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27Russian Journal of Nematology, 2017, 25 (1), 71 - 76
Molecular characterisation of the new strain of Steinernema vulcanicum Clausi, Longo, Rappazzo, Tarasco & Vinciguerra, 2011
Mirella Clausi1, Diego Leone1, Salvatore Antonio Raccuia2, Sergei E. Spiridonov3 and
Maria Teresa Vinciguerra1
'Department of Biological, Geological and Environmental Sciences, University of Catania, Via Androne, 81, Catania, CT 95124, Italy; 2Istitute for Agricultural and Forestry System in Mediterranean, National Research Council, Via Empedocle, 58, Catania CT 95128, Italy; 3Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences,
Leninskii Prospect, 33, 119071, Moscow, Russia e-mail: [email protected]
Accepted for publication 11 August 2017
Summary. The sequences of ITS rDNA and D2-D3 expansion segment of LSU rDNA were obtained for the new strain of Steinernema vulcanicum Clausi, Longo, Rappazzo, Tarasco & Vinciguerra, 2011 isolated near Salto-del-Cane on the Etna slope, Italy. The phylogenetic analysis of these two loci revealed contradicting topologies: according to the analysis of ITS rDNA, S. vulcanicum was very close to S. arenarium, whereas in the D2-D3 LSU rDNA analysis S. apuliae was the closest. The relationships with the other species of the 'glaseri '-group are discussed. Key words: EPNs, 'glaseri''-group, 1ST rDNA, Sicily, steinernematids.
The entomopathogenic nematodes (EPN) have been an object of intensive studies owing mainly to their potential as agents of biological control of insect pests (Shapiro-Ilan et al., 2012). In addition to their applied potential, EPN are inviting objects for evolutionary studies (Hunt & Subbotin, 2016). An accumulation of sequence data on EPN makes these nematodes a promising model for studies in phylogeny, phylogeography and evolutionary aspects of the origin of parasitism (Spiridonov & Subbotin, 2016). The genus Steinernema Travassos, 1927 consists of more than 90 described species, whilst some of the genotypes deposited in NCBI GenBank probably represent undescribed species (Hunt & Subbotin, 2016). The rich nucleotide data revealed the subdivision of this genus into several main evolutionary lines: superclades and clades (Spiridonov & Subbotin, 2016). The representatives of different clades differ in the distribution patterns. Some species are quite widespread or even cosmopolitan, like Steinernema feltiae (Filipjev, 1934). The representatives of other clades are less frequent, as they are probably adapted to specific ecological niches. The so-called 'glaseri'-clade is an example of such an EPN group. Though reported
from different corners of the globe, these nematodes are never dominant in fauna of EPN. The nematodes of this group are characterised by the largest size of infective juveniles, the only stage living in soil. One
species of this group, S. vulcanicum Clausi, Longo,
Rappazzo, Tarasco & Vinciguerra, 2011, was described from the mount Etna area of Italy (Clausi et al., 2011). Although the sequence data for three loci were presented in the original description, the absence of the data for D2-D3 expansion segment of 28S rDNA and the partial sequencing in the ITS rDNA region (with only ITS1 rDNA studied) limited the possibilities of its comparison with other Steinernema species. The finding of the new strain of S. vulcanicum during an EPN survey of the Etna slopes in 2015 has enabled the sequencing of these loci and the comparison with the related species to be based on more representative data.
