Introduction
Entomopathogenic nematodes (EPNs), mainly members of the families Steinernematidae and Heterorhabditidae, are natural insect parasites widely used as biological control agents (Tarasco et al. Reference Tarasco, Fanelli, Salvemini, El-Khoury, Troccoli, Vovlas and De Luca2023). Their infective juveniles (IJs) penetrate susceptible hosts and release their symbiotic bacteria, Xenorhabdus or Photorhabdus, which contribute to rapid host mortality and support nematode development and reproduction. This insecticidal activity has led to their use against a wide range of agricultural pests (Dillman and Sternberg Reference Dillman and Sternberg2012; Raypuriya et al. Reference Raypuriya, Ghanghas, Das, Priyadarshini, Yadav, Dose, Mehla and Maheshwari2025; Shapiro-Ilan and Dolinski Reference Shapiro-Ilan, Dolinski and Campos-Herrera2015; Smart Reference Smart1995). Beyond agricultural pest management, EPNs have also been investigated for their potential use in mosquito larval control (Ardpairin et al. Reference Ardpairin, Subkrasae, Dumidae, Janthu, Meesil, Muangpat, Tandhavanant, Thanwisai and Vitta2023; Cardoso et al. Reference Cardoso, Gomes, Dolinski and Souza2015; Dilipkumar et al. Reference Dilipkumar, Ramalingam, Chinnaperumal, Govindasamy, Paramasivam, Dhayalan and Pachiappan2019; Pansri et al. Reference Pansri, Ardpairin, Subkrasae, Dumidae, Homkaew, Wattanachaiyingcharoen, Thanwisai and Vitta2025; Subkrasae et al. Reference Subkrasae, Ardpairin, Dumidae, Janthu, Meesil, Muangpat, Tandhavanant, Thanwisai and Vitta2022; Thanwisai et al. Reference Thanwisai, Muangpat, Meesil, Janthu, Dumidae, Subkrasae, Ardpairin, Tandhavanant, Yoshino and Vitta2022). Their activity against non-insect pests, including slugs and snails, has also been reported (Ardpairin et al. Reference Ardpairin, Subkrasae, Dumidae, Pansri, Homkaew, Meesil, Kumchantuek, Phoungpetchara, Dillman, Pavesi, Bode, Tandhavanant, Thanwisai and Vitta2024; Dumidae et al. Reference Dumidae, Homkeaw, Subkrasae, Ardpairin, Pansri, Polseela and Vitta2024; Hussein Reference Hussein2024; Tunholi et al. Reference Tunholi, Monteiro, da Silva, de Melo Dolinski, dos Santos, de Azevedo Rodrigues, Pinheiro Bittencourt, Pinheiro and Tunholi-Alves2014, Reference Tunholi, Lorenzoni, da Silva, Tunholi-Alves, Boeloni, da Silva, Monteiro, Prata, Pinheiro and Martins2017; Wanitsumran et al. Reference Wanitsumran, Wattanachaiyingcharoen, Vitta, Thanwisai, Kenthao, Lopin, Pansri, Changtor and Wattanachaiyingcharoen2025; Wilson et al. Reference Wilson, Glen, Hughes, Pearce and Rodgers1994). These findings support the continued investigation of native EPN species and isolates for specific target hosts. By 2025, 114 species of Steinernema and 21 species of Heterorhabditis had been reported worldwide from a variety of habitats (Půža and Machado Reference Půža and Machado2024; Půža et al. Reference Půža, Machado and Malan2025). Since then, two additional species of Steinernema (Anes et al. Reference Anes, Patil, Babu, Aarthi, Gotyal, Josephrajkumar, Mhatre, Sajan, Gowda, Sushil and Půža2025; Zhan et al. Reference Zhan, Tian, Li, Yang, Bao, Zhang, Zhang, Shi, Tomalak, Půža and Guo2025) and two species of Heterorhabditis (Machado et al. Reference Machado, Abolafia, Robles, Ruiz-Cuenca, Bhat, Shokoohi, Půža, Zhang, Erb, Robert and Hibbard2025; San-Blas et al. Reference San-Blas, Morales-Montero, Bastidas, Půža and Machado2025) have been described, bringing the current totals to 116 and 23 species, respectively.
