Entomopathogenic nematodes of the families Steinernematidae Travassos, 1927 and Heterorhabditidae Poinar, 1976 are obligate lethal pathogens of insects with a worldwide distribution (Adams et al., 2007; Hominicki, 2002; Spiridonov and Subbotin, 2016). These organisms are commercially produced and used as biological control of insect pest populations (Shapiro-Ilan et al., 2002).
The family Steinernematidae is divided into seven clades:
In the case of the Steinernematidae, detailed knowledge about the biodiversity and occurrence of this family is important not only scientifically. Since some entomopathogenic nematodes seem to be highly host-specific, every described species of
For light and scanning electron microscope observations, different life stages of
Reproductive isolation of
DNA was extracted from three single virgin first-generation females of nematodes using a DNeasy Blood and Tissue Kit (Qiagen, Germany). PCR amplification of the internal transcribed spacer (ITS) region of rDNA, the D2D3 region of 28 S rDNA, and the mitochondrial
Details on taxa used in the molecular analyses.
GeneBank accession no. | ||||
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Species | Isolate name/geographic origin | ITS rDNA | 28S rDNA |
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B1, England | AF331899 | ||
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The Netherlands | AY171298 | ||
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China | DQ375757 | ||
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Serbia | AF331904 | ||
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Yugoslavia | AF121048 | ||
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OB, Cameroon | JX985267 | ||
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Russia | AY171282 | ||
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Tibet, China | EF431959 | EF520284 | |
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141-C, South Africa | EU740970 | GU004534 | |
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Costa Rica | EF187017 | ||
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Bodega Bay, USA | AF331906 | ||
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SN, USA | AF121050 | ||
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3, Portugal | JQ423217 | ||
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NC, USA | AF331908 | ||
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NJ, USA | AF122015 | ||
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G6, China | DQ105794 | ||
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VK-2013, India | KC252604 | ||
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Sardinia, Italy | EU421129 | EU421130 | |
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Monsanto, USA | GU569051 | GU569068 | |
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73, USA | AY171265 | ||
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Westphalia, Germany | AY230175 | AF331896 | AY943990 |
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Altai 35, Russia | AY171270 | ||
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Nash, UK | AY230176 | ||
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Italy | AY230174 | ||
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Iceland | AY171248 | ||
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20F, Portugal | JN683825 | ||
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D, Switzerland | AY171258 | ||
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Russia | AY171264 | ||
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HkHm22, Japan | AB243442 | ||
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Skr-LUB, Lublin, Poland | KY819012 | ||
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B2, UK | AY230161 | ||
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20F, Portugal | JN683829 | ||
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Quebec, Canada | GU569053 | ||
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SKR S11-50, Poland | MW647848 | MW647849 | MW647850 |
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Hamakita, Japan | AB243440 | ||
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N22, Japan | AY943991 | ||
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Japan | AF331897 | ||
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AiAt199, Japan | AB243441 | ||
Species | Isolate name/geographic origin | GeneBank accession no. | ||
ITS rDNA | 28S rDNA |
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Korea, South Korea | AF122017 | EF439651 | |
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Mt. Chiri, South Korea | AY943994 | ||
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F2, South Africa | KP325084 | ||
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Oregon, USA | AF122019 | ||
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OS-10, USA | AF331891 | AY943995 | |
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Vietnam | AY355441 | GU569057 | |
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New Jersey, USA | AY172023 | ||
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Chile | FJ263673 | ||
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S16/019, Poland | MG543845 | MG547576 | MG547572 |
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B, Germany | AY171255 | ||
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B3, UK (type) | AY230162 | ||
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Texas, USA | EF152568 | EF152569 | |
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LFS65, China | GU994201 | GU994202 | |
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F, Germany | AY171268 | ||
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Turkey | GU569059 | GU569075 | |
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LFS8, China | JN171593 | ||
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LFS40, China | GU994202 | ||
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Yunnan, China | FJ666052 | FJ666053 | |
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N2 Bristol, USA | NC001328 | ||
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X03680 | X03680 |
LSID: 051B950B-081C-4FD9-A8C2-22E7106C29BE.
(Figures 1–8; Tables 1–6).
Body straight or slightly abdominally curved when heat-relaxed, tapering gradually from the base of esophagus to the anterior end and from anus to the distal end. Second-stage cuticle present shortly after leaving the host body, with six labial and four cephalic papillae, but lost in storage after a few days/weeks (depending on the temperature). Cephalic region continuous with body smooth, truncate-conical, with four cephalic papillae and prominent amphidal apertures (Figs 1A and 2B). Mouth and anus closed (Fig. 2B,F). Cuticle with prominent striation along almost the whole body (Fig. 2B,D,F). Lateral fields beginning as a single line close to the anterior end, increasing to eight ridges, posteriorly gradually reduced to four (anus level) and two (phasmid level) (Fig. 2D,F). Deirids not visible. Esophagus with narrow corpus, slightly swollen metacorpus, isthmus surrounded by nerve ring (Fig. 2A,C). Excretory pore in the middle between anterior end and basal bulb (Figs 1A and 2A). Hemizonid distinct, between nerve ring and esophagus base. Cardia present. Bacterial vesicle well developed, with visible rod-shaped bacteria (Fig. 2C). Tail conical, tapering gradually. Phasmids distinct, located 40% of tail length, posterior to anus. Hyaline portions comprising ca. 1/3 of tail length (Figs 1E and 2E,F).
