Multigene Sequence-Based and Phenotypic Characterization Reveals the Occurrence of a Novel Entomopathogenic Nematode Species, Steinernema anantnagense n. sp.
This work is licensed under the Creative Commons Attribution 4.0 International License.
Entomopathogenic nematodes (EPNs) of the genus Steinernema (Travassos, 1927) are among the most important biological control agents used in agriculture to control insect pests. The nematodes of this genus are associated with entomopathogenic bacteria of the genus Xenorhabdus, carried in a specialized receptacle structure hosted in the digestive tract of the free-living infective juveniles (IJs) (Chaston et al., 2011). The infective juveniles search for insects, and once inside the hosts, they release their bacterial symbiont into the hemocoel. Bacteria kill the insect hosts via toxins, enzymes, and insecticidal compounds produced during bacteria multiplication, making these symbiotic organisms highly valuable pest management tools in sustainable and eco-friendly agriculture.
The genus Steinernema is wider in terms of the number of species, when compared to other known EPN genera, with more than 125 valid species that have been described from different geographical regions, except Antarctica (Hominick et al., 1996; Bhat et al., 2020a; Machado et al., 2022; Malan et al., 2023). On the basis of the sequences of the internal transcribed spacer (ITS) region of the rRNA, the species of the genus Steinernema have been phylogenetically divided into 12 multiple species clades: “Affine”, “Bicornutum”, “Cameroonense”, “Carpocapsae”, “Costaricense”, “Feltiae”, “Glaseri”, “Karii”, “Khoisanae”, “Kushidai”, “Longicaudum” and “Monticola”; and three monospecies clades: S. neocurtillae, S. unicornum, and S. rarum (Spiridonov et al., 2016). The “Kushidai” clade currently contains three species, which are: S. kushidai (Mamiya, 1988), S. akhursti (Qiu et al., 2005), and S. populi (Tian et al., 2022), which are characterized by the average size of the IJs (body length of 700–1000 µm).
In order to characterize the prevalence and distribution of EPNs in Indian soils, a survey was conducted in the Pir Panjal Range, in the Kashmir region of the Indian subcontinent. As a result of this survey, several nematode populations were recovered, including three isolates: Steiner_6, Steiner_7, and Steiner_8. Initial molecular characterization suggests that these three isolates are conspecific and represent a new species in the genus Steinernema. In this study, we describe Steinernema anantnagense n. sp. based on morphological observations and morphometric analysis using light microscopy (LM) and scanning electron microscopy (SEM), as well as molecular studies based on genetic sequences of ribosomal RNA and mitochondrial genes. Self-crossing and cross-hybridization experiments were also used. In addition, we isolated and characterized the symbiotic bacterium associated with S. anantnagense n. sp.
Materials and Methods
Nematode survey and collection
Steinernema anantnagense n. sp. Steiner_6, Steiner_7, and Steiner_8 nematodes were isolated from soil samples collected in the Pir Panjal Range of Kashmir Valley, India using Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) larvae as a bait insect. The isolates Steiner_6, Steiner_7, and Steiner_8 were collected in the Waghama area of Bijbehara Anantnag of the union territory of Jammu and Kashmir (GPS coordinates: 33.828914, 75.100091; 1606 m above the sea level) from soils around roots of willow, walnut, and apple intercrops, respectively, in areas adjoining district Anantnag, India. The insect cadavers recovered from soil samples were washed with ddH2O, sterilized with 0.1% NaOCl2, and nematode IJs were recovered from them by the White trap method (White, 1927). The IJs were sterilized with 0.1% NaOCl2 and stored in 250 mL tissue culture flasks in Biological Oxygen Demand incubator at 15°C. The new species has been registered in the ZooBank at urn:lsid:zoobank.org:pub:210D5242-2C15-437F-8D57-B00EECD98B85.
Morphological and morphometrical characterization
Different life stages of S. anantnagense n. sp. were obtained from infected Galleria mellonella larvae exposed to 100 IJs/insects in a 15 cm-diameter Petri dish lined with moistened Whatman number 1 filter paper and kept in the dark at 25°C. The wax moth larvae died within 48 h after inoculation. After they died, the insect cadavers were transferred to a modified White trap (Kaya & Stock, 1997) and incubated at 25°C until IJs emerged. First- and second-generation adult nematodes were obtained by dissecting infected G. mellonella cadavers in Ringer’s solution after 3–4 and 6–7 days of infection, respectively. Infective juveniles (IJs) were collected after they emerged from G. mellonella cadavers in White traps (White, 1927). Nematodes were killed with water at 60°C, fixed in 4% formalin solution (4 mL formaldehyde, 1 mL Glycerol, 95 mL ddH2O), dehydrated by the Seinhorst method (Seinhorst, 1959), and transferred to anhydrous glycerin. Nematodes were, after that, picked with a peacock feather and mounted on permanent glass slides with extra layers of paraffin wax to prevent the flattening of the nematodes as described (Bhat et al., 2022). Morphometric measurements were taken using the Nikon DS-L1 image acquisition software mounted on a phase-contrast microscope (Nikon Eclipse 50i) in μm. Light microscopy photographs were captured using a Nikon Eclipse 80i microscope (Olympus, Tokyo, Japan) equipped with differential interference contrast optics (DIC) and a Nikon Digital Sight DS-U1 camera. For the scanning electron microscopy (SEM), nematodes preserved in 4% formalin were re-hydrated in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried with liquid CO2, mounted on SEM stubs with a carbon tape, coated with gold in sputter coater, and observed with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany) (Abolafia, 2015). All micrographs were processed using Adobe® Photoshop® CS. Morphological characters of closely related species were taken from the original publications. The terminology used for the morphology of stoma and spicules follows the proposals by De Ley et al. (1995) and Abolafia and Peña-Santiago (2017a), respectively, and the terminology for pharynx follows the proposals by Bird and Bird (1991) and Baldwin and Perry (2004).
Self-crossing and cross-hybridization experiments
Self-crossing and cross-hybridization experiments were carried out using G. mellonella larvae hemolymph as described by Kaya & Stock, 1997 with minor modifications. To this end, drops of hemolymph obtained from surface-sterilized G. mellonella larvae were placed in sterile Petri dishes (35×10 mm). Hemolymph drops were treated with a small amount of phenylthiourea to prevent melanization. Then 40–60 surface-sterilized IJs (0.1% NaOCl for 30 min, followed by thrice rinse through sterile distilled water) were added to the hemolymph drops. Then, Petri dishes were wrapped in moistened tissue paper and kept in plastic bags at 25°C (room temperature). Petri dishes were observed daily for the presence of males and virgin females. Then, males and virgin females in the ratio of 3:3 were placed separately in fresh hemolymph drops and were crossed with adults of the opposite sex of the other species. Controls consist of crosses of identical isolates; some females were kept without males to check their virginity (n=30). The Petri dishes were observed daily for 15 days to determine the production of offspring. Experiments were conducted twice under the same conditions. The following species were crossed: Steinernema anantnagense n. sp. (Steiner_6, Steiner_7, and Steiner_8), S. ichnusae Sardinia, S. litorale Aichi, S. weiseri, S. akhursti Akh, S. citrae, S. cholashanense GARZE, S. feltiae P1, S. silvaticum, S. africanum RW14-M-C2a-3, and S. xueshanense DEQ.
