterorhabditis (Nematoda: Heterorhabditidae). Nematologica 33, 1–9. Akhurst, R. J., and Bedding, R. A. 1978. A simple crossbreeding technique to facilitate species determination in the genus Neoaplectana. Nematologica 24, 328–330. Amarasinghe, L. D., Hominick, W. A., Briscoe, B. R., and Reid, A. P. 1994. Occurrence and distribution of entomopathogenic nematodes (Rhabditida: Heterorhabditidae and Steinernematidae) in Sri Lanka. J. Helminthol. 68, 277–286. Bedding, R. 1990. Logistics and strategies for introducing entomopathogenic nematode technology into developing countries. In ‘‘Entomopathogenic Nematodes in Biological Control’’ (R. Gaugler and H. K. Kaya, Eds.), pp. 23–61. CRC press, Boca Raton, FL. Curran, J. 1990. Molecular techniques in taxonomy. In ‘‘Entomopathogenic Nematodes in Biological Control (R. Gaugler and H. K. Kaya, Eds.), pp. 63–74. CRC Press, Boca Raton, FL. Curran, J., and Webster, J. M. 1989. Genotypic analysis of Heterorhabditis isolates from North Carolina. J. Nematol. 21, 140–145. Dix, I., Burnell, A. M., Griffin, C. T., Joyce, S. A., Nugent, M. J., and Downes, M. J. 1991. The identification of biological species in the genus Heterorhabditis (Nematoda: Heterorhabditidae) by crossbreeding second generation amphimictic adults. Parasitology 104, 509–518. De Doucet, M. M. A., and Gabarra, R. 1994. On the occurrence of Steinernema glaseri Steiner, 1929 (Steinernematidae) and Heterorhabditis bacteriophora Poinar, 1976 (Heterorhabditidae) in Catalogne, Spain. Fundam. Appl. Nematol. 17, 441–443. Dunphy, G. B., and Webster, J. M. 1989. The monoxenic culture of Neaoplectana carpocapsae DD136 and Heterorhabditis heliotidis. Rev. Ne’matol. 12, 113–123. Gardner, S. L., Stock, S. P., and Kaya, H. K. 1994. A new species of Heterorhabditis from the Hawaiian Island. J. Parasitol. 80, 100– 109. Gaugler, R. 1988. Ecological considerations in the biological control of soil inhabiting insects with entomopathogenic nematodes. Agric. Ecosystems Environ. 24, 351–360. Griffin, C. T., Joyce, S. A., Dix, I., Burnell, A. M., and Downes, M. J. 1994. Characterization of the entomopathogenic nematode Heterorhabditis (Nematoda:Heterorhabditidae) from Ireland and Britain by molecular and cross-breeding techniques, and the occurrence of the genus in these islands. Fundam. Appl. Nematol. 17, 245–254. Hara, A. H., Gaugler, R., Kaya, H. K., and Lebeck, L. M. 1991. Natural populations of entomopathogenic nematodes (Rhabditida: Heterorhabditidae, Steinernematidae) from the Hawaiian iselands. Environ. Entomol. 20, 211–216. Hashmi, G., Glazer, I., and Gaugler, R. 1996. Molecular comparisons of entomopathogenic nematodes using randomly amplified polymorphic DNA (RAPD) markers. Fundam. Appl. Nematol. 19, 399–406. Hashmi, G., Hashmi, S., and Gaugler, R. 1997. Molecular advances in entomopathogenic nematode research. In ‘‘Recent Research Developments in Entomology’’ (S. Pandalai, Ed.), pp. 161–186. Research Signpost. Hominick, W. M., Reid, A. P., and Briscoe, B. R. 1995. Prevalence and
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habitat specificity of steinernematid and heterorhabditid nematodes isolated during soil surveys of the UK and The Netherlands. J. Helminthol. 69, 27–32. Joyce, S. A., Burnell, A. M., and Power, T. O. 1994. Characterization of Heterorhabditis isolates by PCR amplification of mtDNA and rDNA genes. J. Nematol. 26, 260–270. Li, X. F., and Wang, G. H. 1989. Preliminary investigation on the distribution of Steinernematidae and Heterorhabditidae in Fujian, Guangong and Hainan. Chin. J. Zool. 24, 1–4. Liu, J., and Berry, R. E. 1996. Heterorhabditis marelatus n. sp. (Rhabditida: Heterorhabditidae) from Oregon. J. Invertebr. Pathol. 67, 48–54. Nei, M., and Li, W. H. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 74, 5267–5273. Nickle, W. R., Drea, J. J., and Coulson, J. R. 1988. Guidelines for introducing beneficial insect-parasitic nematodes into United States. Ann. Appl. Nematol. 2, 50–56. Poinar, G. O., Jr. 1979. ‘‘Nematodes for Biological Control of Insects.’’ CRC Press. Boca Raton, FL. Poinar, G. O., Jr. 1986. Entomopathogenic nematodes in biological plant and health protection. In ‘‘Entomogenous Nematodes’’ (J. M. Frenz, Ed.), Vol. 32, pp. 95–121. Fischer Verlag, Stuttgart, Germany. Poinar, G. O., Jr. 1990. Taxonomy and Biology of Steinernematidae and Heterorhabditidae. In ‘‘Entomopathogenic Nematodes in Biological Control’’ (R. Gauglar and H. K. Kaya, Eds.), pp. 23–61. CRC Press, Boca Raton, FL. Poinar, G. O., Jr. 1993. Origins and phylogenetic relationships of the entomophilic rhabditids, Heterorhabditis and Steinernema. Fundam. Appl. Nematol. 16, 332–338. Poinar, G. O., Jr., Karunaker, G. K., and Hastings, D. 1992. Heterorhabditis indicus n.sp. (Rhabditida: Nematoda) from India: Separation of Heterorhabditis species by infective juveniles. Fundam. Appl. Nematol. 15, 467–472. Power, T. O., Adams, B., and Burnell, A. 1995. Molecular diagnostics and taxonomy of Heterorhabditis. In ‘‘Proceeding of the Second International Symposium on Entomopathogenic Nematodes and Their Symbiotic Bacteria, October 15–17, Hawaii. Smith, P. H., Groenen, J. T. M., and De Raay, G. 1991. Characterization of Heterorhabditis isolates using DNA restriction length polymorphism. Rev. Nematol. 14, 445–453. Sneath, P. H. A., and Sokal, R. R. 1973. ‘‘Numerical Taxonomy.’’ Freeman, San Francisco. Stock, S., Strong, D., and Gardner, S. L. 1996. Identification of Heterorhabditis (Nemata: Heterorhabditidae) from California with a new species isolated from the larvae of the ghost moth Hepialis californicus (Lepidoptera: Hepialidae) from the Bodega Bay Natural Preserve. Fundam. Appl. Nematol. 19, 585–592. Sulston, J., and Hodgkin, J. 1988. Methods. In ‘‘The nematode Caenorhabditis elegans (W. B. Wood, Ed), pp. 587–606. Cold Spring Harbor, NY. Welsh, J., and McClelland, M. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213–7218. Williams, G. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A., and Tingly, S. V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531–6535. Williams, B. D., Schrank, B., Huynh, C., Shownkeen, R., and Waterston, R. H. 1992. A genetic mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged sites. Genetics 131, 609–624.
72, 185–189 (1998)
IN984778
Genetic Diversity in Insect-Parasitic Nematodes (Rhabditida: Heterorhabditidae) Ghazala Hashmi1 and Randy Gaugler Department of Entomology, Rutgers University, New Brunswick, New Jersey 08903 Received December 19, 1996; accepted March 25, 1998
Little is known about the genetic structure of various species and populations of entomopathogenic nematodes. We determined genetic variability within and among isolates of seven Heterorhabditis species using random amplified polymorphic DNA (RAPD) markers. We used 10 random primers which were previously identified as useful to quantify genetic variability among these species. Mean percentage similarity among the individuals of conspecific species was 96.25%, whereas, the mean value among different isolates for three species was 83.8%. Mean percentage similarity among different species was 31.3%. The banding patterns produced by RAPDs positively correlated with described morphological classification; however, H. hawaiiensis could not be separated from H. indicus, or H. marelatus from H. hepialius. RAPD profiles placed an unidentified isolate (IS5) with H. indicus. r 1998 Academic Press Key Words: insect parasitic nematode; Heterorhabditis species; genetic variability; RAPD markers.