Body C- or J-shaped when heat-relaxed. Cuticle with faint transverse striation visible in SEM (Fig. 3A–D). Lateral fields not observed. Cephalic region smooth, rounded, with four cephalic and six smaller labial papillae and slit-like amphid openings (Fig. 3A,B). Stoma shallow, funnel-shaped, cheilorhabdions prominent. Esophagus with cylindrical procorpus, slightly swollen metacorpus, and narrower isthmus surrounded by nerve ring located anteriorly to basal bulb. Excretory pore anterior to nerve ring, close to metacorpus (Figs 1B and 4A). Cardia prominent. Anterior deirids similar to genital papillae in shape and size (Fig. 3A). Posterior deirids usually located anteriorly, just before the first pair of genital papillae. Testis monorchic, reflexed. Spicule with two ribs and velum not reaching spicule tip (Figs 1F,G, 4E). Gubernaculum boat-shaped in lateral view, with ventrally curved manubrium (Figs 1H and 4F,G). Typically 23 genital papillae present, comprising 11 pairs and 1 single precloacal midventral. Additional papillae – if occur – usually before posterior deirids. Phasmid openings between ventral last pair of genital papillae. Tail terminus with mucron (Figs 1F, 3D and 4B).
Similar to first-generation male but shorter and more slender. Excretory pore located more posteriorly. Tail relatively longer, with prominent mucron (Table 1; Fig. 4C,D).
Morphometrics (in μm) of different developmental stages of
First generation | Second generation | |||||
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Males | Females | Males | Females | Infective juveniles | ||
Character | Holotype | Paratypes | Paratypes | Paratypes | Paratypes | Paratypes |
Body length [L] | 1,565.3 | 1,461 ± 22.1 (1,205.7–1,635.3) | 4,628 ± 46.4 (4,244.0–5,014.0) | 946 ± 13.8 (817.5–1,093.8) | 2,120 ± 51.5 (1,640.6–2,753.2) | 843.0 ± 13.9 (708.2–964.5) |
Greatest body width [W] | 143.0 | 155.1 ± 2.7 (123.8–177.7) | 209.6 ± 3.4 (181.3–261.3) | 70.1 ± 1.1 (54.9–79.5) | 126.6 ± 3.3 (88.9–146.6) | 27.4 ± 0.5 (23.0–31.9) |
Anterior end to excretory pore [EP] | 88.4 | 80.4 ± 1.5 (63.5–92.4) | 84.4 ± 2.1 (61.4–101.6) | 69.8 ± 1.5 (59.0–84.6) | 72.1 ± 1.3 (57.3–88.4) | 55.9 ± 0.8 (44.4–64.2) |
Anterior end to nerve ring [NR] | 121.9 | 126.0 ± 1.5 (112.0–138.1) | 146.7 ± 1.2 (132.5–157.6) | 97.7 ± 0.8 (86.4–105.8) | 113.7 ± 1.1 (102.5–124.6) | 102.6 ± 1.4 (82.6–117.9) |
Anterior end to esophagus [ES] | 155.9 | 157.2 ± 1.1 (147.6–169.6) | 184.7 ± 1.0 (173.2–193.9) | 120.5 ± 1.1 (109.0–128.7) | 145.8 ± 1.4 (130.3–158.5) | 138.4 ± 0.5 (122.5–150.5) |
Testis reflection | 461.6 | 452.1 ± 9.5 (359.3–537.7) | – | 202.6 ± 13.2 (84.9–379.2) | – | – |
