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Identification of Symbiotic Bacteria (Photorhabdus and Xenorhabdus) from the Entomopathogenic Nematodes Heterorhabditis marelatus and Steinernema oregonense Based on 16S rDNA Sequence
Journal of Invertebrate Pathology 77, 87–91 (2001) doi:10.1006/jipa.2001.5007, available online at http://www.idealibrary.com on
Identification of Symbiotic Bacteria (Photorhabdus and Xenorhabdus) from the Entomopathogenic Nematodes Heterorhabditis marelatus and Steinernema oregonense Based on 16S rDNA Sequence Jie Liu,* Ralph E. Berry,* and Michael S. Blouin† *Department of Entomology and †Department of Zoology, Oregon State University, Corvallis, Oregon 97331-2914 E-mail: [email protected] Received January 19, 1999; accepted January 19, 2001; published online March 6, 2001
microbial substances that inhibit the growth of a wide range of microorganisms (Forst and Nealson, 1996). Because the association between nematodes and bacteria is of fundamental importance for nematode infectivity, mass reproduction, and registration as biological control agents, it is important to verify which species of the bacteria are found in association with different entomopathogenic nematode species in nature. Two new species of entomopathogenic nematodes were described recently from the Pacific Northwest of North America: Heterorhabditis marelatus and Steinernema oregonense (Liu and Berry, 1996a,b). The relationships between the symbionts of these nematodes and known species of Photorhabdus and Xenorhabdus are unclear. Comparison of 16S rRNA gene sequences has proved extremely useful for species identification and phylogenetic reconstruction for symbiotic bacteria (Rainey et al., 1995; Suzuki et al., 1996; Brunel et al., 1997; Liu et al., 1997; Szallas et al., 1997). Our objectives here were to obtain partial 16S rRNA sequence from the symbionts in different life stages of the nematodes H. marelatus and S. oregonense and to use these sequence data to determine how similar the new isolates are to previously described species of Photorhabdus or Xenorhabdus.
Two species of entomopathogenic nematodes, Heterorhabditis marelatus and Steinernema oregonense, were described recently from the west coast of North America. It is not known whether the bacterial symbionts of these nematodes are also unique. Here we compared partial 16S rRNA sequences from the symbiotic bacteria of these two nematodes with sequence from previously described Photorhabdus and Xenorhabdus species. The 16S sequence from the new Xenorhabdus isolate appears very similar to, although not identical to, that of X. bovienii, the common symbiont of S. feltiae. The new Photorhabdus isolate appears to be very distinct from other known Photorhabdus species, although its closest affinities are with the P. temperata group. We also verified a monoxenic association between each isolate and its nematode by amplifying and sequencing bacterial 16S sequence from crushed adult and juvenile nematodes and from bacterial cultures isolated from infected hosts. © 2001 Academic Press Key Words: Photorhabdus; Xenorhabdus; Heterorhabditis marelatus; Steinernema oregonense; 16S rDNA.
INTRODUCTION
Entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) are widely used in biological control of insect pests (Boemare et al., 1997). These nematodes are characterized by their mutualistic relationship with symbiotic bacteria. The bacterial symbionts are carried monoxenically throughout the whole intestine of the infective juveniles in Heterorhabditidae or in a special vesicle in infective juveniles in Steinernematidae (Poinar, 1966; Bird and Akhurst, 1983; Endo and Nickle, 1991). The nematodes provide protection and transportation for their bacterial symbionts. The bacterial symbionts in turn kill the host and establish and maintain suitable conditions for nematode reproduction, providing nutrients and anti-
MATERIALS AND METHODS
Nematodes Nematodes H. marelatus OH10 strain and S. oregonense OS21 strain were obtained from soil samples collected from Seaside and Grants Pass, Oregon, in 1993 (Liu and Berry, 1995). H. marelatus samples NP1 and SJ2 were isolated from soil samples collected from Newport, Oregon, and Florence, Oregon, respectively. Different stages of the nematodes including females, males, and infective juveniles were obtained from larvae of greater wax worms, Galleria mellonella (L.), using the methods described by Poinar (1975). 87
0022-2011/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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TABLE 1 Symbiotic Bacteria Strains, a Their Hosts, and DNA Sequence Genbank Accession Nos. Bacterial species
Bacterial strain
Nematode host
Accession No.