Nematode molecular characterization and phylogenetic analyses
Genomic DNA was extracted from single virgin females as described (Bhat et al., 2023). Briefly, several virgin females were first washed with Ringer’s solution and then with PBS buffer and then individually transferred into sterile PCR tubes (0.2 mL), each containing 20 μL extraction buffer (17.6 μL nuclease-free dH2O, 2 μL 5X PCR buffer, 0.2 μL 1% Tween, and 0.2 μL proteinase K). The buffers with single virgin females were frozen at −20°C for 60 min or overnight and then immediately incubated in a water bath at 65°C for 1.2 h, followed by incubation at 95°C for 10 min. The lysates were cooled on ice and centrifuged at 6500 × g for 2 min. The following primers were used for PCR reactions: the internal transcribed spacer regions (ITS1-5.8S-ITS2) were amplified using primers 18S: (5′-TTGATTACGTCCCTGCCCTTT-3′) (forward), and 28S: (5′-TTTCACTCGCCGTTACTAAGG-3′) (reverse) (Vrain et al., 1992). The D2D3 regions of 28S rRNA were amplified using primers D2F: 5′-CCTTAG TAACGGCGAGTGAAA-3′ (forward) and 536: 5′-CAGC TATCCTGAGGAAAC-3′ (reverse) (Nadler et al., 2006). The 12S mitochondrial gene was amplified using the primers 505F: 5′-GTTCCAGAATAATCGGCTAGAC-3′ (forward) and 506R: 5′-TCTACTTTACTACAACTTACT CCCC-3′ (reverse) (Nadler et al., 2006) and the cytochrome oxidase subunit I (COI) gene was amplified using the universal primers LCO-1490 (5′-GGTCAACAAA TCATAAAGATATTGG-3′) (forward) and HCO-2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (reverse) (Folmer et al., 1994). The 25 µL PCR reactions consisted of 12.5 µL of Dream Taq Green PCR Master Mix (Thermo Scientific, USA), 0.5 µL of each forward and reverse primer at 10 µm, 2 µL of DNA extract and 9.5 µL of nuclease-free distilled water. The PCR reaction was performed using a thermocycler with the following settings: for ITS and D2-D3 markers, 1 cycle of 5 min at 94°C followed by 37 cycles of 30 sec at 94°C, 30 sec at 50°C, 1 min 30 s at 72°C, and by a single final elongation step at 72°C for 10 min. For the 12S marker, the PCR protocol included initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 45 s, followed by a final extension at 72°C for 15 min. For the COI marker, the PCR program was as follows: one cycle of 94°C for 2 min followed by 37 cycles of 94°C for 30 s, 51°C for 45 s, 72°C for 2 min, and a final extension at 72°C for 12 min. PCR was followed by electrophoresis (40 min, 130 V) of 10 μL of PCR products in a 1% TBA (tris–boric acid–EDTA) buffered agarose gel stained with SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, California, USA) (Bhat et al., 2020b). PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and sequenced using reverse and forward primers by Sanger sequencing (Bioserve Ltd., Hyderabad, India). Obtained sequences were manually edited and trimmed using BioEdit and deposited in the NCBI under the accession numbers: OQ40749, OQ407497, and OQ407497 for ITS; OQ407498, OQ407499, and OQ407500 for 28S; OQ404917, OQ407535, and OQ407536 for mtCOI; and OQ407491, OQ407492, and OQ407493 for mt12S.
To obtain genomic sequences of nematodes that belong to all the validly described species closely related to S. anantnagense n. sp., we searched the database of the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990). Steinernema monticola (AB698756, GU395647, AY943994, and AY944020) was used as an outgroup in ITS, D2D3, mtCOI, and mt12S based phylogenetic trees. The resulting sequences were aligned with MUSCLE (v3.8.31) (Edgar, 2004) and used to reconstruct phylogenetic relationships by the Maximum Likelihood method based on the following nucleotide substitution models: Hasegawa-Kishino-Yano model (HKY+G) (ITS), Tamura–Nei (TN93+G+I) (D2–D3 & COI), and Tamura 3-parameter (T92+G) (12S). To select the best substitution models, best-fit nucleotide substitution model analyses were conducted in MEGA 11 (Nei & Kumar, 2000; Tamura et al., 2021). The trees with the highest log likelihood are shown. The percentages of trees where the associated taxa clustered together are displayed next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor–Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. In some cases, a discrete Gamma distribution (+G) was used to model evolutionary rate differences among sites and the rate variation model allowed for some sites to be evolutionarily invariable (+I). The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Graphical representation and edition of the phylogenetic trees were performed with Interactive Tree of Life (v3.5.1) (Chevenet et al., 2006; Letunic and Bork, 2016).
Symbiotic relationships
The Xenorhabdus entomopathogenic bacteria associated with S. anantnagense n. sp. Steiner_6, Steiner_7, and Steiner_8 nematodes were isolated as described previously (Machado et al., 2018; Machado et al., 2019). Briefly, G. mellonella (n = 10) larvae were exposed to 100 nematode infective juveniles. Two to three days later, insect cadavers were surface–sterilized with 0.1% sodium hypochlorite solution and cut open with a sharp blade. Sterile polypropylene inoculation loops were inserted into the cadaver, and the loops were then streaked on LB agar plates and incubated at 28°C for 24–48 h. Xenorhabdus–like colonies were sub-cultured until monocultures were obtained. The strains were further sub-cultured and maintained on LB agar plates at 28°C. To establish their taxonomic identities, we reconstructed phylogenetic relationships based on whole genome sequences of the isolated bacteria and all the different species of the genus Xenorhabdus (Machado et al., 2023) and genomic sequences were obtained as described by Machado et al. (2021). Genome sequences were deposited in the National Centre for Biotechnology Information, and accession numbers are listed in Table S3. Phylogenetic relationships were reconstructed based on the assembled genomes and the genome sequences of all validly published species of the genus with publicly available genome sequences as described by Machado et al. (2023). Whole genome sequence similarities were calculated by the digital DNA-DNA hybridization (dDDH) method using the recommended formula 2 of the genome-to-genome distance calculator (GGDC) web service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) (Auch et al., 2010, 2020; Meier-Kolthoff et al., 2013, 2014).
Results and Discussion
Three populations of Steinernema nematodes, Steiner_6, Steiner_7 and, Steiner_8, were isolated from agricultural soils in the Bijbehara area of district Anantnag, India. Analysis of several taxonomically relevant markers show that Steiner_6, Steiner_7, and Steiner_8 are conspecific, belong to the “Feltiae–Kushidai–Monticola” superclade or “Kushidai” clade, are closely related to S. akhursti, S. populi, S. sangi, and S. kushidai, and represent a new species, for which we propose the name Steinernema anantnagense n. sp. To describe this new species, we compared this species with other closely related species at the molecular and morphological levels and conducted cross-hybridization and self-crossing experiments. As all three populations are identical at the molecular level, we selected Steiner_7 for in-depth morphological and morphometrical identification.
Morphometrics of the IJs and adult generations of Steinernema anantnagense n. sp. (Steiner_7). All characters are in µm (except n, ratios and percentages) and given as mean ± s.d. (range).