Early EPN surveys in Thailand were conducted in the 1990s and documented native Steinernema and Heterorhabditis isolates for biological control (Tangchitsomkid and Sontirat Reference Tangchitsomkid and Sontirat1998). An important step in the study of Thai EPNs was the description of S. siamkayai Stock, Somsook & Reid, Reference Stock, Somsook and Reid1998 from western Thailand by Stock et al. (Reference Stock, Somsook and Reid1998), the first EPN species described from Thailand. Subsequent surveys covered different habitats and regions, including national parks, natural forests, fruit orchards, roadside soils, and agricultural fields. Furthermore, the use of molecular markers, particularly the ITS and D2–D3 regions of rDNA, along with morphological characteristics, has improved EPN identification and revealed greater diversity in the country (Ardpairin et al. Reference Ardpairin, Muangpat, Sonpom, Dumidae, Subkrasae, Tandhavanant, Thanwisai and Vitta2020, Reference Ardpairin, Subkrasae, Dumidae, Janthu, Meesil, Muangpat, Tandhavanant, Thanwisai and Vitta2023; Subkrasae et al. Reference Subkrasae, Ardpairin, Dumidae, Janthu, Meesil, Muangpat, Tandhavanant, Thanwisai and Vitta2022; Thanwisai et al. Reference Thanwisai, Muangpat, Meesil, Janthu, Dumidae, Subkrasae, Ardpairin, Tandhavanant, Yoshino and Vitta2022; Vitta et al. Reference Vitta, Fukruksa, Yimthin, Deelue, Sarai, Polseela and Thanwisai2017; Yooyangket et al. Reference Yooyangket, Muangpat, Polseela, Tandhavanant, Thanwisai and Vitta2018). More recently, Baniya et al. (Reference Baniya, Subkrasae, Ardpairin, Anesko, Vitta and Dillman2024) described S. adamsi Baniya, Subkrasae, Ardpairin, Anesko, Vitta & Dillman, Reference Baniya, Subkrasae, Ardpairin, Anesko, Vitta and Dillman2024 from northern Thailand, adding another species to the EPN fauna of the country.
During sampling in a cassava field in Chai Nat Province, Central Thailand, a novel Steinernema isolate was recovered using the insect-baiting technique. Based on morphological, morphometric, and molecular evidence, we describe this isolate as S. chainatense n. sp., representing a distinct lineage within the genus.
Materials and methods
Collection and examination of nematodes
The strain of a steinernematid nematode eCCN3.3_TH was originally recovered from cassava field soil in Mueang Chai Nat District, Chai Nat Province, Central Thailand (15° 11′ 18″ N, 100° 13′ 39″ E) during a field sampling campaign in July 2022. Approximately 500 g of bagged soil was delivered to the Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand. Recovery of the nematodes was performed by insect-baiting with Galleria mellonella L. larvae (Lepidoptera, Pyralidae), based on the protocol by Bedding & Akhurst (Reference Bedding and Akhurst1975). IJs were harvested from infected larvae using modified White traps (White Reference White1927). For further morphological and molecular identification, live specimens were transferred to the Laboratory of Entomopathogenic Nematodes, University of South Bohemia, České Budějovice, Czech Republic.
Steinernema chainatense n. sp. was propagated in last-instar larvae of G. mellonella following Poinar (Reference Poinar1979). Adults of the first and second generations were recovered by dissecting infected cadavers approximately three and five days after host death, respectively. Nematodes representing all developmental stages were killed by gentle heating, fixed in triethanolamine formalin (TAF) (Courtney et al. Reference Courtney, Polley and Miller1955), and transferred to glycerine according to Seinhorst (Reference Seinhorst1959). For microscopic examination, specimens were mounted in fresh glycerine under coverslips supported by stainless steel wires of a diameter slightly greater than that of the nematodes. Morphometric measurements were obtained from 20 adults of each generation and from IJs by light microscopy using Cell D image analysis software (Olympus Soft Imaging Solutions GmbH, Germany).
Scanning electron microscopy (SEM)
First- and second-generation adults and IJs were prepared for SEM following Nguyen and Smart (Reference Nguyen and Smart1995, Reference Nguyen and Smart1997). Specimens were fixed for 24 h at 4–6 °C in 4% formalin buffered with 0.1 M sodium cacodylate (pH 7.2), followed by post-fixation in 2% osmium tetroxide for 12 h at 25 °C. After dehydration through an ascending ethanol series, the nematodes were critical-point dried using liquid CO₂, mounted on SEM stubs, and sputter-coated with gold. Observations were made using a Verios 5 UC scanning electron microscope (Thermo Fisher Scientific).
Molecular analysis
Genomic DNA from IJs of S. chainatense n. sp. was extracted using the Phire Tissue Direct PCR Master Mix Kit (Thermo Scientific, Vilnius, Lithuania) according to the manufacturer’s instructions. Molecular characterisation was performed by PCR amplification targeting two rDNA regions, the D2–D3 expansion segments of 28S rDNA and the internal transcribed spacer (ITS) region, and the mitochondrial cytochrome c oxidase subunit I (COI), using an Applied Biosystems thermal cycler (Applied Biosystems, Pittsburgh, PA, USA). The D2–D3 region was amplified using primers D2F (5′-CCTTAGTAACGGCGAGTGAAA-3′) and 536R (5′-CAGCTATCCTGAGGAAAC-3′) (Nguyen Reference Nguyen, Nguyen and Hunt2007) with the following thermal conditions: initial denaturation at 94 °C for 7 min; 30 cycles of denaturation at 94 °C for 60 s, annealing at 50 °C for 60 s, and extension at 72 °C for 60 s; followed by a final extension at 72 °C for 10 min. The ITS region was amplified using primers 18S (5′-TTGATTACGTCCCTGCCCTTT-3′; forward) and 28S (5′-TTTCACTCGCCGTTACTAAGG-3′; reverse) (Vrain et al. Reference Vrain, Wakarchuk, Levesque and Hamilton1992) with thermal conditions consisting of an initial denaturation at 94 °C for 7 min; 35 cycles of 94 °C for 60 s, 50 °C for 60 s, 72 °C for 60 s; and final extension at 72 °C for 7 min (Nguyen Reference Nguyen, Nguyen and Hunt2007). The COI region was amplified using primers EPNCOI_F1 (5′-CCTACTATGATTGGTGGTTTTGGTAATTG-3′; forward) and EPNCOI_R2 (5′-GTAGCAGCAGTAAAATAAGCACG-3′; reverse) (Kanzaki and Futai Reference Kanzaki and Futai2002) with thermal conditions consisting of an initial denaturation at 94 °C for 5 min; 35 cycles of 94 °C for 50 s, 55 °C for 50 s, 72 °C for 60 s; and final extension at 72 °C for 7 min. Subsequently, the PCR products were analysed by gel electrophoresis on a 1% (w/v) agarose gel in Tris–borate–EDTA (TBE) buffer (90 mM Tris-base, 90 mM boric acid, 2 mM EDTA) at 100 V for 30 min. The PCR products were purified using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s protocol. The purified products were sent to Macrogen, Inc. (Seoul, South Korea) for sequencing.
