Russian Journal of Nematology, 2012, 20 (2), 97-112
Steinernema changbaiense sp. n. (Rhabditida: Steinernematidae), a new species of entomopathogenic nematodes from Northeast China Juan Ma1,2, Shulong Chen1, Patrick De Clercq2, Richou Han4 and Maurice Moens2,3
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: [email protected] 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) µm, distance from head to excretory pore of 61 (58-68) µm, tail length of 77 (71-85) µm, 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) µm long and a gubernaculum of 40 (33-46) µm 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: Сross hybridisation, D2D3 LSU rDNA, entomopathogenic nematodes, ‘feltiae-kraussei- oregonense’ group, ITS rDNA, morphology.
The widely distributed entomopathogenic species have been described (Nguyen, 2011). The nematodes (EPN) have been found in all inhabited biocontrol efficacy of EPN is variable and depends continents and a range of ecologically diverse on nematode species and strains (Lewis & Grewal, habitats (Hominick, 2002). The families 2005). Further, environmental conditions may affect Heterorhabditidae Poinar, 1976 and survival, reproductive potential and virulence of the Steinernematidae Filipjev, 1934, which are EPN strains. Indigenous isolates of EPN may thus characterised by carrying specific symbiotic bacteria be more suitable against local insect pests. To obtain of the genus Photorhabdus and Xenorhabdus, suitable species and strains for the implementation respectively, are the most commonly studied of integrated pest management strategies, we carried families because of their economic importance. out an extensive survey on EPN in North China. They have a wide range of insect hosts (Shapiro-Ilan During this survey ten EPN species were collected et al., 2002) and are considered to have great (Ma et al., 2010) along with several undescribed potential in biological control of insect pests Steinernema species. Two of these latter species (Ehlers, 1996). were described as S. tielingense and S. xinbinense The number of species in both families has (Ma et al., 2012a; Ma et al., 2012b). We here rapidly increased in recent years. Currently more describe a third species as S. changbaiense sp. n. than 60 steinernematid and 16 heterorhabditid named after the Changbai Mountains, from which
97 Juan Ma et al. foothills the type population was collected. ATA TGC TTA AGT TCA GCG GGT-3’ were Steinernema changbaiense sp. n. differs in used for the amplification and sequencing of the morphology and morphometrics from the described rDNA fragment containing the internal transcribed EPN species, and molecular studies show that this spacer regions ITS1 and ITS2, and the 5.8S rRNA isolate is closely related to species in the ‘feltiae- gene. A second rDNA fragment containing the D2- kraussei-oregonense’ group (Spiridonov et al., 2004). D3 expansion segment of the 28S rRNA gene was amplified using the forward primer D2F 5’-CCT MATERIALS AND METHODS TAG TAA CGG CGA GTG AAA-3’ and the reverse primer 536 5’-CAG CTA TCC T GA GGA Nematode isolation. The nematode isolate was AAC-3’ (Nguyen et al., 2006). The methods recovered from soil samples collected in the vicinity reported by Nguyen (2007) were used for PCR of Benxi County, Liaoning province, China during a amplification. The same primers were used for survey in April, 2008. The soil samples (ca 400 sequencing procedure. The resulting sequences were cm3) were composed of 4-6 sub-samples randomly edited and analysed using software packages taken at least 5 m apart at a depth of 2-20 cm. Sub- Chromas 2.23 (Technelysium, Tewantin, samples were placed in polyethylene bags, mixed, Queensland, Australia) and BioEdit7.0.4.1 (Hall, transported to the laboratory and stored under cool 1999) and deposited in GenBank. The obtained conditions (15 ± 2°C) until processing. sequences were compared with sequences of Entomopathogenic nematodes were extracted from Steinernema species available in GenBank (NCBI). soil by the Galleria mellonella L. (Lepidoptera: The internal transcribed spacer regions (ITS- Pyralidae) baiting method (Bedding & Akhurst, rDNA) and the partial 28S ribosomal DNA (D2-D3 1975). Infective juveniles (IJ) were collected from segment) sequences of the new species and the cadavers using White traps (White, 1927) and corresponding nucleotide sequences of other were multiplied on a new batch of G. mellonella. representatives of Steinernema available in Morphological observations. Nematodes were GenBank were aligned using Clustal X1.83 re-cultured in last instar larvae of G. mellonella. (Thompson et al., 1997). Phylogenetic analyses Only IJ collected during the week after their first (Maximum parsimony and Neighbour-Joining) of emergence from the insect cadavers were used for sequences data were done using PAUP* v 4.0b10 identification. First and second generation adults (Swofford, 2001). Trees were evaluated statistically were collected by dissecting the cadavers 5 and 10- by bootstrap analysis based on 1,000 resamplings of 11 days after nematode infection, respectively. Light the dataset. The resulting trees were visualised by microscopy (LM) and scanning electron microscopy using MrEnt 2.0 (Zuccon, 2010). Caenorhabditis (SEM) studies were done as reported by Ma et al. elegans (EU131007) was applied as outgroup during (2012a). Measurements were done and morphology calculation of the trees based on ITS sequences; was examined using an Olympus BX51 compound Cervidellus