13 Gradinarov_173 7-01-2013 9:34 Pagina 173 Nematol. medit. (2012), 40: 173-180 173 DISTRIBUTION OF ENTOMOPATHOGENIC NEMATODES OF THE GENUS HETERORHABDITIS (RHABDITIDA: HETERORHABDITIDAE) IN BULGARIA D. Gradinarov1*, E. Petrova**, Y. Mutafchiev***, O. Karadjova** * Department of Zoology and Anthropology, Faculty of Biology, Sofia University “St. Kliment Ohridski”, 8 Dragan Tzankov Blvd., 1164 Sofia, Bulgaria ** Institute of Soil Science, Agrotechnology and Plant Protection “N. Pushkarov”, Division of Plant Protection, Kostinbrod, Bulgaria *** Institute of Biodiversity and Ecosystem Research, 2 Gagarin Str., 1113 Sofia, Bulgaria Received: 21 September 2012; Accepted: 21 November 2012. Summary. The results from studies on entomopathogenic nematodes of the genus Heterorhabditis Poinar, 1976 (Rhabditida: Het- erorhabditidae) in Bulgaria, conducted during 1994-2010 are summarized. Of the 1,227 soil samples collected, 3.5% were positive for the presence of Heterorhabditis spp. Specimens belonging to the genus were obtained from 43 soil samples collected at 27 lo- calities in different regions of the country. Heterorhabditids were established at altitudes from 0 to 1175 m, in habitats both along the Black Sea coast and inland. The prevalent species was H. bacteriophora Poinar, 1976. Its identity was confirmed by detailed morphometric studies and molecular analyses of four recently obtained isolates. Inland, H. bacteriophora prefers alluvial soils in river valleys under herbaceous and woody vegetation. It was also found in calcareous soils with pronounced fluctuations in the temperature and water conditions. The presence of the species H. megidis Poinar, Jackson et Klein, 1987 in Bulgaria needs further confirmation. Key words: Heterorhabditis bacteriophora, morphology, molecular identification, habitat preferences. Entomopathogenic nematodes (EPNs) (Rhabditida: processing of 1,227 soil samples collected during the pe- Steinernematidae, Heterorhabditidae) are obligate para- riod November, 1994 to October, 2010 from different sites of a wide range of soil insects. They are subject to regions in Bulgaria. The samples were collected at alti- intensive faunistic research worldwide as prospective tudes ranging from 0 to 2690 m, in diverse habitats: agents for biological pest control (Hominick, 2002). from coastal to alpine, with herbaceous or woody vege- The presence of three species of the genus Heterorhab- tation (coniferous and deciduous forests) and agro- ditis Poinar, 1976 has been reported and confirmed in ecosystems (Table I). Most of the samples were compos- Europe: H. bacteriophora Poinar, 1976, H. megidis ite, each consisting of four sub-samples from a 6 m × 6 Poinar, Jackson et Klein, 1987 and H. downesi Stock, m area that were mixed before processing. The nema- Griffin et Burnell, 2002 (Smits et al., 1991; Stock et al., todes were isolated from the soil using the ‘nematode- 2002). In Bulgaria, faunistic studies on entomopatho- bait’ method (Bedding and Akhurst, 1975) with larvae genic nematodes started in 1994 and until now H. bacte- of the greater wax moth Galleria mellonella L. (Lepi- riophora and seven species of Steinernema Travassos, doptera: Pyralidae). 