Sequences were edited and compared with those available in GenBank using the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI). For each DNA region, the obtained sequences were aligned with those of other members of the glaseri-group using the MAFFT online service (Katoh et al. Reference Katoh, Rozewicki and Yamada2019) with the Q-INS-i algorithm (Katoh and Toh Reference Katoh and Toh2008), followed by manual refinement in BioEdit (Hall Reference Hall1999). Pairwise sequence comparisons were performed in MEGA 7.0 (Kumar et al. Reference Kumar, Stecher and Tamura2016).
Phylogenetic relationships were inferred in MEGA 7.0 using the maximum likelihood (ML) method under the general time reversible (GTR) model (Nei and Kumar Reference Nei and Kumar2000). Among-site rate variation was modelled using a discrete gamma distribution with five rate categories (+G), while allowing for a proportion of evolutionarily invariant sites (+I). Branch support was assessed by bootstrap analysis with 1000 replicates. Steinernema affine (Bovien, 1937) Wouts, Mráček, Gerdin & Bedding, 1982 and Steinernema akhursti Qiu, Hu, Zhou, Mei, Nguyen & Pang, 2005 were used as outgroups.
Results
Steinernema chainatense n. sp.
Steinernema chainatense n. sp. A: First-generation male, tail with spicules, lateral view; B: Second-generation male, tail with spicules, lateral view; C: First-generation male, tail with arrangement of genital papillae; D: First-generation female, vulval region; E: Second-generation female, vulval region; F: First-generation female, tail; G: Second-generation female, conical tail; H–I: Infective juvenile. H: Anterior region with excretory pore; I: Tail with anus and hyaline region. (Scale bars: A–C and G–I = 50 μm; D–F = 100 μm).
Figure 1. Long description
The drawing consists of nine panels labeled A through I.
Panel A shows a first-generation male tail in lateral view, featuring a curved spicule and a gubernaculum near the cloacal opening. A vertical scale bar of 50 micro m is on the left.
Panel B displays a second-generation male tail in lateral view, showing thinner spicules compared to the first generation. A vertical scale bar of 50 micro m is on the left.
Panel C illustrates a first-generation male tail with a ventral view of the genital papillae arrangement, showing multiple pairs of small circular sensory organs along the tail. A vertical scale bar of 50 micro m is present.
Panel D depicts the vulval region of a first-generation female, showing a protruding vulva and internal eggs. A vertical scale bar of 100 micro m is on the left.
Panel E shows the vulval region of a second-generation female, with a less prominent vulva and smaller internal eggs. A vertical scale bar of 100 micro m is on the left.
Panel F shows the tail of a first-generation female, ending in a blunt point with internal intestinal structures visible.
Panel G displays the conical tail of a second-generation female, tapering more sharply than the first generation. A vertical scale bar of 50 micro m is on the left.
Panel H shows the anterior region of an infective juvenile, highlighting the mouth, esophagus, and the excretory pore located mid-way down the esophageal region. A vertical scale bar of 50 micro m is on the left.
Panel I illustrates the tail of an infective juvenile, showing the anus and a distinct hyaline region at the very tip. A vertical scale bar of 50 micro m is on the left.
Steinernema chainatense n. sp. SEM of infective juvenile, female, and male. A–E: Infective juvenile. A: Head region with four papillae and amphid openings (a); B: Lateral field in mid-body (ridges numbered 1–8); C: Anterior region with excretory pore; D: Tail region with anus and phasmid opening (arrow), subventral view; E: Tail region with anus and phasmid opening (arrow), lateral; F: First-generation female, vulva; G–H: First-generation male. G: Tail with a portion of genital papillae (numbered), lateral view; H: Tail region with genital papillae (numbered) and spicules.
Figure 2. Long description
A collection of eight S E M micrographs labeled A through H.
* Panel A: Close-up of the infective juvenile head region showing four papillae and two amphid openings labeled ‘a’. Scale bar is 5 micrometers.
* Panel B: Mid-body of an infective juvenile showing the lateral field with eight distinct longitudinal ridges numbered 1 through 8. Scale bar is 10 micrometers.
