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The first record of entomopathogenic nematodes (Rhabiditiae: Steinernematidae and Heterorhabditidae) in natural ecosystems in Lebanon: A biogeographic approach in the Mediterranean region
Journal of Invertebrate Pathology 107 (2011) 82–85
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
The first record of entomopathogenic nematodes (Rhabiditiae: Steinernematidae and Heterorhabditidae) in natural ecosystems in Lebanon: A biogeographic approach in the Mediterranean region Elise Noujeim a,⇑, Carla Khater b, Sylvie Pages c,d, Jean-Claude Ogier c,d, Patrick Tailliez c,d, Mouïn Hamze a, Olivier Thaler c,d a
National Council for Scientific Research – CNRS, P.O.Box 11-8281, Ryad El Solh, 59, Zahia Selman Street, Beirut, Lebanon National Council for Scientific Research – CNRS, Remote Sensing Center, 59, Zahia Selman Street, Beirut, Lebanon c INRA, Laboratoire d’Ecologie Microbienne des Insectes et interactions hôte-Pathogène, Place Eugène Bataillon, 34000 Montpellier, France d Université Montpellier 2, Laboratoire d’Ecologie Microbienne des Insectes et interactions hôte-Pathogène, Place Eugène Bataillon, 34000 Montpellier, France b
a r t i c l e
i n f o
Article history: Received 30 September 2010 Accepted 11 January 2011 Available online 15 January 2011 Keywords: Steinernema Heterorhabditis Xenorhabdus Photorhabdus Wooded ecosystem Herbaceous ecosystem
a b s t r a c t A survey of entomopathogenic nematodes in Lebanon was conducted for the first time during 2008–2009. Samples were collected on the coastal strip and in nine vegetation types extending from the coastal line to 3088 m above sea level. Wooded and herbaceous ecosystems were considered for sampling purposes. A total of 570 samples were taken, out of which 1% were positive for entomopathogenic nematodes. Approximately, 15.8% out of the 19 sites sampled revealed entomopathogenic nematodes presence (representing three samples). Two entomopathogenic nematodes species Heterorhabditis bacteriophora and Steinernema feltiae were recovered, and identification of their symbiotic bacteria revealed the presence of a Xenorhabdus bovienii, Photorhabdus temperata subsp. thracensis, Photorhabdus luminescens subsp. kayaii and Photorhabdus luminescens subsp. Laumondii. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
2. Material and methods
The entomopathogenic nematodes-EPNs of the families Heterorhabditidae Poinar, and Steinernematidae Filipjev, are parasites of soil-dwelling insects that occur in natural and agricultural soils around the world. Their intestine hosts symbiotic bacteria essential for parasitic success. EPNs were recovered from all continents except Antarctica (where no EPNs were found). Biogeographic assessments of EPNs in the Eastern Mediterranean basin have been conducted in several countries (Glazer et al., 1991; Hazir et al., 2003; Canhilal et al., 2006; Stock et al., 2008; Salame et al., 2010). This paper presents the first survey for EPNs in Lebanon and contributes to filling the knowledge gap for EPN distribution around the Mediterranean basin. This is one of the rare studies that assess the presence of EPNs in natural ecosystems (Garcia Del Pino and Palomo, 1996), following a large-scale biogeographic approach with respect to vegetation levels.
2.1. Study area, sampling strategy and entomopathogenic nematode recovery
⇑ Corresponding author. Fax: +961 1 822639. E-mail address: [email protected] (E. Noujeim). 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.01.004
Surveys were conducted in Lebanon during the humid season (November 2008–June 2009). Samples were collected along a transect extending from 0 to 3088 m above sea level and representing 31% of the total area of Lebanon. Samples were taken in natural ecosystems (wooded and herbaceous) representing the coastal strip and nine vegetation levels with the exception of one vegetation level where no natural wooded ecosystem existed. Specifically, soil samples were taken from 10 herbaceous sites (note, the abbreviation for each site follows a hyphen): Ghalbine-GH, Salaata-SH, Bejje-BH, Tanourine Tahta-TTH, Tanourine-TH, BcharreBH, Ainata-AH, Barqa-BAH, Sbouba-SBH, Qaa-QH, and nine wooden sites: Salaata-SW, Bkirki-BW, Daroune-DW, Tanourine Tahta-TTW, Laqlouq-LW, Bcharre-BW, Ainata-AW, Barqa-BAW, Sbouba-SBW. Within each site, 30 soil samples were collected randomly at a depth of 0–30 cm using a hand shovel. Approximately, 0.5 kg of soil from each sample was placed in plastic boxes in contact with 10 Galleria mellonella larvae based on the insect baiting technique described by Bedding and Akhurst (1975). Boxes were transported in
83
29
27–28
103
103 88 100 68–86 42–51 70–92 53–67 60 ± NA 34–47
736–950
72 91 100 100 75 ± 9 72 ± 4 51 ± 10 50 ± 3 91 ± 9 87 ± 5 61 ± 6 63 ± 2.5 840 ± 12 934 ± 32 64 ± 6 65 ± 6 43 ± 7 43 ± 4
66 ± 5 75 ± 5 33 65–77 LIB14 LIB21 104 Steinernema feltiae (Poinar, 1990) TTH LW LB21
All measurements are in micrometers; data are given as the ranges, means, and standard deviation; p represents the p-values (Wilcoxon test) CFU: colony forming units; NA: data not available in original description. EP, distance from anterior end to excretory pore; D% = EP/distance from anterior end to base of esophagus%; E% = EP/TL%; TBL, maximum body length; TL, tail length.
