Oscheius myriophila (Nematoda: Rhabditida) isolated in sugar cane soils in Mexico with potential to be used as entomopathogenic nematode

Morphological and morphometric identification was performed using an optical microscope equipped with a micrometric eyepiece. The nematodes were extracted through the dissection of wax worm larvae, and the IJs were collected from the white trap according to the method proposed by Nguyen and Smart (1996).

For identification with molecular techniques, three hundred IJs were frozen in liquid nitrogen and macerated with a plastic pestle. Their DNA was purified with a Puregene^® DNA Purification Kit following the manufacturer’s instructions. An 850 bp fragment corresponding to the D2D3 region of the 23S rDNA gene was PCR amplified with the primers pair D2F (5′CCTTAGTAACGGCGAGTGAAA-3′) and 536 (5′-CAGCTATCCTGAGGGAAAC-3′) (Nguyen et al., 2006). The reaction was conducted in a final volume of 50 µl containing each primer at 0.5 µM, 0.16 mM dNTPs, 1.5 mM MgCl₂, 2 units of Taq DNA polymerase (Thermo Fisher Scientific Inc.) and 1X reaction buffer. The PCR product was verified by electrophoresis in a 1% agarose gel, and the bands were excised and purified using a QIAquick^® Gel Extraction kit (QIAGEN, Germany). The PCR conditions were 94 °C for 2 min initial denaturation; 37 cycles of 30 s of denaturation at 94 °C, 45 s of hybridization at 48 °C and extension at 72 °C for 90 s; with a final extension of 72 °C for 5 min. The PCR product was sent to the Biotechnology Institute of the National Autonomous University of Mexico for sequencing. The D2D3 region was analyzed using the BLAST program of the GenBank database of the National Institutes of Health.

For the extraction of symbiotic bacteria from MC5-2014, two methods were used. In the first method, 5000 freshly emerged IJs were obtained from the white trap, and were placed in Eppendorf tubes. The tubes were centrifuged at 1,000 revolutions per minute (rpm) for 7 min, and the supernatant was discarded. Thereafter, washes were conducted with 5% sodium hypochlorite, and three washes were performed with sterile distilled water (Lee and Stock, 2010). The IJ nematodes were macerated in a mortar and pestle for a period of 30 min and plated in solid Luria-Bertani (LB) and HCT (Bacto Tryptone (Difco) 5; Casamino acids (Difco) 2; pH adjusted to 7.5) media. After sterilization, culture medium consisting of KH₂ PO₄, 3.4 g/L; MgSO₄.7H₂ O, 0.012 g/L; MnSO₄.4H₂ O, 0.003 g/L; ZnSO₄.7H₂ O, 0.0028 g/L; Fe(SO₄)3.7H₂ O, 0.02 g/L; CaCl₂.2H₂ O, 0.147 g/L; and glucose, 3 g/L was used. For the second method of extraction from hemolymph, 10 larvae of G. mellonella at the 5th instar were infected with 10 IJs from a stock solution of MC5-2014 nematodes. After 96 hr, larvae with characteristics corresponding to death caused by EPNs were selected, and the outer parts of the larvae were disinfected with 70% alcohol for 10 min, followed by drying for 2 min inside a laminar flow hood (Woodring and Kaya, 1988).

Subsequently, a 0,3 ml insulin syringe whith a 31 × 6 mm needle were used to extract the hemolymph by inserting the needle between the 6th and 7th inter-segments of the larvae. An aliquot of the hemolymph was plated on solid LB and HCT culture media, and the plates were incubated for 24 hr at 27 °C. All colonies were sampled, and the cross-streak technique was used to obtain single colonies. The bacteria were identified by biochemical methods using Gram staining, motility tests, and assays of protease activity and lecithinase and antibiotic production as suggested by Boemare et al. (1997) and Akhurst (1980). For molecular identification of the isolated bacteria, the cells were incubated in LB liquid medium for 12 h at 30 °C, and DNA extraction was then carried out with the Easy-DNA™ kit following the instructions recommended by the manufacturer. Then, amplification of a partial 16S rRNA region (600 bp) was performed by PCR using the primer 63f (5′-CAG GCC TAA CAC ATG CAA GTC-3′) (Nübel et al., 1996). The PCR conditions were 95 °C for 3 min initial denaturation; 37 cycles of 35 s of denaturation at 95 °C, 42 s of hybridization at 59 °C and extension at 72 °C for 1 min 30 s; with a final extension of 72 °C for 5 min. The PCR products were identified at the Biotechnology Institute of the National Autonomous University of Mexico with the primer 63 f. The sequences were analyzed using the BLAST program of the GenBank database, National Institutes of Health.

Pathogenicity bioassays of bacterial strain MC5-2014 were carried out in 10 larvae of the 5th stage of Galleria mellonella, and the treatments were carried out in triplicate. The bacterial isolates were cultured in solid LB medium for 18 hr at 27 ± 2 °C. The surfaces of the larvae were previously disinfected with 70% ethanol and inoculated by injection using 10 µl of the bacterial suspension at a concentration of 1 × 10⁵ cells. The bacterial concentration was obtained via volumetric quantification using a Neubauer chamber.