Xenorhabdus sp. X. beddingii X. bovienii X. bovienii X. japonicus X. nematophilus X. nematophilus X. poinarii
OS21 (this study) DMS4764 ATCC35271 DSM4766 SK-1 AN/5 DSM3370 DSM4768
Steinernema oregonense Steinernema sp. S. feltiae S. feltiae S. kushidai S. carpocapsae S. carpocapsae S. glaseri
AF09816 D78006 D78007 X82252 D78008 D78009 X82251 D78010
Photorhabdus sp. Photorhabdus sp. Photorhabdus sp. P. luminescens luminescens P. luminescens luminescens P. luminescens laumondii P. luminescens laumondii P. luminescens akhurstii P. luminescens akhurstii P. asymbiotica P. temperata subgroup IIa P. temperata subgroup IIa P. temperata P. temperata
NP1 (this study) OH10 (this study) SJ2 (this study) Hb T Hm,1° and 2° V16 HP88 IS5 FRG04 T ATCC 49950 PE87.3 X1Nach T Meg WX2
Heterorhabditis marelatus H. marelatus H. marelatus H. bacteriophora (Australia) H. bacteriophora (U.S.A.) H. bacteriophora (Australia) H. bacteriophora (U.S.A.) H. indica (Israel) H. indica (Guadeloupe) Human clinical specimen H. megidis (Netherlands) H. megidis (Russia) H. megidis (U.S.A.) Heterorhabditis sp. (U.S.A.)
AF079811 AF079812 AF079813 X82248 Z76742 Z76741 Z76743 Z76745 AJ007359 Z76755 Z76748 AJ007405 Z76750 Z76746
a Sources of Photorhabdus reference sequences are detailed in Fischer-Le Saux et al. (1999) and Szallas et al. (1997). Xenorhabdus reference sequences are from Rainey et al. (1995; X82251–52) and Suzuki et al. (1996; D78006-10).
Symbiotic Bacteria Bacteria in the genera Photorhabdus and Xenorhabdus are considered the main symbionts of entomopathogenic nematodes and to be the species primarily responsible for the death of the insect hosts and the growth of the nematodes within the insect cadaver. However, a number of other bacterial species have been isolated from nematodes or infected hosts (Lysenko and Weiser, 1974; Aguillera et al., 1993; Jackson et al., 1995), and entomopathogenic nematodes can feed and reproduce on a number of other bacterial species (Aguillera and Smart, 1993; Jackson et al., 1995). These findings may be laboratory artifacts because the other bacterial species have not been linked to field-collected nematodes. Therefore, in order to verify that the bacterial DNA we sequenced was indeed the symbiotic associate of our nematodes, we amplified and sequenced 16S DNA from crushed nematodes of different life stages (infective juveniles, male and female adults) of each nematode species and from pure bacteria isolated from infected insect hosts. The technique described by Poinar (1966) was used to isolate symbiotic bacteria of the nematodes H. marelatus and S. oregonense indirectly from the hemocoel of greater wax worms, G. mellonella (L.) within 48 h of infection by the nematodes. The bacteria were purified by selecting a single colony from nutrient agar (Difco Laboratories, Detroit, MI). Bacterial strains were cultured at room temperature for 48 h in brain heart
FIG. 1. Unrooted maximum parsimony tree from Photorhabdus partial 16S sequences. Scale bar equals one step. Numbers indicate bootstrap support for interior nodes. Neighbor joining tree gave identical topology and similar relative branch lengths.
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IDENTIFICATION OF SYMBIOTIC BACTERIA FROM NEMATODES
TABLE 2 Uncorrected Percentage of Sequence Difference between Isolates (A) Photorhabdus
1. Photorhabdus sp. (oh10) 2. Photorhabdus sp. (sj2) 3. P. temp (wx2) 4. P. temp (meg) 5. P. asymbiotica 6. P. temp (xlnach) 7. P. temp (pe87) 8. P. lum (v16) 9. P. lum (hp88) 10. P. lum (hb) 11. P. lum (hm1) 12. P. lum (frg04) 13. P. lum (is5)
1
2
3
4
5
6
7
8
9
10
11
12
— 0.0048 0.0405 0.0389 0.0454 0.0405 0.0438 0.0535 0.0535 0.0519 0.0535 0.0423 0.0487
— 0.0422 0.0405 0.0470 0.0422 0.0454 0.0551 0.0551 0.0503 0.0519 0.0423 0.0470
— 0.0048 0.0227 0.0276 0.0276 0.0324 0.0276 0.0324 0.0324 0.0341 0.0389
— 0.0243 0.0259 0.0292 0.0340 0.0292 0.0340 0.0340 0.0358 0.0405
— 0.0178 0.0178 0.0178 0.0178 0.0194 0.0211 0.0244 0.0292
— 0.0032 0.0259 0.0259 0.0211 0.0227 0.0195 0.0276
— 0.0259 0.0259 0.0211 0.0227 0.0195 0.0276
— 0.0097 0.0211 0.0227 0.0163 0.0227
— 0.0211 0.0194 0.0179 0.0243
— 0.0048 0.0162 0.0211
— 0.0179 0.0211
— 0.0081
(B) Xenorhabdus
1. 2. 3. 4. 5. 7. 8.
