2.1 Nematode isolate and symbiotic bacterium culture
The S. feltiae isolate Lican Ray (LR) was recovered from soil obtained from an oak forest near the city Lican Ray (39º 28´12´´S; 72º 7´12´´W) by baiting the soil with late instar waxmoth larvae, Galleria mellonella, in glass flasks with volumes of 500 cm3 (13). The flasks were closed and kept at 20 °C for 96 h, after which dead larvae were moved to modified White traps (14), and emerging infective juveniles (IJ) were removed in a drop of water and used to infect fresh waxmoth larvae to increase the population. Emergent IJ were then stored in tap water at 10 °C.
To retrieve symbiotic bacteria, a pool of IJ was surface-sterilized in 2% NaClO for 3 min, washed thoroughly with sterile water and crushed to release the bacteria. One aliquot of the homogenate was streaked onto plates with nutrient agar supplemented with 0.004% (w/v) triphenyltetrazolium chloride and 0.025% (w/v) bromothymol blue, pH 7 (NBTA plates) (15, 16). On this solid culture medium, Xenorhabdus colonies exhibit a typical green blue color, which allows them to be distinguished from potential contaminants (17). After 48 h of incubation at 28 °C, colonies corresponding to the symbiotic phenotype were isolated and preserved at -80 °C in nutrient broth supplemented with 20% glycerol.
2.2 Morphological and morphometric studies
First and second adult generations and IJ were collected at random from infected insect larvae (14). Males and females were collected on the fourth and eighth day after inoculation of G. mellonella for the first and second generations, respectively, while IJ were collected within two days after emergence. For descriptive purposes, 25 specimens for each stage were fixed in TAF and processed to glycerin by Seinhorst´s rapid method (18). Morphological and morphometric parameters suggested by Hominick et al. (19) were analyzed using an Axiocam MRC in a Zeiss Axioimager A.1 light microscope.
For scanning electron microscopy, adults were obtained from dead G. mellonella larvae and washed three times in buffer M9, the same as the IJ recovered from White traps. All nematodes were relaxed in 60 °C water, fixed in 8% glutaraldehyde and mounted according to the methodology of Koppenhöfer and Stock (20). Scanning was performed using a Philips XL microscope with an SES DS-130 at 20 kV accelerating voltage.
2.3 Molecular characterization
2.3.1 Nematode isolate
DNA was extracted from single females using the extraction method of Williams et al. (21). Single nematodes were collected in PCR tubes with WLB buffer containing 10 mg/ml proteinase K and frozen for at least 10 min at –80° C. The tubes were quickly placed in a water bath at 65° C and then incubated at this temperature for 90 min to allow digestion by proteinase K. Finally, proteinase K was inactivated by heating to 95° C for 15 min, and the tubes were centrifuged to separate the supernatant.
PCR was performed to amplify the large ribosomal subunit (LSU) 28S rDNA using forward primer no. 391 (5´-AGCGGAGGAAAAGAAACTAA-3´) (22) and D3B (5´-TCGGAAGGAACCAGCTACTA-3´) reverse primer (23). One fragment of rDNA that includes the internal transcribed spacer ITS-1, the 5.8S subunit and ITS-2 was PCR amplified using the primer pair 93 (5´-TTGAACCGGGTAAAAGTCG-3´) and 94 (5´-TTAGTTTCTTTTCCTCCGCT-3´) (24). In both PCRs, a volume of 2 µl DNA was used as template in a 50 µl reaction mix that contained 0.5 µM of each primer, 200 µM dNTP and 1 unit of Taq DNA Polymerase Recombinant (Invitrogen) along with 1.5 mM MgCl2 final concentration. Amplifications were performed in a BIOER-LifePro Thermal Cycler. To amplify the LSU fragment, the PCR mix was denatured at 94° C for 3 min, followed by 33 cycles of 94° C for 30 sec, 52° C for 30 sec and 72° C for 1 min, and a final extension of 7 min at 72° C. A similar PCR program was used for the ITS region, adjusting the annealing temperature to 60° C. The amplified fragments were separated by electrophoresis on 1% agarose (w/v) gels using 1X TBE buffer at 100 V for 1 h, then purified using an E.Z.N.A. Gel Extraction Kit (OMEGA Bio-tek). PCR products were sequenced (Macrogen, USA) using internal primers. Forward 502 (5´-CAAGTACCGTGAGGGAAAGTTGC-3´) and reverse 503 (5´-CCTTGGTCCGTGTTTCAAGACG-3´) primers were used for 28S; forward 533 (5´-CAAGTCTTATCGGTGGATCAC-3´) and reverse 534 (5´-GCAATTCACGCCAAATAACGG-3´) were used for ITS fragment (25).
2.3.2 Symbiotic bacteria
DNA was extracted from 1.5 ml of overnight Miller’s LB Broth (10 g/L Tryptone, 10 g/L NaCl, 5 g/L Yeast Extract) culture, using a GenElute Bacterial Genomic DNA kit (Sigma, Sigma-Aldrich). Universal primers that amplify nearly the full-length 16S rDNA from many bacterial genera were used: 27f (5’-AGAGTTTGATCATGGCTCAG-3’) and 1492r (5’-TACGGTTACCTTGTTACGACTT-3’) (26). The reaction was carried out in a final volume of 30 µl containing 1 µl DNA, 1 µM of each primer, 200 µM dNTP and 1 unit Taq DNA Polymerase Recombinant (Invitrogen). PCR parameters consisted of an initial denaturation at 94° C for 3 min, 35 cycles of 94° C for 50 s, annealing at 58° C for 50 s and 72° C extension for 50 s followed by a final extension at 72° C for 7 min. PCR products were visualized, purified and sequenced as previously mentioned.
