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47Russian Journal of Nematology, 2012, 20 (2), 97-112
Steinernema changbaiense sp. n. (Rhabditida:
Steinernematidae), a new species of entomopathogenic nematodes from Northeast
China
1 2 1 2 4 2 3
Juan Ma ' , Shulong Chen , Patrick De Clercq , Richou Han and Maurice Moens '
1 Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Science / IPM Centre of Hebei Province,
Baoding 071000, China
2 Department of Crop Protection, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
3 Institute of Agriculture and Fisheries Research, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium;
e-mail: maurice.moens@ilvo.vlaanderen.be 4 Guangdong Entomological Institute, Guangzhou 510260, China
Accepted for publication 20 May 2012
Summary. During an extensive survey for entomopathogenic nematodes in north China, a new species was recovered from soil samples; it is described herein as Steinernema changbaiense sp. n. This nematode was obtained from soil samples by the insect-baiting technique using last instar larvae of Galleria mellonella (Lepidoptera: Pyralidae). Steinernema changbaiense sp. n. is closely related to species in the ''feltiae-kraussei-oregonense' group. It is characterised by morphometrics of the infective juvenile (IJ) with a body length of 757 (730-790) ^m, distance from head to excretory pore of 61 (58-68) ^m, tail length of 77 (71-85) ^m, E % = 80 (73-88), and a lateral field pattern 2, 5, 6, 4, 2. The male of the first generation can be recognised by a spicule of 63 (56-70) ^m long and a gubernaculum of 40 (33-46) ^m long; the spicule is slender, the velum short. The first generation female has a symmetrical, slightly protruding vulva and a ratio D (%) of 49 (35-55). The new species differs distinctly from related species such as S. ashiunense, S. robustispiculum and S. monticolum by a different number of ridges in the lateral fields and the distance from the head to excretory pore of the IJ, and the body length, morphology of spicules and gubernaculum of the male. Cross hybridisation tests showed that these species are reproductively isolated. The sequences analyses of the internal transcribed spacers (ITS) of rDNA and D2-D3 domain of the LSU regions confirmed the new species identity.
Key words: Cross hybridisation, D2D3 LSU rDNA, entomopathogenic nematodes, 'feltiae-kraussei-oregonense' group, ITS rDNA, morphology.
The widely distributed entomopathogenic nematodes (EPN) have been found in all inhabited continents and a range of ecologically diverse habitats (Hominick, 2002). The families Heterorhabditidae Poinar, 1976 and Steinernematidae Filipjev, 1934, which are characterised by carrying specific symbiotic bacteria of the genus Photorhabdus and Xenorhabdus, respectively, are the most commonly studied families because of their economic importance. They have a wide range of insect hosts (Shapiro-Ilan et al., 2002) and are considered to have great potential in biological control of insect pests (Ehlers, 1996).
The number of species in both families has rapidly increased in recent years. Currently more than 60 steinernematid and 16 heterorhabditid
species have been described (Nguyen, 2011). The biocontrol efficacy of EPN is variable and depends on nematode species and strains (Lewis & Grewal, 2005). Further, environmental conditions may affect survival, reproductive potential and virulence of the EPN strains. Indigenous isolates of EPN may thus be more suitable against local insect pests. To obtain suitable species and strains for the implementation of integrated pest management strategies, we carried out an extensive survey on EPN in North China. During this survey ten EPN species were collected (Ma et al., 2010) along with several undescribed Steinernema species. Two of these latter species were described as S. tielingense and S. xinbinense (Ma et al., 2012a; Ma et al., 2012b). We here describe a third species as S. changbaiense sp. n. named after the Changbai Mountains, from which
foothills the type population was collected. Steinernema changbaiense sp. n. differs in morphology and morphometries from the described EPN species, and molecular studies show that this isolate is closely related to species in the feltiae-kraussei-oregonense' group (Spiridonov et al., 2004).
MATERIALS AND METHODS
Nematode isolation. The nematode isolate was recovered from soil samples collected in the vicinity of Benxi County, Liaoning province, China during a survey in April, 2008. The soil samples (ca 400 cm3) were composed of 4-6 sub-samples randomly taken at least 5 m apart at a depth of 2-20 cm. Sub-samples were placed in polyethylene bags, mixed, transported to the laboratory and stored under cool conditions (15 ± 2°C) until processing. Entomopathogenic nematodes were extracted from soil by the Galleria mellonella L. (Lepidoptera: Pyralidae) baiting method (Bedding & Akhurst, 1975). Infective juveniles (IJ) were collected from the cadavers using White traps (White, 1927) and were multiplied on a new batch of G. mellonella.
Morphological observations. Nematodes were re-cultured in last instar larvae of G. mellonella. Only IJ collected during the week after their first emergence from the insect cadavers were used for identification. First and second generation adults were collected by dissecting the cadavers 5 and 1011 days after nematode infection, respectively. Light microscopy (LM) and scanning electron microscopy (SEM) studies were done as reported by Ma et al. (2012a). Measurements were done and morphology was examined using an Olympus BX51 compound microscope.
Cross hybridisation. The modified hanging-blood assay described by Kaya and Stock (1997) was used for checking reproductive compatibilities of the new species with S. monticolum Stock, Choo & Kaya, 1997, S. tielingense Ma, Chen, Li, Han, Khatri-Chhetri, De Clercq & Moens, 2012, S. feltiae (Filipjev, 1934) Wouts, Mracek, Gerdin & Bedding, 1982 and S. hebeiense Chen, Li, Yan, Spiridonov & Moens, 2006. All of these species were represented by isolates obtained from North China (Ma et al., 2010). No other species were available for cross breeding. Twenty replications were performed for each cross-breeding test combination. Self-breeding controls were done under the same conditions.