* Panel C: Anterior region of an infective juvenile showing the tapered head and the excretory pore. Scale bar is 10 micrometers.
* Panel D: Subventral view of the infective juvenile tail region. A white arrow points to the phasmid opening near the anus. Scale bar is 10 micrometers.
* Panel E: Lateral view of the infective juvenile tail region. A white arrow points to the phasmid opening. Scale bar is 20 micrometers.
* Panel F: High-magnification view of the vulva of a first-generation female. Scale bar is 50 micrometers.
* Panel G: Lateral view of a first-generation male tail. Genital papillae are numbered 4 through 12. The spicule is labeled ‘s’. Scale bar is 50 micrometers.
* Panel H: Ventral view of a first-generation male tail curved in a C-shape. Genital papillae are numbered 1 through 12, and the spicules are visible at the tip. Scale bar is 100 micrometers.
Steinernema chainatense n. sp. LM of male, female, and infective juvenile. A–B: First-generation male. A: Whole body; B: Tail with spicules and gubernaculum. C, E: First-generation female; C: Vulval region; E: Tail region. D, F: Second-generation female. D: Vulval region; F: Tail region. G, H: Infective juvenile. G: Anterior region showing head and excretory pore (arrow); H: Tail with anus and hyaline region. (Scale bars: A, C, E = 100 μm; B, G, H = 20 μm; D, F = 50 μm).
Figure 3. Long description
A multi-panel L M micrograph set.
* Panel A: Whole body of a first-generation male, curved in a J-shape with a 100 mu m scale bar.
* Panel B: Close-up of the male tail showing amber-colored spicules and gubernaculum. Scale bar is 20 mu m.
* Panel C: Vulval region of a first-generation female showing a protruding lip. Scale bar is 100 mu m.
* Panel D: Vulval region of a second-generation female with a less pronounced protrusion. Scale bar is 50 mu m.
* Panel E: Tail region of a first-generation female, tapering to a point. Scale bar is 100 mu m.
* Panel F: Tail region of a second-generation female. Scale bar is 50 mu m.
* Panel G: Anterior region of an infective juvenile. An arrow points to the excretory pore located below the head. Scale bar is 20 mu m.
* Panel H: Tail of an infective juvenile showing the anus and a clear hyaline region at the distal tip. Scale bar is 20 mu m.
Measurements
Morphometric characters of Steinernema chainatense n. sp. All measurements are in μm and in the form: mean ± s.d. (range)
Table 1. Long description
The table presents measurements in micrometers as mean plus or minus standard deviation, with ranges in parentheses. The columns are organized as follows: Males (Holotype, First generation Paratypes, Second generation Paratypes), Females (First generation Paratypes, Second generation Paratypes), and Infective juvenile (Paratypes).
Key data points include:
* Body length (L): Holotype male is 1697. First generation females are the largest at 5211 plus or minus 893. Infective juveniles are 871 plus or minus 40.
* Pharynx length (E): Ranges from 132 in juveniles to 174 in first generation females.
* Tail length (T): Increases from 29 in first generation males to 77 in infective juveniles.
* Spicule length (S L): 91 in holotype male, 88 in first generation paratypes, and 69 in second generation paratypes.
* Gubernaculum length: 57 in holotype male and 60 in first generation paratypes.
* Ratios: D percent (E P / E S times 100) ranges from 82 in first generation males to 54 in infective juveniles. E percent (E P / T times 100) is highest in first generation males at 430 and lowest in infective juveniles at 93.
* Specific juvenile characters: Hyaline tail length (H T) is 43 plus or minus 3.9, and H percent (H T / T times 100) is 55 plus or minus 3.1.
Comparative morphometrics of first-generation males of Steinernema chainatense n. sp. and other species of the glaseri-group. All measurements are in μm as mean (range). Abbreviations are as defined in Table 1
Table 2. Long description
A data table with seven columns: species, S L, G L, W, D%, S W%, and G S%. All measurements are in micrometers as mean and range.
* S. chainatense n. sp.: S L 88 (80–95), G L 60 (57–64), W 113 (102–156), D% 82 (75–88), S W% 178 (143–215), G S% 68 (63–76).
* S. aciari: S L 86 (75–95), G L 56 (48–65), W 104 (88–125), D% 76 (69–88), S W% 204 (180–240), G S% 65 (57–77).
* S. adamsi: S L 62 (44–69), G L 37 (28–59), W 70 (56–90), D% 68 (61–80), S W% 127 (93–143), G S% 61 (43–100).
* S. apuliae: S L 72 (64–80), G L 50 (46–54), W 104 (86–124), D% 81 (75–92), S W% 160 (150–180), G S% 70 (60–80).
* S. arenarium: S L 76 (63–93), G L 55 (49–60), W 188 (184–219), D% 93 (83–102), S W% 210, G S% 65 (60–66).
* S. australe: S L 72 (55–78), G L 45 (36–51), W 75 (61–97), D% 71 (59–87), S W% 172 (118–196), G S% 62 (46–71).
* S. bakwenae: S L 80 (66–89), G L 51 (44–56), W 104 (72–126), D% 89 (67–104), S W% 194 (129–228), G S% 63 (56–79).