1927 have been reported (Shishiniova et al., 2000; Both morphological and molecular identification was Gradinarov et al., 2011). Greatest attention has been performed on four Heterorhabditis isolates from differ- paid to species of the genus Steinernema and important ent localities in SW Bulgaria (cultures BGKB-1/2007, information on the localities in which heterorhabditids BGZG-24B/2009, BGSP-2B/2010 and BGSP-5/2010, are established has not yet been published. listed in Table II). The identification of previously col- The aim of the present study was to i) systematize the lected isolates of the genus was based only on morpho- data on the distribution of Heterorhabditis in the coun- logical and morphometrical characters, using micro- try, ii) perform morphological and molecular identifica- scope slides from the collection of the Department of tion of the available live cultures of Heterorhabditis Zoology and Anthropology, Sofia University. from Bulgaria, and iii) analyze the inland habitat prefer- The morphological identification was performed on ences of the genus Heterorhabditis in Bulgaria. the basis of characters of third stage infective juveniles and male individuals (Poinar, 1976; Poinar and Georgis, 1990; Nguyen and Smart, 1995; Stock et al., 2002). MATERIALS AND METHODS Males were obtained by dissection of parasitized G. mellonella larvae in 0.9% NaCl solution on the ninth The present paper summarizes the results from the day after invasion. The nematodes were fixed in 4% formaldehyde, transferred to glycerol (simple evapora- tion method, after Poinar, 1975), mounted within paraf- fin rings on microscope slides and measured at magnifi- 1 Corresponding author: [email protected] cations of 12×40 and 12×100 under an Olympus BX41 13 Gradinarov_173 7-01-2013 9:34 Pagina 174 174 microscope. The characters and ratios used for morpho- Soil Science, Agrotechnologies and Plant Protection, in logical identification were: body length (L), greatest order to confirm the results of the morphological identi- body width (W), distance from anterior end to nerve fication. For DNA isolation, water suspensions contain- ring (NR), distance from anterior end to excretory pore ing 800 live infective juveniles (IJs) per ml, of each iso- (EP), oesophagus length (OES), body width at oesopha- lates reared on G. melonella under laboratory condi- gus base (Woes), reflection of testis from anterior end tions for years, were used. For each isolate, 1 ml of sus- (TR), tail length (T), body width at cloaca (Wcl), spicule pension was transferred to an Eppendorf tube and cen- length (SP), gubernaculum length (GB), body width at trifuged at 14,000 rpm (18,300 × g) for 1 minute. The anus (ABW), ratios a (L/W), b (L/ES), c (L/T), d resulting precipitate was homogenized with a micropes- (EP/OES), e (EP/T), and GS (GB/SP). The microscope tle. DNA was extracted with QIAGEN DNeasy Blood slides prepared from the four cultures are deposited in and Tissue Kit following the Bench Spin-Column Proto- the collection of the Department of Zoology and An- col for Animal Tissues. A modification was made to the thropology, Sofia University (slides BGKB-1/1-16, two DNA elution steps in the instructions of the manu- BGZG-24B/1-18, BGSP-2B/1-16 and BGSP-5/1-20). facturer, where, instead of 200 µl of AE Buffer for each Molecular identification of the four isolates was per- step, 20 µl and 10 µl were used, respectively, in order to formed at the Division of Plant Protection, Institute of obtain higher DNA concentrations. The resulting DNA Table I. Distribution by altitude and by habitat types of Heterorhabditis in Bulgaria. Positive