Molecular characteristics and phylogeny analysis. Total genomic DNA was extracted from single IJ using the method of Joyce et al. (1994). The forward primer TW81 5'-GTT TCC GTA GGT GAA CCT GC-3' and the reverse primer AB28 5'-
ATA TGC TTA AGT TCA GCG GGT-3' were used for the amplification and sequencing of the rDNA fragment containing the internal transcribed spacer regions ITS1 and ITS2, and the 5.8S rRNA gene. A second rDNA fragment containing the D2-D3 expansion segment of the 28S rRNA gene was amplified using the forward primer D2F 5'-CCT TAG TAA CGG CGA GTG AAA-3' and the reverse primer 536 5'-CAG CTA TCC T GA GGA AAC-3' (Nguyen et al., 2006). The methods reported by Nguyen (2007) were used for PCR amplification. The same primers were used for sequencing procedure. The resulting sequences were edited and analysed using software packages Chromas 2.23 (Technelysium, Tewantin, Queensland, Australia) and BioEdit7.0.4.1 (Hall, 1999) and deposited in GenBank. The obtained sequences were compared with sequences of Steinernema species available in GenBank (NCBI).
The internal transcribed spacer regions (ITS-rDNA) and the partial 28S ribosomal DNA (D2-D3 segment) sequences of the new species and corresponding nucleotide sequences of other representatives of Steinernema available in GenBank were aligned using Clustal X1.83 (Thompson et al, 1997). Phylogenetic analyses (Maximum parsimony and Neighbour-Joining) of sequences data were done using PAUP* v 4.0b10 (Swofford, 2001). Trees were evaluated statistically by bootstrap analysis based on 1,000 resamplings of the dataset. The resulting trees were visualised by using MrEnt 2.0 (Zuccon, 2010). Caenorhabditis elegans (EU131007) was applied as outgroup during calculation of the trees based on ITS sequences; Cervidellus alutus (AF331911), together with Panagrellus redivivus (AF331910), were used as outgroups for calculation of the trees based on D2-D3 sequences.
DESCRIPTION
Steinernema changbaiense sp. n. (Figs 1-6)
Measurements: See Table 1.
First generation male. Body curved ventrally, tapering toward anterior end, mostly C-shaped, rarely J-shaped in heat-relaxed specimens. Cuticle with very faint transverse striae. Head truncated, almost continuous with body contour. Six labial and four cephalic papillae present. Amphids located laterally between labial and cephalic circle of papillae. Stoma short, shallow, cheilorhabdions distinct, sclerotised. Pharynx muscular; procorpus cylindrical, metacorpus slightly swollen. Nerve ring surrounding isthmus at anterior part of valvated basal
Fig. 1. Steinernema changbaiense sp. n. A, B: Infective juvenile. A: Pharyngeal region showing basal bulb and bacterial chamber; B: Tail region. C, D, I, J: First generation male. C: Head and pharyngeal region; D: Tail region; I: Spicule; J: Gubernaculum. E: Second generation male, tail region. F, G: First generation female, tail region. F: old female; G: young female. H: Second generation female tail region. (Scale bars: A, B, D, E, I, J = 10 ^m; F, G, H = 50 ^m; C = 20 ^m.).
Fig. 2. Steinernema changbaiense sp. n. SEM and LM pictures of infective juvenile. A: Lateral field showing the change from two to five ridges in anterior part; B: Lateral field showing five ridges and middle one dividing into two, thus making six in total; C: Lateral field showing six ridges; D: Head region showing amphidial aperture (a) and cephalic papillae (c); E: Tail region showing gradual reduction in number of ridges so as to become two after phasmid level; F: Tail region showing phasmid and lateral field; G: Pharyngeal region showing basal bulb and bacterial vesicle (BV); H: Tail region. (Scale bars: A-C, E-H = 10 ^m; D = 1 ^m.)
bulb. Cardia prominent. Excretory pore always located anterior to nerve ring (ranging from 52 to 66% of the length from anterior body end to base of pharynx). Intestine generally with a wide lumen. Distinct, papilliform deirid situated at the pharyngeal bulb to nerve ring. Well developed and ventrally reflexed testis. Paired symmetrical, moderately to strongly curved and slender spicules, golden/yellowish in colour. Spicule length/width ratio (SL/SPW) = 7.0 (5.2-8.5). Head (manubrium) of spicules, usually elongate (spicule head length/width = 1.2-1.5), slightly offset from shaft, calomus very short. Lamina slender and well curved, with two internal sclerotised ribs, tapering slightly posteriorly. Spicule terminus blunt. Rostrum not well developed, velum short. In lateral view, gubernaculum boat-shaped to almost straight, tapering gradually posteriorly, cuneus V-shaped, corpus with two wings opening posteriorly. Twenty-
three genital papillae comprising 11 pairs and a single mid-ventral papilla located just anterior to cloacal opening. Seven pairs located precloacal (six pairs subventral and one pair lateral), one pair adcloacal and three pairs postcloacal, consistently near tail terminus (one subdorsal, two at tail tip). Tail bluntly conoid, usually concave at ventral side. Short, projection-like mucron mostly present, ca 1.5-3.2 ^m long. Phasmids located just behind the cloacal opening.
Second generation male. Similar to firstgeneration, but smaller in size (Table 1). Spine-like tail mucron much longer than in the first generation, ca 4.4-9.5 ^m long. Spicules and gubernaculum shorter and thinner.
First generation female. Body size variable, usually C-shaped on heat relaxation. Cuticle only faintly striated. Lateral field not observed. Head truncate or slightly round, tapering anteriorly, continuous with
Table 1. Morphometries of Steinernema changbaiense sp. n. All measurements are in ^m and in the in the form: mean ± s.d. (range).