X. beddingi X. japonicu X. nematoph (AN5) X. nematoph (DSM) X. poinarii X. bovienii Xenorhabdus sp. (OS21)
1
2
3
4
5
7
— 0.0312 0.0362 0.0344 0.0313 0.0476 0.0558
— 0.0198 0.0181 0.0329 0.0395 0.0543
— 0.0016 0.0314 0.0378 0.0493
— 0.0313 0.0377 0.0492
— 0.0280 0.0379
— 0.0164
infusion broth (Difco Laboratories). Cultures were harvested by centrifugation and were washed three times with sterilized deionized water. DNA Isolation Nematodes in different stages were washed three times with sterilized deionized water. A single nematode was pulverized in 5% Chelex 100 (Bio-Rad Laboratories, Hercules, CA) with a pestle then incubated overnight in a water bath at 60°C. After centrifuging, supernatant was used as template DNA in PCR. A DNA extraction kit was used to isolate DNA from the bacteria directly (Liu et al., 1997). DNA Amplification and Sequencing The partial 16S rDNA fragment (about 630 bp) was amplified by PCR from bacterial cultures and from total DNA isolated from adult and juvenile stages of each nematode. DNA amplification was repeated five times for each stage and species of nematode. DNA from Escherichia coli was used as positive control for PCR. We used Brosius et al.’s (1978) primers 16S-F (forward) and 16S-R1 (reverse) for PCR and for sequencing. PCR and purification conditions were described previously (Liu et al., 1997). Purified PCR products were sequenced directly by using a DyeDeoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) and run on an ABI 377 DNA se-
quencer. DNA sequences were determined from both strands and from multiple, independently amplified templates on each stage of the nematodes. Sequences obtained during this study are deposited in GenBank under Accession Nos. AF079811 to AF079816. Reference Sequences Previously published 16S rRNA sequences from Xenorhabdus beddingii, X. nematophilus, X. bovienii, X. japonicus, and X. poinarii were used as standards with which to compare the sequence of our bacterial isolate from S. oregonense (Table 1). Sequences from each of the major groups of Photorhabdus identified by Fischer-Le Saux et al. (1999) were used as standards with which to compare the sequence of our isolate from H. marelatus (Table 1). These groups include Photorhabdus luminescens subgroups luminescens, akhurstii, and laumondii (associates of nematodes from tropical climates), P. asymbiotica (human pathogen), and the two subgroups within P. temperata (associates of nematodes from temperate zones). Sequence Alignments and Phylogenetic Analysis The Photorhabdus analysis was based on a total of 616 bp of clean sequence, and the Xenorhabdus analysis was based on 609 bp. Sequences were aligned by eye. Unrooted trees were constructed by maximum parsimony using an exhaustive search (Xenorhabdus)
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FIG. 2. Unrooted maximum parsimony tree from Xenorhabdus partial 16S sequences. Scale bar equals one step. Numbers indicate bootstrap support for interior nodes. Neighbor joining tree gave identical topology and similar relative branch lengths.
or branch and bound heuristic search (Photorhabdus) and by neighbor joining using Kimura 2-parameter distances. All analyses were conducted using PAUP* (Swofford, 1998). RESULTS AND DISCUSSION
All amplifications produced a single band of about 630 bp. More intense bands and more reliable sequences were obtained from DNA isolated from pure bacterial cultures and from adult nematodes than from DNA from infective juveniles. The DNA sequences from different stages of the nematodes were identical. Thus it appears that the bacterial isolates we sequenced are indeed symbiotic with their respective nematode species. The bacterial sequences from H. marelatus isolates OH10 and NP1 were identical and differed from those of the SJ2 isolate by three substitutions. The bacterial sequences from H. marelatus aligned clearly, and without gaps, with those of the other Photorhabdus species. The isolate from S. oregonense aligned clearly, and without gaps, with those of the other Xenorhabdus species.