alutus (AF331911), together with microscope. Panagrellus redivivus (AF331910), were used as Cross hybridisation. The modified hanging- outgroups for calculation of the trees based on D2- blood assay described by Kaya and Stock (1997) D3 sequences. was used for checking reproductive compatibilities of the new species with S. monticolum Stock, Choo DESCRIPTION & Kaya, 1997, S. tielingense Ma, Chen, Li, Han, Khatri-Chhetri, De Clercq & Moens, 2012, S. feltiae Steinernema changbaiense sp. n. (Filipjev, 1934) Wouts, Mráček, Gerdin & Bedding, (Figs 1-6) 1982 and S. hebeiense Chen, Li, Yan, Spiridonov & Measurements: See Table 1. Moens, 2006. All of these species were represented First generation male. Body curved ventrally, by isolates obtained from North China (Ma et al., tapering toward anterior end, mostly C-shaped, 2010). No other species were available for cross rarely J-shaped in heat-relaxed specimens. Cuticle breeding. Twenty replications were performed for with very faint transverse striae. Head truncated, each cross-breeding test combination. Self-breeding almost continuous with body contour. Six labial and controls were done under the same conditions. four cephalic papillae present. Amphids located Molecular characteristics and phylogeny laterally between labial and cephalic circle of analysis. Total genomic DNA was extracted from papillae. Stoma short, shallow, cheilorhabdions single IJ using the method of Joyce et al. (1994). distinct, sclerotised. Pharynx muscular; procorpus The forward primer TW81 5’-GTT TCC GTA GGT cylindrical, metacorpus slightly swollen. Nerve ring GAA CCT GC-3’ and the reverse primer AB28 5'- surrounding isthmus at anterior part of valvated basal
98 Steinernema changbaiense sp. n. from Northeast China
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 µm; F, G, H = 50 µm; C = 20 µm.).
99 Juan Ma et al.
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 μm; D = 1 μm.) bulb. Cardia prominent. Excretory pore always three genital papillae comprising 11 pairs and a located anterior to nerve ring (ranging from 52 to single mid-ventral papilla located just anterior to 66% of the length from anterior body end to base of cloacal opening. Seven pairs located precloacal (six pharynx). Intestine generally with a wide lumen. pairs subventral and one pair lateral), one pair Distinct, papilliform deirid situated at the adcloacal and three pairs postcloacal, consistently pharyngeal bulb to nerve ring. Well developed and near tail terminus (one subdorsal, two at tail tip). ventrally reflexed testis. Paired symmetrical, Tail bluntly conoid, usually concave at ventral side. moderately to strongly curved and slender spicules, Short, projection-like mucron mostly present, ca golden/yellowish in colour. Spicule length/width 1.5-3.2 µm long. Phasmids located just behind the ratio (SL/SPW) = 7.0 (5.2-8.5). Head (manubrium) cloacal opening. of spicules, usually elongate (spicule head Second generation male. Similar to first- length/width = 1.2-1.5), slightly offset from shaft, generation, but smaller in size (Table 1). Spine-like calomus very short. Lamina slender and well tail mucron much longer than in the first generation, curved, with two internal sclerotised ribs, tapering ca 4.4-9.5 µm long. Spicules and gubernaculum slightly posteriorly. Spicule terminus blunt. Rostrum shorter and thinner. not well developed, velum short. In lateral view, First generation female. Body size variable, gubernaculum boat-shaped to almost straight, usually C-shaped on heat relaxation. Cuticle only tapering gradually posteriorly, cuneus V-shaped, faintly striated. Lateral field not observed. Head truncate corpus with two wings opening posteriorly. Twenty- or slightly round, tapering anteriorly, continuous with
100 Steinernema changbaiense sp. n. from Northeast China
Table 1. Morphometrics of Steinernema changbaiense sp. n. All measurements are in µm and in the in the form: mean ± s.d. (range).
Character First generation Second generation Infective juvenile Male Female Male Female Holotype Paratypes Paratypes 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) × 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) × 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) × 100) 151 149 ± 15 – 169 ± 20 – – (114-156) (130-203) GS% ((GL / SP) × 100) 62 65 ±4.7 – 59 ± 5.4 – – (54-72) (51-66) H% ((HT / T) × 100) – – – – – 45 ± 3.8 (41-49) body contour, with six distinct labial papillae. Four Vagina short, leading into paired uteri. Rectum cephalic papillae and amphidial apertures narrow, anal opening distinct. Tail of mature inconspicuous. Stoma short, about 4-7µm long and females blunt, conoid, shorter than anal body 10-13µm wide. Cheilorhabdions well sclerotised. diameter, tail tip with a small papilla-like mucron; Morphology of pharynx similar to male. Excretory in younger specimens sometimes enlarged to sharp pore located anterior to nerve ring. Cardia projection. Postanal swelling usually not present in prominent, protruding into intestinal lumen. Genital mature females. system amphidelphic, ovaries reflexed dorsally. Second generation female. Similar to first Vulva mostly symmetrical, slightly (2.5-5.0 µm) generation in general morphology, but shorter and protruding from body contour; sometimes well narrower (Table 1). Body arcuate when heat- protruding and non-protruding forms were observed. relaxed. Vulva with protruding, symmetrical lips. Vulva opening slightly posterior to mid-body. Tail conical extended in a mucron at the end.