Strain Bar 86 of S. marcescens was used as a positive control, while the Escherichia coli ATCC strain and distilled water were used as negative controls. The injected larvae were placed in 60 × 15 mm Petri dishes with a meridian diet (bee honey 97.5 ml, glycerol 120 ml, wheat bran (sterile) 37.5 ml, rice cereal 300 g, yeast 75 g). The pathogenicity and the changes in the coloration of the cuticle were evaluated at 24 and 48 hr. The results were subjected to analysis of variance (ANOVA) and the Tukey test at the 0.05 probability. The SAS statistical package was applied.

The virulence bioassays of the bacteria isolated from the MC5-2014 strain were performed on 30 larvae of G. mellonella in the 5th instar, and the treatment was carried out in triplicate. Bacterial strains were cultured in solid LB medium for 18 hr at 28 ± 2 °C prior to quantification with the help of a Neubauer camera; volumetric calculations were performed and verified, and four concentrations were used to determine virulence for G. mellonella (10⁵, 10³, 50, and 5 cells). The surfaces of the larvae were previously disinfected with 70% ethanol. The same positive and negative controls mentioned above were used. The larvae were placed in 60 × 15 mm Petri dishes with sterile filter paper with a meridian diet for G. mellonella. The percentage of mortality and the change in the coloration of the cuticle were evaluated at 24 and 48 hr. Virulence (LC₅₀) was determined by PROBIT analysis using the POLO PLUS version 1.0 program (LeORa Software LLC).

Virulence was determined using 6, 10, 14, 18, or 20 infected juveniles (IJs) plus the control, which contained only sterile distilled water. Virulence was determined by obtaining the average lethal concentration (LC₅₀) via PROBIT analysis using the POLO PLUS version 1.0 program. (LeOra Software LLC).

Female: Small-sized nematodes. Slender body 760 to 1160 µm long slightly curved ventrally after fixation. Cuticles are finely annulated with fine longitudinal striations. The head is continuous with the body contour, with six separate well-developed lips. Pore-like amphids on the lateral lips. Cylindrical pharyngeal body with cheilorhabdions that are not notable. The median bulb was absent. The metacorpus is swollen and pharyngeal. The isthmus is distinct, cylindrical, and slightly narrow. Nerve rings usually surround the mid portion of the isthmus. Excretory pores are conspicuously located at the level of the basal bulb. The excretion duct is circularized and curved anteriorly, then posteriorly. Vulva near the mid body and lips protruding.

Male: Similar to the female in general morphology except for a smaller size of 720 to 910 µm in length. The body is straight after fixation. Spicules are paired, separate, symmetrical, and slightly curved ventrally with hooked tips. Head of the spicules with a rounded anterior end, lamina expanded in their proximal part and velum prominent. Bursa leptoderan and eight genital papillae (rays) of different lengths are with a 1 + 1 + 3 + 3 arrangement. There were 3 precloacal papillae and 6 postcloacal papillae. Papillae 8 and 7 form 6 groups, and pairs 5, 4, and 3 form the second postcloacal group. Papillae two and one are separated near the base or back of the spicules. The spicules are paired and separate. They are slightly curved and have a triangular head and a rounded tip. The lengths of the spicules are between 30 and 80 µm. The gubernaculum is ventrally flattened, and it follows the contour of the spicules. The proximal tip is curved upward. The length of the gubernaculum is 20 to 30 µm.

Habitat and locality: soil around the roots of sugar cane (Saccharum officinarum) in Morelos, Mexico.

The male, female and IJ specimens were deposited in the Nacional Collection of Helminthes of the Biology Institute of the National Autonomous University of Mexico (CNHE/UNAM) with registration number 11085.

Diagnosis: O. myriophila belongs to the insectivora group and exhibits a cylindrical pharynx, a body with a leptoderan bursa and eight genital papillae of different lengthsarrangedin a 1 + 1 + 3 + 3 formation.

O. sacchari was isolated from sugar cane soil and belongs to the insectivora group. It presents similarity in the morphology of the anterior part that is a characteristic of the genus. O. sacchari is similar to O. myriophila in the body length of males (average: 843 vs 862 µm), but exhibits a different body width (average 61.6 vs 28 µm) and longer spicules (average: 41 vs 50 µm). Posterior part arrangement with the papillae in a 1 + 1 + 1 + 3 + 3. In females, shorter body lengths (average: 1607 vs 964.5 µm) and narrower body widths (average: 37 vs 88 µm) were observed.

O. necromenus is similar to O. myriophila in the body length of males (average: 843 vs 790 µm), but those of females differ (average: 964.6 vs 1179 µm) as does their body width (average 28-46 µm), and they exhibit longer spicules and a longer gubernaculum (average: 41 vs 40; 22 vs 15 µm, respectively). Detailed species comparisons for the genus of Oscheius are given in Table 1.

Based on the morphometric characteristics of males and females that are commonly used for the identification of nematodes, 10 females and 10 males were observed under a microscope (Nikon eclipse e200), and morphometric measurements were performed (Table 1). The maximum and minimum ranges of the body length were obtained: L, body width: W, spicules, distance from the part before the vulva, and length of eggs. The average values were calculated.