For the Xenorhabdus samples a single most parsimonious tree was obtained whose topology is identical to that of the distance (neighbor joining) tree (Fig. 2). The isolate from S. oregonense appears closely related to, although not identical to the sample of X. bovienii (Fig. 2, Table 2). For the Photorhabdus samples, a single most parsimonious tree was obtained, whose topology is identical to that of the distance (neighbor joining) tree (Fig. 1). The isolates from H. marelatus appear very distinct from the known Photorhabdus species, being the most divergent samples in the data set (4 –5% sequence difference from the other species vs 2– 4% among the other species; Table 2). Their closest affiliation appears to be with P. temperata (Fig. 1; Table 2). It is interesting that the new Xenorhabdus isolate was most similar to X. bovienii (Fig. 2). X. bovienii is a symbiont of S. feltiae, a common nematode species in North America. This result is consistent with the expectation that S. oregonense would harbor a native North American bacterium. Similarly, the new Photorhabdus isolate is most similar to P. temperata isolates that are symbionts of H. megidis from North America (Tables 1 and 2). That H. marelatus is most closely related to temperate zone nematodes such as H. megidis (Adams et al., 1998; Liu et al., 1999) is consistent with the apparent affinities of its bacterium. That H. marelatus is also a very distinct nematode is consistent with the apparent distinctness of its bacterium. Further work is needed (e.g., phenotypic studies, complete 16S sequence, DNA–DNA hybridization) to test whether either new isolate should be considered a new species. ACKNOWLEDGMENTS We are grateful to G. O. Poinar, Jr., A. F. Moldenke, and H. K. Luh for insightful discussion and technical assistance. This study was supported by the Agricultural Research Foundation and the Department of Entomology, Oregon State University. DNA sequence data were analyzed with a high-speed computer system donated by an Intel Higher Education Grant.
REFERENCES Adams, B. J., Burnell, A. M., and Powers, T. O. 1998. A phylogenetic analysis of Heterorhabditis (Nemata: Rhabditidae) based on internal transcribed spacer 1 DNA sequence data. J. Nematol. 30, 22–39. Aguillera, M. M., Hodge, N. C., Stall, R. E., and Smart, G. C., Jr. 1993. Bacterial symbionts of Steinernema scapterisci. J. Invertebr. Pathol. 62, 68 –72. Aguillera, M. M., and Smart, G. C., Jr. 1993. Development, reproduction, and pathogenicity of Steinernema scapterisci in monoxenic culture with different species of bacteria. J. Invertebr. Pathol. 62, 289 –294. Bird, A. F., and Akhurst, R. J. 1983. The nature of the intestinal vesicle in nematodes of the family Steinernematidae. Int. J. Parasitol. 13, 599 – 606.
IDENTIFICATION OF SYMBIOTIC BACTERIA FROM NEMATODES Boemare, N., Givaudan, A., Brehelin, M., and Laumond, C. 1997. Symbiosis and pathogenicity of nematode-bacterium complexes. Symbiosis 22, 21– 45. Brosius, J., Palmer, M. L., Kennedy, P. J, and Noller, H. F. 1978. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 75, 4801– 4805. Brunel, B., Givaudan, A., Lanois, A., Akhurst, R. J., and Boemare, N. 1997. Fast and accurate Identification of Xenorhabdus and Photorhabdus species by restriction analysis of PCR-amplified 16S rRNA genes. Appl. Environ. Microbiol. 63, 574 –580. Endo, B. Y., and Nickle, W. R. 1991. Ultrastructure of the intestinal epithelium, lumen and associated bacteria in Heterorhabditis bacteriophora. Proc. Helminthol. Soc. Wash. 58, 202–212. Fischer-Le Saux, M., Viallard, V., Brunel, B., Normand, P., and Boemare, N. E. 1999. Polyphasic classification of the genus Photorhabdus and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov., and P. asymbiotica sp. nov. Int. J. Syst. Bacteriol. 49, 1645–1656. Forst, S., and Nealson, K. 1996. Molecular biology of the symbioticpathogenic bacteria Xenorhabdus spp., and Photorhabdus spp. Microbiol. Rev. 60, 21– 43. Jackson, T. J., Wang, H. Y., Nugent, M. J., Griffin, C. T., Burnell, A., and Dowds, B. C. A. 1995. Isolation of insect pathogenic bacteria, Providencia retteri, from Heterorhabditis spp. J. Appl. Bacteriol. 78, 237–244. Liu, J., and Berry, R. E. 1995. Natural distribution of entomopathogenic nematodes (Rhabditida: Heterorhabditidae and Steinernematidae) in Oregon soils. Environ. Entomol. 24, 159 –163. Liu, J., and Berry, R. E. 1996a. Heterorhabditis marelatus n. sp. (Rhabditida: Heterorhabditidae) from Oregon. J. Invertebr. Pathol. 67, 48 –54.