101 Juan Ma et al. ’
et al. et (2008) (2007) (2006) (2005) . (1997) . (2006) (2012) et al. et al. et al . (2009) et al . (1991) et al . (2003) et al et al et ek ek ek ek ek ek et al . (2005) č č č et al. á á á Ma Qiu Mr Poinar (1990) Reference Reference Mr Mr Yashida (2005) (2005) Yashida Present study study Present (2011) feltiae-kraussei-oregonense na na (1996) Liu & Berry na Stock na na Khatri-Chhetri Khatri-Chhetri na Chen na 1.1 1.0 Filipjev (1934), 1.0 Phanal. et 1.9 1.0 Phanal. et 1.4 1.4 1.0 2.6) 2.6) 1.6) 1.6) 1.5-1.9 Nguyen 1.2-1.4 Nguyen 1.5-2.0 Steiner (1923), 1.5-2.0 Steiner m L/W m L/W 2.1 (17- 2.1 1.3 (1.2- 1.3 (1.2-1.9) (1.2-1.9) (0.9-1.1) (0.9-1.3) (0.9-1.3) (1.5-2.5) Manubriu
79 68 59 75 63 95) 85) 82) 77) 85) 81) 70) 74) 72) 79) 90) (na) (na) (na) 76 (58- 71 (61- 73 (59- 71 (65- 80 (70- 71 (62- 60 (50- 64 (52- 65 (54- 70 (64- 80 (60- (52-61) (62-84) (43-73) 151 110 (na) (na) (na) 172) 130) 144) 212) 200) 203) 167) 240) 200) 150) 273) 156) 150) 170) 131 (93- 113 (99- 115 (92- 191 (176- 180 (140- 157 (127- 149 (128- 180 (150- 174 (154- 140 (120- 226 (197- 149 (114- 129 (111- 140 (120- generation st sp. n. and related‘ species of the sp. 45 45 75) 78) 87) 64) 85) 61) 73) 60) 61) 56) 66) 63) 59) 66) (na) (na) 73 (64- 71 (64- 80 (73- 60 (50- 64 (50- 56 (52- 67 (58- (44-56) 56 56 33 33 61 61 49 41 45 39 39 64 (52-59) (29-37) (49-70) (41-56) (34-47) (39-53) (32-45) (58-68) 53 (46-57) 49 (39- 45 (35-54) 55 (49- 53 (44-64) 40 (34- 37 (25-43) (25-43) 37 40 (33-46) 50 58 (52- 41 (36-44) 56 (50- 46 (38-50) 51 (48- 50 (39-57) 55 (46- 71 71 49 88 88 76 70 55 55 60 75 75 72) 80) 65) 70) 65) 63) 87) 100) 90 (85- 68 (62- 59 (50- 63 (56- 58 (51- 57 (51- 79 (65- (42-53) (79-98) (49-59) (67-89) (65-73) (66-91) (65-77) (55-66) form:mean (range). Datafor new species bold. in Steinernema changbaiense na 70 (61- na 70 47 38 52 59 64) 55) 51) 47) 39) 42) 49) 62) 50) 68) 44) (na) 58 (53- 51 (46- 44 (37- 39 (33- 36 (30- 45 (41- 54 (49- 43 (32- 61 (47- 34 (27- (35-40) (49-56) (53-64) 78 78 78 81 81 77 80 80 (34- 36 95 94) 94) 88) 88) 85) 85) 88) 87) 86) 80) 89) 89) 84) 84) 100 (na) (na) 88 (85- 81 (76- 78 (70- 80 (73- 75 (67- 76 (63- 72 (65- 78 (60- (70-90) (69-86) (76-91) (73-86) 73 (68– (90-110) 47 55 50 60) 51) 53) 53) 50) 53) 50) 58) 55) 56) 50) 50) 56) 51) (na) (na) 50 (40- 50 45 (42- 45 49 (46- 49 (45- 47 (46- 51 46 (43- 46 (48- 53 (44- 51 (43- 46 (44- 47 (40- 45 46 (36- 46 (47-61) (46-52) 49 (44–
70 70 79 81 81 85) 92) 80) 75) 79) 76) 85) 68) 92) 95) 71) 91) 78) 81 (74- 87 (80- 73 (60- 73 (68- 73 (60- 71 (66- 77 (71- 60 (49- 75 (68- 77 (71- 66 (63- 71 (64- (64-78) (63-86) (70-92) 83 (72– 66 66 63 69 69 67 62 62 59 59 59) 67) 65) 68) 62) 51) 69) 61) 55 (51- 61 (58- 57 (43- 56 (50- 58 (54- 48 (43- 55 (46- (60-72) (50-66) (64-73) (60-72) (53-67) (59-65) (56-60) (52-62) 61 (54– 61 group. All measurements are in µm and in the in the L* EP T D% E% H% SP GL D% SW% GS% GS% SW% D% GL SP H% E% D% T EP L* 980 951 915 909 860 849 843 812 775 756 800) 790) 828) 778) 821) 710) 768 (720- 757 (730- 740 (586- 712 (642- 706 (612- 658 (610- (824-979) (768-929) (736-950) (727-909) (770-835) (705-838) (732-796) (834–988) (820-1110) (797-1102)
Comparative morphometrics of infective juveniles and first generation males of *Abbreviations as in Table 1. na: 1. data not available. Table *Abbreviationsin as Species IJ Male 1 Species IJ Male S. oregonense S. kraussei S. tielingense S. litorale S. xueshanense S. feltiae S. cholashanense S. akhursti everestense S. S. ashiunense S. changbaiense n. sp. S. texanum S. weiseri robustispiculum S. S. monticolum S. hebeiense Table 2.