Character First generation Second generation Infective juvenile Paratypes
Male Female Paratypes Male Female
Holotype Paratypes Paratypes Paratypes
n - 20 20 20 20 20
L 1452 1455± 165 4368 ± 439 1081 ± 49 2470 ± 367 757 ± 34
(1140-1697) (3273-5113) (990-1300) (2025-3240) (730-790)
a 12 13 ± 1.1 17 ± 2.1 13 ± 1.5 15 ± 1.6 22±0.8
(11-14) (14-23) (11-16) (12-17) (21-23)
b 9.5 10 ± 1.0 22 ± 1.7 7.5 ± 0.7 12 ± 1.3 6.5 ± 0.3
(8.9-12) (19-24) (6.3-8.8) (10-14) (6.0-7.1)
c 43 45 ± 6.5 98 ± 25 39 ± 5.0 47 ± 7.2 9.8 ± 0.6
(33-54) (73-149) (32-47) (39-58) (8.7-11)
c' 0.8 0.8 ± 0.07 0.7 ± 0.2 0.9 ± 0.1 1.2 ± 0.2 4.1 ± 0.3
(0.6-0.9) (0.4-0.8) (0.7-1.0) (0.9-1.6) (3.7-4.6)
V - - 56 ± 2.4 - 57 ± 2.1 -
(53-60) (55-61)
Max. body diam. 117 126 ± 20 261 ± 31 81 ± 9.6 175 ± 43 35 ± 1.6
(96-139) (201-308) (72-96) (140-230) (32-38)
Nerve ring (NR) 103 98 ± 10 140 ± 11 109 ± 7.7 144 ± 11 93 ± 4.4
(85-120) (125-160) (98-117) (130-156) (88-100)
Pharynx length (ES) 153 145 ± 20 205 ± 8.9 144 ± 10 195 ± 15 117 ± 5.1
(130-159) (190-223) (131-160) (179-218) (109-125)
Excretory pore (EP) 88 84 ± 4.9 97 ± 12 80 ± 6.2 104 ± 9 61 ± 3.7
(74-92) (80-110) (74-94) (90-112) (58-68)
Tail length (T) 34 34 ± 3.3 50 ± 8.7 28 ±3.4 52 ± 5.4 77 ± 4.4
(27-41) (30-63) (23-33) (44-58) (71-85)
Anal body diam. (ABD) 43 42 ± 4.3 69 ± 7.2 32 ± 2.3 43 ± 7.1 19 ± 1. 0
(37-50) (57-80) (29-37) (35-56) (17-20)
Testis reflexion 447 499 ± 76 - 158 ± 43 - -
(390-530) (117-217)
Spicule length (SP) (along the 66 63 ± 4.8 - 53 ± 6.2 - -
arch) (56-70) (40-61)
Spicule width 8.9 9 ± 1.5 - 7.8 ± 1.4 - -
(7.2-12) (6.3-10)
Gubernaculum length (GL) 41 40 ± 3.7 - 31 ± 2.7 - -
(33-46) (27-34)
Gubernaculum width 4.9 5.9 ± 1.0 - 4.3 ± 0.7 - -
(4.7-7.6) (3.1-4.9)
Head to vulva - - 2418±260 - 1405 ± 194 -
(2010-2668) (1113-1845)
Hyaline tail length (HT) - - - - - 35 ± 4.0
(30-40)
E% ((EP / T) x 100) 274 269 ± 35 224 ± 44 276 ± 39 210 ± 29 80 ± 6.1
(203-318) (182-261) (220-360) (188-250) (73-88)
D% ((EP / ES) x 100) 60 58 ± 4.9 49 ± 7.2 56 ± 6.1 52 ± 5.7 53 ± 3.6
(52-66) (35-55) (46-66) (44-56) (48-58)
SW% ((SP / ABD) x 100) 151 149 ± 15 - 169 ± 20 - -
(114-156) (130-203)
GS% ((GL / SP) x 100) 62 65 ±4.7 - 59 ± 5.4 - -
(54-72) (51-66)
H% ((HT / T) x 100) - - - - - 45 ± 3.8
(41-49)
body contour, with six distinct labial papillae. Four cephalic papillae and amphidial apertures inconspicuous. Stoma short, about 4-7^m long and 10-13^m wide. Cheilorhabdions well sclerotised. Morphology of pharynx similar to male. Excretory pore located anterior to nerve ring. Cardia prominent, protruding into intestinal lumen. Genital system amphidelphic, ovaries reflexed dorsally. Vulva mostly symmetrical, slightly (2.5-5.0 ^m) protruding from body contour; sometimes well protruding and non-protruding forms were observed. Vulva opening slightly posterior to mid-body.
Vagina short, leading into paired uteri. Rectum narrow, anal opening distinct. Tail of mature females blunt, conoid, shorter than anal body diameter, tail tip with a small papilla-like mucron; in younger specimens sometimes enlarged to sharp projection. Postanal swelling usually not present in mature females.
Second generation female. Similar to first generation in general morphology, but shorter and narrower (Table 1). Body arcuate when heat-relaxed. Vulva with protruding, symmetrical lips. Tail conical extended in a mucron at the end.
Table 2. Comparative morphometries of infective juveniles and first generation males of Steinernema changbaiense sp. n. and related species of the 'feltiae-kraussei-oregonense'
group. All measurements are in (im and in the in the form: mean (range). Data for new species in bold.
Species IJ Male 1st generation Reference
L* EP T D% E% H% SP GL D% SW% GS% Manubriu mL/W
S. oregonense 980 66 70 50 (40- 100 47 71 56 73 (64- 151 79 na Liu & Berry (1996)
(820-1110) (60-72) (64-78) 60) (90-110) (na) (65-73) (52-59) 75) (na) (na)
S. krawsei 951 63 79 47 80 38 49 33 45 110 68 1.5-2.0 Steiner (1923),
(797-1102) (50-66) (63-86) (na) (na) (35-40) (42-53) (29-37) (na) (na) (na) Mracek et at. (1991)
S. tielingense 915 69 81 (74- 55 88 (85- 58 (53- 88 61 71 (64- 191 (176- 73 (59- 1.4 Ma etal. (2012)
(824-979) (64-73) 85) (47-61) 94) 64) (79-98) (49-70) 78) 212) 82) (1.2-1.9)
S. litorale 909 61 (54- 83(72- 49(44- 73 (68- 34 (27- 75 53 (44-64) 40 (34- 174 (154- 71 (62- 1.9 Yashida (2005)
(834-988) 69) 91) 56) 84) 44) (67-89) 56) 200) 81) (1.5-2.5)
S. xueshanense 860 67 87 (80- 50 78 51(46- 76 49 80 (73- 131 (93- 76 (58- 1.1 Mracek etal. (2009)
(768-929) (60-72) 92) (46-52) (70-90) 55) (66-91) (41-56) 87) 172) 95) (0.9-1.3)
S. feltiae 849 62 81 45 (42- 78 44 (37- 70 41 60 (50- 113 (99- 59 1.0 Filipjev (1934),
(736-950) (53-67) (70-92) 51) (69-86) 51) (65-77) (34-47) 64) 130) (52-61) Poinar (1990)
S. cholashanense 843 62 73 (60- 49 (46- 81 39 (33- 55 39 64 (50- 115 (92- 71 (61- 1.5-1.9 Nguyen et al. (2008)
(727-909) (59-65) 80) 53) (76-91) 47) (49-59) (32-45) 85) 144) 85)
S. akhursti 812 59 73 (68- 47 (45- 77 52 90 (85- 64 56 (52- 180 (140- 71 (65- 1.0 Qiu et al. (2005)
(770-835) (56-60) 75) 50) (73-86) (49-56) 100) (58-68) 61) 200) 77) (0.9-1.1)
S. everestense 775 55 (46- 71 (64- 46 (36- 78 (60- 61 (47- 79 (65- 50 (39-57) 55 (46- 226 (197- 64 (52- na Khatri-Chhetri et al.