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Liu, J., and Berry, R. E. 1996b. Steinernema oregonensis n. sp. (Rhabditida: Steinernematidae) from Oregon, U.S.A. Fundam. Appl. Nematol. 19, 375–380. Liu, J., Berry, R. E., and Blouin, M. S. 1999. Molecular differentiation and phylogeny of entomopathogenic nematodes (Rhabditida: Heterorhabditidae) based on ND4 gene sequences of mitochondrial DNA. J. Parasitol. 85, 709 –715. Liu, J., Berry, R. E., Poinar, G. O., Jr., and Moldenke, A. F. 1997. Phylogeny of Photorhabdus and Xenorhabdus species and strains as determined by comparison of partial 16S rRNA gene sequences. Int. J. Syst. Bacteriol. 47, 948 –951. Lysenko, O., and Weiser, J. 1974. Bacteria associated with the nematode Neoaplectana carpocapsae and the pathogenicity of this complex for Galleria mellonella larvae. J. Invertebr. Pathol. 24, 332–336. Poinar, G. O., Jr. 1966. The presence of Achromobacter nematophilus in the infective stage of a Neoaplectana sp. (Steinernematidae: Nematoda). Nematologica 12, 105–108. Poinar, G. O., Jr. 1975. “Entomogenous Nematodes, a Manual and Host List of Insect–Nematode Associations.” E. J. Brill, Leiden, Netherlands. Rainey, F. A., Ehlers, R. U., and Stackebrandt, E. 1995. Inability of the polyphasic approach to systematics to determine the relatedness of the genera Xenorhabdus and Photorhabdus. Int. J. Syst. Bacteriol. 45, 379 –381. Suzuki, T., Yabusaki, H., and Nishimura, Y. 1996. Phylogenetic relationships of entomopathogenic nematotophilic bacteria: Xenorhabdus spp., and Photorhabdus sp. J. Basic Microbiol. 5, 351–354. Swofford, D. L. 1998. “PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods),” Version 4. Sinauer Associates, Sunderland, MA. Szallas, E., Koch, C., Fodor, A., Burghardt, J., Buss, O., Szentirmai, A., Nealson, K. H., and Stackebrandt, E. 1997. Phylogenetic evidence for the taxonomic heterogeneity of Photorhabdus luminescens. Int. J. Syst. Bacteriol. 47, 402– 407.
Identification of Symbiotic Bacteria (Photorhabdus and Xenorhabdus) from the Entomopathogenic Nematodes Heterorhabditis marelatus and Steinernema oregonense Based on 16S rDNA Sequence Jie Liu,* Ralph E. Berry,* and Michael S. Blouin† *Department of Entomology and †Department of Zoology, Oregon State University, Corvallis, Oregon 97331-2914 E-mail: [email protected] Received January 19, 1999; accepted January 19, 2001; published online March 6, 2001
microbial substances that inhibit the growth of a wide range of microorganisms (Forst and Nealson, 1996). Because the association between nematodes and bacteria is of fundamental importance for nematode infectivity, mass reproduction, and registration as biological control agents, it is important to verify which species of the bacteria are found in association with different entomopathogenic nematode species in nature. Two new species of entomopathogenic nematodes were described recently from the Pacific Northwest of North America: Heterorhabditis marelatus and Steinernema oregonense (Liu and Berry, 1996a,b). The relationships between the symbionts of these nematodes and known species of Photorhabdus and Xenorhabdus are unclear. Comparison of 16S rRNA gene sequences has proved extremely useful for species identification and phylogenetic reconstruction for symbiotic bacteria (Rainey et al., 1995; Suzuki et al., 1996; Brunel et al., 1997; Liu et al., 1997; Szallas et al., 1997). Our objectives here were to obtain partial 16S rRNA sequence from the symbionts in different life stages of the nematodes H. marelatus and S. oregonense and to use these sequence data to determine how similar the new isolates are to previously described species of Photorhabdus or Xenorhabdus.
