Molecular characterization

DNA was extracted from single females. Each female was transferred into a sterile Eppendorf tube (500 μl) with 20 μl of extraction buffer (17.7 μl of ddH₂O, 2 μl of 10 × PCR buffer, 0.2 μl of 1% tween, and 0.1 μl of proteinase K). Buffer and nematode were frozen at −20°C for 20 min and then immediately incubated at 65°C for 1 h, followed by 10 min at 95°C. The lysates were cooled on ice, then centrifuged (2 min, 9,000 g) and 2 μl of supernatant was used for PCR (Mráček et al., 2014).

A fragment of rDNA containing the ITS regions (ITS1, 5.8S, ITS2) was amplified using primers 18S: 5′-TTGATTACGTCCCTGCCCTTT-3′ (forward), and 28S: 5′-TTTCACTCGCCGTTACTAAGG-3′ (reverse) (Vrain et al., 1992). The other fragment containing D2-D3 expansion segments of the 28S rDNA was amplified using primers D2F: 5′-CCTTAGTAACGGCGAGTGAAA-3′ (forward) and 536: 5′-CAGCTATCCTGAGGAAAC-3′ (reverse) (Nguyen, 2007).

All PCR products were sequenced and for the ITS region, five sequence chromatograms were checked for the presence of intraindividual variability (Půža et al., 2015). The sequences were deposited in GenBank under accession numbers {"type":"entrez-nucleotide","attrs":{"text":"KT373857","term_id":"922576516","term_text":"KT373857"}}KT373857 (ITS sequence) and {"type":"entrez-nucleotide","attrs":{"text":"KT580950","term_id":"955472478","term_text":"KT580950"}}KT580950 (28S sequence). The sequences were edited and compared with those present in GenBank by means of a Basic Local Alignment Search Tool (BLAST) of the National Centre for Biotechnology Information. An alignment of our samples together with sequences of related steinernematid species were produced for ITS and 28S regions using default ClustalW parameters in MEGA 6.0 (Tamura et al., 2013) and optimized manually in BioEdit (Hall, 1999). Pairwise distances were computed using MEGA 6.0 (Tamura et al., 2013).

The phylogenetic trees of the ITS and 28S genes were obtained by the minimum evolution method (Rzhetsky and Nei, 1992) in MEGA 6.0 (Tamura et al., 2013). Steinernema nepalense and Steinernema scapterisci were used as outgroup. The minimum evolution tree was searched using the close-neighbor-interchange (CNI) algorithm (Nei and Kumar, 2000). The neighbor-joining algorithm (Saitou and Nei, 1987) was used to generate the initial tree. The evolutionary distances were computed using the p-distance method (Nei and Kumar, 2000) and are expressed as the number of base differences per site.