(705-838) 61) 78) 51) 89) 68) 87) 66) 273) 74) (2011)
S. ashiunense 768 (720- 55 (51- 71 (66- 46 (43- 78 (70- 36 (30- 59 (50- 37 (25-43) 50 149 (128- 63 1.0 Phan etal. (2006)
800) 59) 76) 50) 85) 42) 65) (44-56) 167) (43-73)
S. changbaiense n. sp. 757 (730- 61 (58- 77 (71- 53 (48- 80 (73- 45 (41- 63 (56- 40 (33-46) 58 (52- 149 (114- 65 (54- 1.3 (1.2- Present study
790) 67) 85) 58) 88) 49) 70) 66) 156) 72) 1.6)
S. texanum 756 59 73 (60- 51(46- 81(76- 59 60 45 67 (58- 157 (127- 75 1.2-1.4 Nguyen et al. (2007)
(732-796) (52-62) 79) 53) 88) (53-64) (55-66) (39-53) 73) 203) (62-84)
S. weiseri 740 (586- 57 (43- 60 (49- 51 (44- 95 36 (34- 68 (62- 53 (46-57) 49 (39- 180 (150- 80 (70- 2.1 (17- Mracek et al. (2003)
828) 65) 68) 55) 39) 72) 60) 240) 85) 2.6)
S. robustispiculum 712 (642- 56 (50- 75 (68- 46 (43- 75 (67- 54 (49- 58 (51- 41(36-44) 56 (50- 129(111- 70 (64- 1.0 Phan etal. (2005)
778) 68) 92) 56) 87) 62) 65) 63) 150) 79)
S. monticolum 706 (612- 58 (54- 77 (71- 47 (44- 76(es- na 70 (61- 45 (35-54) 55 (49- 140 (120- 60 (50- na Stock et al. (1997)
821) 62) 95) 50) se) 80) 61) 150) 70)
S. hebeiense 658 (610- 48 (43- 66 (63- 45 (40- 72 (65- 43 (32- 57 (51- 46 (38-50) 51 (48- 140 (120- 80 (60- na Chen etal. (2006)
710) 51) 71) 50) 80) 50) 63) 59) 170) 90)
*Abbreviations as in Table 1. na: data not available.
Infective juvenile. Body of heat-relaxed specimens almost straight or slightly curved, slender, slightly tapering towards anterior and posterior ends. Body cuticle with fine transverse striae. Lateral field beginning with one cuticular depression or line at 7-8 annules from anterior end. At annule 12-13 two additional lines appearing posteriorly forming two thin ridges. Near excretory pore, number of ridges in lateral fields increasing from two to five. The five ridges remain unchanged for a short distance, then the central ridge divides into two over a short distance after deirid, making a total of six, the maximum number of ridges in lateral field. Two submarginal ridges more distinct than the others. Number of ridges remaining unchanged over long distance. Two central ridges disappear forming four ridges between anus and phasmids. A very short distance posterior to phasmid only two prominent ridges are observed. With the above arrangement, the lateral field formula is 2, 5, 6, 4, 2. Cephalic region gently rounded, continuous with body contour. Stoma closed. Labial papillae not observed; four cephalic papillae distinct; amphidial apertures pore-like. Excretory pore anterior to nerve ring. Hemizonid distinct, located anterior to base of basal bulb, 46-57 ^m behind the excretory pore. Pharynx long and narrow, slightly swollen metacorpus, narrower isthmus and valvate basal bulb. Cardia present. Nerve ring encircling narrow isthmus. Bacterial pouch dorsally displaced, just posterior to cardia, elongate (4-5 ^m in width, 16-22 ^m in length), containing rod-shaped bacteria. Tail long conoid with pointed terminus, hyaline portion occupying less than half of tail length. Phasmid pore-like, located 30-40% of tail length posterior to anus.
Type host and locality. The natural host is unknown as the species was isolated by baiting a soil sample taken in Benxi County, Liaoning province (40.89° N, 124.03° E; altitude 300 m a.s.l.). The type locality is characterised by hills with shrubs, grasses, locust tree and pines. The soil is humid, loamy, under dense grass and tree leaves with moderate shading by pines.
Type material. Holotype male and four male paratypes, six female paratypes and ten infective juvenile paratypes are deposited in the nematode collection of the Institute of Zoology of Ghent University, K.L. Ledeganckstraat 35, Ghent, Belgium. Slides with ten first generation male paratypes, nine first generation female paratypes and twelve infective juvenile paratypes are also deposited in the Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences, Baoding, Hebei, China.
Diagnosis and relationship. Steinernema changbaiense sp. n. is characterised by morphology and morphometries of IJ and adults. Infective juveniles have a body length of 757 (730-790) ^m, the distance from anterior end to the excretory pore averages 61 (58-64) ^m; the lateral field pattern is 2, 5, 6, 4, 2, the tail is 77 (71-85) ^m long and the hyaline portion occupies 41-49 % the tail length. The first generation males have slender and moderately curved golden spicules with a short velum. The gubernaculum is boat shaped in lateral view; the corpus has two wings opening posteriorly. The length of the spicules and gubernaculum of first generation males is 63 (56-70) ^m and 40 (33-46) ^m, respectively. Genital papillae total 23 and comprise 11 pairs and a single mid-ventral papilla. Females have a protruding symmetrical vulva and a blunt tail tip with pointed mucron.