Two species of entomopathogenic nematodes, Heterorhabditis marelatus and Steinernema oregonense, were described recently from the west coast of North America. It is not known whether the bacterial symbionts of these nematodes are also unique. Here we compared partial 16S rRNA sequences from the symbiotic bacteria of these two nematodes with sequence from previously described Photorhabdus and Xenorhabdus species. The 16S sequence from the new Xenorhabdus isolate appears very similar to, although not identical to, that of X. bovienii, the common symbiont of S. feltiae. The new Photorhabdus isolate appears to be very distinct from other known Photorhabdus species, although its closest affinities are with the P. temperata group. We also verified a monoxenic association between each isolate and its nematode by amplifying and sequencing bacterial 16S sequence from crushed adult and juvenile nematodes and from bacterial cultures isolated from infected hosts. © 2001 Academic Press Key Words: Photorhabdus; Xenorhabdus; Heterorhabditis marelatus; Steinernema oregonense; 16S rDNA.
INTRODUCTION
Entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) are widely used in biological control of insect pests (Boemare et al., 1997). These nematodes are characterized by their mutualistic relationship with symbiotic bacteria. The bacterial symbionts are carried monoxenically throughout the whole intestine of the infective juveniles in Heterorhabditidae or in a special vesicle in infective juveniles in Steinernematidae (Poinar, 1966; Bird and Akhurst, 1983; Endo and Nickle, 1991). The nematodes provide protection and transportation for their bacterial symbionts. The bacterial symbionts in turn kill the host and establish and maintain suitable conditions for nematode reproduction, providing nutrients and anti-
MATERIALS AND METHODS
Nematodes Nematodes H. marelatus OH10 strain and S. oregonense OS21 strain were obtained from soil samples collected from Seaside and Grants Pass, Oregon, in 1993 (Liu and Berry, 1995). H. marelatus samples NP1 and SJ2 were isolated from soil samples collected from Newport, Oregon, and Florence, Oregon, respectively. Different stages of the nematodes including females, males, and infective juveniles were obtained from larvae of greater wax worms, Galleria mellonella (L.), using the methods described by Poinar (1975). 87
0022-2011/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
88
LIU, BERRY, AND BLOUIN
TABLE 1 Symbiotic Bacteria Strains, a Their Hosts, and DNA Sequence Genbank Accession Nos. Bacterial species
Bacterial strain
Nematode host
Accession No.
Xenorhabdus sp. X. beddingii X. bovienii X. bovienii X. japonicus X. nematophilus X. nematophilus X. poinarii
OS21 (this study) DMS4764 ATCC35271 DSM4766 SK-1 AN/5 DSM3370 DSM4768
Steinernema oregonense Steinernema sp. S. feltiae S. feltiae S. kushidai S. carpocapsae S. carpocapsae S. glaseri
AF09816 D78006 D78007 X82252 D78008 D78009 X82251 D78010
Photorhabdus sp. Photorhabdus sp. Photorhabdus sp. P. luminescens luminescens P. luminescens luminescens P. luminescens laumondii P. luminescens laumondii P. luminescens akhurstii P. luminescens akhurstii P. asymbiotica P. temperata subgroup IIa P. temperata subgroup IIa P. temperata P. temperata
NP1 (this study) OH10 (this study) SJ2 (this study) Hb T Hm,1° and 2° V16 HP88 IS5 FRG04 T ATCC 49950 PE87.3 X1Nach T Meg WX2
Heterorhabditis marelatus H. marelatus H. marelatus H. bacteriophora (Australia) H. bacteriophora (U.S.A.) H. bacteriophora (Australia) H. bacteriophora (U.S.A.) H. indica (Israel) H. indica (Guadeloupe) Human clinical specimen H. megidis (Netherlands) H. megidis (Russia) H. megidis (U.S.A.) Heterorhabditis sp. (U.S.A.)
AF079811 AF079812 AF079813 X82248 Z76742 Z76741 Z76743 Z76745 AJ007359 Z76755 Z76748 AJ007405 Z76750 Z76746
a Sources of Photorhabdus reference sequences are detailed in Fischer-Le Saux et al. (1999) and Szallas et al. (1997). Xenorhabdus reference sequences are from Rainey et al. (1995; X82251–52) and Suzuki et al. (1996; D78006-10).