23 3
Photorhabdus luminescens kayaï, CIP 108428, Hazir, Turkey Photorhabdus temperate thracensis CIP 108426, Hazir, Turkey Xenorhabdus bovieni LB14 Xenorhabdus bovieni
Xenorhabdus bovienii, FR44, FranceF3
20 103
TTW
LIB03 LIB04 LIB27 Heterorhabiditis bacteriophora (Poinar, 1976)
10
30 19 23 23 103 103 103 103 Photorhabdus Photorhabdus Photorhabdus Photorhabdus Trinidad 92 63 88 95 100 100 100 100 104 ± 6 100 ± 11 103 ± 4 103–130 84 ± 5 86 ± 4 77 ± 4 76–92 107 ± 8 103 ± 10 96 ± 7 83–112 104 ± 6 103 ± 5 99 ± 5 87–110 638 ± 30 571 ± 19 594 ± 24 512–671 111 ± 9 112 ± 7 109 ± 5 117 22 ± 3 23 ± 2 22 ± 2 18–25
EP D% GUL
TTH
39 ± 3 43 ± 2 41 ± 3 36–44
PS% TBL SpL
TL
Infective juveniles (n = 10) 1st generation males (n = 10) Isolate/species name
EPNs were recovered from both herbaceous and wooded ecosystems, in six out of 570 samples and from three out of 19 sites sampled. The three different sites, characterized by a low human disturbance, are located in the area of Tanourine Tahta (TTH; TTW) and Laqlouq (LW). TTH represents an herbaceous ecosystem dominated by low vegetation; the area is located over an old abandoned agricultural terrace situated at the bottom of the valley and is in proximity to a river surrounded by apple and walnut trees. TTW is a wooded formation located in close proximity to TTH and dominated by oak woodland. LW is a wooden formation
Site name
3. Results
Table 1 EPNs morphometric characteristics and pathogenicity of EPNs and their symbiotic bacteria.
2.3. Isolation, molecular classification and characterization of symbiotic bacteria from entomopathogenic nematodes Symbiotic bacteria were isolated from the hemolymph of G. mellonella cadavers that were exposed to nematodes 48 h earlier. Bacterial colonies showing the typical morphology phenotypic characters of the genera Photorhabdus and Xenorhabdus were subjected to a taxonomic classification based on comparison of their 16S rRNA gene sequences with those of type strains as described by Tailliez et al. (2006). The pathogenicity of bacterial isolates was determined by measuring LT50. Approximately, 103 CFU were injected into the hemocoel of each of 20 G. mellonella with the exception of strain LB21 where 104 CFU were needed to obtain an LT50. Statistical tests were carried out using SPSS version 18.0 (Chicago, USA) to construct a life table. Survival curves were compared using Gehan’s generalized Wilcoxon test. This test compares survival time among groups.
% of dead larvae 48 h after infection D%
E%
Entomopathogenic nematodes (n = 40 per Galleria mellonella)
Symbiotic bacteria
Nematode isolates were identified by studying the morphometric traits based on Nguyen and Smart (1996). Infective juveniles and first-generation males were extracted directly from the cadavers, heated in Ringer solution, fixed in TAF, and processed to glycerin (Kaya and Stock, 1997). Morphological observations were made using a Leica DMR microscope. Partial sequences of the nematode’s 28S rRNA genes (28S, D2-D3 domains) and of their internal transcribed spacer regions (ITS) were compared to those available in GenBank to selected representative Heterorhabditis and Steinernema species. Nematode pathogenicity was also analyzed by exposing each of the 25 G. mellonella larvae to 40 IJs from each EPN isolate in a perforated Eppendorf tubes lined with filter paper and with 250 lL of Ringer’s solution. There were three replicates per treatment. Cadavers were then placed on White traps at 22– 24 °C, and the number of emerging IJs was counted. Parasitic success (PS), representing the ability of nematodes to reproduce inside G. mellonella larvae, was calculated by counting the number of White trap containing IJs versus the total number of White traps (Sicard et al., 2003).