INTRODUCTION
At present little is known about the genetic structure of entomopathogenic nematodes. The knowledge of within species diversity is fundamental to understanding genetic structure, which in turn can be used in population genetic studies as well as for identification. Various field populations have been collected throughout the world and each has been designated as an ‘‘isolate’’ or ‘‘strain.’’ No knowledge about the genetic structure of these isolates is available. Molecular markers generated with various methods can be used efficiently to determine the genetic variability within and among populations (Curran and Webster, 1989). Protein and DNA markers have been implemented in entomopathogenic nematode research. Most of these 1 To whom correspondence should be addressed at Department of Entomology, Rutgers University, New Brunswick, NJ 08903. Fax: (732) 932-7229. E-mail: [email protected].
studies have been centered on the search of molecular markers for species identification (for review see Curran, 1990; Hashmi et al., 1997). High levels of genetic variability among Heterorhabditis isolates have been reported using both isozyme and DNA markers (Akhurst et al., 1987; Curran and Webster, 1989; also review by Curran, 1990; Gardner et al., 1994; Joyce et al., 1994; Liu and Berry, 1996; Hashmi et al., 1996; Smith et al., 1991). The genus Heterorhabditis comprises nine species: H. argentinensis Stock, H. bacteriophora Poinar, H. brevicaudis Liu, H. hawaiiensis Gardner, Stock, and Kaya, H. indicus Poinar, Karunakar, and Hastings, H. megidis Poinar, Jackson, and Klein, H. zealandica Poinar, H. marelatus Liu and Berry, and H. hepialius Stock, Strong, and Gardner. Entomopathogenic nematode species have traditionally been characterized using a combination of morphological traits and cross breeding (Akhurst and Bedding, 1978; Dix et al., 1991; Poinar, 1986, 1990). Because of the hermaphrodite nature of Heterorhabditis species, unique morphological characters are difficult to observe (Curran and Webster, 1989; Curran, 1990; Poinar, 1990). In spite of low morphological diversity, considerable genetic diversity exists in this genus (Akhurst, 1987; Curran and Webster, 1989; Curran, 1990; Hashmi et al., 1996; Smith et al., 1991). Random amplified polymorphic DNA (RAPD) markers (Williams et al., 1990; Welsh and McClelland, 1990) have been used for the study of genetic diversity of Heterorhabditis and Steinernema species (Hashmi et al., 1996). These techniques also were used for the description of new species (Gardner et al., 1994; Liu and Berry, 1996; Stock et al., 1997). Hashmi et al. (1996) reported different levels of polymorphism (percentage similarity: 24–96%) among Heterorhabditis populations collected from different parts of the world. Therefore, the population genetic structure may differ among different Heterorhabditis species. In the present study we investigated within and among genetic diversity of various isolates belonging to seven Heterorhabditis species.
