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Rhabditida: Steinernematidae) Rioja Strain Isolated from Bibio Hortulanus (Diptera: Bibionidae) in Spain R

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Rhabditida: Steinernematidae) Rioja Strain Isolated from Bibio Hortulanus (Diptera: Bibionidae) in Spain R Journal of Nematology 38(1):68–75. 2006. © The Society of Nematologists 2006. Morphological and Ecological Characterization of Steinernema feltiae (Rhabditida: Steinernematidae) Rioja Strain Isolated from Bibio hortulanus (Diptera: Bibionidae) in Spain R. Campos-Herrera,M.Escuer,L.Robertson, and C. Gutie´rrez Abstract: A new strain of Steinernema feltiae (Rhabditida: Steinernematidae) was isolated in La Rioja (Spain) from larvae of Bibio hortulanus (Diptera: Bibionidae). A comparative morphometric analysis of this new strain and four additional S. feltiae isolates was performed. Although significant differences in morphometric measurements were observed, PCR-RFLP profiles and sequence analysis of the ITS region of rDNA confirmed the identity of the new strain as A2 RFLP type of S. feltiae. A comparative morpho- metric study among nematodes from three hosts, Galleria mellonella (Lepidoptera: Pyralidae), Spodoptera littoralis (Lepidoptera: Noctuidae) and B. hortulanus, was conducted. Ecological characterization of the Rioja isolate was performed in G. mellonella larvae. Larval mortality was 75.3 and 78.12% in penetration and sand column assays, respectively, and the percentage of penetrating infective juveniles was 12.0 and 2.8% in these assays. Larval mortality in the one-on-one bioassay was 4.2 %, and in exposure-time bioassays, it was 50% at 11.25 hours. Relationships between morphometric characteristics and host mortality are discussed for this new strain of entomopathogenic nematode. Key words: molecular markers, natural host, Spain, Steinernema feltiae, virulence. Entomopathogenic nematodes (EPN) in the Hetero- (Diptera: Bibionidae) were collected from the edge of rhabditidae and Steinernematidae are lethal parasites a potato (Solanum tuberosum L.) field. Dead B. hortula- of insects that are widely distributed in soils throughout nus larvae were washed and placed individually in the world (Hominick et al., 1996). Their association White traps to collect infective juveniles (IJ) (White, with bacteria of the genera Xenorhabdus (for steinerne- 1927). Mortality and establishment of IJ in each larva of matids) and Photorhabdus (for heterorhabditids) makes B. hortulanus were assessed. Live B. hortulanus larvae them highly virulent (Boemare et al., 1997; Boemare, were placed in Petri dishes to obtain adult flies to con- 2002). Their ability to actively locate their insect hosts, firm their identity. their high reproductive potential, the possibility to Morphological identification and comparative biometric mass produce them, and their harmless impact on ver- study: One larva from B. hortulanus producing nema- tebrates and plants make these organisms highly suit- tode progeny (4,215 IJ) was chosen to ensure that only able for the development of environmentally friendly a single EPN species was isolated (Alatorre-Rosas and alternatives in the control of insect pests (Gaugler and Kaya, 1990; de Doucet et al., 1998). Twenty-five infec- Kaya, 1990; Kaya and Gaugler, 1993; Burnell and Stock, tive juveniles (IJ) were recovered for morphometric 2000; Gaugler, 2002). Because survival and virulence of study and the rest were used for in vivo culture on Gal- entomopathogenic nematode species and strains are leria mellonella L. (Lepidoptera: Pyralidae) and Spodop- influenced by environmental conditions, a survey was tera littoralis Boisdv. (Lepidoptera: Noctuidae) (Wood- conducted to identify EPN strains in La Rioja, Spain. A ring and Kaya, 1988). Infective juveniles were fixed and new steinernematid strain of Steinernema feltiae (Filipjev) processed in glycerine following the method described was isolated from a natural host. We present results of by Steinhorst (1966). Semi-permanent slides were pre- the ecological characterization of this new strain and pared following the procedures described by Gómez et discuss its morphological and molecular characteriza- al. (2004). Three insect hosts, B. hortulanus, S. littoralis tion. and G. mellonella, were considered. Twenty-five IJ from each host and twenty-five first generation males (FGM) Material and Methods of each host, S. littoralis and G. mellonella, were consid- ered for morphometric comparisons. Morphological EPN isolation and host larvae mortality study: A survey observations and measurements were performed using was conducted in spring 2001 in La Rioja (Northern a Leica DML microscope equipped with DIC optics. Spain), where 149 last instar larvae of Bibio hortulanus L. Identification was carried out following the keys of Ad- ams and Nguyen (2002) and Hominick et al. (1997). Received for publication May 17, 2005. The new strain was compared with other S. feltiae Dpto. Agroecologia Centro de Ciencias Medioambientales, CSIC. C/Serrano 115 dpdo. Madrid, 28006 Spain. strains: Spanish strains M47 and M65 (isolated from two This research was supported by