MATERIAL AND METHODS
The soil sample no. 41 was collected on 3 June 2015 at Salto del Cane, Italy by Dr Mirella Clausi. The EPN were obtained from soil with Galleria-baiting and then kept in the laboratory as a suspension of infective juveniles. The DNA was
extracted from single juveniles according to Holterman et al. (2006). The worm-lysis solution was prepared immediately before DNA extraction containing 950 ^l of a mixture of 2 ml of 1M NaCl, 2 ml of 1M Tris-HCl (pH 8) plus 5.5 ml of deionized water plus 10 ^l of mercaptoethanol and 40 ^l of proteinase K (20 mg ml1). Nematodes were transferred individually to 25 ^l of sterile water in a tube and after addition of 25 ^l of worm-lysis solution, the tubes were incubated at 65°C for 90 min. The tubes with homogenate were then incubated at 99°C for 5 min to deactivate proteinase K and 0.8-1.2 ^l of homogenate was used as PCR template. PCR reactions were performed using Encyclo Plus PCR kit (Evrogen®, Moscow, Russia) according to the manufacturer's manual. Two pairs of primers were used for amplification of ITS rDNA region: those proposed by Curran & Driver (1994): TW81 (GTT TCC GTA GGT GAA CCT GC) -AB28 (ATA TGC TTA AGT TCA GCG GGT) and the primers of Vrain et al. (1992): Vrain_F (TTG ATT ACG TCC CTG CCC TTT) and Vrain_R (TTT CAC TCG CCG TTA CTA AGG). The latter pair was more effective as they ensured the sequencing of the flanking parts of ITS rDNA region. PCR cycling parameters included primary denaturation at 94°C for 3 min followed by 34 cycles 94°C for 30 s, 55°C for 30 s and 72°C for 1 min, followed by post-amplification extension at 72°C for 5 min. Primer pair D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3') and D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') was used to amplify D2-D3 expansion segment of LSU rDNA fragment (Nunn, 1992). PCR cycling parameters included primary denaturation at 94°C for 3 min followed by 34 cycles 94°C for 30 s, 57°C for 30 s and 72°C for 1 min, followed by post-amplification extension at 72°C for 5 min. PCR reaction products were visualised in agarose gel and bands were excised for DNA extraction with Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA). Samples were directly sequenced using the same primers as used for primary PCR reactions. Obtained sequences were deposited in NCBI GenBank as MF491477 for ITS rDNA sequence and MF491515 for D2-D3 LSU rDNA sequence. The similar sequences were searched for in NCBI GenBank using BLAST algorithm (Altschul et al., 1990). Sequence alignments were generated using Clustal_X (Thompson et al., 1997) under default values for gap opening and gap extension penalties and analysed using PAUP* 4.0b10 (Swofford, 1998); MEGA 7.0.14 (Kumar et al., 2016) was used to infer phylogeny and estimate the nucleotide differences with three methods of analysis (Maximum Parsimony - MP; Neighbour Joining - NJ; and Maximum Likelihood - ML).
RESULTS
The direct sequencing of ITS rDNA region with primers of Vrain et al. (1992) resulted in the accurate reading of the entire locus. The obtained ITS rDNA sequence of the new strain no. 41 of S. vulcanicum was compared with ITS 1 rDNA sequence of the type strain (deposited as GU929442). The complete identity of comparable 380 bp long DNA fragments was considered as an indication of the conspecificity of the type isolate and strain no 41. The deposited 902 bp long ITS rDNA sequence of strain no. 41 of S. vulcanicum was used in BLAST search. The sequences for all the species of the 'glaseri'-group together with few sequences of other clades (as an outgroup) of the genus Steinernema were used for the phylogenetic analysis. As the starting and finishing positions for these species did not coincide, the flanking parts of some sequences were cut to obtain the regular (rectangular) 832 bp long data matrix (an alignment with 331 parsimony-informative characters). The MP, NJ and ML analyses of this alignment provided the phylograms with the similar topology. The MP tree (Fig. 1) demonstrated that the sequence of S. vulcanicum clustered with different isolates of S. arenarium and even formed a weakly supported clade with the isolate nominally belonging to the same species from Gaza Strip. The monophyletic status of the clade represented by S. arenarium + S. vulcanicum was always strongly supported. The inner subdivision of this clade only revealed the existence of two subclades: those of East European and South European-Mediterranean haplotypes (Fig. 1). The pairwise comparison of sequences of this 'arenarium' clade with S. vulcanicum and some species of 'glaseri'-group demonstrated that the sequence of the nematode from Gaza Strip differed in 32 bp (Table 1) from S. vulcanicum. The intraspecific nucleotide differences in S. arenarium were only slightly lower reaching the level of 29-30 bp between the S. arenarium from Gaza Strip and East European isolates.