185
0022-2011/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
186
HASHMI AND GAUGLER MATERIALS AND METHODS
Nematode Culture The nematode species and isolates tested included: H. bacteriophora (HP88 and E1), H. indicus (EMS-13, Coimbatore), H. hawaiiensis (MG-13), H. megidis (HSH2 and HO1), H. zealandica, H. marelatus (OH10), and H. hepialius (Bodega Bay). Nematode species H. indicus Coimbatore, H. hawaiiensis MG-13, and H. zealandica were obtained from biosys (Columbia, MD). An Egyptian isolate (EMS-13) of H. indicus was provided by M. Shamseldean (Cairo University), IS5 by I. Glazer (Volcani, Israel), H. hepialius by P. Stock (University of California), and DNA of H. marelatus by J. Liu (Oregon State University). Each isolate was passaged through Galleria mellonella, and emerging infective juveniles were collected (Poinar, 1979). The infective juveniles were surface-sterilized and inoculated on lipid agar media (Dunphy and Webster, 1989), seeded with their symbiotic bacteria, Photorhabdus luminescens. All stages of nematodes were collected by centrifugation (Sulston and Hodgkin, 1988) and stored frozen at 220°C until used for DNA extraction. DNA Extraction and RAPD Analysis DNA extraction of nematodes was performed according to the method of Sulston and Hodgkin (1988). The PCR conditions and reagent concentrations were as described previously (Hashmi et al., 1996). Based on the previous study with 80 random primers (Hashmi et al., 1996) 10 primers were chosen. Those primers (OP-A02, OP-A13, OP-A18, OP-C06, OP-C11, OP-H04, OP-H08, OP-H18, OP-H19, OP-S07) were obtained from Operon Technologies, Inc. (Alameda, CA). The amplification products were electrophoresed in 1% w/v, agarose gel in 13 TBE buffer at 1 V/cm for 12–16 h and visualized by staining with ethidium bromide. Reproducibility of DNA profiles was determined by replicating all RAPD reactions at least three times. Reactions were replicated with DNA extracted from different batches of nematodes at different times. DNA Extraction and RAPD Analysis of Single Nematode DNA from single infective stage juvenile was extracted as described for Caenorhabditis elegans by Williams et al. (1992) with the following modifications. Ten nematodes of each species were picked randomly under sterile conditions and individually placed in a drop (15 µl) of nematode lysis buffer on a Petri plate, cut with a syringe, transferred in an Eppendorf tube, and processed. The DNA of each nematode was divided into three aliquots and used in PCR with three different primers (OP-A13, OP-C06, and OP-H19). The PCR conditions were similar to those described for bulk DNA.
Data Analysis Two independent DNA preparations of each species were used except for H. marelatus. Each experiment was replicated at least three times. Genomic DNA was amplified with each of the 10 primers. For single nematode analysis, 10 infective stage juveniles of each species were hand-picked with a needle and DNA was isolated and analyzed as described above. All polymorphic bands were scored for the presence versus absence of a specific amplification product. DNA fragments at low intensities were scored as present only when they were reproducible in repeated experiments. Banding patterns of DNA generated by each primer were analyzed in a pair-wise comparison using the method described by Nei and Li (1979). Percentage similarity was calculated for shared DNA fragments per primer for each species as F 5 2CXY/(TX 1 TY). Where F is the similarity for primer, CXY is the number of common bands produced by both species and TX and TY are the bands generated by isolates X and Y, respectively. Average linkage method (Sneath and Sokal, 1973) was used for cluster analysis on the values of 12F, using the unweighted pair-group method algorithm (UPGMA) and dendrograms were plotted (SAS Institute, 1992). RESULTS
Representative banding patterns generated with individual nematodes of two Heterorhabditis species are shown (Fig. 1). Primer-specific bands generated with individual nematode of each genotype were similar. Some minor differences were observed in band intensities due to differences in DNA concentration of individual nematodes. Although isolates of Heterorhabditis spp. used in this study were from different geographical regions, they showed little variability in the band patterns (Table 1). On average the similarity among isolates of H. bacteriophora, H. indicus, and H. megidis was 83.8%. Overall mean within species similarity for these three Heterorhabditis spp. was 89.75%. High within population similarity (mean 96.2%) was observed with randomly selected individual nematodes, whereas, interspecific similarities based on RAPD markers were 31.3%. The Heterorhabditis species tested were easily distinguished by RAPD analysis (Fig. 2). Similarities between seven Heterorhabditis species ranged from 11.1 to 95% (Table 2) when analyzed for shared DNA fragments. The recently described species H. marelatus (Liu and Berry, 1996) showed a high similarity (95%) with H. hepialius, followed by H. megidis (35.7%), H. bacteriophora (29.6%), H. zealandica (19.0%), and H. indicus (16.6%). H. indicus shared a high similarity with H. hawaiiensis (92.0%). Low levels of similarities were observed between H. indicus and other species
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FIG. 1. RAPD-PCR profiles of two Heterorhabditis species (H. bacteriophora and H. indicus) produced with primer OP-A13 using individual nematodes. (A) DNA banding pattern of DNA isolated from all stages of nematodes (lane 1), DNA isolated from the same population and amplified under similar conditions used for individual nematodes (lane 2), and individual nematode (lanes 3–6) of H. bacteriophora. M, molecular weight marker l DNA cut with HindIII. (B) DNA banding patterns of five individual nematodes (lanes 1–5) of H. indicus. M, molecular weight marker l DNA cut with HindIII and fX174 DNA/HaeIII.