102 Steinernema changbaiense sp. n. from Northeast China Infective juvenile. Body of heat-relaxed Diagnosis and relationship. Steinernema specimens almost straight or slightly curved, changbaiense sp. n. is characterised by morphology slender, slightly tapering towards anterior and and morphometrics of IJ and adults. Infective posterior ends. Body cuticle with fine transverse juveniles have a body length of 757 (730-790) µm, striae. Lateral field beginning with one cuticular the distance from anterior end to the excretory pore depression or line at 7-8 annules from anterior end. averages 61 (58-64) µm; the lateral field pattern is 2, At annule 12-13 two additional lines appearing 5, 6, 4, 2, the tail is 77 (71-85) µm long and the posteriorly forming two thin ridges. Near excretory hyaline portion occupies 41-49 % the tail length. pore, number of ridges in lateral fields increasing The first generation males have slender and from two to five. The five ridges remain unchanged moderately curved golden spicules with a short for a short distance, then the central ridge divides velum. The gubernaculum is boat shaped in lateral into two over a short distance after deirid, making a view; the corpus has two wings opening posteriorly. total of six, the maximum number of ridges in The length of the spicules and gubernaculum of first lateral field. Two submarginal ridges more distinct generation males is 63 (56-70) µm and 40 (33-46) than the others. Number of ridges remaining µm, respectively. Genital papillae total 23 and unchanged over long distance. Two central ridges comprise 11 pairs and a single mid-ventral papilla. disappear forming four ridges between anus and Females have a protruding symmetrical vulva and a phasmids. A very short distance posterior to blunt tail tip with pointed mucron. phasmid only two prominent ridges are observed. A comparison of morphometric data and With the above arrangement, the lateral field morphological features of the new species and formula is 2, 5, 6, 4, 2. Cephalic region gently closely related species is presented in Tables 2 and rounded, continuous with body contour. Stoma 3, respectively. The lateral field formula of S. closed. Labial papillae not observed; four cephalic changbaiense n. sp. is 2, 5, 6, 4, 2; two submarginal papillae distinct; amphidial apertures pore-like. ridges are more distinct than the others. This feature Excretory pore anterior to nerve ring. Hemizonid distinguishes the new species from most of the other distinct, located anterior to base of basal bulb, 46-57 species in the kraussei-feltiae-oregonense group μm behind the excretory pore. Pharynx long and (Table 3). Steinernema changbaiense sp. n. differs narrow, slightly swollen metacorpus, narrower from species with a same number of six lateral isthmus and valvate basal bulb. Cardia present. ridges at mid-body such as S. akhursti Qiu, Hu, Nerve ring encircling narrow isthmus. Bacterial Zhou, Mei, Nguyen & Pang, 2005 and S. jollieti pouch dorsally displaced, just posterior to cardia, Spiridonov, Krasomil-Osterfeld & Moens, 2004 by elongate (4-5 μm in width, 16-22 µm in length), the morphology of the lateral field (prominent containing rod-shaped bacteria. Tail long conoid sublateral pair vs all ridges equally spaced and with pointed terminus, hyaline portion occupying prominent). The new species can also be separated less than half of tail length. Phasmid pore-like, from the related species by morphometric values. located 30-40% of tail length posterior to anus. The body length of IJ of S. changbaiense sp. n. (757 Type host and locality. The natural host is (730-790) µm) is shorter than that of S. oregonense unknown as the species was isolated by baiting a Liu & Berry, 1996 (980 (820-1110) µm), S. akhursti soil sample taken in Benxi County, Liaoning (812 (770-835) µm), S. cholashanense Nguyen, province (40.89º N, 124.03º E; altitude 300 m a.s.l.). Půža & Mráček, 2008 (843 (727-909) µm), S. feltiae The type locality is characterised by hills with (849 (736-950) µm), S. kraussei (Steiner, 1923) shrubs, grasses, locust tree and pines. The soil is Travassos, 1927 (951 (797-1102) µm), S. tielingense humid, loamy, under dense grass and tree leaves (915 (824-979) µm), and S. xueshanense Mráček, with moderate shading by pines. Liu & Nguyen, 2009 (860 (768-929) µm), but it is Type material. Holotype male and four male longer than that of S. hebeiense (658 (610-710) µm). paratypes, six female paratypes and ten infective The spicule length of S. changbaiense sp. n. (63 (56- juvenile paratypes are deposited in the nematode 70) µm) is shorter than that of S. akhursti (90 (85- collection of the Institute of Zoology of Ghent 100) µm), S. everestense Khatri-Chhetri, University, K.L. Ledeganckstraat 35, Ghent, Waeyenberge, Spiridonov, Manandhar & Moens, Belgium. Slides with ten first generation male 2011 (79 (65-87) µm), S. feltiae (70 (65-77) µm), S. paratypes, nine first generation female paratypes and tielingense (88 (79-98) µm) and S. xueshanense (76 twelve infective juvenile paratypes are also (66-91) µm). deposited in the Institute of Plant Protection, Hebei Steinernema changbaiense n. sp. clusters with S. Academy of Agricultural and Forestry Sciences, ashiunense Phan, Takemoto, & Futai, 2006, S. Baoding, Hebei, China. robustispiculum Phan, Subbotin, Waeyenberge &