Tail length [T] | 45.1 | 41.2 ± 0.5 (35.4–45.5) | 46.7 ± 1.6 (32.4–60.9) | 42.3 ± 1.0 (31.7–52.1) | 57.5 ± 1.4 (46.5–72.1) | 75.2 ± 1.1 (64.4–86.4) |
Anal body diameter [ABW] | 50.1 | 54.1 ± 0.6 (49.9–59.2) | 94.0 ± 2.9 (62.1–121.8) | 36.8 ± 0.4 (30.7–40.8) | 54.4 ± 1.5 (43.1–70.7) | 17.3 ± 0.4 (14.6–23.8) |
Spicule length [SL] | 64.2 | 59.8±0.5 (52.6–65.3) | – | 51.2 ± 0.9 (42.5–60.2) | – | – |
Gubernaculum length [GL] | 39.2 | 43.6 ± 0.5 (39.1–50.2) | – | 30.4 ± 0.6 (24.2–39.5) | – | – |
a [L/W] | 10.9 | 9.5 ± 0.1 (8.5–11.0) | 22.2 ± 0.4 (17.4–24.7) | 13.6 ± 0.2 (12.0–16.4) | 16.9 ± 0.4 (14.1–23.2) | 30.9 ± 0.3 (27.2–33.8) |
b [L/ES] | 10.4 | 9.3 ± 0.1 (8.0–10.2) | 25.1 ± 0.2 (23.5–27.2) | 7.9 ± 0.1 (7.2–9.4) | 14.5±0.3 (12.0–18.1) | 6.1 ± 0.1 (5.5–6.9) |
c [L/T] | 34.7 | 35.6 ± 0.5 (31.2–41.9) | 102.0 ± 3.8 (75.4–140.3) | 22.7 ± 0.6 (17.2–28.5) | 37.3 ± 1.1 (24.6–50.1) | 11.2 ± 0.1 (10.5–13.2) |
Hyaline% [(H/T) × 100] | – | – | – | – | – | 33.6 ± 3.9 (22.7–39.9) |
D% [(EP/ES) × 100] | 56.7 | 51.2 ± 0.9 (42.1–59.3) | 45.7 ± 1.1 (35.5–54.2) | 58.0±1.2 (48.1–71.9) | 49.5 ± 0.9 (36.2–58.1) | 40.4 ± 0.4 (35.8–44.8) |
E% [(EP/T) × 100] | 196.0 | 195.8 ± 4.0 (160.2–240.9) | 186.0 ± 7.9 (128.1–266.8) | 167.4 ± 1.2 (128.2–222.8) | 127.2 ± 3.9 (101.4–163.9) | 74.4 ± 0.9 (62.6–85.8) |
SW% [(SL/ABW) × 100] | 128.1 | 110.9 ± 1.5 (97.0–126.9) | – | 139.6 ± 3.2 (105.1–171.4) | – | – |
GS% [(GL/SL) × 100] | 61.1 | 79.1 ± 1.2 (60.8–82.8) | – | 59.6 ± 1.1 (49.5–68.6) | – | – |
V% [(Vulva – anterior end/L) × 100] | – | – | 53.7 ± 0.3 (49.0–56.8) | – | 54.3 ± 0.8 (39.3–59.1) | – |
Note: – = character absent.
Body C-shaped when heat-relaxed and fixed. Cuticle smooth when observed in a light microscope, with faint striation in SEM (Fig. 5B,D,F). Lateral fields not observed. Deirids inconspicuous, difficult to observe even under SEM. Labial region rounded, continuous with the body. Six labial and four cephalic papillae (Fig. 5B). Slit-like amphidial apertures. Cheliorhabdions large, sclerotized. Stoma prominent. Esophagus with cylindrical procorpus, swollen metacorpus, and distinct isthmus. Excretory pore in mid-esophagus region. Nerve ring just anterior to basal bulb (Fig. 5A). Cardia prominent. Gonads amphidelphic, reflexed. Vulva in the form of transverse slit located slightly posterior to mid-body (Table 1). Vulval lips slightly protruding, asymmetrical, with larger posterior lip (Figs 1D and 5C,D). Tail length shorter than body anal diameter, with slight post-anal swelling. Tail terminus with mucron (Figs 1C and 5E,F).
Similar to first-generation female but smaller. Vulva more protruding, with distinct asymmetry between lips. Tail with mucron, but without pronounced post-anal swelling (Table 1).