A comparison of morphometric data and morphological features of the new species and closely related species is presented in Tables 2 and 3, respectively. The lateral field formula of S. changbaiense n. sp. is 2, 5, 6, 4, 2; two submarginal ridges are more distinct than the others. This feature distinguishes the new species from most of the other species in the kraussei-feltiae-oregonense group (Table 3). Steinernema changbaiense sp. n. differs from species with a same number of six lateral ridges at mid-body such as S. akhursti Qiu, Hu, Zhou, Mei, Nguyen & Pang, 2005 and S. jollieti Spiridonov, Krasomil-Osterfeld & Moens, 2004 by the morphology of the lateral field (prominent sublateral pair vs all ridges equally spaced and prominent). The new species can also be separated from the related species by morphometric values. The body length of IJ of S. changbaiense sp. n. (757 (730-790) ^m) is shorter than that of S. oregonense Liu & Berry, 1996 (980 (820-1110) ^m), S. akhursti (812 (770-835) ^m), S. cholashanense Nguyen, Puza & Mracek, 2008 (843 (727-909) ^m), S. feltiae (849 (736-950) ^m), S. kraussei (Steiner, 1923) Travassos, 1927 (951 (797-1102) ^m), S. tielingense (915 (824-979) ^m), and S. xueshanense Mracek, Liu & Nguyen, 2009 (860 (768-929) ^m), but it is longer than that of S. hebeiense (658 (610-710) ^m). The spicule length of S. changbaiense sp. n. (63 (5670) ^m) is shorter than that of S. akhursti (90 (85100) ^m), S. eve res tense Khatri-Chhetri, Waeyenberge, Spiridonov, Manandhar & Moens, 2011 (79 (65-87) ^m), S. feltiae (70 (65-77) ^m), S. tielingense (88 (79-98) ^m) and S. xueshanense (76 (66-91) ^m).
Steinernema changbaiense n. sp. clusters with S. ashiunense Phan, Takemoto, & Futai, 2006, S. robustispiculum Phan, Subbotin, Waeyenberge &
Species IJ Male first generation Male Female first generation Female second
second generation
generation
Lateral line Spicule Genital papillae Mucron Mucron Vulva Post-anal swelling
S. akhursti 2, 6,2 Brown, slightly curved, blunt with aperture on ventral side, velum well developed 22+ 1 P P Slightly protruding lips, double flapped epitygmata Present, not prominent
S. ashitiense 8 Slightly yellowish, strongly curved, length / width ratio = 4 (3.5-4.4), prominent rostrum, velum large 20+ 1 P P Vulva a median transverse slit, protruding from body; no epiptygma A
S. changbaiense 2, 5,6,4,2. Golden/yellowish, moderately 20/22 + 1 P P Symmetrical, slightly protruding P
sp. n. curved, slender, manubrium usually elongate, velum short
S. chotashanense 2, 5, 7, 4, 2 Gold/yellowish, head shape variable mostly rounded, velum bow-like, tip bluntly pointed, rostrum pronounced 22+ 1 P P Mostly protuberant, low epitygmata Mostly present
S. everestense 2, 8, 6, 4, 3 Yellowish/brown, head elongated, velum short but prominent, terminus with projection 22/24 + 1 P P Protuberant vulva, with double flapped low epitygmata Mostly present
S. feltiae 2, 6, 7, 8, 4, 2 Brown, head elongated, velum present, rostrum not developed, blunt terminus with slit ventrally 22/24 + 1 P P Protuberant vulva with low epitygmata P
S. hebeiense 8 Yellow-brownish, head elongated, velum thin 22 + 1 A P Protuberant, epitygmata present na
S. ichnusae 2, 8, 9, 8, 4, 6, 2 or 2, 8, 6, 4, 2 Colourless or slightly yellowish, oblong manubrium 20/22 + 1 Mostly A P Not or slightly protruding Not developed
S. kraussei 8 Colourless, head variable, square laterally, terminus pointed, velum present, rostrum pronounced 22/24 + 1 P P Slightly protruding A
S. monticohim na Brown-orange; manubrium rounded, velum extending from base of manubrium to proximal 2/3 of lamina. 20/22 + 1 P P Not protruding P
S. robustispiculum 8 Yellow-brownish, strongly curved, head as long as wide; rostrum prominent, velum large 22+ 1 P na Protruding from body, without epiptygma na
S. texanum 2, 7,2 Brown, head elongated, velum prominent, terminus blunt 22+ 1 A A Protuberant, small epitygmata P
S. tielingense 2, 6, 7, 8, 2 Yellowish, velum bow-like, terminus blunt. Rostrum not well developed 22+ 1 A Mostly P Vulva symmetrical, slightly protruding, anterior lip larger than posterior A
S. xueshanense 2, 7, 8, 7, 6, 4, 2 Slightly yellowish, rounded to ellipsoid manubrium, velum bow-like 22+ 1 Mostly A P Slightly protruding Absent or slightly developed
Species description reference as per Table 2. A, absent; P, present; na, information not available.
Moens, 2005 and S. monticolum in the phylogenetic analysis of ITS and D2D3 sequences. However, it can be distinguished from these species by the morphology and morphometrics of IJ and adults. Infective juveniles of S. changbaiense sp. n. can be distinguished from S. ashiunense by the lateral field at mid-body (6 vs 5 ridges), the hyaline tail length as % of total tail length of IJ (45 (41-49) vs 36 (30-42) %) and the ratio a (22 (21-23) vs 25 (24-27)). The first generation male of the new species can be recognised by the longer body length (1455 (11401680) vs 1279 (1050-1395) pm) and the number of genital papillae (23 vs 21). Steinernema changbaiense sp. n. can be differentiated from S. robustispiculum by several features of the first generation male such as b ratio (10 (8.9-12) vs 8 (79)), pharynx length (145 (130-159) vs 173 (162-186) pm), length from anterior end to excretory pore (84 (74-90) vs 96 (89-104) pm), SW% (149 (114-156) vs 129 (111-150)) and spicule length/width ratio (7.0 (5.2-8.8) vs 4.5 (3.8-5.6)). First generation females can be distinguished by a body length of 4368 (3273-5113) vs 3206 (2745-3765) pm.
