and 1 A. Fodor Budapest, Hungary SUMMARY

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1 Cross fertility, gnotobiology and molecular identification of steinernematids of long dauer phenotype: comparative evaluation of the results obtained by the GeneGel Excel kit 1 Arvinbayar, B. 1 Szállás, E., 2 Füredi, S., 11 Lengyel, K., 1 Somogyi, E. 1 Saskői A., and 1 A. Fodor 1 Department of Genetics, Eötvös University, Budapest, Hungary 2 Laboratory of Forensic Research, Police Department, Ministry of Inner Affairs, Budapest, Hungary fodorandras@yahoo.com SUMMARY Thirteen Steinernema strains of long dauer phenotypes were compared on the basis of their PCR RFLP profiles based on the GeneGel Excel kit analysis. They were also grown on some Xenorhabdus symbionts isolated from S. glaseri, S. arenarium as well as from unidentified Steinernema isolates, such as SP2, Slovak and Italy. They were also intercrossed to study their species differences. We found that one group of strains belong to the S. glaseri group, characterized by a very similar RFLP profiles and unlimited cross fertility. They grew on each other symbionts. Another group comprised the S. arenarium group of rather similar RFLP patterns and gnotobiological similarity, although their cross fertility has shown limitations. Two strains (Morocco, Palestine) definitely belong to two, different species. The taxonomic positions of the SP2 and the Italy strains are still ambiguous. INTRODUCTION Thirteen Steinernema strains of long dauer phenotypes, potential tools for grub control were studied for their identification within the frame of a COST 850 Workshop. We have studied their growth and development on each others bacterial symbionts (gnotobiological analysis), cross fertility and PCR - RFLP profiles obtained by GeneGel excel kit 1,2. PhastSystem PCR RFLP is an excellent method for identifying entomopathogenic nematodes 3,4 On the other hand, the classic species definition is that those individuals which are capable of producing fertile progeny do belong to the same species. We intended to determine which steinernematids of long dauer phenotype belong to the same species and intended to compare their PCR-RFLP patterns by using the GeneGel Excel kit. Considering the species specific symbiosis of Steinernema and Xenorhabdus (bacterium) strains, as a consequence of it entomopathogenic nematodes could grow and propagate only on their own symbionts, a precondition for the successful cross is to find bacterium on which both parental strain could somehow grow and propagate. We have isolated the Xenorhabdus symbionts of several Steinernema strains and tested the others for growth and reproduction. Finally we found bacterium for each intended crosses.

2 MATERIALS AND METHODS Bacterium strains: Xenorhabdus bacterium has been isolated straight from surface sterilized infective dauer juveniles as has been described by Lucskai. Bacteria were tested on indicator plates and for their catalase negativity and other taxonomic characters. We have isolated Xenorhabdus from the nematode strains KMD15, S. anomali, SP2, Slovak, Morocco and Italy and used also the DSM reference strain of X. poinarii (natural symbionts of S. glaseri). Nematode strains: In our stock collection we have two Steinernema arenarium (anomali) strains: one from N. Simoes (Portugal Azores), one from Ramon Georgis (Biosys, USA). S. glaseri strains NC1 (from Michael G. Klein, USDA-Ohio, USA), NC513, NCLU (from Pierre Abad s collection via Attila Lucskai) and AZ26 (from N. Simoes, Portugal Azores). The Italian Steinernema strain ( Italy ) was kindly provided by Prof. Trigiani Oreste, (Bari, Italy); the Spanish (SP2) one from Fernando Garcia del Pinto (Barcelona, Spain), the Polish strain from Marek Tomalak, Poznan, Poland), the Slovak strain from Zdenek Mracek, Ceske Budejovice, Check Republic, The Russian strain from Sergei Spiridonov Moscow, Russia, the Gel (probably the original Russian isolate) from Z. Mracek; the Morocco strain was kindly provided by Ralf-Udo Ehlers while the Palestine one by Dr. Nair Iraki). For in vitro culturing nematodes, large glass Petri plates of 9 cm diameter were filled with TSY agar medium 2 and one half was seeded with the proper bacterium. A sterile Sartorius filter of 0.2 µm pore size was put on the bacterium free part of the TSY agar. Surface sterilized IJs were then transferred to filter. Couple of hours later, when the nematodes left the filter moved toward the bacterial lawn the filter were removed. If the nematodes are in conform to the bacteria, they molt to L4 and grow to fertile adults and propagate. (Otherwise they may or may not recover and even if they recover they die or become unfertile). For intercross experiments, small plastic Petri plates of 3.5 cm diameter were used with TSY agar. A drop of an overnight bacterium culture was dropped in the center of the plates, one virgin (L4) female from the maternal strain and 1-9 young males from the paternal strain were transferred by platinum wire. The Petri plates were closed by using parafilm and put in a humidity chamber (e.g. glass Petri plates with water). We observed that the males started to court immediately whoever the females were and usually mated successfully. In the control groups, where the males and the females originated from the same strain, the females have become gravid carrying embryos, the majority of which developed to larvae inside their mother (entokia matricida) 6,7. In case of absence of cross fertility the oocytes remained unfertile and no progeny developed. The DNA isolation from single nematodes as well as the PCR amplification of the ITS1 5.8S rdna ITS2 region of the 18S- 5.8S 28S rdna operon was carried out by using the protocol of Triga et al (2000) as described 3. The molecular analysis was conducted according to the manufacturer s original protocol, without any modification. Based on our previous studies 3, we used four enzymes (AluI, MseI, TaqI and RsaI) to cut the DNA and compare the restriction patterns.