Bacterial identification
2.2. Identification and characterization of entomopathogenic nematode isolates
temperata thracensis LB03 luminescens kayaï LB04 luminescens Laumondii LB27 luminescens Laumondii, TT01,
LT50 (h)
coolers to the laboratory (darkness, 23 ± 2 °C) and assessed for mortality during 1 week. After 8 days, all insects were recovered, and parasitized cadavers were individually placed on White traps (Kaya and Stock, 1997) and held at 23 ± 2 °C to allow for the emergence of the infective juvenile nematodes (IJs). Emerging nematodes were pooled for each sample and used to infect fresh G. mellonella larvae for establishment of cultures and to produce nematodes (infective juveniles-IJs and males) used for identification. Soil samples were analyzed for soil texture and organic matter content (analysis was performed by the soil laboratory of the Ministry of Agriculture in Lebanon).
Bacterial dose (CFUs)
E. Noujeim et al. / Journal of Invertebrate Pathology 107 (2011) 82–85
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E. Noujeim et al. / Journal of Invertebrate Pathology 107 (2011) 82–85
located in an old abandoned agricultural terrace and includes a temporary wet area (water pond). Soil textures of sites that were positive for EPNs varied from sandy-loamy to loamy-sandy. Negative sites are dominated by clay and limestone texture. In pathogenicity assays, 100% of the G. mellonella used for EPNs infestation were killed with all the strains tested. Gehan’s generalized Wilcoxon test showed no significant difference among assays of LIB03 (P = 0.908), LIB04 (P = 0.543), LIB27 (P = 0.235), LIB14 (P = 0.996), and LIB21 (P = 0.117). All heterorhabditid isolates are characterized by the IJ’s average body length (571 and 638 lm), and E% (distance from anterior end to excretory pore divided by tail length) ranged between 100% and 104%, spicule length of 39–43 lm, and GUL (gubernaculums length) of 22–23 lm (Table 1). The molecular analysis of the nematode’s 28S + ITS sequences confirmed that they belong to Heterorhabditis bacteriophora (Poinar) (Fig. 1A). Morphometric examination (Table 1), confirmed by molecular analysis, indicated that the two Steinernema isolates, LIB14 and LIB21, belong to Steinernema feltiae (Filipjev). The IJs body length ranged from 840 to 934 lm, excretory pore located at 61–63 lm, spicule length of 66–75 lm, and GUL of 43 lm (Table 1). Steinernematid and heterorhabditid isolates were all found to be pathogenic and capable of reproduction in G. mellonella as shown in Table 1. The taxonomic position of the nematode’s symbiotic bacteria as determined by their 16S rRNA gene sequences (Fig. 1B) indicates that bacterial isolates LB14 and LB21 are c classified within the
Xenorhabdus bovienii group, and bacterial isolates LB03, LB04, and LB27 were classified as Photorhabdus temperata subsp. thracensis, Photorhabdus luminescens subsp. kayaii and Photorhabdus luminescens subsp. Laumondii, respectively. Photorhabdus and Xenorhabdus isolates were pathogenic when injected at 103 cells per G. mellonella larvae. No significant difference was observed between strains P. luminescens Laumondii LB27 and strain TT01 (P > 0.05) nor between X. bovieni strains LB14 and FR44 (P > 0.05). Strains LB03 was less virulent then CIP 108426 (P < 0.05; LT50 = 30 h and 23 h, respectively), and LB04 was more virulent than CIP 108428, (P < 0.05; LT50 = 19 h and 20 h, respectively), LB21 was less virulent than FR44 (P < 0.05; 104 CFU needed to obtain LT50; LT50 = 33 h and 27–28 h, respectively) (Table 1). 4. Discussion The present study described for the first time the isolation and characterization of entomopathogenic nematodes and their symbiotic bacteria in Lebanon. The percentage of positive samples recovered in our study (1.05%) is comparable to other surveys conducted in the region such as in Jordan (Stock et al., 2008), with 0.9% for positive samples, and in Syria with 2.37% positive samples (Canhilal et al., 2006). Other regions of the world tend to have a higher recovery rate for EPNs recovered, e.g., the 50.6% of samples taken in the Czech Republic were reported to be positive for EPNs (Mrácˇek et al., 2005).
Fig. 1. Distance trees showing the phylogenetic position of nematode isolates and their symbiotic bacteria. The trees were constructed using the 28S rRNA gene sequences and the Internal Transcribed Sequences (ITS) for entomopathogenic nematodes (A) and the 16S rRNA gene sequences for symbiotic bacteria (B). The Kimura two-parameter model and the neighbor-joining module of PAUP software were used. Bootstrap values are shown at the nodes (percentages of 1000 replicates). GenBank accession numbers for 28S and ITS sequences for nematodes and 16S for bacteria are given in parentheses, and underlined accession numbers correspond to sequences determined in this study. Caenorhabditis elegans and Proteus vulgaris were used as outgroups. Bars in Fig. 1A and B correspond to 10% and 1% divergence, respectively.