* S. boemarei: S L 79 (64–96), G L 52 (43–65), W 96 (70–122), D% 85 (68–99), S W% 170 (120–240), G S% 70 (50–90).
* S. brazilense: S L 83 (75–89), G L 47 (41–56), W 94 (78–99), D% 65 (57–80), S W% 192 (158–208), G S% 57 (48–65).
* S. caudatum: S L 75, G L 52, W 91, D% 71, S W% 222, G S% 70.
* S. cubanum: S L 58 (50–67), G L 39 (37–42), W 97 (77–117), D% 70, S W% 141, G S% 67.
* S. diaprepesi: S L 79 (71–90), G L 54 (45–61), W 113 (90–145), D% 80 (66–86), S W% 180 (150–200), G S% 69 (59–79).
* S. ethiopiense: S L 73 (69–77), G L 73 (69–77), W 92 (81–99), D% 57 (54–61), S W% 164 (154–175), G S% 67 (63–70).
* S. glaseri: S L 77 (64–90), G L 46 (44–59), W 72 (54–92), D% 70 (60–78), S W% 205 (164–243), G S% 70 (64–84).
* S. guangdongense: S L 86 (80–94), G L 64 (47–73), W 112 (90–135), D% 70 (67–78), S W% 175 (152–216), G S% 75 (59–82).
* S. hattingi: S L 83 (76–92), G L 50 (45–60), W 118 (82–156), D% 86 (81–92), S W% 173 (133–243), G S% 61 (55–69).
* S. hermaphroditum: S L 68 (65–70), G L 48 (47–50), W not available, D% 48 (44–50), S W% 150 (140–160), G S% 71 (70–75).
* S. indicum: S L 66 (60–70), G L 51 (43–58), W 125 (82–188), D% 72 (62–79), S W% 182 (152–380), G S% 78 (65–88).
* S. innovationi: S L 81 (74–91), G L 58 (54–63), W 139 (100–171), D% 82 (67–95), S W% 140 (110–190), G S% 70 (60–80).
* S. jeffreyense: S L 87 (79–95), G L 57 (51–61), W 139 (94–167), D% 47 (34–68), S W% 215 (171–295), G S% 65 (61–71).
* S. karii: S L 83 (73–91), G L 57 (42–64), W 136 (107–166), D% 66 (53–78), S W% 151, G S% 70 (50–80).
* S. keralense: S L 68 (60–72), G L 55 (50–63), W 138 (120–156), D% 76 (62–89), S W% 136 (114–169), G S% 82 (72–92).
* S. khoisanae: S L 85 (70–88), G L 56 (50–63), W 108 (90–132), D% 85 (71–99), S W% 203 (167–227), G S% 70 (60–80).
* S. khuongi: S L 79 (64–99), G L 55 (50–62), W 94 (66–111), D% 68 (60–83), S W% 120 (100–150), G S% 70 (50–100).
* S. kushidai: S L 63 (48–72), G L 44 (39–60), W 97 (75–156), D% 51 (42–59), S W% and G S% not available.
* S. lamjungense: S L 87 (81–94), G L 57 (50–66), W 154 (116–205), D% 61 (44–91), S W% 173 (151–193), G S% 65 (56–77).
* S. leizhouense: S L 82 (75–93), G L 55 (50–60), W 104 (75–143), D% 75 (67–83), S W% 213 (177–251), G S% 67 (60–74).
* S. litchii: S L 86 (76–96), G L 65 (58–72), W 99 (83–119), D% 80 (70–95), S W% 204 (175–230), G S% 76 (71–83).
* S. loci: S L 71 (60–80), G L 46 (40–52), W 85 (60–100), D% 73 (61–80), S W% 190 (170–210), G S% 70 (50–80).
* S. longicaudum: S L 91 (72–108), G L 60 (54–65), W 136 (86–194), D% 75 (56–92), S W% 161 (116–225), G S% 66 (56–88).
* S. phyllophagae: S L 72 (65–77), G L 51 (46–56), W 65 (57–79), D% 75 (68–80), S W% 187 (136–223), G S% 71 (61–77).
* S. puertoricense: S L 78 (71–88), G L 40 (36–45), W 101 (67–148), D% 77, S W% 152, G S% 51.
* S. pui: S L 84 (78–88), G L 62 (58–65), W 137 (118–180), D% 77 (70–91), S W% 152 (140–184), G S% 74 (69–81).
* S. pwaniense: S L 92 (80–97), G L 60 (56–64), W 158 (109–196), D% 85 (71–98), S W% 146 (110–185), G S% 77 (73–83).
* S. riojaense: S L 78 (72–83), G L 48 (43–51), W 127 (98–172), D% 90 (80–105), S W% 155 (115–179), G S% 61 (55–70).
* S. taiwanensis: S L 94 (89–99), G L 68 (65–70), W 112 (92–141), D% 70 (66–75), S W% 213 (210–214), G S% 73 (71–74).
* S. tophus: S L 79 (70–91), G L 59 (47–65), W 162 (112–185), D% 92 (71–103), S W% 120 (100–160), G S% 70 (60–90).
* S. vulcanicum: S L 71 (46–88), G L 50 (32–65), W 110 (48–159), D% 70 (42–90), S W% 140 (90–210), G S% 70 (60–90).