103 Juan Ma et al.
d P A A P A na slightly slightly swelling swelling prominent generation develope Present, not not Present, Mostly present Mostly present Mostly present Mostly present osterior p epiptygma epitygmata epitygmata y; no epiptygma d low epitygmata low epitygmata protruding from apped epitygmata o Slightly protruding Slightly fl b appedepitygmata low Female first generation Female second fl species. protruding, anterior lip larger than protruding, anterior lip larger
P P A Protuberant, small epitygmata Steinernema Steinernema second generation and related
n.
sp. Mucron Mucron Mucron Vulva Post-anal 22 + 1 P na without Protruding from body, 22 + 1 22 + 1 A A Mostly Mostly P P slightly symmetrical, Vulva protruding Slightly Absent or 22 + 1 P P protruding Slightly lips, double 22 + 1 P P Mostly protuberant, 20 + 1 P P a median transverse slit, Vulva 22 + 1 A 22 + 1 A P Protuberant, epitygmata present na 20/22 + 1 P P 22/24 + 1 Symmetrical, slightly protruding P 20/22 + 1 P A Mostly P 20/22 + 1 P Protuberant vulva withlow P protruding or slightly Not P developed Not Notprotruding P apillae 22/24 + 1 P 22/24 + 1 22/24 + 1 P P double Protuberantvulva, with Genital Genital p
d Steinernema changbaiense d t
d Spicule Spicule resent; na, information not available.information na,resent; not develope velum thin thin velum p 2/3 of lamina. 2/3 of lamina. wish, head shape variable with projection h/brown, head elongated, velum extending oblong manubrium oblong manubrium n, head elongated, velum rominent, velum large large velum rominent, elongate, velum short p rominent, terminus blun rominent, terminus p resent, rostrum pronounceresent, rostrum terminus ventrally terminus with slit p rominent rostrum, velum large rostrum, rominent head as long as wide; rostrum headas as wide; long p Comparative morphology of Colourless or slightly yellowish, or slightly Colourless length / width ratio = 4 (3.5-4.4), laterally, terminus terminus pointed, velum laterally, luntly pointed, rostrum pronounce pointed, rostrum luntly blunt. Rostrum not developed well aperture on ventral side, velum well mostly rounded, velum bow-like, tip velum short but prominent, terminus b from base of manubrium to proximal present, rostrum not developed, blunt ellipsoid bow-like manubrium, velum curved, slender, manubriumcurved, usually slender, Table 3.Table er Table 2. A, absent; P, P, A, absent; 2. Table er p 8 curved, strongly yellowish, Slightly 8 head elongated, Yellow-brownish, 8 strongly curved, Yellow-brownish, IJ Male first generation Male na Brown-orange; manubrium rounded, manubrium na Brown-orange; 2, 8, 6, 4, 3 Yellowis Lateral line line Lateral ence as 2, 5, 6, 4, 2. 2. 4, 6, 5, 2, moderately Golden/yellowish, r or 2, 8, 6, 4, 2 2, 8, 9, 8, 4, 6, 2 2 6, 4, 8, 9, 8, 2, 2, 7, 7,8, 6, 4, 2 rounded yellowish, Slightly to tion refe p 2, 5, 7, 4, 2 Gold/yello 2, 6, 7, 8, 2 velum bow-like, terminus Yellowish, 2, 7, 2 Brown, head elongated, velum 8 Colourless, headvariable, square 2, 6, 2 Brown, curved,slightly blunt with ecies descri 2, 6, 7, 8, 4, 2 Brow p S S. akhursti S. ashiuense S. changbaiense sp. n. S. cholashanense S. everestense S. feltiae S. hebeiense S. ichnusae S. kraussei S. monticolum S. robustispiculum S. texanum S. tielingense S. xueshanense Species
104 Steinernema changbaiense sp. n. from Northeast China