Characters
First Generation
Second Generation
Infective Juveniles (paratypes)
Male (holotype)
Male (paratypes)
Female (paratypes)
Male (paratypes)
Female (paratypes)
n
1
20
20
20
20
20
Body length (L)
1279
1618 ± 246 (1223–1899)
3765 ± 441 (2327–4872)
1068 ± 76 (899–1168)
2081 ± 200 (1761–2437)
789 ± 35 (749–834)
a (L/BD)
6.6
8.3 ± 1.1 (6.4–9.8)
23 ± 1.2 (17–29)
19 ± 2.6 (15.1–25.3)
14.7 ± 1.5 (12.1–17.4)
22 ± 1.9 (19–24)
b (L/NL)
7.0
9.2 ± 1.4 (7.0–11.3)
17.0 ± 2.4 (13–21)
7.1 ± 0.6 (5.9–8.2)
11.0 ± 1.0 (9.6–13.5)
6.0 ± 0.4 (5.5–6.7)
c (L/T)
35
49 ± 9.0 (34–64)
100 ± 18 (61–122)
43 ± 5.8 (31–52)
54 ± 6.6 (43–67)
13.8 ± 1.8 (12.2–16.4)
c’ (T/ABW)
1.2
1.1 ± 0.2 (0.8–1.6)
0.7 ± 0.1 (0.5–0.8)
0.9 ± 0.2 (0.6–1.4)
1.0 ± 0.1 (0.8–1.3)
1.8 ± 0.2 (1.6–2.1)
V (VA/L×100)
–
53 ± 1.7 (50–58)
–
53 ± 1.7 (50–57)
–
Max. body diam. (MBD)
193
194 ±11.3 (167–211)
434 ± 30 (314–409)
57 ± 7.3 (42–63)
143 ± 16 (123–173)
37 ± 3.6 (32–42)
Lip region width
10.2
10.1 ± 1.9 (8.2–12.2)
14.4 ± 1.9 (11.1–18.3)
8.4 ± 1.6 (6.1–12.1)
11.4 ± 1.7 (8.9–14.6)
4.6 ± 2.0 (3.7–5.8)
Stoma length
12.3
13.8 ± 1.6 (10.1–16.9)
16.4 ± 2.4 (12.4–22.1)
11.7 ± 1.1 (10.1–14.1)
14.1 ± 2.3 (11.1–18.1)
10.7 ± 2.0 (8.6–13.8)
Procorpus length
66
57 ± 4.1 (51–66)
65 ± 6.0 (55–76)
47 ± 3.9 (41–53)
54 ± 3.8 (49–65)
43 ± 5.4 (37–52)
Metacorpus length
33
34 ± 1.7 (30–36)
44 ± 4.6 (38–50)
30 ± 2.4 (25–35)
37 ± 2.4 (33–43)
26 ± 2.7 (23–29)
Isthmus length
40
42 ± 4.0 (37–50)
53 ± 6.4 (45–68)
37 ± 1.5 (35–40)
49 ± 3.7 (43–55)
36 ± 2.8 (31–39)
Bulb length (BL)
31
30 ± 2.2 (25–35)
43 ± 4.5 (33–50)
25 ± 2.7 (21–30)
35 ± 2.9 (30–39)
17 ± 1.8 (16–20)
Bulb width (EBW)
26
25 ± 1.6 (21–27)
35 ± 3.5 (27–41)
21 ± 1.9 (18–25)
28 ± 2.7 (25–33)
11 ± 1.7 (9.1–13.9)
Pharynx length (PL)
170
162 ± 5.9 (150–172)
206 ± 14 (182–232)
139 ± 5.0 (129–146)
175 ± 6.4 (164–191)
121 ± 8.0 (109–133)
Nerve ring – ant. end (NR)
109
112 ± 6.8 (103–129)
165 ± 12.1 (143–182)
91 ± 7.0 (75–104)
109 ± 11.9 (88–127)
63 ± 8.4 (54–71)
Excretory pore – ant. end (EP)
114
111 ± 10 (88–124)
112 ± 11.9 (92–140)
80 ± 9.2 (62–98)
92 ± 8.1 (82–112)
55 ± 6.7 (45–62)
Width at excretory pore (WEP)
58
61 ± 3.4 (49–65)
137 ± 11.3 (123–170)
35 ± 3.6 (30–42)
60 ± 6.7 (51–74)
22 ± 2.4 (19–25)
Neck length (stoma+pharynx, NL)
182
176 ± 6.4 (165–185)
223 ± 14 (196–255)
151 ± 5.0 (142–159)
189 ± 6.5 (177–204)
132 ± 8.3 (120–143)
Body width at neck base
79
86 ± 5.8 (76–96)
217 ± 23 (195–264)
41 ± 3.9 (36–50)
95 ± 8.5 (82–107)
29 ± 2.9 (24–32)
Testis reflexion
630
631 ± 37 (576–689)
–
522 ± 32 (481–568)
–
–
Vagina length
–
–
31 ± 3.1 (24–36)
–
24 ± 3.3 (19–30)
–
Body width at vulva
–
–
386 ± 53 (309–498)
–
164 ± 25 (124–193)
–
Vulva – ant. end (VA)
–
–
1989 ± 214 (1247–2252)
–
1105 ± 101 (961–1295)
–
Vulva – post. end (VP)
–
–
1776 ± 242 (1080–2038)
–
976 ± 110 (801–1161)
–
Rectum length
–
–
35 ± 4.0 (29–44)
–
21 ± 2.5 (17–24)
13.8 ± 1.9 (11–16)
Anal body diam. (ABD)
31
32 ± 3.9 (25–36)
86 ± 7.1 (77–110)
29 ± 4.1 (21–35)
41 ± 6.8 (31–53)
20 ± 1.7 (18–22)
Tail length (T)
37
34 ± 2.4 (29–39)
38 ± 4.2 (32–49)
26 ± 3.9 (21–35)
49 ± 3.9 (38–53)
58 ± 6.7 (49–66)
Hyaline part of tail (H)
–
–
–
–
–
16.3 ± 4.1 (11.8–23.7)
Spicule length (SL)
66
64 ± 4.6 (56–70)
–
44 ± 2.6 (40–49)
–
–
Gubernaculum length (GL)
34
36 ± 3.8 (31–43)
–
25 ± 2.3 (21–29)
–
–
Stoma length/lip region width
1.2
1.4 ± 0.2 (0.9–1.8)
1.2 ± 0.2 (0.9–1.8)
1.4 ± 0.3 (0.9–1.9)
1.3 ± 0.3 (0.9–1.8)
2.4 ± 0.5 (1.6–3.0)
Nerve ring % (NR/NL×100)
60
64 ± 5.3 (56–78)
74 ± 7.1 (64–86)
61 ± 4.6 (52–68)
58 ± 6.6 (46–67)
48 ± 7.8 (38–58)
Excretory pore % (EP/NL×100)
62
63 ± 6.1 (50–74)
50 ± 4.6 (42–60)
53 ± 6.6 (41–68)
49 ± 4.1 (43–58)
42 ± 4.4 (35–48)
Rectum% (R/ABD×100)
–
–
0.4 ± 0.1 (0.3–0.6)
0.5 ± 0.1 (0.3–0.8)
0.7 ± 0.1 (0.6–0.9)
D% (EP /NL×100)
62
63 ± 6.1 (49–74)
50 ± 4.9 (43–60)
53 ± 6.6 (41–68)
49 ± 4.1 (43–58)
42 ± 4.4 (35–48)
E% (EP/T×100)
311
333 ± 43 (256–403)
5.7 ± 1.2 (4.5–9.5)
319 ± 59 (219–460)
240 ± 36 (201–362)
96 ± 12.8 (74–113)
SW% (SL/ABD×100)
217
208 ± 39 (154–297)
–
2.3 ± 0.4 (1.8–3.4)
GS% (GL/SL×100)
51
57 ± 6.7 (46–70)
–
0.6 ± 0.1 (0.4–0.7)
H% (H/T ×100)
–
–
–
–
–
28 ± 5.8 (20–36)
– = characters absent.