The deposited 599 bp long sequence of D2-D3 expansion segment of LSU rDNA enabled the BLAST-search for related sequences in NCBI GenBank. As well as in the first analysis of LSU rDNA data for this species, a wider set of related species was involved in the analysis. The obtained 493 bp long alignment contained 111 parsimony-informative characters. The sequence of S. vulcanicum strain no. 41 was always clustering with that for the type isolate of S. apuliae (Fig. 2). The pair of these sequences was always strongly supported in all three methods of analysis forming a
S. loci GQ497740
100
90
93
100
S. phyllophagae FJ410327
100
80
93
i-S. cubanum AY230166 ~~h- Steinernema sp. GUI 74002 Steinernema sp. GUI74004 £g/aser/EU048543 glaseri GUI73998 S. glaseri GU395635 ii— S. glaseri KM016416 "L S. glaseri KMO16411
-Steinernema boemarei F J152414
I-S. apuliae HQ416968
Steinernema sp. '213' AY171285
100
82
100
H
-steinernema sp. zu ay I
S. arenarium AY230160 (Russia)
S. arenarium KU 194613 (Ukraine) S. arenarium KU194612 (Russia) S. arenarium KY818704 (Poland) S. arenarium KU 194608 (Slovenia) S. arenariumKU 194611 (Morocco) S. arenarium KU194607 (Slovenia) S. arenarium HM160094 (Bulgaria) S. arenarium HM160095 (Bulgaria) Steinernema vulcanicum strain #41
S. arenarium KC633190 (Gaza Strip) S. australe FJ235125 S. diaprepesi GUI73996
T- Rt
~~diaprepesi GUI73994 S. lamjungense HM000101 S. longicaudum AY230177 guangdongense AY170341 Steinernema sp. KX405171 Steinernema sp. GU395622
100
4 Steinernema sp. JF834533
S. hermaphroditum JQ687355 -S.jeffreyense KC897093
{
100
S. khoisanae EU727170 S. khoisanae DQ314287 Steinernema sp. JN651413 Steinernema sp. JN6 51412
10
Fig.1. Phylogenetic relationships of Steinernema vulcanicum as inferred from an analysis of complete sequences of ITS rDNA. Maximum parsimony analysis: of 832 total characters 195 - parsimony-informative, gaps as "missing". Bootstrap values are indicated on branches.
S. nyetense JX985264 S. cameroonense JX985265
71
100
84
58
S. lamjungense HM000102
S. apuliae GU569044 • S. apuliae KU194621 " Steinernema vulcanicum strain #41
S. arenarium KU194617
rS. arenarium AF331892 S. arenarium HM160092 S. arenarium HM160093 S. arenarium KU194619 S. arenarium KU 194620 S. arenarium KU194618 Steinernema sp. EU177771 S. boemarei GU569046
— S. phyllophagae FJ666054
4 S. cubanum AF331889
S. longicaudum AF331894 S. glaseri KU 180692 S. glaseri GU177832 . S. glaseri JQ362414 » S. glaseri GU177831 •Steinernema sp. GU177837
H
98
I Steinernema sp. GUI77836 1 Steinernema sp. GU1778^ Steinernema sp. GUI 7783$ S. diaprepesi GU569048
S. puertoricense AF331903
ùteiner
■|_T s. die ■HZ"
Steinernema sp. FJ235126 S. brazilense FJ410326
100
100
71
Steinernema sp. GU395636 Steinernema sp. JQ687357 Steinernema sp. KT358813 S. fc/77/AF331902. Steinernema sp. JN651411 Steinernema sp. JN651410 S. hermaphroditum AY598358 —I S. scarabaeiPH172023
1 S. hermaphroditum JQ687356 Steinernema sp. GU395652 • Steinernema sp. GU395642 S. longicaudum GU569054 S. longicaudum GU395645 Steinernema sp. AF331901 S. tophus KJ701240 S. ieffreyense KP164866
1
S. khoisanae GU569052 S. khoisanae DQ314289 p S. aciari GU395637 I p Steinernema sp. GU395643 |rS. jowgz GU569057 1 Steinernema sp. KX405173 S. schliemanni HM77811J
S. everestense HM000104 Steinernema sp. GU395638 S. akhursti KF289902 S.weiseri FJ165549 Steinernema sp. GU395650 S. xueshanense FJ666053 Steinernema sp. GU395649 • S. unicornum GUI91462 » S. neocurtillae FJ263674
Fig.2. Phylogenetic relationships of Steinernema vulcanicum as inferred from an analysis of D2-D3 expansion segment of LSU rDNA. Maximum parsimony analysis: of 493 total characters 111 - parsimony-informative, gaps as "missing". Bootstrap values are indicated on branches.