Moens, 2005 and S. monticolum in the phylogenetic Infective juveniles of the new species can be analysis of ITS and D2D3 sequences. However, it distinguished by the hyaline tail length as % of total can be distinguished from these species by the tail length (45 (41-49) vs 54 (49-62) %) and the morphology and morphometrics of IJ and adults. number of ridges in the lateral field at mid-body (6 Infective juveniles of S. changbaiense sp. n. can be vs 8). When compared to S. monticolum, S. distinguished from S. ashiunense by the lateral field changbaiense sp. n. is recognised by the location of at mid-body (6 vs 5 ridges), the hyaline tail length as the excretory pore opening in IJ (posterior mid- % of total tail length of IJ (45 (41-49) vs 36 (30-42) pharynx vs in anterior 1/3 of pharynx), and certain %) and the ratio a (22 (21-23) vs 25 (24-27)). The characters of first generation males, viz. body length first generation male of the new species can be (1455 (1140-1680) vs 1600 (1360-1840) µm), recognised by the longer body length (1455 (1140- greatest width (126 (96-139) vs 160 (117-206) µm), 1680) vs 1279 (1050-1395) µm) and the number of spicule length (63 (56-70) vs 70 (61-80) µm) and the genital papillae (23 vs 21). Steinernema spicule head (elongate vs round). changbaiense sp. n. can be differentiated from S. Molecular characteristics. Steinernema robustispiculum by several features of the first changbaiense sp. n. is characterised genetically by generation male such as b ratio (10 (8.9-12) vs 8 (7- sequences of ITS (JN865168) and D2D3 regions 9)), pharynx length (145 (130-159) vs 173 (162-186) (JN830950) of rDNA. The sequence length of the µm), length from anterior end to excretory pore (84 ITS region, including primers TW81 and AB28 is (74-90) vs 96 (89-104) µm), SW% (149 (114-156) 748 bp. The length of the ITS1 + 5.8S + ITS2 vs 129 (111-150)) and spicule length/width ratio sequence is 670 bp, ITS1 = 262 bp, ITS2 = 251 bp, (7.0 (5.2-8.8) vs 4.5 (3.8-5.6)). First generation and its composition is: A = 0.27164, C = 0.14328, G = females can be distinguished by a body length of 0.22090, T = 0.36418. The length of the ITS2 sequence 4368 (3273-5113) vs 3206 (2745-3765) µm. 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 μm; F, G = 20 μm.)
105 Juan Ma et al.
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 ITS2 (bp) A (%) C (%) G (%) T (%) Sequence length (bp) (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 Phylogeny. Maximum parsimony analysis of the other species the ‘kraussei-feltiae-oregonense’ ITS regions showed that the alignment resulted in group (Table 4). More interspecific relationships 1029 characters of which 175 are constant, 176 and pairwise distances are shown in Table 5. variable characters are parsimony-uninformative Steinernema changbaiense sp. n. differs from S. and 678 characters are parsimony-informative. robustispiculum, its closest taxon, by 14 bp. The Parsimony and distance based tree-building nucleotide differences between S. monticolum, S. approaches produced almost identical trees. The ashiunense and the new species are 38 bp and 30 bp, phylogenetic relationships between 54 Steinernema respectively. The sequence of the D2D3 region of S. species are presented in Fig. 5 (for MP, tree length = changbaiense sp. n. is 870 bp long and its 4661, CI = 0.4267, RI = 0.6480, RC = 0.2765, HI = composition is: A = 0.24138, C = 0.19425, G = 0.5733). In this consensus tree, S. changbaiense sp. 0.30575, T = 0. 25862. Pairwise distances show that n. is grouped together with S. ashiunense, S. S. changbaiense sp. n. differs from its sister taxon S. robustispiculum and S. monticolum with bootstrap ashiunense by 4 bp. The nucleotide difference support of 100%. For the D2-D3 region, maximum between S. monticolum and the new species is 20 parsimony analysis showed the alignment to result bp. Sequence length and composition of other in 1065 characters of which 465 are constant, 173 species are presented in Table 4 and pairwise taxa variable characters are parsimony-uninformative comparison is given in Table 6. These data confirm and 427 characters are parsimony informative. The that S. changbaiense sp. n. is a new species. phylogenetic relationships between 48 Steinernema