First generation females (n = 20)
Body 2.3–4.9 mm long, and C-shaped after heat relaxation and fixation. Cuticle with poorly visible annuli, with fine transversal incisures. Lateral fields absent. Labial region rounded, and continuous with the adjacent part of the body. Labial plate with six lips that are fused together, each with one labial papilla at the tip and one lower cephalic papilla each except for the lateral lips. Amphid openings present at the lateral lips, close to the labial papilla, with a small transversal slit. Stoma funnel-shaped, shallow, short, and wider at the anterior part. Cheilostom short with rounded and refringent rhabdia; gymnostom scarcely developed with a minute rhabdia; stegostom robust, slightly wider than long, with a funnel-shaped lumen and walls with very minute rhabdia. Pharynx muscular with a subcylindrical procorpus, a somewhat swollen metacorpus, a short and robust isthmus, and a spheroid basal bulb with reduced valves. Nerve ring surrounds the posterior part of the isthmus. Secretory-excretory pore circular, located at the anterior part of the isthmus. Deirids inconspicuous. Cardia short, conoid, and surrounded by intestinal tissue. Intestine tubular without differentiation, with thinner walls at the anterior end. Reproductive system didelphic, amphidelphic, and ovaries are reflexed in dorsal position. Oviducts well developed with glandular spermatheca, and uteri tubular with numerous uterine eggs. Vagina short with muscular walls, vulva protruding in the form of a transverse slit. Rectum 0.3–0.6 times the anal body diameter, with three small rectal glands. Anus well developed. Tail conoid, shorter than body anal diameter, with an acute terminus. Phasmids located at the posterior part of the tail, at 25–30% of the tail length.
Second generation females (n = 20)
Similar to first generation females, but smaller, measuring 1.8–2.4 mm in length. Tail conoid, with an acute terminus, longer than the first generation females.
First generation males (n = 20)
Body 1.2–1.9 mm long, ventrally curved posteriorly, C- or J-shaped when heat killed. General morphology similar to that of females. Reproductive system monorchic, with the testis ventrally reflexed. Spicules paired, symmetrical, ventrally curved with a well-developed manubrium, either rounded or spoon-shaped. Calamus short and narrower, lamina ventrad curved at the anterior part and bears longitudinal ribs, ending in a blunt terminus. Velum indistinct, does not reach the spicule tip, and with no rostrum or retinaculum. Gubernaculum with a rounded manubrium, a fusiform corpus and a narrower and elongated tip, 0.5–0.7 times spicules length. Tail conoid with a rounded terminus bearing a fine mucron. Bursa absent. 11 pairs of genital papillae and a single mid-ventral papilla present, arranged as follows: five pairs (GP1-GP5) subventral precloacal, one pair (GP6) lateral precloacal, one single (MP) midventral precloacal, two pairs (GP7-GP8) sub-ventral ad-cloacal, one pair (GP9) subdorsal postcloacal and two pairs (GP10-GP11) postcloacal at terminus. Phasmids terminal, located laterally between the last pair of genital papillae.
Second generation males (n = 20)
Morphology of second generation males similar to that of the first generation males, but smaller, 0.8–1.2 mm in length. Tail with long, straight and robust mucron. Spicules curved ventrally, with a rhomboid shaped manubrium, slightly broader than the calamus, and a lamina curved ventrally at the anterior part. Ventral velum very reduced, and two longitudinal lateral ribs present. Gubernaculum with a slightly ventrad curved manubrium that is rounded and ventrad bent, slightly fusiform corpus and a narrower and slender terminus. Arrangement of genital papillae and phasmids similar to that of first generation males.
Infective juvenile (L3 stage) (n = 20)
IJ body 0.7–0.8 mm long, almost straight or slightly curved body shape when heat-killed. Body tapers gradually at both extremes, cuticle with transverse incisures, well-developed annuli. Lateral fields begin as a single line close to the anterior end, and increase to eight ridges before gradually reducing to five and then two near the anus and phasmid levels, respectively. Lip region slightly narrower than the adjacent part of the body, six amalgamated lips; with smaller lateral lips, six reduced labial and four prominent cephalic papillae. Amphidial apertures pore-like, oral opening triangular with a noticeable margin. Stoma reduced and tubular with a small lumen, consisting of a short cheilostom and an elongated gymno-stegostom. Pharynx elongated and narrow, with a very long corpus; a slightly narrower isthmus, and a pyriform basal bulb with reduced valves. Nerve ring surrounds the isthmus, excretory pore located at the metacorpus level. Hemizonid present. Deirids inconspicuous. Cardia conoid. Intestine bears a bacterial sac at the anterior part. Rectum long, almost straight, with very short cuticular and elongated cellular parts, anus distinct. Genital primordium located at the equatorial region, tail conoid, tapering gradually to a pointed terminus, with a longer cellular part than the hyaline part and an irregular cellular-hyaline junction. Phasmids located at 37–45% of the tail length.
Life cycle
Steinernema anantnagense n. sp. is a highly pathogenic nematode species that can be easily reared on G. mellonella larvae at a temperature ranging from 18–24°C. The life cycle of this new species is similar to the life cycle of other Steinernema species. When G. mellonella larvae are exposed to 50–100 infective juveniles (IJs), they die within 24–48 h. The first- and second-generation adults of S. anantnagense n. sp. can be found in the insect cadavers 3–4 and 5–6 days after infection, respectively. The pre-infective juveniles leave the host body, mature for a few days, and then migrate to the water traps after 10–15 days.
Type host and locality
The type hosts of Steinernema anantnagense n. sp. are unknown as the nematodes of this genus can infect different species of insects, and were obtained from soil samples using the insect baiting technique (Bedding and Akhurst 1975; White 1927). Steinernema anantnagense n. sp. Steiner_6, Steiner_7, and Steiner_8 nematodes were isolated, using the Corcyra cephalonica baiting method, from soil samples collected around the roots of willow, walnut, and apple trees in the Anantnag district of the Union Territory of Jammu and Kashmir, India (GPS coordinates: Lat. 33.828914°, Long. 75.100091°, 1606 m above sea level).
Type material
The type material for Steinernema anantnagense n. sp. are Steiner_7 nematode populations. For each stage (holotype and paratypes), including first-generation males and females, second-generation males and females, and infective juveniles, six permanent slides were prepared and deposited in the National Nematode Collection of India, located at the Indian Agricultural Research Institute (IARI) in New Delhi, India. Additionally, some permanent slides (paratypes) (n = 15) were deposited at the nematode collection of the Department of Animal Biology, Plant Biology and Ecology at the University of Jaén in Spain (IND001-01 – IND001-15). Live cultures of these nematodes are maintained at the Division of Entomology, Faculty of Agriculture, Sher-e-Kashmir University of Agricultural Science & Technology of Kashmir, India.
Etymology
The species name is derived from the location Anantnag, a District in the Union Territory of Jammu and Kashmir, India, where the nematode specimens used in this study to describe the new species were obtained.
Cross-hybridization experiments
Mating experiments were carried out to determine the reproductive isolation of S. anantnagense n. sp. by pairing males and females of this species with individuals from other Steinernema species, including S. ichnusae, S. litorale, S. weiseri, S. akhursti, S. citrae, S. cholashanense, S. feltiae, S. silvaticum, S. africanum, and S. xueshanense. No offspring were produced when S. anantnagense n. sp. nematodes were allowed to interact with nematodes of the any of the above mentioned species, indicating that S. anantnagense n. sp. is reproductively isolated. Cross tests were also conducted between males and females of Steiner_6, Steiner_7, and Steiner_8 to determine their conspecific status. The results showed that fertile offspring were produced, confirming that they belong to the same species. Controls were also carried out, which included self-crossed species, and offspring were observed in all of them. However, no progeny were observed in the single-female control plates.