3 RESULTS Gnotobiology: The Palestine strain could not be grown on any of the Xenorhabdus isolates. Morocco strain could grow almost all the bacterium, but the growth was best on its own symbionts. For crosses only the KMD15 and the Anomali bacterium plates could be used. Data are summarized in Table 1. Table 1 Growth and propagation of Steinernema strains on each others symbionts Xenorhabdus: strains: X. X. sp. X. sp. X. sp. X. sp. X. sp Steinernema isolates: poinarii KMD11 anomali SP2 Italy Morocco S. glaseri NC NCLU NC Kmd AZ S. anomali Polish Slovak Gel Russian SP Italy Morocco Not +++ tested Palestine Data show, that there are hardly any growth of the majority of the strains could be observed on any artificial symbionts. Of the S. glaseri symbionts only a selected line of Kmd15 bacteria could serve as food for some other strains, while the symbionts of S. anomali (Azores) could moderately be used to grow one or two generations of other steinernematids. Palestine strain could not be grown any other bacteria, therefore we could not test them in intercross studies. The molecular identification of the bacterial symbionts is in progress. Results of crossbreeding studies: On the basis of the classic species definition, EPN strains NC1, Kmd15, NC513, NCLU, and AZ 26 do belong to the same (S. glaseri) species, while strains Anomali, Gel, Russian comprise another species (S. arenarium). On the basis of cross fertility, neither SP2, nor Italy, nor Polish, nor Slovak could be sorted into any of these two species for awhile, in spite of some the similarities between the molecular markers (see later). Morocco does definitely belong to another (unknown) species. Because of the absences of the Palestine bacterium, and because the Palestine nematode is unwilling to grow in any other Xenorhabdus tested, we could not carry on intercross experiments with this EPN strain, but seems completely different from the others. Results are summarized in Table 2.

4 Table 2 The results of cross breeding tests Maternal strains: NC1 NC LU NC 513 AZ 26 Kmd15 Paternal strains: Anomali Gel Rus sian Sp 2 Italy Polish Morocco S. g. NC NCLU NC AZ Kmd S. anomali Polish Gel Russian SP Italy Morocco Results of the molecular analysis: Results are demonstrated on figures 1-3 (GenePhore PCR-RFLP analysis 1, 2 & 3. Fig.1. GenePhore PCR-RFLP Analysis 1. M M M M = Molecular marker 7 S. anomali (Azores) /RsaI (Fragments sizes: 120, 130, 190) 8 Kmd15 / RsaI (Fragments sizes: 130, 210) 9 S. anomali (Azores) /MseI (Fragments sizes: 120, 250) 10 Kmd15 /MseI (Fragments sizes: 120, 300) 11 S. anomali (Azores) /TaqI (Fragments' size: 100, 110,125,210, 260) 12 Kmd15 /TaqI (Fragments' size: 100, 110, 125, 150, 230) Fig.1. of the RsaI, MseI and TaqI PCR-RFLP restriction profiles of the ITS1 5.8S ITS2 regions of the 18S 5.8.S 28S operons of entomopathogenic nematode strains of long