Mating attempts were observed between
The new species was characterized by analysis of the morphology and morphometrics of IJs and adults (Table 1). IJs have a body length of 843 μm (708–965), a body diameter of 27 (23–32) μm, and a tail length of 75 (64–86) μm. The distance from the anterior end to the excretory pore is 56 (44–64) μm and to the esophagus base is 138 (123–151) μm, D% = 40 (36–45), E% = 74 (63–86). The lateral field in the mid-body region has 8 ridges, and the hyaline part of the tail occupies ~1/3 of its length. The first-generation male is characterized by a spicule length of 60 (53–65) μm and by a gubernaculum length of 44 (39–50) μm. The spicule manubrium is almost as long as it is wide, the shaft is short, and the velum expands from the calomus to the end of the ventral rib. The tail of both generation males is mucronated. The first- and second-generation females of
The species belongs to the
Comparative morphometrics of third-stage infective juveniles of
Morphometric character a | ||||||||||||
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Species | L | W | EP | NR | ES | T | a | b | c | D% | E% | Reference |
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589 (424–662) | 26 (22–31) | 46 (42–50) | 76 (70–84) | 111 (106–120) | 50 (44–59) | 22.5 (19–25) | 5.3 (4.9–5.9) | 11.7 (10–13) | 41 (38–44) | 92 (NA) | Mamiya, (1988) |
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658 (610–710) | 26 (23–28) | 48 (43–51) | 78 (73–83) | 107 (100–111) | 66 (63–71) | 26 (24–28) | 6.2 (5.7–6.7) | 10 (9.4–11) | 45 (40–50) | 72 (65–80) | Chen et al. (2006) |
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670 (631–728) | 33 (31–38) | 25 (20–30) | 54 (46–69) | 94 (81–103) | 54 (51–59) | 20 (17–23) | 6.1 (7.1–7.9) | 12 (11–13) | 42 (25–50) | 44 (35–56) | Uribe–Lorío et al. (2007) |
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694 (635–744) | 30 (28–31) | 51 (46–53) | 86 (75–90) | 116 (109–125) | 73 (65–78) | 24 (21–25) | 6.1 (5–7) | 9.7 (8–11) | 44 (40–47) | 71 (65–78) | Ma et al. (2012) |
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711 (625–820) | 23 (20–28) | 60 (53–65) | NA | 123 (115–135) | 68 (60–73) | 31 (25–34) | 5.7 (4.9–6.4) | 10.5 (9.0–11.7) | 48 (46–50) | 88 (NA) | Spiridonov et al. (2004b) |
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737 (673–796) | 25 (22–28) | 52 (47–58) | 80 (74–86) | 110 (101–121) | 67 (61–73) | 29 (27–33) | 6.7 (6.2–7.4) | 11 (10–12) | 48 (43–57) | 79 (70–86) | Malan et al. (2016) |
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740 (586–828) | 25 (24–29) | 57 (43–65) | 84 (72–92) | 113 (95–119) | 60 (49–68) | 29 (25–33) | 6.6 (5.7–7.2) | 12 (10–14) | 51 (44–55) | 95 (NA) | Mráček et al. (2003) |
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753 (704–784) | 35 (30–40) | 52 (46–54) | 91 (78–97) | 127 (120–138) | 81 (76–89) | 22 (19–25) | 5.9 (5.6–6.3) | 9.3 (8.7–10.2) | 40 (36–44) | 62 (56–70) | Phan et al. (2001) |
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754 (623–849) | 26 (23–28) | 56 (49–64) | 98 (83–108) | 125 (118–137) | 71 (63–81) | 30 (25–34) | 6.0 (5.1–7.1) | 15 (13–14) | 44 (39–58) | 110 (85–132) | Stokwe et al. (2011) |
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756 (732–796) | 30 (29–34) | 59 (52–62) | 92 (84–102) | 115 (111–120) | 73 (60–79) | 25 (22–27) | 6.5 (6.2–7.0) | 10 (9.6–12.5) | 51 (46–53) | 81 (76–88) | Nguyen et al. (2007) |
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812 (770–835) | 33 (33–35) | 59 (55–60) | 90 (83–95) | 119 (115–123) | 73 (68–75) | 24 (23–26) | 6.8 (6.6–7.2) | 11 (10–12) | 47 (45–50) | 77 (73–86) | Qiu et al. (2005) |
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– |
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843 (727–909) | 30 (26–35) | 62 (59–65) | 87 (72–97) | 125 (110–138) | 73 (60–80) | 28 (24–34) | 6.8 (6.1–7.2) | 12 (10–14) | 49 (46–53) | 81 (76–91) | Nguyen et al., (2008) |
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849 (766–928) | 29 (22–32) | 63 (58–67) | 113 (108–117) | 136 (130–143) | 86 (81–89) | 30 (27–34) | 6.4 (5.8–6.8) | 10 (9.4–11) | 46 (44–50) | 74 (67–81) | Nguyen et al., (2007) |
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860 (670–975) | 30 (26–35) | 62 (51–73) | 96 (75–109) | 121 (100–141) | 75 (63–86) | 29 (23–33) | 7.3 (6.3–7.7) | 11.4 (9.9–13.1) | 50 (46–56) | – | Sturhan et al. (2005) |
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860 (768–929) | 30 (29–33) | 67 (60–72) | 91 (81–96) | 135 (130–143) | 87 (80–92) | 28 (26–32) | 6.4 (5.8–7.0) | 9.9 (9.0–11) | 50 (46–52) | 78 (70–90) | Mrácˇek et al. (2009) |
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866 (767–969) | 31 (27–35) | 63 (59–68) | 102 (94–108) | 138 (119–148) | 81 (76–89) | 28 (24–32) | 6.3 (5.6–6.9) | 11 (8.8–12) | 46 (42–49) | 77 (68–83) | Tarasco et al. (2008) |
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909 (834–988) | 31 (28–33) | 61 (54–69) | 96 (89–104) | 125 (114–133) | 83 (72–91) | 29.5 (27–31) | 7.3 (6.7–7.9) | 11 (9.7–11.9) | 49 (44–56) | 73 (68–84) | Yoshida (2004) |
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915 (824–979) | 35 (32–38) | 69 (64–73) | 98 (90–105) | 128 (120–135) | 81 (74–85) | 26 (23–28) | 7 (6–8) | 11 (9–13) | 55 (47–61) | 88 (85–94) | Ma et al. (2012) |
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951 (797–1,102) | 33 (30–36) | 63 (50–66) | 105 (99–111) | 134 (119–145) | 79 (63–86) | 29 (NA) | 7.1 (NA) | 12.1 (NA) | 47 (NA) | 80 (NA) | Nguyen et al. (2007) |
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980 (820–1,110) | 34 (28–38) | 66 (60–72) | NA | 132 (116–148) | 70 (64–78) | 30 (24–37) | 7.6 (6–8) | 14 (12–16) | 50 (40–60) | 100 (90–110) | Liu and Berry (1996) |
Notes: Measurements are given in μm and in the form: mean (range). aabbreviations as in Table 1, NA = data not available.