Infective juveniles of the new species can be distinguished by the hyaline tail length as % of total tail length (45 (41-49) vs 54 (49-62) %) and the number of ridges in the lateral field at mid-body (6 vs 8). When compared to S. monticolum, S. changbaiense sp. n. is recognised by the location of the excretory pore opening in IJ (posterior mid-pharynx vs in anterior 1/3 of pharynx), and certain characters of first generation males, viz. body length (1455 (1140-1680) vs 1600 (1360-1840) pm), greatest width (126 (96-139) vs 160 (117-206) pm), spicule length (63 (56-70) vs 70 (61-80) pm) and the spicule head (elongate vs round).
Molecular characteristics. Steinernema changbaiense sp. n. is characterised genetically by sequences of ITS (JN865168) and D2D3 regions (JN830950) of rDNA. The sequence length of the ITS region, including primers TW81 and AB28 is 748 bp. The length of the ITS1 + 5.8S + ITS2 sequence is 670 bp, ITS1 = 262 bp, ITS2 = 251 bp, and its composition is: A = 0.27164, C = 0.14328, G = 0.22090, T = 0.36418. The length of the ITS2 sequence is longer than that of S. ashiunense, S. robustispiculum
Fig. 3. Steinernema changbaiense sp. n. SEM and LM pictures of males. A-F: First generation. A: Head region showing four cephalic (c), six labial papillae (l) and excretory pore (EP); B: Posterior region of male showing arrangement of pairs 1-5 of genital papillae; C: Posterior part of tail region showing genital papillae pairs 5-11 and a single precloacal papilla (v); D: Gubernaculum; E: Spicules; F: Tail region. G: Second generation male, tail region. (Scale bars: A-E = 10 pm; F, G = 20 pm.)
Table 4. Sequence lengths and composition of ITS rDNA and D2-D3 region of 28S rRNA gene of Steinernema species
closely related to S. changbaiense sp. n. (bold font,).
Species ITS1 (bp) ITS2 (bp) A (%) C (%) G (%) T (%) Sequence length (bp)
ITS regions
S. akhursti 271 295 0.24066 0.17981 0.23098 0.34855 723
S. ashiuense 261 245 0.26284 0.14804 0.22054 0.36405 662
S. cholashanense 265 303 0.24803 0.17517 0.22345 0.35334 725
S. feltiae 275 298 0.24795 0.16438 0.21644 0.37123 730
S. everestense 271 299 0.23930 0.18156 0.23250 0.34663 727
S. hebeiense 260 292 0.25983 0.15309 0.21629 0.37079 712
S. ichnusae 265 318 0.24128 0.17155 0.21757 0.36960 717
S. kushidai 279 304 0.23108 0.18243 0.24054 0.34595 740
S. weiseri 265 297 0.25171 0.16553 0.22025 0.36252 731
S. monticolum 264 245 0.26577 0.15165 0.22823 0.35435 666
S. oregonense 267 298 0.24205 0.17704 0.22268 0.35823 723
S. robustispiculum 262 249 0.26796 0.14820 0.22305 0.36078 668
S. texanum 263 286 0.24221 0.16997 0.21530 0.37252 706
S. changbaiense sp. n. 262 251 0.27164 0.14328 0.22090 0.36418 670
S. tielingense 265 320 0.24528 0.18059 0.22237 0.35175 742
S. xueshanense 264 293 0.23810 0.17087 0.22549 0.36555 714
D2D3 region
S. ashiuense 0.24203 0.19244 0.30697 0.25856 847
S. cholashanense 0.24742 0.19588 0.30241 0.25430 873
S. hebeiense 0.25459 0.18693 0.29702 0.26147 872
S. feltiae 0.24683 0.19377 0.30219 0.25721 867
S. ichnusae 0.24853 0.19578 0.30481 0.25088 853
S. kraussei 0.25000 0.19329 0.30093 0.25579 864
S. kushidai 0.24683 0.18454 0.30334 0.26528 867
S. monticolum 0.24769 0.19097 0.30324 0.25810 864
S. oregonense 0.24770 0.19585 0.30415 0.25230 868
S. texanum 0.24912 0.19532 0.30292 0.25263 855
S. changbaiense sp. n. 0.24138 0.19425 0.30575 0.25862 870
S. tielingense 0.2494 0.1968 0.2998 0.2540 874
S. xueshanense 0.24710 0.19490 0.30278 0.25522 862
and S. monticolum, but much shorter than that of all other species the 'kraussei-feltiae-oregonense' group (Table 4). More interspecific relationships and pairwise distances are shown in Table 5. Steinernema changbaiense sp. n. differs from S. robustispiculum, its closest taxon, by 14 bp. The nucleotide differences between S. monticolum, S. ashiunense and the new species are 38 bp and 30 bp, respectively. The sequence of the D2D3 region of S. changbaiense sp. n. is 870 bp long and its composition is: A = 0.24138, C = 0.19425, G = 0.30575, T = 0. 25862. Pairwise distances show that S. changbaiense sp. n. differs from its sister taxon S. ashiunense by 4 bp. The nucleotide difference between S. monticolum and the new species is 20 bp. Sequence length and composition of other species are presented in Table 4 and pairwise taxa comparison is given in Table 6. These data confirm that S. changbaiense sp. n. is a new species.
Phylogeny. Maximum parsimony analysis of the ITS regions showed that the alignment resulted in 1029 characters of which 175 are constant, 176 variable characters are parsimony-uninformative and 678 characters are parsimony-informative. Parsimony and distance based tree-building approaches produced almost identical trees. The phylogenetic relationships between 54 Steinernema species are presented in Fig. 5 (for MP, tree length = 4661, CI = 0.4267, RI = 0.6480, RC = 0.2765, HI = 0.5733). In this consensus tree, S. changbaiense sp. n. is grouped together with S. ashiunense, S. robustispiculum and S. monticolum with bootstrap support of 100%. For the D2-D3 region, maximum parsimony analysis showed the alignment to result in 1065 characters of which 465 are constant, 173 variable characters are parsimony-uninformative and 427 characters are parsimony informative. The phylogenetic relationships between 48 Steinernema
Table 5. Pairwise distances of the ITS regions of closely related species to Steinernema changbaiense sp. n. Below diagonal: total character differences; above diagonal: mean character differences (adjusted for missing data). Data for new species in bold.