Symbiotic Bacteria Bacteria in the genera Photorhabdus and Xenorhabdus are considered the main symbionts of entomopathogenic nematodes and to be the species primarily responsible for the death of the insect hosts and the growth of the nematodes within the insect cadaver. However, a number of other bacterial species have been isolated from nematodes or infected hosts (Lysenko and Weiser, 1974; Aguillera et al., 1993; Jackson et al., 1995), and entomopathogenic nematodes can feed and reproduce on a number of other bacterial species (Aguillera and Smart, 1993; Jackson et al., 1995). These findings may be laboratory artifacts because the other bacterial species have not been linked to field-collected nematodes. Therefore, in order to verify that the bacterial DNA we sequenced was indeed the symbiotic associate of our nematodes, we amplified and sequenced 16S DNA from crushed nematodes of different life stages (infective juveniles, male and female adults) of each nematode species and from pure bacteria isolated from infected insect hosts. The technique described by Poinar (1966) was used to isolate symbiotic bacteria of the nematodes H. marelatus and S. oregonense indirectly from the hemocoel of greater wax worms, G. mellonella (L.) within 48 h of infection by the nematodes. The bacteria were purified by selecting a single colony from nutrient agar (Difco Laboratories, Detroit, MI). Bacterial strains were cultured at room temperature for 48 h in brain heart
FIG. 1. Unrooted maximum parsimony tree from Photorhabdus partial 16S sequences. Scale bar equals one step. Numbers indicate bootstrap support for interior nodes. Neighbor joining tree gave identical topology and similar relative branch lengths.
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TABLE 2 Uncorrected Percentage of Sequence Difference between Isolates (A) Photorhabdus
1. Photorhabdus sp. (oh10) 2. Photorhabdus sp. (sj2) 3. P. temp (wx2) 4. P. temp (meg) 5. P. asymbiotica 6. P. temp (xlnach) 7. P. temp (pe87) 8. P. lum (v16) 9. P. lum (hp88) 10. P. lum (hb) 11. P. lum (hm1) 12. P. lum (frg04) 13. P. lum (is5)
1
2
3
4
5
6
7
8
9
10
11
12
— 0.0048 0.0405 0.0389 0.0454 0.0405 0.0438 0.0535 0.0535 0.0519 0.0535 0.0423 0.0487
— 0.0422 0.0405 0.0470 0.0422 0.0454 0.0551 0.0551 0.0503 0.0519 0.0423 0.0470
— 0.0048 0.0227 0.0276 0.0276 0.0324 0.0276 0.0324 0.0324 0.0341 0.0389
— 0.0243 0.0259 0.0292 0.0340 0.0292 0.0340 0.0340 0.0358 0.0405
— 0.0178 0.0178 0.0178 0.0178 0.0194 0.0211 0.0244 0.0292
— 0.0032 0.0259 0.0259 0.0211 0.0227 0.0195 0.0276
— 0.0259 0.0259 0.0211 0.0227 0.0195 0.0276
— 0.0097 0.0211 0.0227 0.0163 0.0227
— 0.0211 0.0194 0.0179 0.0243
— 0.0048 0.0162 0.0211
— 0.0179 0.0211
— 0.0081
(B) Xenorhabdus
1. 2. 3. 4. 5. 7. 8.