Diagnosis and relationships of Steinernema anantnagense n. sp.
Steinernema anantnagense n. sp. is characterized by adults with a short stoma, rounded cheilorhabdia, and a robust pharynx with a round basal bulb. Females of the first generation are between 2.3–4.9 mm in length, with didelphic-amphidelphic reproductive system, and possess a shorter conoid tail bearing a short mucron in the first generation (c = 61–122, c′ = 0.5–0.8) and a longer conoid tail with thin mucron in the second generation (c = 43–67, c′ = 0.8–1.3). Males are smaller, between 1.2–1.9 mm in length, with a reproductive system that is monorchid and that has ventrally curved spicules bearing rounded manubrium in the first generation and rhomboid manubrium in the second generation, gubernaculum is fusiform in the first generation and anteriorly ventrad bent in the second generation, tail is conoid and slightly ventrally curved with a minute mucron in the first generation (c = 34–64, c′ = 0.8–1.6) and with a longer and more robust mucron in the second generation (c = 31–52, c′ = 0.6–1.4). The infective juveniles have a nearly straight body (0.7–0.8 mm length), poorly developed stoma and pharynx, lateral fields with eight longitudinal ridges and a conoid-elongate tail (49–66 µm, c = 12–16, c′ = 1.6–2.1) with a hyaline posterior part.
Steinernema anantnagense n. sp. belongs to a group of species known as the “Kushidai-clade”, and presents several traits common to this group. Several of the morphological and morphometric traits of the IJs and adults overlap with those of other species in the “Kushidai-clade”. However, several distinct morphological and morphometrical characteristics can differentiate S. anantnagense n. sp. from these closely related species (Tables 2–4).
Comparison of morphometrics of the third-stage infective juveniles of Steinernema anantnagense n. sp. with other members of “Feltiae-Kushidai” clade. Measurements are in μm except n, ratio and percentage. Data for new species is in bold.
Comparison of morphometrics of the first-generation males of Steinernema anantnagense n. sp. with other members of “Feltiae-Kushidai” clade. Measurements are in μm except n, ratio and percentage. Data for new species is in bold.
Species
n
L
BD
EP
NR
NL
T
SL
GL
a
B
c
c′
D%
SW%
GS%
Mucron
S. akhursti
20
1350–1925
115–150
93–113
120–163
168–205
30–40
85–100
58–68
NA
NA
NA
NA
52–61
140–200
65–77
P
S. anantnagense n. sp.
20
1223–1899
167–211
88–124
103–129
165–185
29–39
56–70
31–43
6–10
7–11
34–64
0.8–1.6
49–74
154–297
46–70
P
S. africanum
15
977–1400
65–131
69–109
79–104
132–147
34–46
65–76
32–49
9–12
7–12
25–34
0.9–1.1
52–74
144–197
49–68
P
S. cholashanense
20
1070–1778
73–204
75–135
91–126
135–173
29–43
60–71
32–45
9–24
8–11
36–51
0.6–0.9
50–85
92–144
61–85
P
S. citrae
20
1028–1402
87–113
64–92
92–119
123–155
17–31
57–80
32–59
NA
NA
NA
NA
47–67
156–233
48–89
P
S. feltiae
25
1414–1817
60–90
110–126
NA
164–180
37–43
65–77
34–47
NA
NA
NA
NA
51–64
99–130
52–61
P
S. hebeiense
20
1036–1450
74–98
58–73
78–93
118–132
24–35
51–63
38–50
12–17
8–11
30–49
0.6–0.9
48–59
120–170
60–90
A
S. ichnusae
20
1151–1494
73–204
94–108
NA
135–173
33–48
64–67
43–46
20–29
7–9
29–39
0.8–0.9
59–65
120–162
64–69
A
S. jollieti
12
1296–1952
98–135
83–110
NA
110–168
24–38
55–70
45–60
12–19
8–14
53–86
NA
53–83
NA
NA
A
S. kraussei
20
1200–1600
110–144
73–99
95–122
137–178
36–44
42–53
29–37
11
9 NA
NA
NA
NA
NA
NA
P
S. kushidai
20
1200–1900
75–156
71–105
120–137
156–189
30–40
48–72
39–60
NA
NA
NA
NA
42–59
NA
NA
A
S. litorale
25
1230–1514
82–111
77–107
94–128
133–163
26–41
67–89
44–64
12–16
8–10
33–56
0.6–0.9
34–56
154–200
62–81
P
S. nguyeni
20
818–1171
58–106
47–71
70–103
112–144
18–25
58–75
30–55
11–15
7–10
38–53
0.6–0.8
38–57
185–279
46–81
P
S. oregonense
20
1560–1820
105–161
95–139
101–133
139–182
24–32
65–73
52–59
NA
NA
NA
0.6 NA
64–75
NA
NA
A
S. populi
25
1258–1514
66–95
95–121
107–143
131–177
39–68
57–77
38–60
15–20
8–10
20–33
0.8–1.5
59–78
107–160
58–82
P/A
S. puntauvense
19
1010–1931
101–139
68–114
104–128
130–159
28–40
71–81
30–40
NA
NA
NA
NA
45–85
140–200
55–75
P
S. sandneri
25
1206–1635
124–178
64–92
112–138
148–170
35–46
53–65
39–50
9–11
8–10
31–42
NA
42–59
97–127
61–83
P
S. sangi
20
1440–2325
120–225
67–99
109–166
150–221
27–42
58–80
34–46
NA
NA
NA
NA
42–63
120–160
50–70
P
S. silvaticum
26
975–1270
52–78
71–92
90–126
116–168
20–47
42–64
30–43
14–20
8–9
24–55
0.8–1.4
45–63
NA
NA
P
S. tielingense
20
1430–2064
111–159
94–133
96–132
145–173
22–33
79–98
49–70
11–18
9–13
57–85
0.3–0.6
64–78
176–212
59–82
A
S. texanum
20
1197–1406
81–116
79–100
94–114
123–147
19–30
55–66
39–53
NA
NA
NA
NA
58–73
127–203
62–84
A
S. xinbinense
20
1133–1440
90–126
57–75
91–120
138–159
30–41
49–62
30–41
11–13
7–9
31–39
0.7–1.0
41–50
114–156
54–72
P
S. xueshanense
20
1313–2040
97–159
113–137
NA
151–175
29–48
66–91
41–60
NA
NA
NA
NA
73–87
93–172
58–95
A
S. weiseri
20
990–1395
84–138
57–84
94–115
134–154
19–32
62–72
46–57
9–12
7–10
36–64
0.6–0.9
39–60
150–240
70–85
A
NA = Not available; P = Present; A = Absent.
Comparison of morphometrics of the first-generation females of Steinernema anantnagense n. sp. with other members of “Feltiae-Kushidai” clade. Measurements are in μm except n, ratio and percentage. Data for new species is in bold.
