Chapter - 4 Molecular Characterization of EPN

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1 Chapter - 4 Molecular Characterization of EPN

2 4. Molecular Characterization of EPN 4.1. Introduction EPN are a group of unique nematodes capable of killing insect pests within 24 to 48 hr of infestation. They belong to two families steinernematidae and heterorhabditidae. The species distinction within the two families containing EPN (Heterorhabditidae and Steinernematidae) is not always clear. The history of nomenclature and the designation of phylogenetic relationships are complex and the subject of ongoing debate (Hominick et al., 1997; Adams and Nguyen 2002; Spiridonov et al., 2004; Nadler et al., 2006). It can be very time consuming to individually examine many dozens of samples and it may also require expensive equipment (e.g. scanning electron microscope) and a great deal of expertise and experience with the morphology of EPN species (Hominick et al., 1997; Adams and Nguyen 2002). For these reasons, more objective and less labour intensive molecular identification techniques are now used in parallel with or in lieu of classical morphological identification or cross-breeding experiments (Stock et al., 2001; Floyd et al., 2002; Yoshida 2003a; Spiridonov et al., 2004; Stock et al., 2004). Several molecular approaches have been used to identify, diagnose, delimit species and assess phylogenetic relationships of EPN and other nematodes (Reid and Hominick, 1992; Gardner et al., 1994; Liu et al., 1997; Reid et al., 1997; Nguyen et al., 2001; Stock et al., 2001; Aisyafaznim et al., 2010). Among them, three molecular methods namely, random amplified polymorphic DNA (RAPD), restriction fragment length polymorphisms (RFLP) and DNA sequencing are being extensively applied. DNA sequence analysis has recently been incorporated into nematode systematics. This molecular approach has been demonstrated to provide more information about variation within and among nematode species than the RFLP approach (Powers et al., 1997; Szalanski et al., 2000). In addition, sequence analysis has shown to be an appropriate approach in assessing phylogenetic relationships at different taxonomic levels and as well as for species delimitation (Adams et al., 1998; Blaxter et al., 1998; Szalanski et al., 2000; Stock et al., 2001). Description of nematodes is basically founded on morphological characters, which are not readily applicable to nematode identification primarily because of overlapping

3 morphometrics and similar morphology (Poinar, 1990; Hominick, 2002). In order to overcome the difficulty of morphology-based identifications, DNA sequences of ribosomal RNA (rrna) genes have been used to identify steinernematids (Szalanski et al., 2000; Nguyen et al., 2001; Stock et al., 2001; Spiridonov et al., 2004) Materials and Methods Materials The kit used is given in appendix (I). Buffers and solutions used are listed in appendix (II). Chemicals and indicators used for the study are listed in appendix (III). Sterile nuclease-free and protease free glass wares and plastic wares were used for the preparation and storage of reagents and to carry out experimental procedures. The nematode isolates used for molecular identification in the study are given in Table 4.1. Galleria mellonella larvae also used for culturing of EPN. Methods Nematode isolates EPN isolates collected from four different places viz. Thiruvananthapuram (N), Kasaragod (K), Bangalore (B) and Hyderabad (H) were selected for the study. These nematode isolates were maintained on last instar larvae of the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae) in the CTCRI laboratory for the last ten years. Infective juveniles emerging from the host insect cadavers were collected and stored in water at 15 C and used in all the experiments. Table 4.1. Place of collection of EPN isolates Name of the Isolates N B K HY Place of Collection Thiruvananthapuram, Kerala Bangalore, Karnataka Kasaragod, Kerala Hyderabad, Andra Pradesh