106 Steinernema changbaiense sp. n. from Northeast China - 0.18333 0.18120 0.05067 0.16940 0.17842 0.17510 0.21909 0.19026 0.18333 0.19640 0.06174 0.04827 0.21467 38 13 14 0.19061 0.18514 0.17886 0.18656 0.18453 0.22636 0.19457 0.18320 0.19616 0.04027 0.02102 0.21563 ew speciesin bold. new speciesin bold. sp. n. 5228 0.19204 0.20615 formissingfor data). Data n Steinernema changbaiense ed formissing data). Datafor Steinernema changbaiense sp. n. 06154 0.05446 0.051640.04572 0.02439 0.00477
racter differences(adjusted on of closelyrelated species to s of closely related species to - 0.013920.01878 0.02204 0.02334 0.02323 0.04033 0.03484 0.016450.03404 0.07466 0.05854 2 3 4 5 6 7 8 9 10 11 12 13 12 11 10 9 7 8 4 5 6 2 3 49 47 41 38 43 48 55 51 46 41 21 - 34 32 23 24 23 32 - 0.04502 0.03601 0.04796 0.07937 0.06716 12 0.07937 16 0.06093 - 0.04796 19 11 0.01290 0.01620 0.01746 0.02320 0.03787 0.03244 0.015260.03165 0.07204 0.05609 0.06846 20 0.03601 14 0.05496 20 - 0.04695 15 0.04502 34 32 23 24 23 32 5 0.00586 0.00703 0.01995 0.02758 0.02817 0.017610.02696 0.06807 0.04893 - 0.07311 20 0.03643 30 28 24 25 24 28 38 6 0.04893 - 0.03052 1714 13 15 16 19 30 31 - - 0.06931 29 9 27 23 22 27 40 0.01048 0.01856 0.02840 0.0289740 26 0.018780.02579 0.06349 0.04535 - 16 0.02645 61 59 55 52 55 61 65 56 59 56 - - 18 0.02100 0.02738 0.02797 0.022300.03165 0.06757 0.05131 - 0.03796 0.03252 0.022330.03169 0.07466 0.05735 - 0.05220 0.049770.04572 0.04167 0.04540 0.05104 0. 1 4 45 43 39 36 39 44 52 47 44 39 20 Pairwise distances of the D2D3 regi
Pairwise distancesof the ITS region
132 136 136 146 129 137 133 160 143 30 129 14 - 146 136 136 132 sp. sp. n. Table 6. 1 2 3 4 5 6 7 8 9 10 11 12 73 65 69 75 76 42 - 0.11111 0.17456 0.1 0.17456 0.1111142 - 76 65 69 75 73 27 - 0.05119 0.04203 0.13099 0.06868 0.08075 0.07702 0.12673 0.10587 0.18019 0.19599 27 0.06868 0.19055 - 0.13099 59 0.04203 41 0.05119 0.18741 57 0.20154 0.18915 0.144300.07928 0.11832 0.08668 34 0.08368 0.12141 - 92 0.13361 93 0.04245 34 88 57 72 0.15167 50 - 0.21296 105 95 0.09328 0.08481 0.14250 0.12071 0.18871 0.20247 61 62 67 0.08253 - 120 93 0.13540 61 50 0.20334 101 96 71 - 122 60 83 93 95 - 0.10719 0.14979 0.18938 0.17579 0.22449 0.22309 0.10408 74 94 - 0.05777 0.10042 0.16364 0.14185 0.19419 0.19629 110 101 110 114 114 132 117 140 118 - 0.24080 0.21751 0.06227 138 138 131 136 137 143 127 140 134 159 146 0.02719 127 - 143 134 121 157 138 18 127 137 136 - 131 143 131 131 127 138 133 132 158 142 46 125 32 140 126 128 129 124 132 Table 5. Table
sp. n. n. sp. 0.09347 0.09245 0.14194 0.12208 0.19301 0.19444 0.07972 0.13199 0.07261 0.07516 0.03431 - S. changbaiense S. cholashanense S. tielingense S. feltiae S. weiseri S. ichnusae S. xueshanense S. hebeiense S. texanum S. kraussei S. kushidai S. monticolum S. ashiuense
SN Species Species SN 1 2 3 4 5 6 7 8 9 10 11 12 13 Below diagonal: total character differences; above diagonal: mean cha Belowdiagonal: total character differences; abovemean diagonal: character differences(adjust S. xueshanense S. cholashanense S. tielingense S. kraussei S. hebeiense S. weiseri S. feltiae texanum S. S. kushidai everestense S. S. ashiunense S. robustispiculum S. changbaiense S. monticolum SN Species SN Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14
107 Juan Ma et al.
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 μm; C-F = 50 μm.)
108 Steinernema changbaiense sp. n. from Northeast China
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).
109 Juan Ma et al.
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).