E. Noujeim et al. / Journal of Invertebrate Pathology 107 (2011) 82–85
Precipitation rate can be correlated to the occurrence of EPNs in the soil. Regions that have relatively dry soil for extended periods, e.g., the eastern part of the Mediterranean basin, have a low prevalence of EPNs in contrast to areas with more abundant humid periods such as the North Mediterranean basin. The sites in Lebanon that were positive for EPNs have higher average soil humidity than the other regions of Lebanon; the former may receive between 1000 and 1500 mm of precipitation per year and benefits from streams, spring thaw, and other water sources. Our sampling methods utilized a biogeographic approach, which was correlated to vegetation levels and based on the sampling strategy of Emelianoff et al. (2008). The low rate of recovery in our study tends to indicate that the biogeographic sampling method based on vegetation levels may be an inappropriate strategy in the eastern part of the Mediterranean area. On the other hand, soil type tends to be one of the determining abiotic factors influencing the presence of EPNs. While Lebanon is dominated by limestone (Dubertret, 1953), our positive sites were characterized by soil texture ranging from sandy-loamy to loamy-sandy. Negative sites were mainly dominated by clay. These results are comparable to other studies (Kung et al., 1990a; Mrácˇek et al., 2005; Campos-Herrera et al., 2008), showing that clay content negatively affects EPNs occurrence even though Hazir et al. (2003) reported Steinernema sp. in a clay loam soil. In future studies, characterizing the insect host species in specific sites (in progress) will bring additional information in reference to the correlation between EPNs presence and the invertebrate fauna in the soil. Furthermore, utilization of other bait insects (besides G. mellonella) should also be carried out because some EPN species such as S. scapterisci Nguyen & Smart and S. kushidai Mamiya show poor infectivity to G. mellonella (Adams and Nguyen, 2002). Also, the isolated symbiotic bacteria, pathogen when injected into G. mellonella, should also serve as the basis for further research to test pathogenicity against insect pests in Lebanon (study in progress against a local insect pest). Acknowledgments The authors acknowledge Dr. Talal Darwish, Dr. Ihab Jomaa, M. Mohamad Bou Daher from the Remote Sensing Center of the National Council for Scientific Research-CNRS-Lebanon for providing the maps needed and helping in soil analysis and interpretation. Thanks to Dr. Gaby Khalaf, Dr. Milak Fakhry and Mr. Elie Najjar from the Marine Research Center of the CNRS-Lebanon, Dr. Nabil Nemer and Mr. Patrick Hayek for their technical assistance. Thanks to Hervé Mauléon for his kind attention and advice. The CNRS-Lebanon is highly acknowledged for providing a PhD scholarship to the first author to conduct this study and to fund the Project.
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References Adams, B.J., Nguyen, K.B., 2002. Taxonomy and systematics. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Oxon, New York, pp. 1–34. Bedding, R.A., Akhurst, R.J., 1975. A simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica 21, 109–110. Campos-Herrera, R., Gómez-Ros, J.M., Escuer, M., Cuadra, L., Barrios, L., Gutiérrez, C., 2008. Diversity, occurrence, and life characteristics of natural EPN population from La Rioja (Northern Spain) under different levels of disturbance associated with agricultural management and their relationships with soil factors. Soil Biol. Biochem. 40, 1474–1484. Canhilal, R., Reid, W., Kutuk, H., El-Bouhssini, M., 2006. Natural occurrence of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) in Syrian Soils. Res. J. Agr. Biol. Sci. 2 (6), 493–497. Dubertret, L., 1953. Carte géologique au 1/50000 de la Syrie et du Liban. 21 feuilles avec notices explicatrices. Ministère des Travaux Publics. L’imprimerie Catholique, Beirut, Liban. Emelianoff, V., Le Brun, N., Pagès, S., Stock, S.P., Tailliez, P., Moulia, C.M., Sicard, M., 2008. Isolation and identification of entomopathogenic nematodes and their symbiotic bacteria from Hérault and Gard (Southern France). J. Invertebr. Pathol. 98, 211–217. Garcia Del Pino, F., Palomo, A., 1996. Natural occurrence of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) in Spanish soils. J. Inver. Pathol. 68, 84–90. Glazer, I., Liran, N., Steinberger, Y., 1991. A survey of entomopathogenic nematodes (Rhabditida) in the Negev desert. Phytoparasitica 19 (4), 291–300. Hazir, S., Keskin, N., Stock, S.P., Kaya, H.K., Ozcan, S., 2003. Diversity and distribution of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) in Turkey. Biodivers. Conserv. 12, 375–386. Kaya, H.K., Stock, S.P., 1997. Techniques in insect nematology. In: Lacey, L.A. (Ed.), Manual of Techniques in Insect Pathology. Academic Press, New York., pp. 281– 324. Kung, S.