– Data not available
Comparative morphometrics of infective juveniles of Steinernema chainatense n. sp. and other species of the glaseri-group. All measurements are in μm as mean (range). Abbreviations are as defined in Table 1
Table 3. Long description
The table consists of 12 columns: Species, L, W, E P, N R, E S, T, a, b, C, D%, and E%. Measurements are in micrometers as mean and range.
* Steinernema chainatense n. sp.: L 871 (798–939), W 27 (24–30), E P 72 (64–81), N R 100 (86–111), E S 132 (121–138), T 77 (68–87), a 32 (28–35), b 6.6 (6–7.1), C 11 (10–13), D% 54 (48–60), E% 93 (78–105).
* S. aciari: L 1113 (975–1250), W 47 (42–53), E P 95 (87–100), N R 114 (106–125), E S 146 (135–157), T 78 (68–88), a 24 (20–27), b 7.9 (7.0–8.3), C 14 (14–16), D% 65 (60–70), E% 123 (113–134).
* S. adamsi: L 880 (775–957), W 35 (32–40), E P 70 (64–75), N R 83 (75–92), E S 103 (90–112), T 73 (62–82), a 25 (22–29), b 8 (8.0–10), C 12 (11–14), D% 68 (61–78), E% 96 (84–111).
* S. australe: L 1316 (1162–1484), W 38 (34–41), E P 110 (95–125), N R 126 (102–143), E S 169 (140–194), T 103 (92–114), a 35 (31–38), b 7.8 (6.6–9.2), C 13 (11–15), D% 65 (57–78), E% 107 (94–122).
* S. glaseri: L 1130 (864–1448), W 43 (31–50), E P 102 (87–110), N R 120 (112–126), E S 162 (158–168), T 78 (62–87), a 29 (26–35), b 7.3 (6.3–7.8), C 15 (14–16), D% 65 (58–71), E% 131 (122–138).
* S. vulcanicum: L 1204 (1081–1323), W 39 (29–55), E P 93 (77–107), N R 117 (98–136), E S 157 (134–173), T 86 (74–100), a 32 (22–39), b 7.7 (6.8–9.2), C 14 (11–17), D% 60 (47–68), E% 109 (83–135).
Other species listed include S. apuliae, S. arenarium, S. bakwenae, S. boemarei, S. brazilense, S. caudatum, S. cubanum, S. diaprepesi, S. ethiopiense, S. guangdongense, S. hattingi, S. hermaphroditum, S. indicum, S. innovationi, S. jeffreyense, S. karii, S. keralense, S. khoisanae, S. khuongi, S. lamjungense, S. leizhouense, S. litchii, S. loci, S. longicaudum, S. puertoricense, S. pui, S. pwaniense, S. riojaense, S. taiwanensis, and S. tophus. Dashes indicate data not available.
- Data not available
Description
Infective juvenile (IJ)
Body slender, slightly C-shaped when heat-killed, averaging 871 μm in length. Head region rounded, continuous with body contour, slightly truncated at anterior end. Lip region with four cephalic papillae and a pair of amphidial apertures. Cuticle distinctly annulated, with striations ca. 1.5 μm wide at mid-body (clearly visible under SEM). Lateral fields with eight ridges (nine lines) at mid-body. Lateral field beginning anteriorly with a single incisure on the second annulus; at about the 10th annulus, two marginal incisures appear, forming two ridges, with the number subsequently increasing to six ridges (seven incisures) near the excretory pore. More posteriorly, two additional ridges arise, resulting in a total of eight ridges. At the level of the phasmids, the number decreases from eight to two ridges, which continue almost to the tail tip. Lateral field formula: 2, 6, 8, 2. Deirids not observed. Hemizonid distinct, located anterior to basal bulb. Pharynx with slender, cylindrical corpus; isthmus narrow, surrounded by nerve ring. Excretory pore situated slightly posterior to mid-pharynx (D% = 54). Cardia conspicuous. Bacterial vesicle typically well developed, ellipsoidal, about 14 μm wide and 28 μm long. Tail conoid, tapering gradually, with dorsal and ventral depressions posterior to phasmids. Hyaline portion long, comprising about 55% of tail length. Anus sickle-shaped, approximately 4–5 μm wide. Phasmids distinct, positioned anterior to mid-tail (at ca. one-third of the tail length), ventral to lateral field.
First-generation male
Body J-shaped in heat-killed specimens, curved ventrally in posterior region and gradually tapering anteriorly. Cuticle finely annulated, with faint transverse striations. Stoma short, wide, and reduced. Excretory pore situated posterior to the nerve ring, at about 82% of the pharynx length on average. Spicules paired, curved, brown, averaging 88 μm in length, with pointed tips. Manubrium elongate, with a length/width ratio of 1.7:1. Calomus indistinct; lamina slightly curved, bearing two internal ribs. Velum distinct, parallel to the lamina, extending from the calomus to the distal part of the lamina. Gubernaculum boat-shaped, with anterior end pointed. Tail short, conoid, tapering gradually to a bluntly rounded terminus. Genital papillae 25 in total, arranged as 12 pairs and a single midventral papilla anterior to the cloaca. Paired papillae distributed as follows: seven subventral precloacal pairs, one sublateral precloacal pair, one subventral adcloacal pair, two sublateral postcloacal pairs, and one subdorsal postcloacal pair.