SN Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14
1 S. xueshanense - 0.03431 0.07516 0.07261 0.13199 0.07972 0.09347 0.09245 0.14194 0.12208 0.19301 0.19444 0.19061 0.18333
2 S. cholashanense 27 - 0.05119 0.04203 0.13099 0.06868 0.08075 0.07702 0.12673 0.10587 0.18019 0.19599 0.17886 0.16940
3 S. tielingense 59 41 - 0.04245 0.13361 0.08368 0.08668 0.07928 0.14430 0.11832 0.18915 0.20154 0.18656 0.17842
4 S. kraussei 57 34 34 - 0.13540 0.08253 0.09328 0.08481 0.14250 0.12071 0.18871 0.20247 0.18453 0.17510
5 S. hebeiense 92 93 95 96 - 0.10408 0.10719 0.14979 0.18938 0.17579 0.22449 0.22309 0.22636 0.21909
6 S. weiseri 57 50 61 60 74 - 0.05777 0.10042 0.16364 0.14185 0.19419 0.19629 0.19457 0.19026
7 S. feltiae 73 65 69 75 76 42 - 0.11111 0.17456 0.15228 0.19204 0.20615 0.18514 0.18120
8 S. texanum 71 61 62 67 105 72 88 - 0.15167 0.12141 0.18741 0.19055 0.18320 0.18333
9 S. kushidai 110 101 114 114 132 117 140 118 - 0.06227 0.21751 0.24080 0.21563 0.21467
10 S. everestense 94 83 93 95 122 101 120 93 50 - 0.20334 0.21296 0.19616 0.19640
11 S. ashiunense 138 131 136 137 143 127 140 134 159 146 - 0.02719 0.04027 0.06174
12 S. robustispiculum 126 127 131 131 143 127 134 121 157 138 18 - 0.02102 0.04827
13 S. changbaiense sp. n. 138 132 136 136 146 129 137 133 160 143 30 14 - 0.05067
14 S. monticolum 132 124 129 128 140 125 133 132 158 142 46 32 38 -
Table 6. Pairwise distances of the D2D3 region of closely related species to Steinernema changbaiense sp. n. Below diagonal: total character differences; above diagonal: mean character differences (adjusted for missing data). Data for new species in bold.
SN Species 1 2 3 4 5 6 7 8 9 10 11 12 13
1 S. changbaiense sp. n. - 0.05220 0.04977 0.04572 0.04167 0.04540 0.05104 0.06154 0.05446 0.05164 0.04572 0.02439 0.00477
2 S. cholashanense 45 - 0.01392 0.01878 0.02204 0.02334 0.02323 0.04033 0.03484 0.01645 0.03404 0.07466 0.05854
3 S. tielingense 43 12 - 0.01290 0.01620 0.01746 0.02320 0.03787 0.03244 0.01526 0.03165 0.07204 0.05609
4 S. feltiae 39 16 11 - 0.00586 0.00703 0.01995 0.02758 0.02817 0.01761 0.02696 0.06807 0.04893
5 S. weiseri 36 19 14 5 - 0.01048 0.01856 0.02840 0.02897 0.01878 0.02579 0.06349 0.04535
6 S. ichnusae 39 20 15 6 9 - 0.02100 0.02738 0.02797 0.02230 0.03165 0.06757 0.05131
7 S. xueshanense 44 20 20 17 16 18 - 0.03796 0.03252 0.02233 0.03169 0.07466 0.05735
8 S. hebeiense 52 34 32 23 24 23 32 - 0.04502 0.03601 0.04796 0.07937 0.06716
9 S. texanum 47 30 28 24 25 24 28 38 - 0.03643 0.04695 0.06846 0.06093
10 S. kraussei 44 14 13 15 16 19 19 30 31 - 0.03052 0.07311 0.05496
11 S. kushidai 39 29 27 23 22 27 27 40 40 26 - 0.06931 0.04893
12 S. monticolum 20 61 59 55 52 55 61 65 56 59 56 - 0.02645
13 S. ashiuense 4 49 47 41 38 43 48 55 51 46 41 21 -
o
O
Bt B
Fig. 4. Steinernema changbaiense sp. n. SEM and LM pictures of females. A-D: First generation. A, C: Vulva region; B, D: Tail region. E-F: Second-generation. E: Vulva region; F: Tail region. (Scale bars: A, B = 10 pm; C-F = 50 pm.)
88 91
100 97
_65_
60
87
97
85
98
100
93
97
86
93 98
83 97
84
63
77 66
100 98
71
91
83
70
100
99
98
99
_Z3_
58
100
100 82
55
73
61 75
100 65
67
JJXL
100
54
97
98 93
96 61
100 68
100 99
IM
100
100
100 69
60
67
62
-C. elegans EU131007
-S. monticolum AE122017
-S. changbaiense sp. n. JN865168
I-S. ashiunense DQ354694
771-S. robustispiculum AY355442
-S. oregonense AY230180
-S. xinbinense JN171593
-S. tielingense GU994201 -S. kraussei AB243442 —S. silvaticum AY2 30162 —S. cholashanense EI431959 —S. xueshanense EJ666052 —S.jollieti AY171265 -5. hebeiense DO]105794 -S. weisen AY 1712 68 -5. liiorale AB243441 —S. ichnusae EU421129 -5. feltiae AE 121050 -S.'sangi AY355441 —S. texanum EF152568 kushidaiAB243440 -S. akhursti DQ375757 -5. exerestense HM000103 —S. scarabaei FJ263673 —S. unicornum G0497167 -5. karii AY230173 -S. aciari AY787660 -S. loci AY355443 -S. thanhi AY355444 -5. brazilense EJ410325 —S. diaprepesi AE 122021 —S. australe EJ235125 —S. gl as er i GU173999 —S. cubanum AY230166 —S. arenarium AY230160 —S. boemarei FJ152414 -S. khoisanae DQ3 14287 —S. longicaudum AY230177 —S. guangdongense. AY170341 —S. neocurtillae AE 122018 —S. scapterisci AY230183 —S. colombiense EU345421 —S. carpocapsae AY171282 —S. siam kavai AF 331917 -5. tami ÄY171280 -5. riobrare DQS35613 —S. pakistanense AY230181 -S. abbasi AY230158 —S. ceratophorum AY230165 —S. bicornutum AY171279 —S. intermedium AY171290 —S. sichuanense DQ884965 -S. affine AY17129S -S. beddmgi AY603397 -S. rarum DQ221116