First-generation males of S
Comparative morphometrics of first-generation males of
Morphometric character a | ||||||||
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Species | SL | GL | W | D% | SW% | GS% | MUC b | n |
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90 (85–100) | 64 (58–68) | 131 (115–150) | 56 (52–61) | 180 (140–200) | 71 (65–77) | P | 20 |
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66 (60–71) | 39 (32–45) | 137 (73–204) | 64 (50–85) | 115 (92–144) | 71 (61–85) | P | 20 |
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65 (57–80) | 44 (32–59) | 103 (87–113) | 58 (47–67) | 198 (156–233) | 68 (48–89) | P | 20 |
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92 (81–101) | 46 (41–51) | 128 (89–157) | 53 (51–66) | 160 (150–170) | 49 (45–55) | A | 19 |
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70 (65–77) | 41 (34–47) | 75 (60–90) | 60 (51–64) | 113 (99–130) | 59 (52–61) | P | 25 |
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57 (51–63) | 46 (38–50) | 86 (74–98) | 51 (48–59) | 140 (120–170) | 80 (60–90) | A | 20 |
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66 (64–67) | 44 (43–46) | 137 (73–204) | 62 (59–65) | 139 (120–162) | 67 (64–69) | A | 20 |
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64 (55–70) | 54 (45–60) | 115 (98–135) | 64 (53–83) | 145 (NA) | 84 (NA) | A | 12 |
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49 (42–53) | 33 (29–37) | 128 (110–144) | 53 (NA) | 110 (NA) | 67 (NA) | P | NA |
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63 (48–72) | 44 (39–60) | 97 (75–156) | 51 (42–59) | 150 (NA) | 70 (NA) | A | 20 |
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75 (67–89) | 53 (44–64) | 96 (82–111) | 40 (34–56) | 174 (154–200) | 71 (62–81) | P | 25 |
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66 (58–75) | 43 (30–55) | 82 (58–106) | 48 (38–57) | 215 (185–279) | 66 (46–81) | P | 20 |
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71 (65–73) | 56 (52–59) | 138 (105–161) | 73 (64–75) | 151 (NA) | 79 (NA) | A | 20 |
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77 (71–81) | 34 (30–40) | 119 (101–139) | 67 (45–85) | 170 (140–200) | 65 (55–75) | P | 19 |
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63 (58–80) | 40 (34–46) | 159 (120–225) | 49 (42–63) | 150 (120–160) | 60 (50–70) | P | 20 |
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51 (42–64) | 37 (30–43) | 65 (52–78) | 60 (45–63) | NA | NA | P | 26 |
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60 (55–66) | 45 (39–53) | 99 (81–116) | 67 (58–73) | 157 (127–203) | 75 (62–84) | A | 20 |
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88 (79–98) | 62 (49–70) | 129 (111–159) | 71 (64–78) | 191 (176–212) | 73 (59–82) | A | 20 |
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68 (62–72) | 53 (46–57) | 112 (84–138) | 49 (39–60) | 180 (150–240) | 80 (70–85) | A | 20 |
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56 (49–62) | 35 (30–41) | 103 (90–126) | 45 (41–50) | 137 (114–156) | 63 (54–72) | P | 20 |
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76 (66–91) | 49 (41–60) | 144 (97–159) | 80 (73–87) | 152 (93–172) | 64 (58–95) | A | 20 |
Notes: Measurements are given in μm and in the form: mean (range). aabbreviations as in Table 1. bMUC = mucron; P = present, A = absent, NA = data not available.