(16.1% with H. megidis and 16.6% with H. zealandica and H. marelatus). DNA profiles generated with an Israeli isolate (IS5) of Heterorhabditis showed a similarity of 89% to H. indicus (data not shown) and it is considered an isolate of this species. H. bacteriophora showed highest similarity with H. zealandica (63%), whereas the similarity with all other species was less than 30%. Four species groups can be identified using cluster analysis (Fig. 3) as follows: (1) H. bacteriophora and H.
TABLE 1 Percentage Similarity within and among Isolates of Heterorhabditis Species Based on Shared DNA Fragments Nematode H. bacteriophora H. hepialius H. hawaiiensis H. indicus H. megidis H. zealandica Average H. bacteriophora H. indicus H. megidis Average Average H. b vs H. z H. b vs H. m
Percent similarity
Nematode
Percent similarity
Within isolates 95.3 98.9 97.2 98.0 93.8 94.3 96.25 6 3.5 Among isolates 83.3 85.2 82.9 83.8 6 1.5 Within species a 89.75 6 3.0 Among species b 63 24
H. b vs H. mar H. b vs H. hep H. b vs H. i H. b vs H. h H. z vs H. m H. z vs H. mar H. z vs H. hep H. z vs H. i H. z vs H. h H. m vs H. mar H. m vs H. hep H. m vs H. i H. m. vs H. h H. mar vs H. hep H. mar vs H. i H. mar vs H. h H. i vs H. h Average
29.6 29.6 22.2 22.2 11.1 19.0 19.0 16.6 16.6 35.7 35.7 16.1 15.0 95.0 16.6 16.6 92.0 31.3 6 5.1
a Within species average of H. bacteriophora, H. indicus and H. megidis. b H. bacteriophora (H.b), H. hepialius (H. hep), H. hawaiiensis (H. h), H. indicus (H. i), H. megidis (H. m), H. marelatus (H. mar), H. zealandica (H. z).
zealandica; (2) H. megidis; (3) H. indicus and H. hawaiiensis, and (4) H. marelatus and H. hepialius. DISCUSSION
Entomopathogenic nematodes show great potential in biological control of insect pests (Gaugler, 1988; Poinar, 1986). Although research has been conducted on the biocontrol abilities of these nematodes (Gaugler, 1988; Nickle et al., 1988; Bedding, 1990), knowledge is scarce about the genetic structure of natural populations. The genetic differentiation among species is the result of the interaction of random chance, migration, and natural selection. Thus, various field isolates are often different from one another at different degrees. DNA and isozyme markers have shown a wide range of similarity among isolates of Heterorhabditis spp. (Akhurst, 1987; Curran and Webster, 1989; Hashmi et al., 1996). These studies suggest the presence of many variants among species collected from different geographical areas. The question is how can we classify the different variants found among these species? Variants
FIG. 2. Comparison of RAPD-PCR banding pattern of six Heterorhabditis species using primer OP-H19. M, molecular weight markers lDNA cut with HindIII and fx174 DNA/HaeIII; 1, H. bacteriophora; 2, H. megidis; 3, H. marelatus; 4, H. hawaiiensis; 5, H. zealandica; 6, H. indicus.