5 dauer phenotypes belonging to the Steinernema arenarium (S. anomali from Nelson Simoes s collection in Portugal Azores) and Steinernema glaseri (KMD 15). The RsaI pattern of S. anomali (7) and consists of a 120, 130 and 190 Dalton fragments and is rather different from that of KMD15 (8), which has a profile of 130 and 210 Dalton fragments. Similarly, the two strains also differ in their MseI and TaqI profiles, respectively. Before we started the intercross experiments, experiments Kmd15 was believed to be belonging to a new species, S. ohioensis (Lucskai and Klein, unpublished) and S. anomali was believed to exist as a separate species. Kmd15, however, can be crossed all the other four S. glaseri strains (NCI, NCLU, NC 513, AZ26), and, as we could see on Fig. 2. and 3, their GenPhore PCR-RFLP restriction profiles are also rather similar. As for our S. anomali strains (the other one came from Biosys, USA) they could cross freely with each other as well as with the Gel and Russian strains, proving, that they belong to the same species, S. arenarium. But the restriction profiles of GEL and Anomali (Biosys) strains are not identical to each other, neither to those of Polish, Italian and SP2 strains either. Since the intercross experiments gave also negative results, we suppose that even if all these strains belong to a large S. arenarium groups, they are not only geographically, but sexually isolated. Fig. 2. GenePhore PCR-RFLP Analysis Molecular Marker 2. S. a. (anomali) (Biosys) / TaqI 3. S. g. (Kmd 15) / TaqI 4. S. g. (NC513) / TaqI 5. St ei ner nema sp. Pol i sh /TaqI 6. S. g. (NC I) / TaqI 7. Molecular Marker 8. Stein ern ema sp. (Morocco)/TaqI 9. S. a. (Gel) /TaqI 10. Az26 /TaqI 11. Steinernemasp. I taly/taqi 12. Molecular Marker 13. S. anomali (Biosys) /Alu 14. S. g. (Kmd 15) /Alu 15. S. g. (NC513) /Alu 16. Steinernema sp. Polish /Alu 17. S. g. (NCI) / Alu 18. Molecular Marker 1 9. Stein ern ema sp. (Morocco) /Alu 20. S. a. anomali (Gel) /Alu 21. S. G. Az26 /Alu 22. Steinernemasp. I taly/alu 23. Molecular Marker Fig. 2. Comparison of the TaqI and AluI PCR-RFLP restriction profiles of the ITS1 5.8S ITS2 regions of the 18S 5.8.S 28S operons of strains belonging to the S. glaseri (S.g.), S. arenarium (S. a.) groups as well as those of taxonomically unidentified Steinernema strains of long dauer phenotypes from Poland (Polish), Italy, (Italy), Spain (SP2) and Morocco. The AluI profiles of KMD15, NC513, NCI seem identical but AZ26 differs in some smaller fragments significantly. The characteristic glaseri pattern, however, cannot be mixed up with any other. The AluI patterns of the Anomali (Biosys) and Gel strains are identical, and

6 that of the Polish strain is very similar to them, almost identical, but the Italian strain, however, seems completely different. The Morocco pattern is completely unique. The TaqI profiles of KMD15, NC513 and AZ26 are unambiguously identical and NCI slightly differ from them. However, all the other RFLP patterns are significantly differ from the glaseri profile. Those of S. anomali (Biosys) and Gel are similar but not identical and the Polish profile differs from both. The Italy just like the Morocco shows unique pattern. Fig. 3 GenePhore PCR-RFLP Analysis S. a. (anomali) (Biosys) / RsaI 14. S. g. Kmd / RsaI 15. S. g. Nc513 / RsaI 16. Steinernema sp. Polish / RsaI 17. S. g. NCI / RsaI 18. Stein ern ema sp. (Morocco) / RsaI 1. Molecular Marker 2. S. a. (anomali) (Biosys) / MseI 3. S. g. (Kmd15) / MseI 4. S. g. (NC 513) / MseI 5. Steinernema sp. Polish / MseI 6. S. g. NCI /MseI 7. Stein ern ema sp. (Morocco)/MseI 8. S. a. (Gel) /MseI 9. S. g. (Az26) / MseI 10. Steinernemasp. I taly/ MseI 11. Steinernema sp. (Slovak) / MseI 12. Stein ern ema sp. (SP 2 ) / MseI 19. Gel / RsaI 20. S. g. sp.(az26) / RsaI 21 Steinernemasp. I taly / RsaI 22. Steinernema sp. Slovak / RsaI 23. Stein ern ema (SP 2 ) / RsaI 24. Molecular Marker Fig. 3. Comparison of the MseI and RsaI PCR-RFLP restriction profiles of the ITS1 5.8S ITS2 regions of the 18S 5.8.S 28S operons of strains belonging to the S. glaseri (S.g.), S. arenarium (S. a.) groups as well as those of taxonomically unidentified Steinernema strains of long dauer phenotypes from Poland (Polish), Italy, (Italy), Spain (SP2), Slovak and Morocco. The MseI profiles of KMD15, NC513, seem identical and very similar to that of AZ26. This enzyme (as we had found before) 3 could not cut the DNA of S. glaseri NCI. The characteristic glaseri pattern, however, cannot be mixed up with neither any other. The MseI patterns of the Anomali (Biosys) and Gel strains are different, since the Gel DNA was not very well digested by this enzyme. The Polish strain was almost identical to that of Anomali. The Italian strain showed completely different pattern of lots of fragments, but was very similar to that of SP2. The pattern of the Slovak strain was something between the Polish and the Italian, but was definitely different from both of them. The Morocco pattern is completely unique.