Natural host unknown. The nematode isolate S17-050 was obtained from sandy-loamy soil samples collected in eastern Poland (51°46’55”N 22°42’35”, 147 m a.s.l.) in 2017. The soil samples were collected in a mixed forest from 0 to 20 cm depth. Nematodes were isolated using a modified live trap method (Bedding and Akhurst, 1975) with the use of
Holotype male, paratype males, paratype infective juveniles, paratype females, and second-generation paratype males and females were deposited in the nematode collection of the Museum and Institute of Zoology, Polish Academy of Sciences, Wilcza 64, Warsaw, Poland (see Table S2 for deposition numbers).
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MIZ PAN WARSZAWA 2-2021/15 |
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MIZ PAN WARSZAWA 2-2021/16 |
Slide no. 17 – |
MIZ PAN WARSZAWA 2-2021/17 |
Slide no. 18 – |
MIZ PAN WARSZAWA 2-2021/18 |
Slide no. 19 – |
MIZ PAN WARSZAWA 2-2021/19 |
As it is known that the molecular diversity in the group of nematodes assigned as
Percentage of similarity (upper triangle) and genetic distance measured by the number of nucleotide substitutions (lower triangle) in the sequences of ITS rDNA regions of
Species | Acc. no. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 |
|
MW078536 | – | 97.0 | 97.7 | 97.7 | 97.5 | 97.5 | 97.2 | 97.7 | 97.5 | 96.6 | 96.5 | 96.3 | 96.0 | 95.0 | 94.9 | 94.5 |
2 |
|
AY230174 | 21 | – | 98.1 | 98.1 | 97.9 | 97.9 | 98.1 | 97.2 | 95.7 | 98.1 | 98.2 | 97.5 | 97.5 | 95.4 | 95.3 | 94.9 |
3 |
|
AY171270 | 17 | 13 | – | 99.2 | 98.5 | 98.5 | 99.6 | 98.6 | 98.2 | 100 | 99.3 | 99.4 | 98.3 | 96.1 | 96.0 | 95.6 |
4 |
|
AY171248 | 16 | 12 | 5 | – | 99.0 | 99.0 | 99,0 | 99.0 | 97.3 | 99.2 | 98.9 | 98.6 | 97.6 | 95.7 | 95.6 | 95.2 |
5 |
|
KY819012 | 18 | 14 | 11 | 6 | – | 99.7 | 98.2 | 99.6 | 96.6 | 98.5 | 98.9 | 97.9 | 97.2 | 95.3 | 95.2 | 94.8 |
6 |
|
AY171264 | 18 | 14 | 11 | 6 | 2 | – | 98.2 | 99.9 | 86.6 | 98.5 | 99.2 | 97.9 | 96.9 | 95.3 | 95.2 | 94.8 |
7 |
|
AB243442 | 20 | 13 | 3 | 6 | 13 | 13 | – | 98.2 | 97.8 | 99.6 | 98.9 | 99.0 | 98.3 | 96.3 | 96.1 | 95.7 |
8 |
|
AY230175 | 17 | 14 | 10 | 6 | 3 | 1 | 13 | – | 96.8 | 98.6 | 99.3 | 98.1 | 96.9 | 95.5 | 95.3 | 94.9 |
9 |
|
JN683825 | 17 | 14 | 1 | 6 | 12 | 12 | 4 | 11 | – | 99.2 | 98.5 | 99.5 | 98.3 | 96.1 | 96.0 | 95.6 |
10 |
|
AY171258 | 17 | 13 | 0 | 5 | 11 | 11 | 3 | 10 | 1 | – | 99.3 | 98.6 | 98.3 | 96.1 | 96.0 | 95.6 |
11 |
|
AY230176 | 18 | 12 | 5 | 7 | 8 | 6 | 8 | 5 | 6 | 5 | – | 98.0 | 97.6 | 95.6 | 95.5 | 95.0 |
12 |
|
MW647848 | 20 | 16 | 3 | 8 | 14 | 14 | 6 | 13 | 4 | 3 | 8 | – | 97.8 | 95.6 | 95.5 | 95.0 |
13 |
|
AY230161 | 28 | 15 | 11 | 15 | 19 | 21 | 11 | 21 | 10 | 11 | 16 | 14 | – | 95.3 | 95.1 | 94.7 |
14 |
|
MG543845 | 34 | 30 | 26 | 28 | 32 | 32 | 25 | 31 | 24 | 26 | 30 | 29 | 31 | – | 99.9 | 99.4 |
15 |
|
AY171255 | 35 | 31 | 27 | 29 | 33 | 33 | 26 | 32 | 25 | 27 | 31 | 30 | 32 | 2 | – | 99.6 |
16 |
|
AY230162 | 37 | 33 | 29 | 31 | 35 | 35 | 28 | 34 | 26 | 29 | 32 | 32 | 34 | 3 | 2 | – |
Percentage of similarity (upper triangle) and genetic distance measured by the number of nucleotide substitutions (lower triangle) in the sequences of ITS rDNA of