for EPNs Positive for Heterorhabditis Altitude ranges / Habitats Samples Number of % positive for % Number of samples EPNs samples 0-450 m altitude 170 28 16.5 11 39.3 Agricultural fields 10 1 10.0 1 100.0 Beaches 16 4 25.0 3 75.0 Coniferous woodlands 4 2 50.0 2 100.0 Deciduous woodlands 63 10 15.8 1 10.0 Meadows 67 10 14.9 4 40.0 Orchards 2 1 50.0 0 0.0 Other 8 0 0.0 - - 500-980 m altitude 508 95 18.7 29 30.5 Agricultural fields 37 15 40.5 11 73.3 Coniferous woodlands 35 4 11.4 1 25.0 Deciduous woodlands 155 32 20.7 5 15.6 Meadows 223 33 14.8 11 33.3 Orchards 25 3 12.0 1 33.3 Other 33 8 24.2 0 0.0 1000-1450 m altitude 295 96 32.5 3 3.1 Coniferous woodlands 48 9 18.8 0 0.0 Deciduous woodlands 123 44 35.8 0 0.0 Meadows 122 43 35.3 3 7.0 Other 2 0 0.0 - - 1500-2690 m altitude 254 115 45.3 0 0.0 Coniferous woodlands 70 28 40.0 0 0.0 Deciduous woodlands 8 3 37.5 0 0.0 Meadows 34 14 41.2 0 0.0 Subalpine meadows 70 26 37.1 0 0.0 Subalpine Juniperus formation 63 43 68.3 0 0.0 Alpine meadows 9 1 11.1 0 0.0 TOTAL 1227 334 27.2 43 12.9 13 Gradinarov_1737-01-20139:34Pagina175 Table II. Localities with establishment of Heterorhabditis in Bulgaria. Locality Collection date Altitude (m) Soil type Habitat Positive samples Source Lozen (42°36'N; 23°29'E) Nov 1994 700 Alluvial Mesophilic meadow 1 Shishiniova et al., 1997 Lozen (42°36'N; 23°29'E) Dec 1994 700 Alluvial Riverbank forest 1 Shishiniova et al., 1997 Svishtov (43°37'N; 25°26'E) Aug 1995 80 Loess Agricultural field 1 Shishiniova et al., 1997 Jeleznitsa (42°32'N; 23°21'E) Sep 1997 1175 Brown forest Mesophilic meadow 3 Shishiniova et al., 2000 Varna (43°14'N; 28°00'E) Jul 1997 60 Grey forest Coniferous forest 2 Shishiniova et al., 2000 Razhdavitsa (42°23'N; 22°42'E) May 1998 550 Cinnamonic Pine forest 1 Shishiniova et al., 2000 Zemen (42°28'N; 22°43'E) Sep 1998 580 Alluvial Riverside meadow 1 Shishiniova et al., 2000 Gorubljane (42°36'N; 23°24'E) Oct 1998 600 Alluvial Walnut orchard 1 New data Zemen (42°28'N; 22°43'E) Apr 1999 620 Calcareous Xerophilic meadow 1 New data Aheloi (42°38'N; 27°39'E) Mar 1999 0-1 Sand Beach 2 New data Ravda (42°38'N; 27°40'E) Mar 1999 0-1 Sand Beach 1 New data Ichtiman (42°28'N; 23°47'E) Mar 2000 650 Alluvial Meadow to an irrigation canal 1 New data Zemen (42°28'N; 22°43'E) Apr 2000 580 Alluvial Meadow 1 New data Razhdavitsa (42°23'N; 22°42'E) Mar 2001 500 Alluvial Riverbank forest 2 New data Stara Kresna (41°47'N; 23°11'E) May 2001 640 Alluvial Meadow to an irrigation canal 1 New data Kresna Gorge (41˚45'N; 23˚09'E) May 2002 200 Alluvial Meadow 1 Gradinarov et al., 2011 Novo Selo (42°11'N; 22°40'E) Jun 2002 850 Alluvial Riverside meadow 1 New data Kokaljane (42°35'N; 23°25'E) Aug 2002 600 Alluvial Mixed deciduous forest 1 New data Kostinbrod (42°48'N; 23°10'E) Aug 2002 550 Alluvial Strawberry, raspberry 11 Gradinarov, 2003 Melnik (41°31'N; 23°24'E) Nov 2002 450 Cinnamonic Meadow 1 New data Blatska (41°32'N; 23°52'E) Apr 2003 590 Alluvial Meadow 1 New data Hadzhidimovo (41°30'N; 23°53'E) Apr 2003 500 Alluvial Riverside meadow 1 New data Kostinbrod (42°48'N; 23°11'E), BGKB-1/2007 Jun 2007 550 Alluvial Riverbank forest 1 New data Gradinarov Zemen (42°28'N; 22°44'E), BGZG-24B/2009 Sep 2009 580 Alluvial Riverside meadow 2 New data Kojucha (41°27'N; 23°15'E) Oct 2009 175 Calcareous Xerophilic meadow 1 New data Katuntci (41°26'N; 23°25'E), BGSP-2B/2010 Oct 2010 150 Alluvial Riverbank forest 1 New data et al.