Second generation male
General morphology similar to that of the first-generation male, but smaller.
First-generation female
In heat-killed specimens, body C-shaped to strongly spirally coiled. Fine cuticular annulations faintly visible under SEM. Head bluntly rounded, slightly tapering anteriorly, continuous with body contour, bearing six labial and four cephalic papillae. Excretory pore situated at the level of the nerve ring. Vulval opening slightly posterior to mid-body, transverse, strongly protruding from body contour and slightly asymmetrical; epiptygma small. Vagina short, leading to paired uteri. Rectum narrow; anal opening distinct, with absent to moderate postanal swelling. Tail of mature females obese, bluntly conoid, shorter than anal body diameter, abruptly tapering to a rounded terminus.
Second generation female
Morphologically similar to first-generation females, but smaller. Vulva symmetrical, slightly protruding from body contour. Tail conical, approximately 1.7 times the anal body diameter in length, gradually tapering to a pointed tip. Postanal swelling weakly developed.
Type material
Holotype 1st generation male, paratype males (20 slides with 60 specimens), paratype females (15 slides with 50 specimens) and IJs (12 slides with 110 specimens) deposited at the EPN collection in the Laboratory of Entomopathogenic Nematodes, Faculty of Agriculture and Technology, University of South Bohemia, České Budějovice, Czech Republic. In addition, a living culture of this strain is maintained at the Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand.
Etymology
The species epithet chainatense refers to Chai Nat Province, Thailand, the type locality of the species.
Molecular characterisation and phylogenetic analysis
Molecular data place S. chainatense n. sp. within the longicaudum clade of the glaseri-group (Figures 4–5). The ITS sequence (GenBank accession number PZ864282) of S. chainatense n. sp. differs from those of other members of the longicaudum clade by 3.6%–10.4%, whereas differences in the D2–D3 (GenBank accession number PZ869493) region range from 1.5% to 3.5%, corresponding to 29–94 bp and 9–28 bp differences, respectively (Table 4). The COI sequence (GenBank accession number PZ864315) is available for only three other species of the longicaudum clade. The COI sequence of S. chainatense n. sp. differs by 11.6% from that of S. longicaudum (AY943992), 10.8% from S. hermaphroditum (AY943987), and 12.1% from S. keralense (PQ248558).
Phylogenetic relationships of Steinernema chainatense n. sp. and other members of the glaseri-group of Steinernema based on analysis of ITS rDNA sequences. Steinernema affine and S. akhursti were used as outgroup taxa. The tree was inferred using the Maximum Likelihood method under the GTR+G+I model. Bootstrap support values based on 1,000 replicates are shown next to the branches. Branch lengths are proportional to the number of substitutions per site.
Figure 4. Long description
The phylogenetic tree branches from a root on the left toward terminal nodes on the right. At the top of the tree, S. chainatense n. sp. (P Z 8 6 4 2 8 2) is clustered with S. taiwanensis with a bootstrap value of 91. This clade is part of a larger group including S. adamsi, S. guangdongense, S. longicaudum, S. lamjungense, S. hermaphroditum, S. pui, and S. keralense.
Moving down the tree, the next major clade includes S. bakwenae, S. jeffreyense, S. khoisanae, S. hattingi, S. tophus, and S. innovationi. Below this, a distinct group contains S. loci, S. leizhouense, S. thanhi, and S. aciari.
The middle section of the tree features a cluster with S. australe, S. brazilense, and S. diaprepesi, followed by a large clade containing S. boemarei, S. arenarium, S. apuliae, S. riojaense, S. phyllophagae, S. glaseri, S. khuongi, and S. cubanum.
Toward the bottom, a separate lineage includes S. indicum, S. pwaniense, S. litchii, S. karii, and S. ethiopiense. The outgroup taxa, S. akhursti and S. affine, are positioned at the very bottom of the tree. Bootstrap values ranging from 55 to 100 are indicated at the nodes. A scale bar at the bottom left represents 0.20 substitutions per site.
Phylogenetic relationships of Steinernema chainatense n. sp. and other members of the glaseri-group of Steinernema based on analysis of D2–D3 expansion segments of 28S rDNA. Steinernema affine and S. akhursti were used as outgroup taxa. The tree was inferred using the Maximum Likelihood method under the GTR+G+I model. Bootstrap support values based on 1,000 replicates are shown next to the branches. Branch lengths are proportional to the number of substitutions per site.
Figure 5. Long description
A Maximum Likelihood phylogenetic tree with horizontal branches. At the bottom left, two outgroup taxa, S. affine (A F 3 3 1 8 9 9) and S. akhursti (K F 2 8 9 9 0 2), branch off first. The main clade, supported by a bootstrap value of 84, contains the glaseri-group.
From top to bottom, the terminal nodes are:
* S. chainatense n. sp. (P Z 8 6 9 4 9 3) in bold, sister to S. lamjungense (H M 0 0 0 1 0 2) with a support value of 50.