-P., Guagler, R., Kaya, H.K., 1990. Soil type and entomopathogenic nematodes persistence. J. Invert. Pathol. 55, 401–406. Mrácˇek, Z., Becˇvárˇ, S., Kindlmann, P., Jersáková, J., 2005. Habitat preference for entomopathogenic nematodes, their insect hosts and new faunistic records for the Czech Republic. Biol. Contr 34, 27–37. Nguyen, K.B., Smart, G.C., 1996. Identification of entomopathogenic nematodes in the Steinernematidae and Heterorhabditidae (Nemata: Rhabditida). J. Nematol. 28, 286–300. Poinar Jr, G.O., 1976. Description and biology of a new insect parasitic rhabditoid, Heterorhabditis bacteriophora n. gen. n. sp. (Rhabditida; Heterorhabditidae n. fam.). Nematologica 21, 463–470. Poinar Jr, G.O., 1990. Taxonomy and biology of Steinernematidae and Heterorhabditidae. In: Gaugler, R., Kaya, H.K. (Eds.), Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, Florida, pp. 23–61. Salame, L., Glazer, I., Miqaia, N., 2010. Characterization of populations of entomopathogenic nematodes isolated at diverse sites across Israel. Phytoparasitica 38, 39–52. Sicard, M., Le Brun, N., Pagès, S., Godelle, B., Boemare, N., Moulia, C., 2003. Effect of native Xenorhabdus on the fitness of their Steinernema hosts: contrasting types of interaction. Parasitol. Res. 100 (3), 657–659. Stock, S.P., Al Banna, L., Darwish, R., Katbeh, A., 2008. Diversity and distribution of entomopathogenic nematodes (Nematoda: Steinernematidae, Heterorhabditidae) and their bacterial symbionts (c Proteobacteria: Enterobacteriaceae) in Jordan. J. Invertebr. Pathol. 98, 228–234. Tailliez, P., Pages, S., Ginibre, N., Boemare, N., 2006. New insights into diversity in the genus Xenorhabdus, including the description of ten novel species. Int. J. Syst. Evol. Microbiol. 56, 2805–2818.
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
The first record of entomopathogenic nematodes (Rhabiditiae: Steinernematidae and Heterorhabditidae) in natural ecosystems in Lebanon: A biogeographic approach in the Mediterranean region Elise Noujeim a,⇑, Carla Khater b, Sylvie Pages c,d, Jean-Claude Ogier c,d, Patrick Tailliez c,d, Mouïn Hamze a, Olivier Thaler c,d a
National Council for Scientific Research – CNRS, P.O.Box 11-8281, Ryad El Solh, 59, Zahia Selman Street, Beirut, Lebanon National Council for Scientific Research – CNRS, Remote Sensing Center, 59, Zahia Selman Street, Beirut, Lebanon c INRA, Laboratoire d’Ecologie Microbienne des Insectes et interactions hôte-Pathogène, Place Eugène Bataillon, 34000 Montpellier, France d Université Montpellier 2, Laboratoire d’Ecologie Microbienne des Insectes et interactions hôte-Pathogène, Place Eugène Bataillon, 34000 Montpellier, France b
a r t i c l e
i n f o
Article history: Received 30 September 2010 Accepted 11 January 2011 Available online 15 January 2011 Keywords: Steinernema Heterorhabditis Xenorhabdus Photorhabdus Wooded ecosystem Herbaceous ecosystem
a b s t r a c t A survey of entomopathogenic nematodes in Lebanon was conducted for the first time during 2008–2009. Samples were collected on the coastal strip and in nine vegetation types extending from the coastal line to 3088 m above sea level. Wooded and herbaceous ecosystems were considered for sampling purposes. A total of 570 samples were taken, out of which 1% were positive for entomopathogenic nematodes. Approximately, 15.8% out of the 19 sites sampled revealed entomopathogenic nematodes presence (representing three samples). Two entomopathogenic nematodes species Heterorhabditis bacteriophora and Steinernema feltiae were recovered, and identification of their symbiotic bacteria revealed the presence of a Xenorhabdus bovienii, Photorhabdus temperata subsp. thracensis, Photorhabdus luminescens subsp. kayaii and Photorhabdus luminescens subsp. Laumondii. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
2. Material and methods
The entomopathogenic nematodes-EPNs of the families Heterorhabditidae Poinar, and Steinernematidae Filipjev, are parasites of soil-dwelling insects that occur in natural and agricultural soils around the world. Their intestine hosts symbiotic bacteria essential for parasitic success. EPNs were recovered from all continents except Antarctica (where no EPNs were found). Biogeographic assessments of EPNs in the Eastern Mediterranean basin have been conducted in several countries (Glazer et al., 1991; Hazir et al., 2003; Canhilal et al., 2006; Stock et al., 2008; Salame et al., 2010). This paper presents the first survey for EPNs in Lebanon and contributes to filling the knowledge gap for EPN distribution around the Mediterranean basin. This is one of the rare studies that assess the presence of EPNs in natural ecosystems (Garcia Del Pino and Palomo, 1996), following a large-scale biogeographic approach with respect to vegetation levels.