4 DNA Extraction DNA extraction was carried out as per the protocol developed by Pastrick et al. (1995) and modified by Blok et al. (1997) with changes in the phenol/chloroform extraction step. The nematode suspensions were taken in 1.5ml eppendorf tube and surface sterilized with 0.1 ml of 0.1% mercuric chloride. The suspensions were centrifuged at 10,000 g for three min. After centrifugation, the supernatant was discarded and washed the pellet with sterile distilled water. The nematode pellet was dissolved with 100 µl of DNA extraction buffer (100 mm EDTA, 100 mm NaCl, 100 mm Tris, 0.5% SDS, 200 µg proteinase K) and homogenized using a sterile micr opestle with a pinch of sterile glass powder. The tubes were incubated at 55 o C for three hours and after incubation, centrifuged at 10,000 g for 10 min. The supernatant was collected in new eppendorf tube and added 50 µl of buffer saturated phenol and incubated for min at 55 o C with frequently mixing of the samples. Later, 50 µl of chloroform-isoamyl alcohol was added to it and mixed well before centrifuging at 10,000 g for 10 min. The aqueous phase containing DNA was collected and to this 0.6 volume of isopropanol was added and incubated overnight at room temperature for precipitating the DNA. After incubation, this mixture was centrifuged for 10 min at 10,000 g. The supernatant was discarded and the pellet was washed with 70% ethanol. The pellet was dried and dissolved in 50 µl TE buffer (10 mm Tris and 1 mm EDTA; ph 8) and stored at -20 o C. Amplification of Ribosomal DNA and analysis The D2-D3 expansion fragments of the conserved 28S ribosomal DNA (rdna) of EPN were amplified using the specific universal primers D2A FP 5'- ACA AGT ACC GTG AGG GAAAGT TG-3' and D3B RP 5'- TCG GAA GGA ACC AGC TAC TA-3' (De Ley et al., 1999). The PCR was performed in a 25 µl reaction mixture having 2.5 µl of 10x Taq buffer A (Containing 15 mm MgCl 2 ), 2 µl 10mM dntp's (2.5 mm each), 1.5 µl of each primer (20 ng), 4 µl of template DNA, and 1U of Taq DNA polymerase (Bangalore Genei, India) and 13 µl of sterile distilled water. The reaction was carried out in a Eppendorf thermal cycler (Eppendorf AG, Hamburg, Germany) with the thermal cycle programme of 94 C for 2 min (initial denaturation), 30 cycles with 94 C for 1min (denaturation), 55.6 C for 1 min

5 (annealing), 72 C for 1 min 30 sec (extension) and final extension at 72 C for 10 min. The optimum annealing temperature was empirically investigated by gradient PCR starting with a range of annealing temperatures from 50 to 60 C and further PCR amplification was performed at the standardized annealing temperature. Negative control without DNA was also run. The amplified products were resolved on a 1.5% agarose gel containing 0.5 mg ml -1 ethidium bromide. The DNA bands were visualized under UV transilluminator and documented through Gel Doc System (Alpha imager, Alpha Innotech, USA). The size of the amplified product is approximately 600 bp. A 100 bp ladder (Bangalore Genei, India) was used as DNA size marker. In Silico analysis The PCR products were excised from the gel, extracted and purified using QIAquick Gel extraction kit (QIAGEN, Tokyo, Japan).Sequenced the DNA with the amplification primer using the automated sequence facility of Bangalore Genei. Sequences were edited using the BioEdit software (Hall, 1999). The nucleotide sequences were compared with those in the NCBI databases using the Basic Local Alignment Search Tool (BLAST, The sequences obtained for the four nematode isolates were aligned with each other by using Clustal W multiple alignment programme of BioEdit software (Hall, 1999). From the aligned sequences a phylogenetic tree was constructed using the neighbour - joining method (Tajima and Nei, 1984), the data sets were subjected to 100 bootstraps replicates. The tree was constructed using the TREECON software Results Procedures for obtaining PCR amplified products were repeated four times for consistency of results. Amplified products were reamplified to ensure they were reasonably free of foreign products. The PCR amplification with primers D2A FP and D3B RP at an annealing temperature of 55.6 C yielded a fragment of approximately 600 bp for all the four isolates of EPN. No PCR products were obtained in the negative water control. Sequence analysis of the four isolates showed a higher similarity with varying percentage at the nucleotide level with that of the isolate Rhabditis sp.tumian-2007 (Accession No. EU , Liu et al.,

