Mun. Ent. Zool. Vol. 5, Suppl., October 2010

Mun. Ent. Zool. Vol. 5, Suppl., October 2010 1075 MORPHOMETRICS AND MOLECULAR CHARACTERIZATION OF NEW ISOLATE OF ENTOMOPATHOGENIC NEMATODE, HETERORHABDITIS BACTERIOPHORA POINAR, 1976 (NEMATODA, RHABDITIDA) FROM THE NORTH OF IRAN Naser Eivazian Kary* * Department of Plant Protection, Faculty of Science, University of Tarbiat Moallem of Azarbaijan, Tabriz, IRAN. Email: eivazian@azaruniv.ac.ir [Kary, N. E. 2010. Morphometrics and molecular characterization of new isolate of entomopathogenic nematode, Heterorhabditis bacteriophora Poinar, 1976 (Nematoda: Rhabditida) from the North of Iran. Munis Entomology & Zoology, 5, suppl.: 10751084] ABSTRACT: For the first time during the survey of entomopathogenic nematodes in north of Iran, a geographical isolates of Heterorhabditis recovered from soil using Galleria baiting technique. Based on morphological and molecular characterization this isolates was identified as Heterorhabditis bacteriophora NIR1. Morphometric and morphological comparisons of the new isolate with type species and other described isolates from different areas showed that H. bacteriophora NIR1 is morphologically similar to others but differ from type species in the following cases. For infective juvenile, means of body length (582 vs 570 μm), distance from anterior end to nerve ring (86 vs 83 μm) and esophagus length (121 vs 125 μm) and for male, means of body length (910 vs 820 μm), maximum body width (60 vs 43 μm), anal body width (27 vs 23 μm), esophagus length (114 vs 103 μm), distance from anterior end to excretory pore (155 vs 121 μm) and spicule length (42 vs 40 μm) were different from type isolate. Molecular studies of rdna ITS regions showed that the Iranian isolate is closely related to the other described isolates of H. bacteriophora from other area. Sequence length of amplified region of ITSrDNA of NIR1 isolate (819 base pairs) is the same as in type species. Intraspecific relationships among isolates of H. bacteriophora showed that geographic distribution is associated with the genetic and morphologic differences. KEY WORDS: Entomopathogenic nematodes, Heterorhabditis bacteriophora, North of Iran, ITSrDNA. Entomopathogenic nematodes of the family Heterorhabditidae Poinar, 1975 are obligate pathogens of insects. Heterorhabditis harbors bacterial symbiont, Photorhabdus Boemare, Akhurst and Mourant 1993, that kill the insect host and digests tissues, providing suitable conditions for growth and development within the cadaver (Boemare et al., 1993; Forst and Clarke, 2002). In a search for ecologically and economically suitable EPNs isolates for biocontrol programs, identification of genetic and morphologic intraspecies variability is essential factor. The present study is an attempt to evaluate morphology, morphometric and ITSrDNA sequences of new geographical isolates of H. bacteriophora currently isolated from north of IRAN. The internal transcribed spacer region (ITS) of the ribosomal DNA is a wellconserved region controlled by concerted evolution (Baldwin et al., 1995). It has been demonstrated to be highly informative as a taxonomic marker at species level (Subbotin 2006, Adams et al., 2006). Here we used the region to evaluate intraspecies variation between studied isolate and well defined ones.