Species
L
BD
EP
NR
NL
T
A
B
c
c′
V
ABD
D%
Mucron
S. akhursti
5625–9000
200–270
113–138
150–175
213–258
38–63
30*
32*
149*
0.6*
48–53
68–100
NA
P
S. anantnagense n. sp.
2327–4872
314–409
92–140
143–182
196–255
32–49
17–29
13–21
61–122
0.5–0.8
50–58
77–110
43–60
P
S. africanum
2469–4215
154–194
67–111
79–130
170–201
35–55
13–27
13–24
51–104
0.7–1.0
50–57
37–70
32–62
P
S. cholashanense
3232–6363
156–332
111–148
176–223
181–231
46–70
13–23
18–32
62–119
0.6–1.0
50–57
54–105
29–65
P
S. citrae
2038–4019
137–212
54–90
130–179
189–220
33–60
NA
NA
NA
NA
50–59
43–79
27–46
P
S. feltiae
3095–3774
170–254
68–97 **
70–97 **
197–304
39–70
14–20
12–17
49–88
0.7–1.2 *
44–57
47–62
40–54 *
P
S. hebeiense
3972–4254
142–245
48–95
88–123
133–158
133–158
17–25
21–29
67–129
0.5–0.9
50–57
45–65
36–66
A
S. ichnusae
4547–6186
242–323
106–156
NA
215–262
51–79
17–24
21–26
68–113
0.6–1.0
51–57
70–94
47–63
P
S. jollieti
3746–6030
219–298
96–136
NA
184–310
31–55
15–24
19–31
72–185
NA
44–56
NA
52
P
S. kraussei
2500–5400
153–288
66–99
127–146
178–205
33–59
17
22
88
NA
54
39–50
45
P
S. kushidai
2100–4700
54–59
78–105
111–144
204–255
30–45
NA
NA
NA
NA
54–59
54–84
37–46
A
S. litorale
3930–5048
175–215
65–105
130–165
185–213
25–60
21–26
20–26
78–157
0.5–0.9
0.5–0.9
55–75
33–57
P
S. nguyeni
2290–5361
130–216
49–98
84–139
137–194
20–67
15–30
15–30
53–165
0.6–1.1
52–63
130–216
30–56
A
S. oregonense
4400–6200
217–268
217–268
129–162
186–220
28–46
NA
NA
NA
NA
46–56
42–79
43–57
A
S. populi
4038–13762
217–531
90–178
150–213
213–278
41–88
18–36
19–50
75–182
0.5–0.9
45–60
60–157
36–65
A
S. puntauvense
3687–8335
181–221
51–85
123–146
141–206
41–66
NA
NA
NA
NA
51–55
57–102
25–45
P
S. sandneri
4244–5014
181–261
61–102
132–158
173–194
32–61
17–25
24–27
75–140
NA
49–57
62–122
36–54
P
S. sangi
4830–7200
270–360
80–121
140–170
216–240
36–62
NA
NA
NA
NA
43–530
84–140
35–51
P
S. silvaticum
1520–3290
50–175
50–175
50–175
121–188
33–79
15–41
10–18
34–80
1.0–1.8
44–57
26–53
33–79
A
S. texanum
2720–3623
130–202
78–107
111–135
160–189
30–52
NA
NA
NA
NA
50–55
50–71
NA
A
S. tielingense
4028–8538
200–307
82–103
111–144
186–263
40–69
17–32
21–45
72–158
0.5–0.9
49–54
56–92
32–49
A
S. xinbinense
3025–5121
159–200
70–87
106–141
167–192
30–53
19–25
17–26
79–123
0.5–0.8
46–57
50–67
38–45
P
S. xueshanense
4181–8181
182–343
117–148
NA
196–274
43–66
NA
NA
NA
NA
52–62
38–72
NA
P
S. weiseri
3780–5940
202–263
75–86
108–154
162–226
38–59
17–29
22–31
87–156
0.5–0.8
50–58
51–80
NA
P
NA = Not available; P = Present; A = Absent.
Steinernema anantnagense n. sp. and S. akhursti (Qiu et al., 2005) morphologically differ in several traits. In the case of IJs, the distance from the head to the nerve ring is shorter in S. anantnagense n. sp. (54–71) compared to S. akhursti (83–95 μm), and the tail is shorter (49–66 μm) in S. anantnagense n. sp. than in S. akhursti (68–75 μm). The ratio of body length to tail (c) is greater in S. anantnagense n. sp. (12–16) than in S. akhursti (10–12), while the ratio of tail to body length (c′) is lower in S. anantnagense n. sp. (1.6–2.1) than in S. akhursti (3.3–3.7). Additionally, S. anantnagense n. sp. has a smaller H% value (20–36) compared to S. akhursti (49–56) (Table 2). The first-generation males of S. anantnagense n. sp. have a larger body diameter (167–211 μm) and much shorter spicule and gubernaculum (56–70 μm and 31–43 μm, respectively) compared to S. akhursti (body diameter: 115–150 μm, spicule: 85–100 μm, gubernaculum: 58–68 μm) (Table 3). The first-generation females of the two species also differ in some morphometric measurements (Table 4).
Steinernema anantnagense n. sp. differs from S. populi (Tain et al., 2022) in IJ body length (0.75–0.83 vs. 0.97–1.17 mm), the distance from anterior end to excretory pore (45–62 vs. 70–86 μm) and to nerve ring (54–71 vs. 98–113) μm), tail length (49–66 vs. 55–72 μm) and lower a, b, c and c′ ratios and lower D% (Table 2). The first-generation males of the new species differ from those of S. populi in body diameter (167–211 vs. 66–95 μm), tail length (29–39 vs. 39–68 μm), lower a and c ratios, and mucron (always present vs. present or absent) (Table 3). The first-generation females of the new species differ from those of S. populi in mucron (present vs. absent) and other characters (Table 4).
Steinernema anantnagense n. sp. can be distinguished from S. kushidai (Mamiya, 1988) by several morphological features. The body length of IJs in S. anantnagense n. sp. is longer (0.75–0.83 mm) than in S. kushidai (0.42–0.66 mm), and the distance from the anterior end to the nerve ring is shorter (54–71 μm) in S. anantnagense n. sp. compared to S. kushidai (70–84 μm). Additionally, the neck length is longer (120–143 μm), and the ratio c is higher (12–16) in S. anantnagense n. sp. than in S. kushidai (neck length: 106–120 μm, c: 10–13) (Table 2). The first-generation males of S. anantnagense n. sp. can be distinguished from S. kushidai by having a larger body diameter (167–211 vs. 75–156 μm), a shorter tail (29–39 vs. 40 μm), and the presence of a mucron, while S. kushidai does not have a mucron (Table 3). The first-generation females of S. anantnagense n. sp. also have a larger body diameter (314–409 μm), a longer distance from the anterior end to the nerve ring (143–182 μm), and the presence of a mucron, which are all different from S. kushidai (54–59 μm, 111–144 μm, and absent mucron, respectively) (Table 4).
In comparison to S. sangi (Phan et al., 2001), S. anantnagense n. sp. has a longer body length of IJs (0.75–0.83 vs. 0.70–0.78 mm), a shorter distance from the anterior end to the nerve ring (54–71 vs. 78–97 μm), a shorter tail length (49–66 vs. 76–89 μm), and greater c ratio, greater E%, and lower H% (12–16 vs. 9–10, 74–113 vs. 56–70, and 20–36 vs. 44–52, respectively) (Table 2). There are also minor differences in some characters between the first-generation males and females of the new species and those of S. sangi, which are presented in Tables 3 and 4, respectively.