6 The isolates Hy, K, B and N showed 94%, 90%, 90% 91% respectively. Based on the identity similarity matrix data constructed, the isolates B, Hy, K and N showed similarity percentage of 85.7, 89.0, 84.5 and 67.7 respectively with Rhabditis. sp. Tumian The phylogenetic tree was constructed using the neighbour-joining method of the TREECON software with 100 bootstrap replicates indicated that these isolates were closely related to Rhabditis sp.tumian (Accession No. EU ). Among the four isolates N, B and K appear to be highly similar as compared to the isolate Hy. The partial sequences of the D2-D3 expansion segments of the conserved 28S ribosomal DNA were deposited with the NCBI GenBank database under the accession number HM (isolate B), HM (isolate N), HM (isolate Hy) and HM (isolate K). Fig.4.1. PCR Amplification of D2-D3 expansion fragment of the 28S rdna region of four EPN isolates K, B, N and Hy; Lane M: 100 bp DNA Ladder (Bangalore Genei, Bangalore); Lane -ve: Negative control for PCR

7 Fig.4.2. Phylogenetic analysis of four EPN isolates using the nucleotide sequence of D2-D3 expansion fragment of the 28S rdna region

8 Fig.4.3. Multiple sequence alignment of the D2-D3 expansion fragments of the28s rdna region of four EPN isolates

9 4.4. Discussion This work is mainly focussed on the isolation and molecular-based identification of four EPN strains belonging to Rhabditis sp. In this study sequenced the D2-D3 expansion fragments of the conserved 28S ribosomal DNA of four EPN isolates and analyzed the phylogenetic relationships. The 28S rdna of nematodes contain relatively conserved core elements, as well as 12 variable expansion domains (Chilton et al., 2003). Among the variable expansion domains, the D domain is commonly used to resolve phylogenetic relationships at lower taxonomic levels and to develop species-specific primers to separate closely related species due to existence of high levels of genetic divergence rates between different lineages within this domain (Al Banna et al., 1997; Duncan et al., 1999; Subbotin et al., 2000; Al Banna et al., 2004). The D2-D3 expansion domains of the nuclear 28S rdna subunit are sequence region that has been successfully used for diagnosing Pratylenchus species as well as other Phytoparasitic nematodes (Mizuku et al., 1997; Handoo et al., 2001; Inserra et al., 2001). The D2-D3 data give additional insight into the long debated origin of heteroderidae (including cyst-forming) and Meloidogyne (root-knot nematodes) that is pertinent to ongoing model systems for understanding pathways for pathogenesis (Baldwin et al., 2004). In the recent years, comparative analysis of the D2-D3 28S rdna expansion segment sequences has become a popular tool to differentiate cryptic species which are morphologically identical (or with some overlapped morphological variation) but genetically distinct (Subbotin et al., 2005). The D2-D3 28S rdna expansion segment is the most rapidly evolving coding region of the rdna and is flanked by highly conserved sequences and can distinguish taxa at species level. In order to clarify the taxonomic status of entomopathogenic nematode isolates, we characterized the D2-D3 expansion segment of large subunit of nuclear DNA. Sequencing demonstrated a higher similarity of our EPN isolates to Rhabditis sp. Tumian (2007) (Accession No. EU ). Based on the

10 morphologic and morphometric characterisitics, Mohandas et al. (2004) identified this nematode as a new EPN, belonging to genus Rhabditis and sub genus Oscheius. In our study, we extracted the DNA from nematodes using mechanical tissue homogenization and proteinase K digestion. Recently, a new method was proposed based on the chemical lysis of nematodes in sodium hydroxide (Floyd et al., 2002). Because nematodes are digested completely, the method may increase the DNA extraction efficiency, reduce the time for extraction and exclude the most critical step of mechanical homogenization. Direct sequencing of PCR products provides full characterization of amplified target DNA. The comparison of newly obtained sequences from samples with those published or deposited in the GenBank is a most reliable approach for molecular identification. Molecular techniques have made EPN systematics a lot more exciting, and will continue to do so in the future. However, it would be a mistake to replace traditional (morphological) methods with molecular techniques. Instead the combination of both approaches offer a more resourceful perspective for resolving a variety of questions in nematode taxonomy and particularly for EPN Conclusion Based on molecular data, four EPN isolates were identified. The molecular results in the present study indicated that these nematodes belong to the family rhabditidae.