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can be used to describe the limits and distribution of a local population. Various names used to describe variants of entomopathogenic nematode species include isolate, strain, or line. Knowledge of the patterns of genetic difference within all the relevant species is essential for their use as efficient biological control agents. Our study attempted to quantify the variations among Heterorhabditis species. Several DNA preparations of two isolates each of H. bacteriophora, H. megidis, and H. indicus were used to determine similarity among isolates within the same species. Mean percentage similarity of 83.8% was observed among these isolates. An unidentified isolate IS5 was similar (89%) to H. indicus, indicating that more than 80% similarity defined the same genotype among Heterorhabditis spp. Percentage similarity determined by the size of the DNA fragments generated with RAPD also indicated high similarity (mean 96.25%) within isolates. These results are not surprising with Heterorhabditis species considering their life cycle, where the first generation is hermaphroditic and thus individuals in a population can be closely related. These data suggest that heterorhabditids are locally inbred populations with genetic difference between isolates from different geographical regions. Results of the genetic diversity study showed high similarity (63.0%) of shared DNA fragments between H. bacteriophora and H. zealandica, whereas similarity with all other species was less than 30%. H. hawaiiensis has been reported close to H. indicus in morphological characters (Gardner et al., 1994). These two species also showed high similarity (92%) at the DNA level. RAPD profiles generated with several primers produced similar banding patterns for these two species. Because of the high similarities we consider that H. hawiiensis is conspecific with H. indicus. These findings are in agreement with other molecular studies where these species have shown high similarities (Power et al., 1995). H. marelatus and H. hepialius also showed high similarities (92%). Differences if observed were only in band intensities with many markers and may represent the same species. Comparative studies both at morphological and molecular levels are needed to
TABLE 2 Genetic Similarities for Shared DNA Fragments among Heterorhabditis Species Identified with RAPD Markers Species
1
2
3
4
5
6
7
H. bacteriophora H. zealandica H. megidis H. marelatus H. hepialius H. indicus H. hawaiiensis
100 63.0 24.0 29.6 29.6 22.2 22.2
100 11.1 19.0 19.0 16.6 16.6
100 35.7 35.7 16.1 15.0
100 95.0 16.6 16.6
100 16.6 16.6
100 92.0
100
FIG. 3. Unweight pair-group method algorithm (UPGMA) dendrogram showing estimated average genetic distance between Heterorhabditis species. Average linkage cluster analysis was performed on the values of 12F as described under Materials and Methods. Hb, H. bacteriophora; Hz, H. zealandica; Hm, H. megidis, Hmer, H. meralatus; Hhep, H. hepialus; Hi, H. indicus; Hha, H. hawaiiensis.
resolve the status of these species. The high similarities among these two species groups can be attributed to specific environment and habitat preference. Although heterorhabditids have been isolated from different geographical regions (Poinar, 1990; De Doucet and Gabarra, 1994; Smith et al., 1991; Li and Wang, 1989), they are frequently distributed in similar habitats (Hara et al., 1991; Griffin et al., 1994; Amarasinghe et al., 1994; Poinar, 1993). Habitat specificity has been suggested for steinernematids collected from the United Kingdom and The Netherlands (Hominick et al., 1995). In our studies, two isolates of H. indicus and one of H. hawaiiensis, collected from different geographical areas with similar habitats (Gardner et al., 1994; Poinar et al., 1992; M. Shamseldean, personal communication), showed high similarities. This explanation seems quite plausible since H. hepialius and H. marelatus also were isolated from coastal areas with similar environmental conditions (Stock et al., 1996; Liu and Berry, 1996). Although more comparative data are needed on the genetic structure of different populations of the same species, our data suggest that the presence of a population in an area is closely linked with genotype-environmental interactions. These populations can be adapted to specific hosts and environments; therefore similar genotypes are present in a particular habitat. Determination of biological and genetic variability within and among species is important in order to develop an efficient biological control program. An understanding of population level and species level variability will be important for designing strategies for monitoring the release of new isolates, with subsequent recovery and positive identification of released isolates. The present study is the first report of the quantification of genetic diversity among Heterorhabditis species that will be useful in understanding the genetic structure of these nematodes.
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