7 DISCUSSION On the basis of the RFLP pattern studied Morocco strain shows no similarities with any other strains and probably comprise a well defined species. Italy, Sp2, Slovak and Polish strains have unique patterns, completely different from those of the Glaseri group but showing some similarities to the patterns of the members (Gel, Anomali) of the arenarium group. It is even hard to say that we found and arenarium profile, when using different restriction enzymes. It might be concluded that there are a uniform Glaseri and an non-homogenous Arenarium group, but we do not think, that this definition would lead too far. As for the cross breeding experiments we should carefully exclude those barriers of the successful meeting which are due to the capabilities of growing the parental strains on each other symbionts before making any definitive conclusions. In fact all nematode strains we studied mated intensively each other, with or without success, but in the control plates the crosses were always successful. Our molecular data are in conform with both the gnotobiological as well as to the cross breeding results. On the basis of these results we can declare, that there is a Steinernema glaseri species of little heterogeneity and there is a heterogeneous S. arenarium group of strains, some of which are separated both geographically, sexually as well as filogenetically. For awhile the question, whether Steinernema sp. Polish, Steinernema sp. SP2, Steinernema sp. Slovak, Steinernema sp. Italy belong to different biological species but to the same phylogenetic species (S. arenarium). We can say for sure, however, that both Steinernema sp. Morocco and Steinernema sp. Palestine are well defined species. We may also conclude that the cross fertility data and the data of the RFLP analysis are in rather good agreement and could be used together (and with other taxonomic methods) for species determination within the entomopathogenic nematode Steinernema genus. It can be seen, that the AluI, TaqI, of those strains which belong to the S. glaseri species (Kmd15, Az26, S. glaseri, NC513, NCLU) is practically identical, but there are slight differences between their MseI and RsaI patterns. Interestingly all the patterns of Gel and Anomali differed (the Russian was not analyzed). Hardly any similarities of the patterns of the different strains were found. References Al-Samarrai, T.H., Zhang, N., Lamont I. L., Martin, L., Kolbe, J., Wilsher, M., Morris, A J. and Schmied, J. (2000). Simple and inexpensive but highly discriminating method for computer, assisted DNA fingerprinting. J. Clin. Microbiol. 38, Sambrook, J., E., Fritsch, F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual, 3 rd edition, Cold Spring Harbor Laboratory, CSH. Triga, D., H. Pamjav, T. Vellai, A. Fodor, and Z. Buzás Gel electrophoretic restriction fragment length polymorphism analysis of DNA derived from individual nematodes, using the PhastSystem. Electrophoresis 20, Pamjav, H., D. Triga, Z. Buzás, T. Vellai, A. Lucskai, B. Adams, A.P. Reid, A. Burnell, C. Griffin, I. Glazer, M. Klein, and A. Fodor. (1999) Novel application of PhastSystem PAGE using RFLP-ITS patterns of individuals for molecular identification of EPNs. Electrophoresis 20, Forst S, Dowds B, Boemare N, Stackebrandt E. (1997).Xenorhabdus and Photorhabdus spp.: bugs that kill bugs. Annu. Rev Microbiol. 51, 47-72

8 Ehlers R-U. (2001). Mass production of entomopathogenic nematodes for plant protection. Appl Microbiol Biotechnol. 56, Han, R. and Ehlers, R.-U. (2000). Pathogenicity, development, and reproduction of Heterorhabditis bacteriophora and Steinernema carpocapsae under axenic in vivo conditions. J. Invertebr Pathol. 75, Lucskai, A. and Klein, M. personal communication