The IJs of S. anantnagense n. sp. displays several distinguishing features from other related species. In comparison to S. cholashanense (Nguyen et al., 2008), the position of the nerve ring is more anterior (54–71 μm vs. 72–97 μm)), and the c′ ratio is lower (1.6–2.1 vs. 3.5–5.0). When compared to S. hebeiense (Chen et al., 2006), S. anantnagense n. sp. has a greater body length (0.75–0.83 vs. 0.61–0.71 mm), larger body diameter (32–42 vs. 23–48 μm), a more anterior position of the nerve ring (54–71 μm vs. 73–83 μm), a longer neck length (120–143 vs. 100–111 μm), a lower a ratio (19–24 vs. 24–28), and a higher c ratio (12–16 vs. 9.4–11). When compared to S. tielingense (Ma et al., 2012), S. anantnagense n. sp. has a shorter body length (0.75–0.83 vs. 0.82–0.98 mm), a more anterior position of the excretory pore and nerve ring (45–62 μm and 54–1 μm, respectively, as opposed to 64–73 μm and 90–105 μm, respectively), a shorter tail (49–66 vs. 74–85 μm), smaller ratios of a, b, and c′ and H% (19–24, 5.5–6.7, 1.6–2.1, and 20–36, respectively, as opposed to 27–31, 6.7–7.9, 3.5–4.6, and 53–64, respectively), but a longer c ratio (12–16 vs. 10–12). Compared to S. xinbinense (Ma et al., 2012), S. anantnagense n. sp. has a greater body length (0.75–0.84 vs. 0.64–0.74 mm), larger body diameter (32–42 μm vs. 28–31 μm), a smaller distance from the anterior end to the nerve ring (54–71 μm vs. 75–90 μm), a shorter tail (49–66 vs. 65–78 μm), a longer c ratio (12–16 vs. 8–11), a smaller c′ ratio (1.6–2.1 vs. 3–5), and a longer E% (74–113 vs. 65–78). Steinernema anantnagense n. sp. can be differentiated from S. xueshanense (Mrácek et al., 2009) by a smaller distance from the anterior end to the excretory pore and nerve ring (45–62 vs. 60–72 μm and 54–71 vs. 81–96 μm, respectively), a shorter tail length (49–66 vs. 80–92 μm), lower ratios of a and c′ (19–24 vs. 26–32 and 1.6–2.1 vs. 3.8–5.1, respectively), and lower H% (20–36 vs. 46–55). In addition, the position of the IJs nerve ring in the new species is more anterior (54–71 μm) compared to S. feltiae (Nguyen, 2007) (108–117 μm), and it also has a shorter tail length (49–66 vs. 81–89 μm) (Table 3).
Nematode molecular characterization
The ITS regions of S. anantnagense n. sp. (Steiner_6, Steiner_7, and Steiner_8) are each 730 bp in length, consisting of ITS1 (278 bp), 5.8S (157 bp), and ITS2 (295 bp). Compared to other related species, the ITS region of S. anantnagense n. sp. shows differences of 19–117 bp, resulting in sequence similarity values of 78–97% (Table 5). Similarly, the D2-D3 expansion segments of the 28S rRNA gene of S. anantnagense n. sp. differ from those of other species by 5–35 bp, resulting in sequence similarity values of 95–99% (Table 6). In addition, the mitochondrial COI exhibit differences of 65–90 bp, resulting in 82–87% sequence similarity values, respectively (Table S1). Further, the mitochondrial 12S genes also exhibit 33–82 bp differences, resulting in sequence similarity values of 79–92%, respectively (Table S2). When these sequences were compared with sequences in the NCBI database using BLAST search, we observed that the top hit record for the ITS was 97.24% with S. akhursti (DQ375757) from China, for the D2D3 was 99.42% with S. akhursti (KF289902) from China, for mtCOI was 88.23% with S. sangi (MF621239) from India, and for mt12S rRNA was 92.34% with S. kushidai (AP017467) from Japan. Taken together, these observations suggest that S. anantnagense n. sp. represents a new taxonomic entity within the “Kushidai” clade, as evidenced by the lower sequence similarity scores between this species and all other known species, thus supporting its novel taxonomic status.
Pairwise distances in base pairs of the ITS rRNA regions among closely related Steinernema species and Steinernema anantnagense n. sp. Data for new species is in bold.
Species (ITS rRNA)
S. anantnagense n. sp. OQ407490
S. akhursti DQ375757
S. kushidaiAB243440
S. cholashanense EF431959
S. oregonense AY230180
S. sangi AY355441
S. texanum EF152568
S. xueshanense FJ666052
S. populi MZ367621
S. jollieti AY171265
S. xinbinense JN171593
S. weiseri KJ696685
S. tielingense GU994201
S. africanum ON041031
S. kraussei AY230175
S. citrae EU754718
S. silvaticum AY230162
S. litorale AB243441
S. ichnusae EU421129
S. nguyeni KP325084
S. feltiae AY230169
S. hebeiense DQ105794
S. monticola AB698756
S. anantnagense n. sp. OQ407490
19
53
72
72
72
72
73
74
74
80
83
85
86
86
87
87
90
91
92
101
116
117
S. akhursti DQ375757
97
51
75
74
73
77
76
65
75
82
86
89
89
90
87
91
93
95
94
102
119
115
S. kushidai AB243440
92
93
95
89
96
103
90
94
89
97
104
101
103
108
110
104
107
110
110
122
128
130
S. cholashanense EF431959
89
88
85
61
74
58
58
106
69
68
72
71
72
83
70
82
73
74
84
75
112
116
S. oregonense AY230180
89
88
86
91
47
51
33
99
19
31
43
46
39
52
42
45
56
49
52
51
94
115
S. sangi AY355441
89
88
85
88
93
57
58
98
57
54
43
45
60
53
60
61
48
48
54
47
88
103
S. texanum EF152568
89
88
83
91
92
91
53
95
61
60
66
67
59
75
55
69
74
69
77
68
110
118
S. xueshanense FJ666052
89
88
86
91
95
91
92
99
41
43
46
54
47
60
44
54
57
53
60
54
86
114
S. populi MZ367621
89
90
86
83
84
84
85
84
105
103
110
111
112
109
113
115
117
116
112
124
145
123
S. jollieti AY171265
88
88
86
89
97
91
91
94
83
48
52
54
55
64
55
58
63
56
62
58
95
115
S. xinbinense JN171593
88
87
85
90
96
92
91
94
84
93
45
50
32
58
40
38
58
57
57
50
92
116
S. weiseri KJ696685
87
86
83
89
94
94
90
93
82
92
93
17
59
32
60
65
26
27
34
27
80
114
S. tielingense GU994201
87
86
84
89
93
93
90
92
82
92
93
98
63
46
65
66
30
28
47
32
88
117
S. africanum ON041031
87
86
84
89
94
91
91
93
82
92
95
91
91
64
45
35
72
68
64
62
98
124
S. kraussei AY230175
86
86
82
87
92
92
88
91
82
90
91
95
93
90
65
69
44
46
19
40
89
120
S. citrae EU754718
86
86
82
89
94
91
92
94
82
92
94
91
90
93
90
55
73
70
64
60
98
118
S. silvaticum AY230162
86
86
83
87
93
91
89
92
81
91
95
90
90
95
90
92
76
70
68
68
101
126
S. litorale AB243441
86
85
82
89
92
93
88
91
80
90
91
96
96
89
93
89
88
38
45
34
79
118
S. ichnusae EU421129
86
85
82
88
93
93
89
92
81
92
92
96
96
90
93
89
89
94
45
25
87
122
S. nguyeni KP325084
85
85
82
87
92
92
88
91
81
90
91
95
93
90
97
90
90
93
93
41
86
117
S. feltiae AY230169
84
84
80
88
92
93
89
92
79
91
92
96
95
91
94
91
90
95
96
94
79
119
S. hebeiense DQ105794
81
80
78
82
85
86
82
86
75
85
86
88
86
84
86
85
84
88
86
87
88
135
S. monticola AB698756
78
79
76
78
79
81
78
79
77
79
79
79
78
77
78
78
77
78
77
78
78
74
Below diagonal: percentage similarity; above diagonal: total character difference.