1076 Mun. Ent. Zool. Vol. 5, Suppl., October 2010 MATERIALS AND METHODS Collection of soil samples: About 81 soil samples were collected from different cultivated and noncultivated areas of north of Iran during 2009. Each soil sample was a composite of nearly 20 random subsamples taken distantly located from each other using a small shovel and at a depth of 030 cm. The soil was thoroughly mixed on a plastic sheet and half of each sample was used for EPNs extraction. Nematodes isolation and propagation: EPNs were recovered from soil samples using insect baiting method described by Bedding and Akhurst (1975). The traps were checked every two days and dead larvae from each container were set up in modified white trap (Kaya and Stock, 1997) to collect emerging infective juvenile and replaced by fresh ones. To verify collected nematodes pathogenicity and establishment of new cultures, emerging nematodes were pooled for each sample and used to infect fresh G. mellonella larvae. Nematode identification Morphological characterization: For morphological studies, nematodes were examined live or heat killed in 60ºC Ringer s solution. All nematodes used in this study were reared in G. mellonella larvae. Twenty G. mellonella larvae were exposed to about 1000 infective juveniles in a Petri dish lined with two moistened filter papers at room temperature (25± 3ºC). For isolating mature females and males of the first and second generations, the infected larvae were dissected in Ringer s solution 4 and 7 days after infection, respectively. The following abbreviations have been used in the text or tables: L= total body length; ABW= anal body width; EP: excretory pore position; Es: oesophagus length; GuL= gubernaculums length; MBW: maximum body width; NR= nerve ring position; ; SpL= spicule length (measured along the curvature in a line along the centre of the spicule); ratio a= L/MBW; ratio b= L/Es; ratio c= L/TL; ratio d= EP/Es 100; ratio e= EP/TL 100; GS= GuL/SpL 100; StL= stoma length; StW= stoma width; SW=SpL/ABW; TL= tail length (measured with considering the extra cuticular sheath of the second stage juvenile). Molecular characterization Total genomic extraction and ITSrDNA amplification were done as described by Eivazian Kary et al. (2009). Amplified products were purified using a Qiagen Purification kit (Qiagen, Leusden, The Netherlands). Purified DNA was sequenced in IBMPCNRS, France. The DNA sequences were edited with Chromas 2.01 and aligned using Clustal X 1.64 (Thompson et al., 1997) with the (ITS15.8SITS2) sequences of other Heterorhabditis bacteriophora species obtained from GenBank. RESULTS AND DISCUSSION Description Adults. Head truncate or slightly rounded (Fig. 2A,E; 3B and 4B). Six distinct protruding pointed lips surrounding oral aperture. Each lip bears one labial papilla. Amphids located posterior to labial papillae (Fig. 2A, E). Cheilorhabdions seen as lightly refractile areas lining anterior (noncollapsed) portion of stoma. Oesophagus rhabditoid. Corpus cylindrical; metacarpus not differentiated. Isthmus short. Basal bulb pyriform with reduced valve. Nerve ring located in middle of isthmus. Excretory pore usually posterior to the basal bulb (Fig. 3B).

Mun. Ent. Zool. Vol. 5, Suppl., October 2010 1077 Infective stage juveniles: Mouth and anus closed; pharynx and intestine collapsed; tail pointed (Fig. 1D); cuticle with longitudinal striae (Fig. 1C); cells of a rodshaped bacterium occur in the ventricular portion of the intestine; body initially covered with the enclosing 2nd stage cuticle, which is lost soon after the juveniles leave the host cadaver. Hermaphrodite (First generation) (Fig. 2E and 4). Intestine with few large cells. Lateral fields not seen, phasmids inconspicuous. Vullva near midbody. With ovotestis. Vulva located near middle of body. Vulva open, protruding outward and functional for oviposition. Tail pointed. Amphimictic female. Similar to hermaphroditic females but different in head and vulva region: head subconical, lip region often faintly set off from remainder of head, vulva nonfunctional for oviposition, often covered with a hardened deposit. The vulva appears to be nonfunctional after mating and does not protrude outward, often surrounded by a hardened deposit. Gonads amphidelphic, reflexed. Anal region slightly protruding. Males (Fig 3. A): Anterior region similar to female, but smaller (Fig. 2A). Monorchic. Testis single, anteriorly reflexed (Fig. 3D) leading into a seminal vesicle containing sperm cells; vas deferens well developed. Spicules paired, symmetrical and separate; shape of capitulum variable, from pointed to flat (Fig. 2C, 3E,F). Gubernaculum with the proximal portion curving ventrally between the spicules. Bursa peloderan, open, supported normally by ten pairs of papillae: a small anterior pair, two pair adjacent to the spicules and six pairs distal to the anal opening and one pair at the tip. Morphometric characterization The morphometric data of the H. bacteriophora NIR1 is presented in the Table 1. Based on morphological and morphometric data, NIR1 isolate was found similar to the type isolate and they could be considered as conspecies. But some differences were obtained between them in the means of body length (582 vs 570 μm), distance from anterior end to nerve ring (86 vs 83 μm) and esophagus length (121 vs 125 μm) of infective juvenile and for male, means of body length (910 vs 820 μm), maximum body width (60 vs 43 μm), anal body width (27 vs 23 μm), esophagus length (114 vs 103 μm), distance from anterior end to excretory pore (155 vs 121 μm) and spicule length (42 vs 40 μm) were different from type isolate. DNA characterization Sequences and multiple alignments: The sequence lengths, flanked by the two primers TW81 and AB28 of the ITS regions of H. bacteriophora strain NIR1, is 819 base pairs and has the same length in HP88 isolate. Sequences alignment and constructed tree (Fig. 5) showed that Iranian isolate differ from its closest taxon (IRA10) by three bp. Pairwise distances of H. bacteriophora NIR1 from other isolates are presented in Tables 2. The phylogenetic relationships among the 10 isolates of H. bacteriophora are presented in Figure 5. The nine isolates of H. bacteriophora comprise a monophyletic group by analysis of the ITS region. In this clade, the seven strains, NIR1, IRA10, NJ, Aqaba beach, LIB 27 HM, X7 and Muaggar form a monophyletic group representing the sister group to strain HP88 and 20C. This topology shows that the NIR1 and IRA10 (the isolate from Northwest of IRAN)