2.1. Study area, sampling strategy and entomopathogenic nematode recovery
⇑ Corresponding author. Fax: +961 1 822639. E-mail address: [email protected] (E. Noujeim). 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.01.004
Surveys were conducted in Lebanon during the humid season (November 2008–June 2009). Samples were collected along a transect extending from 0 to 3088 m above sea level and representing 31% of the total area of Lebanon. Samples were taken in natural ecosystems (wooded and herbaceous) representing the coastal strip and nine vegetation levels with the exception of one vegetation level where no natural wooded ecosystem existed. Specifically, soil samples were taken from 10 herbaceous sites (note, the abbreviation for each site follows a hyphen): Ghalbine-GH, Salaata-SH, Bejje-BH, Tanourine Tahta-TTH, Tanourine-TH, BcharreBH, Ainata-AH, Barqa-BAH, Sbouba-SBH, Qaa-QH, and nine wooden sites: Salaata-SW, Bkirki-BW, Daroune-DW, Tanourine Tahta-TTW, Laqlouq-LW, Bcharre-BW, Ainata-AW, Barqa-BAW, Sbouba-SBW. Within each site, 30 soil samples were collected randomly at a depth of 0–30 cm using a hand shovel. Approximately, 0.5 kg of soil from each sample was placed in plastic boxes in contact with 10 Galleria mellonella larvae based on the insect baiting technique described by Bedding and Akhurst (1975). Boxes were transported in
83
29
27–28
103
103 88 100 68–86 42–51 70–92 53–67 60 ± NA 34–47
736–950
72 91 100 100 75 ± 9 72 ± 4 51 ± 10 50 ± 3 91 ± 9 87 ± 5 61 ± 6 63 ± 2.5 840 ± 12 934 ± 32 64 ± 6 65 ± 6 43 ± 7 43 ± 4
66 ± 5 75 ± 5 33 65–77 LIB14 LIB21 104 Steinernema feltiae (Poinar, 1990) TTH LW LB21
All measurements are in micrometers; data are given as the ranges, means, and standard deviation; p represents the p-values (Wilcoxon test) CFU: colony forming units; NA: data not available in original description. EP, distance from anterior end to excretory pore; D% = EP/distance from anterior end to base of esophagus%; E% = EP/TL%; TBL, maximum body length; TL, tail length.
23 3
Photorhabdus luminescens kayaï, CIP 108428, Hazir, Turkey Photorhabdus temperate thracensis CIP 108426, Hazir, Turkey Xenorhabdus bovieni LB14 Xenorhabdus bovieni
Xenorhabdus bovienii, FR44, FranceF3
20 103
TTW
LIB03 LIB04 LIB27 Heterorhabiditis bacteriophora (Poinar, 1976)
10
30 19 23 23 103 103 103 103 Photorhabdus Photorhabdus Photorhabdus Photorhabdus Trinidad 92 63 88 95 100 100 100 100 104 ± 6 100 ± 11 103 ± 4 103–130 84 ± 5 86 ± 4 77 ± 4 76–92 107 ± 8 103 ± 10 96 ± 7 83–112 104 ± 6 103 ± 5 99 ± 5 87–110 638 ± 30 571 ± 19 594 ± 24 512–671 111 ± 9 112 ± 7 109 ± 5 117 22 ± 3 23 ± 2 22 ± 2 18–25
EP D% GUL
TTH
39 ± 3 43 ± 2 41 ± 3 36–44
PS% TBL SpL
TL
Infective juveniles (n = 10) 1st generation males (n = 10) Isolate/species name
EPNs were recovered from both herbaceous and wooded ecosystems, in six out of 570 samples and from three out of 19 sites sampled. The three different sites, characterized by a low human disturbance, are located in the area of Tanourine Tahta (TTH; TTW) and Laqlouq (LW). TTH represents an herbaceous ecosystem dominated by low vegetation; the area is located over an old abandoned agricultural terrace situated at the bottom of the valley and is in proximity to a river surrounded by apple and walnut trees. TTW is a wooded formation located in close proximity to TTH and dominated by oak woodland. LW is a wooden formation
Site name
3. Results
Table 1 EPNs morphometric characteristics and pathogenicity of EPNs and their symbiotic bacteria.