2.3.3 Phylogenetic analysis
The DNA sequences of the isolate Lican Ray were compared with those present in GenBank by means of the basic local alignment search tool (BLAST) of the National Center for Biotechnology Information (NCBI). The 28S and ITS sequences and corresponding reference nucleotide sequences of Steinernema spp., including “feltiae group”, available in GenBank were aligned with the default parameters of Clustal W (27). The alignments were manually edited using BioEdit (28). The 16S rRNA sequence of the symbiotic bacteria was aligned to corresponding sequences of Xenorhabdus spp.
Phylogenetic relationships were determined by using maximum likelihood (ML) based on the Tamura-Nei model (29) via the program Molecular Evolutionary Genetics Analysis Version 6.0 (MEGA 6) (30). Trees were constructed by clustering of associated taxa based on 1,000 replicates in a bootstrap test. The newly obtained sequences were submitted to the NCBI GenBank database under accession numbers indicated in bold on the phylogenetic trees. The dataset was also analyzed using Bayesian inference (BI) with MrBayes 3.1.2 (31). The best fitted model of DNA evolution was obtained using jModelTest 0.1.1 (32) with the Akaike information criterion. The GTR+G model (ITS and 28S) and GTR+G+I models (16S) were selected. Two independent runs were performed simultaneously on the data, each one using one cold and three heated chains. After 5 million generations, the average standard deviation of split frequencies between the two independent runs at completion were 0.005 (ITS and 16S) and 0.006 (28S). After discarding 25% of burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) are given on appropriate nodes. Trees were visualized using TreeView (33). The newly obtained sequences were submitted to the NCBI GenBank database under the accession numbers indicated in bold on the phylogenetic trees.
2.4 Determination of some optimal parameters
The effects of temperature, lethal concentration and different water levels in the substrate on the mortality of G. mellonella larvae, penetration rate and reproduction of the nematode were determined. These data were useful to determine the main characteristics of the life cycle under optimal conditions.
2.4.1 Temperature
The optimal temperature for insect mortality (days after inoculation, DAI), penetration rate, time to emerge from the insect cadaver (emergence days) and offspring production was determined. One hundred IJ were applied in 0.2 mL of water per G. mellonella larvae in Petri dishes (3.5 cm diam) with a filter paper on the bottom. They were covered with a plastic bag to maintain humidity and stored in an incubator at different temperatures (5, 10, 15, 20, 25 and 30 ºC). Each treatment had five replicates, each consisting of a group of four plates, distributed in a random design. Insects were checked for five days to determine mortality. The level of IJ invaders was measured by dissecting cadaver one day after death and counting the number of specimens inside. Offspring were determined by counting the total number of emerged IJ from dead larvae in a modified White trap. Nematodes were recovered and stored at 10 ºC in Falcon tubes every day until no more were detected.
2.4.2 Lethal concentration
The effect of the different inoculum densities was determined for the same parameters as assessed previously as well as the penetration efficacy (%); this effect was evaluated by applying 0.2 mL of water containing 0, 10, 20, 40, 80 and 240 IJ per Petri dish containing one G. mellonella larvae, thus employing the same conditions used previously. Each treatment had five replicates, with each replicate consisting of a group of four plates, and the replicates were distributed in a random design. Plates were stored as indicated above at 20 °C, the optimal temperature determined by the previous assay. Penetration efficacy (%) was calculated according to the formula of Kaya and Stock (14), which relates number of nematodes recovered from dissection to those inoculated. Recovered nematodes were maintained as indicated above.
2.4.3 Water content
Substrates containing three water levels were assessed to determine IJ movement at the permanent wilting point, field capacity and saturation. The substrate consisted of a steamed mixture of uniform proportions of sand, agricultural soil and organic matter, with a content of clay, silt and sand of 13.2, 22.9 and 63.9%, respectively. The texture was sandy loam. Petri dishes (3.5 cm diam, with a filter paper) containing 4.5 g of the different substrates were inoculated with 120 IJ/0.2 mL of water. This nematode density was selected according to the previous assay. After 30 min, one G. mellonella larva was placed in each petri dish, kept in the conditions indicated previously and incubated at 20 °C. Each treatment had five replicates, each consisting of a group of four plates, distributed in a random design. The same parameters determined before were used to evaluate water content.
The three experiments were repeated twice under the same conditions.
2.5 Data analysis
For all the assays, mortality was corrected according to Abbot´s formula (34). The control percentage data was arcsine transformed (angular transformation), and normality and variance homogeneity were verified prior to performing an ANOVA using the program Minitab V 15. The other variables were analyzed with no transformations. A Tukey test was performed in case the ANOVA showed significant differences at p<0.05.
To determine lethal concentration (LC), mortality at 48 h was considered. Data were analyzed using a probit test using the Probit Program V 1.5, calculating the CL50 and CL90.