1078 Mun. Ent. Zool. Vol. 5, Suppl., October 2010 are two closest groups and Iran 8 are genetically different from other strains of H. bacteriophora, but the difference is not enough for new species. Molecular studies revealed that Iranian population is an isolate of H. bacteriophora and most of its morphologic and morphometric characters are also similar to the mentioned isolates. Therefore, the differences are considered as intraspecific variations. There are very rare reported data on the intraspecific morphological and molecular variations of H. bacteriophora, however, our molecular studies are recently available. Spiridonov et al. (2004) indicated sequence differences between 13 H. bacteriophora isolates from Germany, Russia, UK, Belgium, Iceland, Scotland and Switzerland, usually varied between 111 bp (up to 1%) but reached 21 bp (2.8%) between the UK (B2) and the Moscow isolates. Developing elaborated tools to discriminate isolates based on intraspecific variation are necessary at least from two practical views. First, in the case of using exotic isolate in biological control programs, a fast and accurate methods is needed for monitoring it and second, in mass production of EPNs distinguishing morphological or molecular characters that link with desirable characters in IJs such as high virulence against the insect(s) target and ease of culture can reduce significantly strain development costs. ACKNOWLEDGEMENTS This work was supported from the Azarbaijan University of Tarbiat Moallem. We are grateful to K. Shams for helping in this study. LITERATURE CITED Adams, B. J., Fodor, A., Koppenhöfer, H. S., Stackebrandt, E., Stock, S. P. & Klein, M. G. 2006. Biodiversity and systematics of nematodebacterium entomopathogens. Biological control, 37: 32 49. Baldwin, B. G., Campbell, C. S., Porter, J. M. & Sanderson, M. J. 1995. Utility of nuclear ribosomal DNA internal transcribed spacer sequences in phylogenetic analysis of angiosperms. Annals of the Missouri Botanical Garden, 82: 274277. Bedding, R. A. & Akhurst, R. J. 1975. A simple technique for detection of insect parasitic rhabditid nematodes in soil. Nematologica, 21: 109110. Boemare, N. E., Akhurst, R. J. & Mourant, R. G. 1993. DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov. International Journal of Systematic Bacterilogy, 43: 249255. Eivazian Kary, N., Niknam, G., Griffin, C. T., Mohammadi, S. A. & Moghaddam, M. 2009. A survey of entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae (Nematoda: Rhabditida) in the northwest of Iran. Nematology, 11 (1): 107116. Forst, S. & Clarke, D. 2002. Bacterianematode symbioses. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. pp. 5577. New York, NY 10016, USA. Kaya, H. K. & Stock, S. P. 1997. Techniques in insect nematology. In: Lacey, L. A. (Ed.) Manual of techniques in insect pathology. Biological Techniques series. San diego, London: Academic press, pp. 281324. Poinar, G. O., Jr. 1975. Description and Biology of a new insect parasitic rhabditoid, Heterorhabditis bacteriophora n. gen. n. sp. (Rhabditida: Heterorhabditidae n. fam.). Nematologica, 21: 463470. Spiridonov, S. E., KrasomilOsterfeld, K. & Moens, M. 2004. Steinernema jollieti sp. n. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from the American Midwest. Russ. J. Nematol., 12: 85 95.

Mun. Ent. Zool. Vol. 5, Suppl., October 2010 1079 Subbotin, S. A., Vierstraete, A., De Ley, P., Rowe, J., Waeyenberge, L., Moens, M. & Vanfleteren, J. R. 2001. Phylogenetic relationships within the cystforming nematodes (Nematoda, Heteroderidae) based on analysis of sequences from the ITS region of ribosomal DNA. Molecular Phylogenetics and Evolution, 21: 116. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Researc, 25: 4876 4882. Figure 1. Ensheathed infective juvenile of Heterorhabditis bacteriophora NIR1, A,B) Anterior region showing hemizonid and dorsal tooth; C), Middle part with longitudinal ridge; D) Tail.