2.3. Isolation, molecular classification and characterization of symbiotic bacteria from entomopathogenic nematodes Symbiotic bacteria were isolated from the hemolymph of G. mellonella cadavers that were exposed to nematodes 48 h earlier. Bacterial colonies showing the typical morphology phenotypic characters of the genera Photorhabdus and Xenorhabdus were subjected to a taxonomic classification based on comparison of their 16S rRNA gene sequences with those of type strains as described by Tailliez et al. (2006). The pathogenicity of bacterial isolates was determined by measuring LT50. Approximately, 103 CFU were injected into the hemocoel of each of 20 G. mellonella with the exception of strain LB21 where 104 CFU were needed to obtain an LT50. Statistical tests were carried out using SPSS version 18.0 (Chicago, USA) to construct a life table. Survival curves were compared using Gehan’s generalized Wilcoxon test. This test compares survival time among groups.
% of dead larvae 48 h after infection D%
E%
Entomopathogenic nematodes (n = 40 per Galleria mellonella)
Symbiotic bacteria
Nematode isolates were identified by studying the morphometric traits based on Nguyen and Smart (1996). Infective juveniles and first-generation males were extracted directly from the cadavers, heated in Ringer solution, fixed in TAF, and processed to glycerin (Kaya and Stock, 1997). Morphological observations were made using a Leica DMR microscope. Partial sequences of the nematode’s 28S rRNA genes (28S, D2-D3 domains) and of their internal transcribed spacer regions (ITS) were compared to those available in GenBank to selected representative Heterorhabditis and Steinernema species. Nematode pathogenicity was also analyzed by exposing each of the 25 G. mellonella larvae to 40 IJs from each EPN isolate in a perforated Eppendorf tubes lined with filter paper and with 250 lL of Ringer’s solution. There were three replicates per treatment. Cadavers were then placed on White traps at 22– 24 °C, and the number of emerging IJs was counted. Parasitic success (PS), representing the ability of nematodes to reproduce inside G. mellonella larvae, was calculated by counting the number of White trap containing IJs versus the total number of White traps (Sicard et al., 2003).
Bacterial identification
2.2. Identification and characterization of entomopathogenic nematode isolates
temperata thracensis LB03 luminescens kayaï LB04 luminescens Laumondii LB27 luminescens Laumondii, TT01,
LT50 (h)
coolers to the laboratory (darkness, 23 ± 2 °C) and assessed for mortality during 1 week. After 8 days, all insects were recovered, and parasitized cadavers were individually placed on White traps (Kaya and Stock, 1997) and held at 23 ± 2 °C to allow for the emergence of the infective juvenile nematodes (IJs). Emerging nematodes were pooled for each sample and used to infect fresh G. mellonella larvae for establishment of cultures and to produce nematodes (infective juveniles-IJs and males) used for identification. Soil samples were analyzed for soil texture and organic matter content (analysis was performed by the soil laboratory of the Ministry of Agriculture in Lebanon).
Bacterial dose (CFUs)
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located in an old abandoned agricultural terrace and includes a temporary wet area (water pond). Soil textures of sites that were positive for EPNs varied from sandy-loamy to loamy-sandy. Negative sites are dominated by clay and limestone texture. In pathogenicity assays, 100% of the G. mellonella used for EPNs infestation were killed with all the strains tested. Gehan’s generalized Wilcoxon test showed no significant difference among assays of LIB03 (P = 0.908), LIB04 (P = 0.543), LIB27 (P = 0.235), LIB14 (P = 0.996), and LIB21 (P = 0.117). All heterorhabditid isolates are characterized by the IJ’s average body length (571 and 638 lm), and E% (distance from anterior end to excretory pore divided by tail length) ranged between 100% and 104%, spicule length of 39–43 lm, and GUL (gubernaculums length) of 22–23 lm (Table 1). The molecular analysis of the nematode’s 28S + ITS sequences confirmed that they belong to Heterorhabditis bacteriophora (Poinar) (Fig. 1A). Morphometric examination (Table 1), confirmed by molecular analysis, indicated that the two Steinernema isolates, LIB14 and LIB21, belong to Steinernema feltiae (Filipjev). The IJs body length ranged from 840 to 934 lm, excretory pore located at 61–63 lm, spicule length of 66–75 lm, and GUL of 43 lm (Table 1). Steinernematid and heterorhabditid isolates were all found to be pathogenic and capable of reproduction in G. mellonella as shown in Table 1. The taxonomic position of the nematode’s symbiotic bacteria as determined by their 16S rRNA gene sequences (Fig. 1B) indicates that bacterial isolates LB14 and LB21 are c classified within the
Xenorhabdus bovienii group, and bacterial isolates LB03, LB04, and LB27 were classified as Photorhabdus temperata subsp. thracensis, Photorhabdus luminescens subsp. kayaii and Photorhabdus luminescens subsp. Laumondii, respectively. Photorhabdus and Xenorhabdus isolates were pathogenic when injected at 103 cells per G. mellonella larvae. No significant difference was observed between strains P. luminescens Laumondii LB27 and strain TT01 (P > 0.05) nor between X. bovieni strains LB14 and FR44 (P > 0.05). Strains LB03 was less virulent then CIP 108426 (P < 0.05; LT50 = 30 h and 23 h, respectively), and LB04 was more virulent than CIP 108428, (P < 0.05; LT50 = 19 h and 20 h, respectively), LB21 was less virulent than FR44 (P < 0.05; 104 CFU needed to obtain LT50; LT50 = 33 h and 27–28 h, respectively) (Table 1). 4. Discussion The present study described for the first time the isolation and characterization of entomopathogenic nematodes and their symbiotic bacteria in Lebanon. The percentage of positive samples recovered in our study (1.05%) is comparable to other surveys conducted in the region such as in Jordan (Stock et al., 2008), with 0.9% for positive samples, and in Syria with 2.37% positive samples (Canhilal et al., 2006). Other regions of the world tend to have a higher recovery rate for EPNs recovered, e.g., the 50.6% of samples taken in the Czech Republic were reported to be positive for EPNs (Mrácˇek et al., 2005).