52,77,62
100,100,99
► O 1 77 AY170341
100,87,85
St. ////I IMOOC) 1 O 1
- fcfoDQ3 14287 ^ tophus KJ701241
innovation! KJ578793 — WzzAY230173
100,100,100 —— AS*.
S. a c /
98,100,11
83,99,81
¿S*. / yVF7 1 2202 1
iS. FJ410325
aS. FJ235 125
AY171288
-1100,100,83
100,100,100 AY230166
j^hyZZoj^hcxs^*^ FJ410327
95 99,90
77,86,61 100,85,86
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aS*. Z^o^maT-^i FJ 1 524 1 4 aS*. AY230160
//7 f Z^J'T 4L* //1C/ V^ // //V' //
strain #41
aS. JX985267
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Fig.3. Phylogenetic relationships of Steinernema vulcanicum as inferred from an analysis of concatenated ITS rDNA and D2-D3 LSU rDNA. The bootstrap values are indicated near the nodes in the format: MP, NJ, ML. The number of pseudoreplicates: 1000 for MP and NJ, and 500 for ML. Model for ML analysis Hasegawa-Kishino-Yano with gamma distribution (HKY+G). Scale - the number of substitutions per site in NJ analysis.
wider group with S. arenarium and other 'glaseri'-group sequences. The pairwise sequence analysis revealed a 1 bp difference with S. apuliae and 5-9 bp differences with S. arenarium and S. boemarei isolates (Table 2). In attempt to resolve inner phylogenetic nodes of the 'glaseri'--group of Steinernema, the so-called 'concatenated' sequences were combined and analysed (the data for ITS and D2-D3 LSU rDNA were united into a single sequence). In the obtained 1399 bp long alignment, 443 characters were found to be parsimony-informative. As the possibility to combine such concatenated sequences was only open for some representatives of 'glaseri'--group, fewer species were included into analysis (Fig. 3). The resulting phylogram has demonstrated the presence of the strongly supported clade of four species, i.e., S.
apuliae + S. arenarium + S. boemarei + S. vulcanicum.
DISCUSSION
The position of S. vulcanicum in the 'glaseri'-group of the genus Steinernema was different in the analyses of ITS rDNA and D2-D3 LSU rDNA. In the former, this species has found its place within the S. arenarium clade while in the latter it formed the clade with S. apuliae. The ITS rDNA data are more informative bringing more characters for comparison of these closely related species than D2-D3 LSU rDNA data. Our knowledge of intra-specific nucleotide differences in steinernematids is still not sufficient. When in some steinernematid clades the isolates can differ in 2.5% of the nucleotides of ITS rDNA, other clades demonstrate
much higher level of stability of ITS rDNA sequences in the same species (Spiridonov et al., 2004). Yet another urgent problem is the primary morphological identification of a steinernematid sample. It is expected that the sequence deposited in NCBI GenBank under a binomial name had been described based on the traditional methods of identification. However, the morphology of steinernematids is quite variable, the taxonomic value of separate morphological characters is questionable, and the species concept itself is underdeveloped for these nematodes. Having in mind these circumstances and contradicting indications from ITS and LSU rDNA data, we consider the species independence of S. vulcanicum to be the optimal at the moment.
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Clausi, M., Leone, D., Raccuia, S.A., Spiridonov, S.E., M. T. Vinciguerra. Молекулярная характеристика нового изолята Steinernema vulcanicum Clausi, Longo, Rappazzo, Tarasco & Vinciguerra, 2011.
Резюме. Получены последовательности ITS rDNA и D2-D3 сегмента большой субъединицы рибосомы (LSU rDNA) нематод нового изолята (№ 41) Steinernema vulcanicum Clausi, Longo, Rappazzo, Tarasco & Vinciguerra, 2011. Новый изолят был выделен из почвы близ Salto-del-Cane на склонах Этны в Сицилии (Италия). Филогенетический анализ этих двух локусов рибосомальной ДНК дал несколько различающиеся топологии: по последовательности ITS rDNA S. vulcanicum ближе всего к S. arenarium, тогда как по D2-D3 LSU rDNA этот вид ближе всего к S. apuliae. Обсуждаются взаимоотношения штейнернематид группы 'glaseri'.