1080 Mun. Ent. Zool. Vol. 5, Suppl., October 2010 Figure 2. SEM of Heterorhabditis bacteriophora NIR1, A) Anterior region of male showing cephalic (CP) and labial (LP) papilla; B,C,D) Posterior end of male showing spicule (Sp); bursa (B) and ribs (R); E) Hermaphrodite female`s head showing LP, CP and amphid (A); F) Anterior region showing smooth head and dorsal tooth.

Mun. Ent. Zool. Vol. 5, Suppl., October 2010 1081 Figure 3. Male: A) Body Shape; B) Anterior end showing excretory pore(ep); C) Nerve ring (NR); D) Testis reflection (TR); E&F)Posterior end showing spicule (Sp), gubernaculums (Gu), bursa and ribs.

1082 Mun. Ent. Zool. Vol. 5, Suppl., October 2010 Figure 4. Hermaphrodite Female: A) Body shape; B) Anterior end; C) Head and labial papilla; D) Vulva. Muaggar EU200360 X7 EU435140 LIB27 HM140691 8 Aqaba beach EU200361 NJ AY170328 34 IRA10 EU598227 92 H. bacteriophora NIR1 HP88 EF043438 20C EU074157 Iran8 FJ653914 20 Figure 5. Phylogenetic relationship among several H. bacteriophora strains based on the sequences of the ITS region by Maximum parsimony method.

Mun. Ent. Zool. Vol. 5, Suppl., October 2010 1083 Table 1. Morphometrics of Heterorhabditis bacteriophora strain NIR1, measurments are in micrometer and in the form: mean ± standard deviation (range). Character First generation Hermaphroditic Female Second generation Male Female Infective juvenile n 20 22 20 20 4712±288 910±74 2548±162 582±10 L (805 (41025145) 1040) (23122585) (562594) a 25±2 19±1 16±1 25±1.3 (2427) (1520) (1517) (1621) b 26±1.4 9±1 12±1 4.6±0.2 (2428) (79) (1013) (44.7) c 244±17 25±4 46±3 6.3±0.3 (209248) (2541) (4149) (5.56.5) Body diam.(w) 265±28 60±5 177±6 24±1.4 (229325) (5475) (162190) (2227) Excretory pore (EP) 235±25 155±10 131±5 103±4 (210266) (137179) (127145) (97104) Nerve ring (NR) 148±19 78±4 97±3 86±4 (139169) (7386) (84109) (7590) Pharynx length (ES) 211±12 114±5 142±8 121±6 (185220) (105120) (135144) (106128) Tail length (T) 79±12 39±4 67±3 92±5 (5998) (2744) (6070) (86102) Anal body diam. 58±7 27±3 29±4 15±1 (ABW) (5365) (2030) (2630) (1417) Spicule length (SpL) 42±5 (3150) Spicule width (SpW) 7±1 (58) Gubernaculum 21±2 length (GL) (1724) D% 44±5 131±8 39±5 84±2 (4050) (120148) (3444) (8087) E% 408±29 398±48 154±23 108±7 (375438) (375558) (133164) (98116) SW 162±27 (135220) GS 50±4

1084 Mun. Ent. Zool. Vol. 5, Suppl., October 2010 Muaggar EU200360 Table 2. Pairwise distances of H. bacteriophora NIR1 with several isolates of species. NJ AY170328 0.0106 1 2 3 4 5 6 7 8 9 LIB27 HM140691 0.0000 0.0106 Aqaba beach EU200361 Iran8 FJ653914 IRA10 EU598227 H. bacteriophora NIR1 0.0000 0.0106 0.0000 0.9120 0.9062 0.9120 0.9120 0.0053 0.9062 0.0053 0.0053 0.9236 0.0133 0.0242 0.0133 0.0133 0.9513 0.0080 X7 EU435140 0.0000 0.0106 0.0000 0.0000 0.9120 0.0053 0.0133 HP88 EF043438 20 CEU074157 0.0026 0.0133 0.0026 0.0026 0.9062 0.0079 0.0160 0.0026 0.0106 0.0215 0.0106 0.0106 0.9265 0.0160 0.0242 0.0106 0.0133