Fig. 1. Distance trees showing the phylogenetic position of nematode isolates and their symbiotic bacteria. The trees were constructed using the 28S rRNA gene sequences and the Internal Transcribed Sequences (ITS) for entomopathogenic nematodes (A) and the 16S rRNA gene sequences for symbiotic bacteria (B). The Kimura two-parameter model and the neighbor-joining module of PAUP software were used. Bootstrap values are shown at the nodes (percentages of 1000 replicates). GenBank accession numbers for 28S and ITS sequences for nematodes and 16S for bacteria are given in parentheses, and underlined accession numbers correspond to sequences determined in this study. Caenorhabditis elegans and Proteus vulgaris were used as outgroups. Bars in Fig. 1A and B correspond to 10% and 1% divergence, respectively.
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Precipitation rate can be correlated to the occurrence of EPNs in the soil. Regions that have relatively dry soil for extended periods, e.g., the eastern part of the Mediterranean basin, have a low prevalence of EPNs in contrast to areas with more abundant humid periods such as the North Mediterranean basin. The sites in Lebanon that were positive for EPNs have higher average soil humidity than the other regions of Lebanon; the former may receive between 1000 and 1500 mm of precipitation per year and benefits from streams, spring thaw, and other water sources. Our sampling methods utilized a biogeographic approach, which was correlated to vegetation levels and based on the sampling strategy of Emelianoff et al. (2008). The low rate of recovery in our study tends to indicate that the biogeographic sampling method based on vegetation levels may be an inappropriate strategy in the eastern part of the Mediterranean area. On the other hand, soil type tends to be one of the determining abiotic factors influencing the presence of EPNs. While Lebanon is dominated by limestone (Dubertret, 1953), our positive sites were characterized by soil texture ranging from sandy-loamy to loamy-sandy. Negative sites were mainly dominated by clay. These results are comparable to other studies (Kung et al., 1990a; Mrácˇek et al., 2005; Campos-Herrera et al., 2008), showing that clay content negatively affects EPNs occurrence even though Hazir et al. (2003) reported Steinernema sp. in a clay loam soil. In future studies, characterizing the insect host species in specific sites (in progress) will bring additional information in reference to the correlation between EPNs presence and the invertebrate fauna in the soil. Furthermore, utilization of other bait insects (besides G. mellonella) should also be carried out because some EPN species such as S. scapterisci Nguyen & Smart and S. kushidai Mamiya show poor infectivity to G. mellonella (Adams and Nguyen, 2002). Also, the isolated symbiotic bacteria, pathogen when injected into G. mellonella, should also serve as the basis for further research to test pathogenicity against insect pests in Lebanon (study in progress against a local insect pest). Acknowledgments The authors acknowledge Dr. Talal Darwish, Dr. Ihab Jomaa, M. Mohamad Bou Daher from the Remote Sensing Center of the National Council for Scientific Research-CNRS-Lebanon for providing the maps needed and helping in soil analysis and interpretation. Thanks to Dr. Gaby Khalaf, Dr. Milak Fakhry and Mr. Elie Najjar from the Marine Research Center of the CNRS-Lebanon, Dr. Nabil Nemer and Mr. Patrick Hayek for their technical assistance. Thanks to Hervé Mauléon for his kind attention and advice. The CNRS-Lebanon is highly acknowledged for providing a PhD scholarship to the first author to conduct this study and to fund the Project.
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