X. beddingi X. japonicu X. nematoph (AN5) X. nematoph (DSM) X. poinarii X. bovienii Xenorhabdus sp. (OS21)
1
2
3
4
5
7
— 0.0312 0.0362 0.0344 0.0313 0.0476 0.0558
— 0.0198 0.0181 0.0329 0.0395 0.0543
— 0.0016 0.0314 0.0378 0.0493
— 0.0313 0.0377 0.0492
— 0.0280 0.0379
— 0.0164
infusion broth (Difco Laboratories). Cultures were harvested by centrifugation and were washed three times with sterilized deionized water. DNA Isolation Nematodes in different stages were washed three times with sterilized deionized water. A single nematode was pulverized in 5% Chelex 100 (Bio-Rad Laboratories, Hercules, CA) with a pestle then incubated overnight in a water bath at 60°C. After centrifuging, supernatant was used as template DNA in PCR. A DNA extraction kit was used to isolate DNA from the bacteria directly (Liu et al., 1997). DNA Amplification and Sequencing The partial 16S rDNA fragment (about 630 bp) was amplified by PCR from bacterial cultures and from total DNA isolated from adult and juvenile stages of each nematode. DNA amplification was repeated five times for each stage and species of nematode. DNA from Escherichia coli was used as positive control for PCR. We used Brosius et al.’s (1978) primers 16S-F (forward) and 16S-R1 (reverse) for PCR and for sequencing. PCR and purification conditions were described previously (Liu et al., 1997). Purified PCR products were sequenced directly by using a DyeDeoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) and run on an ABI 377 DNA se-
quencer. DNA sequences were determined from both strands and from multiple, independently amplified templates on each stage of the nematodes. Sequences obtained during this study are deposited in GenBank under Accession Nos. AF079811 to AF079816. Reference Sequences Previously published 16S rRNA sequences from Xenorhabdus beddingii, X. nematophilus, X. bovienii, X. japonicus, and X. poinarii were used as standards with which to compare the sequence of our bacterial isolate from S. oregonense (Table 1). Sequences from each of the major groups of Photorhabdus identified by Fischer-Le Saux et al. (1999) were used as standards with which to compare the sequence of our isolate from H. marelatus (Table 1). These groups include Photorhabdus luminescens subgroups luminescens, akhurstii, and laumondii (associates of nematodes from tropical climates), P. asymbiotica (human pathogen), and the two subgroups within P. temperata (associates of nematodes from temperate zones). Sequence Alignments and Phylogenetic Analysis The Photorhabdus analysis was based on a total of 616 bp of clean sequence, and the Xenorhabdus analysis was based on 609 bp. Sequences were aligned by eye. Unrooted trees were constructed by maximum parsimony using an exhaustive search (Xenorhabdus)
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LIU, BERRY, AND BLOUIN
FIG. 2. Unrooted maximum parsimony tree from Xenorhabdus partial 16S sequences. Scale bar equals one step. Numbers indicate bootstrap support for interior nodes. Neighbor joining tree gave identical topology and similar relative branch lengths.
or branch and bound heuristic search (Photorhabdus) and by neighbor joining using Kimura 2-parameter distances. All analyses were conducted using PAUP* (Swofford, 1998). RESULTS AND DISCUSSION
All amplifications produced a single band of about 630 bp. More intense bands and more reliable sequences were obtained from DNA isolated from pure bacterial cultures and from adult nematodes than from DNA from infective juveniles. The DNA sequences from different stages of the nematodes were identical. Thus it appears that the bacterial isolates we sequenced are indeed symbiotic with their respective nematode species. The bacterial sequences from H. marelatus isolates OH10 and NP1 were identical and differed from those of the SJ2 isolate by three substitutions. The bacterial sequences from H. marelatus aligned clearly, and without gaps, with those of the other Photorhabdus species. The isolate from S. oregonense aligned clearly, and without gaps, with those of the other Xenorhabdus species.
For the Xenorhabdus samples a single most parsimonious tree was obtained whose topology is identical to that of the distance (neighbor joining) tree (Fig. 2). The isolate from S. oregonense appears closely related to, although not identical to the sample of X. bovienii (Fig. 2, Table 2). For the Photorhabdus samples, a single most parsimonious tree was obtained, whose topology is identical to that of the distance (neighbor joining) tree (Fig. 1). The isolates from H. marelatus appear very distinct from the known Photorhabdus species, being the most divergent samples in the data set (4 –5% sequence difference from the other species vs 2– 4% among the other species; Table 2). Their closest affiliation appears to be with P. temperata (Fig. 1; Table 2). It is interesting that the new Xenorhabdus isolate was most similar to X. bovienii (Fig. 2). X. bovienii is a symbiont of S. feltiae, a common nematode species in North America. This result is consistent with the expectation that S. oregonense would harbor a native North American bacterium. Similarly, the new Photorhabdus isolate is most similar to P. temperata isolates that are symbionts of H. megidis from North America (Tables 1 and 2). That H. marelatus is most closely related to temperate zone nematodes such as H. megidis (Adams et al., 1998; Liu et al., 1999) is consistent with the apparent affinities of its bacterium. That H. marelatus is also a very distinct nematode is consistent with the apparent distinctness of its bacterium. Further work is needed (e.g., phenotypic studies, complete 16S sequence, DNA–DNA hybridization) to test whether either new isolate should be considered a new species. ACKNOWLEDGMENTS We are grateful to G. O. Poinar, Jr., A. F. Moldenke, and H. K. Luh for insightful discussion and technical assistance. This study was supported by the Agricultural Research Foundation and the Department of Entomology, Oregon State University. DNA sequence data were analyzed with a high-speed computer system donated by an Intel Higher Education Grant.
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