Unión de Agricultores y Ganaderos de La sites with similar environmental conditions) (García Rioja-Coordinadora de Agricultores y Ganadero (Grant: 2001/2001250), Insti- tuto Nacional de Investigación y Tecnología Agraria y Alimentaria (Grant: del Pino, 1994), 76 UK isolates (Yoshida, 2003) and one RTA03-004-C4-4), and Ministerio de Educación, Cultura y Deportes. It is a isolate from Pakistan (Tabassum and Shahina, 2004). portion of a Ph.D. dissertation by the first author. The authors thank M. Arias, A. Ploeg and A. Bello for their comments on the manuscript, L. Barrios Molecular characterization: PCR-RFLP and sequence for their statistical suggestions, J. Ochoa for allowing us to use his field and analysis of the ITS region of rDNA were performed to J. Jiménez and S. Labrador for their technical support. E-mail: [email protected]. further confirm the identity of the isolate. DNA was This paper was edited by S. Patricia Stock. extracted from a single female following procedures 68 performed in 25-wellassay where plates each IJ (4 wasjusted picked cm individually. by Assays were volumetricThe dilutions, concentration except requiredjuveniles in in were one-on-one each stored at bioassay 10 was ad- performed using last instar posure time, one-on-one andproposed sand by column Glazer assays and were strain Lewis was (2000). assessed Penetration, ex- using a method modi nema weiseri AF192981 ( A1 RFLP Type), AY23078 ( Petri dishes with moist moved. Insects wereAfter rinsed 1, and 2Lewis, incubated and 2000) in 3 followed 5-cm of hr, by ultra-pure eight water a was larvae placed single in were each well randomly (GlazerLewis, re- and 2000). initial IJ that hadetration invaded rate the was insectmales calculated host and (Glazer as females in and the eachunder larva percentage a was of counted. stereoscopic The the incubated microscope pen- for and a further the twovidually days. number in Insects 5-cm of were Petri dissected dishesremove with nematodes moist from theirrecorded surface daily. and Dead placed larvae indi- were250 rinsed µl in tap of waterG. to ultra-pure mellonella water/well.was Larval placed mortality in was each well followed by aperiod. single last instar relative humidity, with threewere repetitions carried over out aand 10 in control) day to the ensure similarfi dark conditions. at All bioassays 22sieve) ± dampened 1 with 150g/well) µl of of sterile ultra-pure sandHounslow, water. Middlesex, (passed UK) The through containing a a thin 2-mm layer mesh (0.8 AY230169 ( considered for comparisons.isolates Accession and numbers ason are: et closely al., related 1994). SequencesITS2 species, from sequences four were generated using(Altschul Clustal W et (Thomp- al.,was 1997). compared Alignments andinto of the analyzed pGEM-T the using vector ITS1-5.8S- andBeverly, BLASTN sequenced. MA). The analysis sequence Theing ITS1-5.8S-ITS2 manufacturer region wasHae III, cloned Hha I, Hincwere performed II, using Hinf the I, enzymes Mnldescribed Alu I I, by and Dra Rsa I, Vrain Eco I,Hominick RI, follow- et et al. al. (1997)fi using (1992). the Restriction primers 18S digests carried and out 26S with 10described µl by of Williams digested et al. females. (1992). PCR PCR ampli- reactions were cation conditions followed procedures described by nal volume was identical in all the treatments (IJ Ecological characterization: Exposure time bioassay: Penetration bioassay: ) (Spiridonov et al., 2004). S. feltiae S. feltiae larva. The control was a 25-well plate with ’ s instructions (New England Biolabs, A2 RFLP type), AY230170 ( SN strain) and AY230167 ( A suspension of 200 IJ in 100 µl A suspension of 400 IJ in 100 µl fi lter paper. The mortality was S. feltiae ° G. mellonella C for 2-3 weeks before use. The virulence of the new MY9, Japanese type), G. mellonella 2 Steinernema feltiae /well) (Sterilin fi larvae. Infective S. weiseri lter paper and fi ° ed from that C and 55% S. feltiae Steiner- , were larva. TABLE 1. Morphometrics of infective juveniles (IJ) and first generation males (FGM) of Steinernema feltiae Rioja strain in G. mellonella, S. littoralis and B. hortulanus. Means, standard deviations and ranges (in parenthesis) are given in µm. Steinernema feltiae IJb FGMc Character/ratioa G. mellonella S. littoralis B. hortulanus ANOVA (df), p G. mellonella S. littoralis T test (df), pd L 783 ± 75.3* (660–914) 645 ± 60.4** (537–754) 874 ± 57.4* (683–895) F = 51.157 (2, 71), p < 0.0001 1220 ± 176.4 (820–1648) 1238 ± 146.7 (970–1583) n.s. W 26 ± 2.9* (20–30) 21 ± 2.3** (17–26) 25 ± 1.3* (22–28) F = 23.386 (2, 70), p < 0.0001 107 ± 20.9 (73–141) 115 ± 20.9 (68–161) n.s. ES 118 ± 8.3* (102–138) 105 ± 11.4** (88–129) 116 ± 9.2* (92–127) F = 14.787 (2, 70), p < 0.0001 138 ± 12.4 (100–152) 141 ± 14.1 (100–168) n.s. NR 93 ± 6.5* (80–105) 76 ± 8.8** (61–99) 90 ± 9.4* (71–101) F = 27.211 (2, 70), p < 0.0001 EP 64 ± 10.6* (50–89) 51 ± 8.5** (41–76) 54 ± 2.4* (50–59) F = 16.786 (2, 69), p < 0.0001 86 ± 9.9 (61–97) 89 ± 13.0 (68–114) n.s. T 72 ± 5.5* (61–80) 64 ± 6.1** (55–76) 71 ± 3.6* (65–79) F = 14.202 (2, 68), p < 0.0001 28 ± 3.5 (21–33) 24 ± 3.7 (15–29) n.s.
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