Pairwise distances in base pairs of the D2D3 fragment of 28S rRNA regions among closely related Steinernema species and Steinernema anantnagense n. sp. Data for new species is in bold.
Species (D2D3 rRNA)
S. anantnagense n. sp. OQ407498
S. akhursti KF289902
S. weiseri FJ165549
S. oregonense GU569055
S. puntauvense EF187018
S. feltiae AF331906
S. ichnusae EU421130
S. africanum OM423154
S. kushidai AF331897
S. tielingense GU994202
S. populi MZ367685
S. xueshanense FJ666053
S. kraussei KC631424
S. jollieti GU569051
S. cholashanense EF520284
S. texanum EF152569
S. xinbinense GU994204
S. citrae MF540676
S. silvaticum KC631426
S. sangi MF620997
S. nguyeni KR815816
S. monticola GU395647
S. anantnagense n. sp. OQ407498
5
12
13
13
13
15
16
17
18
18
18
19
19
23
24
25
25
27
30
31
35
S. akhursti KF289902
99
16
16
17
17
17
18
18
22
20
22
22
21
26
27
29
27
28
27
33
38
S. weiseri FJ165549
98
98
12
5
5
7
6
22
13
18
15
16
9
18
21
19
18
24
32
22
37
S. oregonense GU569055
98
98
98
11
11
13
14
24
10
27
17
8
17
13
22
14
25
19
33
29
38
S. puntauvense EF187018
98
98
99
99
0
4
5
23
10
23
16
13
10
15
20
16
17
21
30
21
40
S. feltiae AF331906
98
98
99
99
100
4
5
23
10
23
16
13
10
15
20
16
17
21
30
21
40
S. ichnusae EU421130
98
98
99
98
99
99
5
25
14
25
17
15
10
19
20
20
17
25
32
21
40
S. africanum OM423154
98
98
99
98
99
99
99
21
13
22
19
16
8
18
22
19
12
26
30
15
37
S. kushidai AF331897
98
98
97
97
97
97
97
97
26
30
26
26
29
28
36
29
30
34
32
33
42
S. tielingense GU994202
98
97
98
99
99
99
98
98
97
27
20
10
18
12
25
12
22
20
36
26
44
S. populi MZ367685
98
97
98
96
97
97
97
97
96
96
27
30
27
30
29
31
32
38
40
37
44
S. xueshanense FJ666053
98
97
98
98
98
98
98
98
97
97
96
20
22
20
25
22
30
28
41
33
45
S. kraussei KC631424
98
97
98
99
98
98
98
98
97
99
96
97
21
13
27
11
27
16
37
31
45
S. jollieti GU569051
98
97
99
98
99
99
99
99
96
98
96
97
97
23
24
22
20
31
35
24
42
S. cholashanense EF520284
97
97
98
98
98
98
98
98
96
98
96
97
98
97
27
10
27
19
38
30
45
S. texanum EF152569
97
96
97
97
97
97
97
97
95
97
96
97
96
97
96
32
32
37
39
35
45
S. xinbinense GU994204
97
96
98
98
98
98
97
98
96
98
96
97
99
97
99
96
30
17
39
34
47
S. citrae MF540676
97
96
98
97
98
98
98
98
96
97
96
96
96
97
96
96
96
37
42
11
49
S. silvaticum KC631426
96
96
97
98
97
97
97
97
95
97
95
96
98
96
98
95
98
95
41
41
50
S. sangi MF620997
96
96
96
96
96
96
96
96
96
95
95
94
95
95
95
95
95
94
95
46
46
S. nguyeni KR815816
96
96
97
96
97
97
97
98
96
97
95
96
96
97
96
95
95
99
95
94
51
S. monticola GU395647
95
95
95
95
95
95
95
95
94
94
94
94
94
94
94
94
94
93
93
94
93
Below diagonal: percentage similarity; above diagonal: total character difference.
Nematode phylogenetic relationships
Phylogenetic reconstructions based on the nucleotide sequences of the internal transcribed spacer (ITS) marker of the rRNA gene, D2D3 expansion segments of the 28S rRNA gene, the cytochrome oxidase subunit I (COI), and the mitochondrial 12S rRNA gene show that S. anantnagense n. sp. Steiner_6, Steiner_7, and Steiner_8 are conspecific and belong to the “Kushidai” clade and the “Feltiae–Kushidai–Monticola” superclade (Figs. 6 and 7). Phylogenetic analyses of all four abovementioned markers clearly separate S. anantnagense n. sp. from all other species. In addition, these phylogenetic reconstructions show that S. anantnagense n. sp. is closely related to other Asian species, including S. akhursti, S. kushidai, and S. populi. No phylogenetic tree was built using the 18S rRNA genetic region because insufficient 18S rRNA gene sequences are publicly available. However, the resulting sequences were deposited in the NCBI databank under the following accession numbers: OQ407498 (Steiner_6), OQ407499 (Steiner_7), and OQ407500 (Steiner_8).
Symbiotic relationships
Phylogenetic reconstructions based on whole genome sequences show that the bacterial symbiont isolated from S. anantnagense n. sp. Steiner_7, named here XENO-2, is closely related to X. japonica DSM 16522T and X. vietnamensis VN01T (Fig. 8). The digital DNA–DNA hybridization (dDDH) values between XENO-2 and X. japonica DSM 16522T, and between XENO-2 and X. vietnamensis VN01T are 51.8% and 40.0%, respectively. These values are below the 70% divergence threshold for prokaryotic species delineation, indicating that XENO-2T represents a novel species within the genus Xenorhabdus (Wayne et al., 1987). This species is formally described elsewhere.
A Side Note on The Nomenclature of Steinernema Monticolum
The term “monticolum” was introduced by Stock et al. (1997) to refer to the geographic origin of the nematodes studied, which were collected in Mount Jiri (Sancheong, Gyeongnam province, Korea). This term is a combination of “monti” referring to “mountain” and “colum” derived from the Latin suffix “cola” meaning “that lives in a place.” However, it should be noted that, as the suffix “cola” is a masculine noun in Latin, it does not have gender variations. Therefore, the correct term to use is “monticola.” The correct usage of this term has been discussed in detail by Nicolson, 1987. In light of this, we propose to refer to this species as Steinernema monticola, as was first used by Choo et al. (1998).
Conclusions
The differences in morphology, morphometry, molecular characteristics, reproductive isolation, and clear phylogenetic distinction support that Steiner_6, Steiner_7, and Steiner_8 represent a new species of entomopathogenic nematodes. We propose to name this species Steinernema anantnagense n. sp. This discovery marks the second new species description in the Steinernema genus from the Indian Subcontinent. Our findings provide valuable insights into the biodiversity and distribution of these biological control agents. Furthermore, our results underscore the importance of accurately characterizing newly described Steinernema species through the inclusion of all three standard rDNA markers (ITS, SSU, and LSU) in combination with the mitochondrial COI gene, in addition to classical taxonomy. We recommend that all future species descriptions follow this approach.