100
100
76
63
ma
100 M.
97
100
100
100
100 100
100
IM
98
90
Fig. 5. Phylogenetic relationships of Steinernema changbaiense sp. n. with 54 Steinernema spp. based on ITS-rDNA sequences. Caenorhabditis elegans (EU131007) was used as outgroup. Numbers at the nodes represent bootstrap proportion for MP (above) and neighbour joining (below) (50% or more).
100 100
66 97
50
51
73
S2 79
64
89
62 75
70 76
100 100
67
98 56
64
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100 100 100 100
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-Panagrellus redivivus AF3 31910 ■S. monticolum EF439651 -S. changbaiense sp.n. JN830950 -S. ashiunense FJ165550 ■S. ever est ens e HM000104 ■S. ichnusae EU421130 S. xueshanense FJ666053 ■S. hebeiense DQ399664 -S. silvaticum DQ3 99663 -S. texcmum EF152569 ■S. xinbinense GU994204 -S. cholashanense EF520284 -S. tielingense GU994202 ■S.kraussei AF331896 -S. oregonense AF331891 -S.feltiae AF331906 -S.weiseri FJ165549 -5. kushidai AF331897 ■S. scarabaei AY172023 ■S. hermaphroditum AY598358 -S.karii AF331902 -5. brazilense FJ410326 ■S. australe FJ235126 -S.puertoricense AF331903 ■S. diaprepesi AY944001 -S. arenarium AF331892 -S. boemarei FJ152415 ■S. glaseri GUI 77831 ■S. cubanum AF3318S9 -S. khoisanae DQ314289 ■S. longicaudum AF331901
- S. guangdongense AY169 5 5 S -S. costaricense EF187017
-S. unicornum GUI 91462 -S. varum AY253296 ■S. neocurtillae FJ263674 -S.riobrave GUI 77834 -5. bicomutum AF331904 ■S. ceratophorum AF3318S8 -S.abbasi FJ935791 -S.websteri EF217326
- S. anatoliense AYS 41761 -5. qffine AF331899
-S. intermedium AF331909 ■S. sichuanense DQ884966 ■S. carpocapsae AF331900 -S. siamkayai AF331907 -S. scapterisci AF331898 ■S. colombiense EU345420
- Cervidellvs alutus AF331911
Fig. 6. Phylogenetic relationships of Steinernema changbaiense sp. n. with 48 Steinernema spp. based on D2-D3 region sequences of the 28S rRNA gene. Panagrellus redivivus (AF331910) and Cervidellus alutus (AF331911) were used as outgroups. Numbers at the nodes represent bootstrap proportion for MP (above) and neighbour joining (below) (50% or more).
species based on D2-D3 region data are presented in Fig. 6 (tree length = 1806, CI = 0.5138, RI = 0.6934, RC = 0.3563, HI = 0.4862). The new species is placed as a sister taxon of S. ashiunense and is also grouped with S. monticolum forming a subgroup within the feltiae group. Affiliation of these species is supported by a bootstrap value of 66% and 97% for MP and NJ, respectively.
Morphological studies and molecular analyses show that S. changbaiense sp. n. is a new species evolving independently from its sister taxa.
Cross-hybridisation. Cross-hybridisation assays between the new species and S. monticolum, S. tielingense, S. feltiae and S. hebeiense yielded no progeny. In the self-cross controls, offspring was produced normally.
Bionomics. Steinernema changbaiense sp. n. has a life cycle comparable to that of other described species of the 'kraussei-feltiae-oregonense' group. Galleria mellonella larvae were killed after 2 days and first generation adults developed after 4 to 5 days at 25°C. Second generation adults developed after 10 days. It usually took more than 12 days for infective juveniles to emerge from the insect cadavers.
ACKNOWLEDGEMENT
This study was supported by the project ENCHIBE (No. ZEIN2007 PR339) from VLIR, Belgium and by the China Agriculture Research System (CARS-11-B-08) and A2012120103. We thank Lieven Waeyenberge for advice and Ms Nancy de Sutter and Jinyue Wang for technical assistance.
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Juan Ma, Shulong Chen, P. De Clercq, Richou Han and M. Moens. Steinernema changbaiense n. sp. (Rhabditida: Steinernematidae) - новый вид энтомопатогенных нематод из северо-восточного Китая. Резюме. Во время обследования почв северного Китая был обнаружен новый вид энтомопатогенных нематод, описываемый как Steinernema changbaiense sp. n. Новый вид обнаружен с помощью живых приманок - личнок вощинной моли Galleria mellonella (Lepidoptera: Pyralidae). Steinernema changbaiense n. sp. относится к группе видов 'feltiae-kraussei-oregonense' и характеризуется морфометрическими признаками инвазионных личинок: длиной тела 757 (730790) pm, расстоянием от головного конца до экскреторной поры 61 (58-68) pm, длиной хвостового конца 77 (71-85) pm, индексом E % = 80 (73-88) и изменением числа ребер в латеральном поле вдоль тела 2, 5, 6, 4, 2. Самцы первого поколения характеризуются наличием спикул длиной 63 (56-70) pm и рулька длиной 40 (33-46) pm. Спикулы тонкие, велюм короткий. Самки первого поколения имеют симметричную слегка выступающую вульву и индекс D (%) = 49 (35-55). Новый вид отличается от таких близких видов как S. ashiunense, S. robustispiculum и S. monticolum иным количеством ребер в латеральных полях инвазионных личинок, а также строением самцов. Эксперименты по скрещиванию и молекулярно-филогенетический анализ последовательностей ITS rDNA и D2-D3 LSU rDNA подтвердили независимый статус S. changbaiense sp. n.