Species | Acc. no. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 |
|
|
– |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
2 |
|
AY230174 |
|
– | 95.2 | 92.1 | 90.4 | 90.5 | 93.8 | 94.9 | 88.6 | 89.2 | 88.6 | 89.1 | 89.4 | 88.0 | 85.2 | 76.0 |
3 |
|
AY230162 |
|
31 | – | 92.0 | 90.4 | 90.2 | 92.6 | 94.6 | 88.8 | 89.4 | 89.2 | 88.9 | 89.6 | 88.0 | 84.8 | 75.8 |
4 |
|
EF431959 |
|
48 | 50 | – | 94.2 | 97.2 | 93.2 | 94.2 | 91.3 | 92.7 | 91.7 | 92.8 | 92.2 | 91.8 | 86.6 | 77.5 |
5 |
|
AF122019 |
|
56 | 59 | 33 | – | 93.5 | 92.0 | 92.1 | 89.9 | 91.5 | 89.7 | 91.2 | 90.4 | 90.4 | 86.8 | 76.6 |
6 |
|
FJ660052 |
|
59 | 63 | 17 | 41 | – | 91.3 | 92.1 | 90.3 | 91.2 | 89.0 | 91.2 | 89.8 | 89.9 | 85.6 | 76.9 |
7 |
|
GU994201 |
|
42 | 51 | 42 | 48 | 56 | – | 94.8 | 89.9 | 89.5 | 89.7 | 90.5 | 90.1 | 89.2 | 86.2 | 77.2 |
8 |
|
JN171593 |
|
34 | 36 | 35 | 47 | 50 | 34 | – | 90.8 | 90.8 | 90.2 | 91.5 | 91.0 | 90.6 | 86.6 | 77.0 |
9 |
|
AF121050 |
|
73 | 74 | 57 | 60 | 61 | 68 | 60 | – | 95.8 | 91.4 | 94.0 | 93.4 | 94.4 | 88.9 | 76.1 |
10 |
|
EU421129 |
|
70 | 71 | 50 | 52 | 55 | 71 | 62 | 28 | – | 91.8 | 95.5 | 93.9 | 85.2 | 88.6 | 76.9 |
11 |
|
AY171265 |
|
73 | 70 | 53 | 61 | 62 | 67 | 63 | 54 | 46 | – | 91.1 | 90.0 | 91.1 | 86.1 | 77.3 |
12 |
|
AY171268 |
|
69 | 73 | 48 | 52 | 55 | 62 | 55 | 39 | 32 | 51 | – | 94.5 | 96.6 | 89.0 | 77.0 |
13 |
|
KP325084 |
|
69 | 70 | 53 | 60 | 61 | 67 | 61 | 47 | 43 | 56 | 39 | – | 92.9 | 87.9 | 76.4 |
14 |
|
AB243441 |
|
78 | 80 | 57 | 59 | 61 | 73 | 63 | 38 | 34 | 46 | 24 | 44 | – | 88.7 | 77.2 |
15 |
|
DQ105794 |
|
97 | 102 | 92 | 84 | 90 | 93 | 90 | 76 | 80 | 82 | 77 | 81 | 74 | – | 74.9 |
16 |
|
AF122017 |
|
139 | 142 | 133 | 134 | 133 | 132 | 134 | 143 | 135 | 120 | 134 | 132 | 132 | 143 | – |
The highest sequence identity of the D2D3 region of the new species was 98.2%, corresponding to 15 nucleotide substitution, in respect to the analyzed
Percentage of similarity (upper triangle) and genetic distance measured by the number of nucleotide substitutions (lower triangle) in the sequences of D2D3 domain of 28 S rDNA of
Species | Acc. no. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 |
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– |
|
|
|
|
|
|
|
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|
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|
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2 |
|
AF331896 |
|
– | 99.8 | 99.8 | 97.2 | 98.2 | 98.9 | 97.6 | 98.2 | 97.9 | 97.4 | 98.1 | 96.2 | 92.6 |
3 |
|
GU569053 |
|
2 | – | 99.5 | 97.2 | 98.6 | 99.1 | 97.8 | 98.5 | 98.1 | 97.6 | 98.1 | 96.6 | 92.7 |
4 |
|
MW647849 |
|
2 | 4 | – | 97.2 | 98.2 | 98.9 | 97.7 | 98.2 | 97.8 | 97.3 | 97.0 | 95.7 | 92.9 |
5 |
|
MG547576 |
|
24 | 24 | 24 | – | 97.2 | 96.8 | 96.1 | 96.6 | 96.1 | 95.7 | 96.7 | 94.7 | 91.1 |
6 |
|
EF520284 |
|
14 | 12 | 14 | 24 | – | 98.4 | 97.5 | 98.1 | 97.7 | 97.2 | 97.8 | 96.4 | 92.7 |
7 |
|
AF331891 |
|
9 | 7 | 11 | 27 | 14 | – | 97.9 | 98.7 | 98.2 | 98.0 | 98.6 | 96.9 | 93.4 |
8 |
|
FJ666053 |
|