110 Steinernema changbaiense sp. n. from Northeast China species based on D2-D3 region data are presented in JOYCE, S.A., REID, A., DRIVER, F. & CURRAN, J. 1994. Fig. 6 (tree length = 1806, CI = 0.5138, RI = 0.6934, Application of polymerase chain reaction (PCR) RC = 0.3563, HI = 0.4862). The new species is methods to identification of entomopathogenic placed as a sister taxon of S. ashiunense and is also nematodes. In: Biotechnology: genetics of grouped with S. monticolum forming a subgroup entomopathogenic nematode-bacterium complex. within the feltiae group. Affiliation of these species (A.M. Burnell, R.-U. Ehlers & J.P. Masson Eds). pp. is supported by a bootstrap value of 66% and 97% 178-187. European Commission, Brussels, Belgium. for MP and NJ, respectively. KAYA, H.K. & STOCK, S.P. 1997. Techniques in insect Morphological studies and molecular analyses nematology. In: Manual of techniques in insect show that S. changbaiense sp. n. is a new species pathology. (L. Lacey Ed.). pp. 313-314. Academic evolving independently from its sister taxa. Press, San Diego, CA, USA. Cross-hybridisation. Cross-hybridisation assays KHATRI-CHHETRI, H.B., WAEYENBERGE, L., SPIRIDONOV, between the new species and S. monticolum, S. S., MANANDHAR, H.K. & MOENS, M. 2011. tielingense, S. feltiae and S. hebeiense yielded no Steinernema everestense n. sp. (Rhabditida: progeny. In the self-cross controls, offspring was Steinernematidae), a new species of produced normally. entomopathogenic nematode from Pakhribas, Bionomics. Steinernema changbaiense sp. n. has Dhankuta, Nepal. Nematology 13: 443-462. a life cycle comparable to that of other described LIU, J. & BERRY, R.E. 1996. Steinernema oregonensis n. sp. species of the ‘kraussei-feltiae-oregonense’ group. (Rhabditida: Steinernematidae) from Oregon, USA. Galleria mellonella larvae were killed after 2 days Fundamental and Applied Nematology 19: 375-380. and first generation adults developed after 4 to 5 LEWIS, E.E. & P. S. GREWAL. 2005. Interactions with plant days at 25°C. Second generation adults developed parasitic nematodes. In: Nematodes as Biocontrol Agents. after 10 days. It usually took more than 12 days for (P.S. Grewal, R.-U. Ehlers & D.I. Shapiro-Ilan Eds). pp. infective juveniles to emerge from the insect 349-362. CABI, New York, USA. MA, J., CHEN, S.L., ZOU, Y.X., LI, H.X., HAN, R.C., DE cadavers. CLERCQ, P. & MOENS, M. 2010. Natural occurrence of ACKNOWLEDGEMENT entomopathogenic nematodes in north China. Russian Journal of Nematology 18: 117-126. This study was supported by the project ENCHIBE MA, J., CHEN, S.L., LI, H.X., HAN, R.C., KHATRI- (No. ZEIN2007 PR339) from VLIR, Belgium and by CHHETRI, H.B., DE CLERCQ, P. & MOENS, M. 2012a. the China Agriculture Research System (CARS-11-B- A new entomopathogenic nematode, Steinernema 08) and A2012120103. We thank Lieven Waeyenberge tielingense n. sp. Rhabditida: Steinernematidae), from for advice and Ms Nancy de Sutter and Jinyue Wang for north China. Nematology 14: 321-338. technical assistance. MA, M., CHEN, S., DE CLERCQ, P., WAEYENBERGE, L., HAN, R. & MOENS, M. 2012b. A new REFERENCES entomopathogenic nematode, Steinernema xinbinense n. sp. (Nematoda: Steinernematidae), BEDDING, R.A. & AKHURST, R.J. 1975. A simple from north China. Nematology 14: 723-739. technique for the detection of insect-parasitic MRÁČEK, Z. 1994. Steinernema kraussei (Steiner, 1923) nematodes in soil. Nematologica 21: 109-110. (Nematoda: Rhabditida: Steinernematidae): CHEN, S., LI, X., YAN, A., SPIRIDONOV, S.E. & MOENS, redescription of its topotype from Westphalia. Folia M. 2006. A new entomopathogenic nematode, Parasitologica 41: 59-64. Steinernema hebeiense sp. n. (Rhabditida: MRÁČEK, Z., STURHAN, D. & REID, A. 2003. Steinernema Steinernematidae), from North China. Nematology 8: weiseri sp. n. (Rhabditida: Steinernematidae), a new 563-574. entomopathogenic nematode from Europe. Systematic EHLERS, R.-U. 1996. Current and future use of nematodes Parasitology 56: 37-47. in biocontrol: Practice and commercial aspects in MRÁČEK, Z., LIU, Q.Z. & NGUYEN, K.B. 2009. regard to regulatory policies. Biocontrol Science and Steinernema xueshanense n. sp. (Rhabditida, Technology 6: 303-316. Steinernematidae), a new species of HALL, T.A. 1999. BioEdit: a user-friendly biological sequence entomopathogenic nematode from the province of alignment editor and analysis program for Windows Yunnan, southeast Tibetan Mts., China. Journal of 95/98/NT. Nucleic Acids Symposium Series 41: 95-98. Invertebrate Pathology 102: 69-78. HOMINICK, W.M. 2002. Biogeography. In: NGUYEN, K.B. 2007. Methodology, morphology and Entomopathogenic nematology (R. Gaugler Ed.). pp. identification. In: Entomopathogenic nematodes: 115-143. CABI Publishing, Wallingford, UK. systematics, phylogeny and bacterial symbionts. (K.B.
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Nguyen & D.J. Hunt Eds). pp. 63-84. Brill, Leiden, case studies in cotton, turf and citrus. In: the Netherlands. Entomopathogenic nematology (R. Gaugler Ed.). pp. NGUYEN, K.B. 2011. Morphology and taxonomy of 333-356. CABI publishing, Wallingford, UK, entomopathogenic nematodes. SPIRIDONOV, S. E., KRASOMIL-OSTERFELD, K. & MOENS,
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 (730- 790) µm, расстоянием от головного конца до экскреторной поры 61 (58-68) µm, длиной хвостового конца 77 (71-85) µm, 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) µm и рулька длиной 40 (33-46) µm. 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.
112