19 | 19 | 19 | 33 | 20 | 18 | – | 98.0 | 97.9 | 97.3 | 98.1 | 96.6 | 92.7 |
9 |
|
AF3311906 |
|
15 | 13 | 17 | 29 | 16 | 11 | 17 | – | 99.3 | 98.8 | 99.4 | 97.2 | 93.3 |
10 |
|
EU421130 |
|
18 | 16 | 18 | 32 | 19 | 14 | 17 | 5 | – | 98.6 | 98.9 | 97.2 | 93.3 |
11 |
|
GU569051 |
|
22 | 20 | 24 | 36 | 23 | 16 | 22 | 9 | 12 | – | 98.9 | 96.7 | 93.1 |
12 |
|
GU569059 |
|
16 | 16 | 18 | 28 | 19 | 12 | 16 | 5 | 8 | 9 | – | 97.1 | 93.7 |
13 |
|
EF152569 |
|
31 | 29 | 31 | 45 | 30 | 26 | 28 | 24 | 23 | 27 | 25 | – | 93.1 |
14 |
|
EF439651 |
|
53 | 53 | 54 | 69 | 56 | 49 | 56 | 51 | 52 | 54 | 49 | 53 | – |
The analysis of the
Percentage of similarity (upper triangle) and genetic distance measured by the number of nucleotide substitutions (lower triangle) in the sequences of
Species | Acc. no. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 |
|
|
– |
|
|
|
|
|
|
|
|
|
|
2 |
|
JN683829 |
|
– | 94.4 | 94.0 | 87.1 | 86.4 | 87.3 | 84.1 | 85.7 | 84.5 | 85.0 |
3 |
|
AY943990 |
|
32 | – | 95.6 | 87.8 | 86.8 | 86.7 | 85.0 | 86.2 | 85.2 | 85.4 |
4 |
|
MW647850 |
|
34 | 25 | – | 86.2 | 86.4 | 86.9 | 83.6 | 85.7 | 84.5 | 85.4 |
5 |
|
MG547572 |
|
73 | 68 | 78 | – | 84.7 | 85.4 | 83.6 | 83.5 | 82.9 | 84.7 |
6 |
|
AY943995 |
|
77 | 75 | 77 | 87 | – | 87.8 | 85.5 | 86.6 | 83.4 | 86.8 |
7 |
|
JQ423217 |
|
72 | 75 | 74 | 83 | 69 | – | 85.9 | 88.0 | 84.0 | 96.9 |
8 |
|
GU569068 |
|
90 | 85 | 93 | 93 | 82 | 80 | – | 85.0 | 84.7 | 84.1 |
9 |
|
GU569075 |
|
81 | 78 | 81 | 99 | 76 | 68 | 85 | – | 82.7 | 84.8 |
10 |
|
AY943991 |
|
88 | 84 | 88 | 97 | 94 | 91 | 87 | 98 | – | 84.5 |
11 |
|
AY943994 |
|
85 | 83 | 83 | 87 | 75 | 74 | 90 | 86 | 88 | – |
The alignment of the analyzed ITS sequences resulted in 870 positions, in which 266 positions were conserved, while 573 positions were variable, including 426 parsimony-informative and 136 singleton ones. The phylogenetic tree based on the ITS sequences shows that
In the case of the D2D3 region sequences, the alignment resulted in 899 positions, in which 611 positions were constant, while 247 positions were variable, including 130 parsimony-informative and 117 singleton ones. The phylogenetic tree based on D2D3 sequences shows that
The alignment in the
Sequence analysis of ITS rDNA and D2D3 expansion segment of 28 S rDNA have been proved useful for estimation of EPN species, by supporting morphological data (Nadler et al., 2006; Nguyen, 2007a, b; Stock et al., 2001). The phylogenetic trees based on ITS, D2D3, and
In turn, phylogenetic analyses of D2D3 have provided evidence that this region has fever ambiguously aligned positions than ITS rDNA, nevertheless it is too conservative to be informative of the relationships between closely related species of the
We also analyzed the sequence of the mitochondrial
In addition, the new species is well supported by the molecular diagnostic traits. Current evidence suggests that finding autapomorphies is useful in delimitation of nematode species for better indication of lineage independence (Adams et al., 2007). The sequence alignments of
In conclusion, the molecular analysis based on ITS rDNA, D2D3 of 28 S rDNA, and