* A cluster containing S. taiwanensis, S. longicaudum, and S. guangdongense (support values 91 and 69).
* S. hermaphroditum and S. adamsi (support 69).
* A group including S. tophus, S. innovationi, S. bakwenae, S. jeffreyense, S. hattingi, and S. khoisanae (support values 91 and 86).
* A clade containing S. litchii, S. pwaniense, S. indicum, S. karii, and S. ethiopiense (support values 99, 52, and 85).
* S. keralense.
* A large sub-clade (support 74) containing S. australe, S. brazilense, S. puertoricense, S. diaprepesi, S. apuliae, S. riojaense, S. arenarium, S. vulcanicum, and S. boemarei.
* A final cluster (support 99) containing S. phyllophagae, S. cubanum, S. khuongi, and S. glaseri (support 86).
* S. leizhouense and S. aciari (support 88) branching near the base of the glaseri-group.
A scale bar at the bottom left indicates 0.050 substitutions per site.
Pairwise nucleotide similarities (below diagonal) and numbers of nucleotide differences (above diagonal) for the ITS and D2–D3 rDNA regions among species of the longicaudum clade within the glaseri-group and other related species of the glaseri-group
Table 4. Long description
The table is split into two vertical sections representing different r D N A regions. Each section lists 15 species/strains on the vertical axis and corresponding numbers 1 through 15 on the horizontal axis.
Top Section (I T S region):
* Species include S. chainatense n. sp., S. taiwanensis, S. adamsi, S. longicaudum, S. guangdongense, S. pui, S. lamjungense, S. hermaphroditum, S. keralense, S. hattingi, S. khoisanae, S. jeffreyense, S. bakwenae, S. tophus, and S. innovationi.
* Nucleotide differences (above diagonal): Values range from 19 (between S. adamsi and S. guangdongense) to 160 (between S. keralense and S. innovationi).
* Percentage similarities (below diagonal): Values range from 80.5 percent (between S. keralense and S. tophus) to 97.7 percent (between S. adamsi and S. guangdongense).
Bottom Section (D 2 to D 3 region):
* Species list is identical to the top section.
* Nucleotide differences (above diagonal): Values are generally lower than the I T S region, ranging from 4 (between S. tophus and S. innovationi) to 37 (between S. longicaudum and S. tophus).
* Percentage similarities (below diagonal): Values are higher than the I T S region, ranging from 94.9 percent to 99.5 percent (between S. tophus and S. innovationi).
In both sections, the diagonal is empty as it represents self-comparison.
Phylogenetic analyses of both rDNA regions clearly separate S. chainatense n. sp. from all other glaseri-group species (Figures 4–5). The ITS-based phylogeny indicates a close relationship with S. taiwanensis, S. adamsi, S. guangdongense, and S. longicaudum, with S. taiwanensis as its sister taxon. In contrast, the D2–D3 tree provides poor resolution of relationships within the glaseri-group. This poor resolution further supports the limited suitability of the D2–D3 region for resolving phylogenetic relationships within Steinernema (Půža and Machado Reference Půža and Machado2024). Overall, molecular data support the recognition of S. chainatense n. sp. as a new species according to the phylogenetic and evolutionary species concepts (Adams Reference Adams1998).
Ecological characterisation
S. chainatense n. sp. was isolated from a cassava field located on a lowland alluvial plain in Chai Nat Province, Central Thailand, at an elevation of approximately 20 m. Sampling was conducted during the mid-rainy season under a tropical wet-and-dry climate. At the time of sampling, the topsoil was dry, with 1% moisture content, a pH of 7.0, and a loamy texture. The natural host of this nematode remains unknown.
Bacterial symbiont
The bacterial symbiont of S. chainatense n. sp. was recently characterised and described as Xenorhabdus thailandensis (Thanwisai et al. Reference Thanwisai, Machado, Bhat, Pidot, Tandhavanant, Subkrasae, Meesil, Ardpairin, Pansri and Vitta2025). This bacterium was also found in association with S. lamjungense (Meesil et al. Reference Meesil, Ardpairin, Sharkey, Pidot, Vitta and Thanwisai2025), another member of the longicaudum clade. To date, all members of the longicaudum clade with known bacterial symbionts are associated with Xenorhabdus species belonging to a distinct clade, including X. griffiniae, X. ehlersii, X. thailandensis, and closely related, as yet undescribed taxa. The bacterial symbionts of S. guangdongense, S. pui , and S. taiwanensis remain unknown.
Diagnosis and relationships
Steinernema chainatense n. sp. can be characterised by a combination of morphological and morphometric characters of IJs and first-generation males. IJs are characterised by a body length of 871 (798–939) μm, a maximum body diameter of 27 (24–30) μm, and a lateral field with eight equally developed ridges at mid-body and a formula of 2, 6, 8, 2. First-generation males possess large, brown spicules, 88 (80–95) μm long, with a well-developed velum. The male genital papillae number 25 in total, comprising 12 pairs and a single midventral papilla. Steinernema chainatense n. sp. is further characterised by sequences of the ITS and D2–D3 regions of rDNA and COI.
Discussion
Based on this combination of characters, S. chainatense n. sp. belongs to the longicaudum clade (sensu Spiridonov and Subbotin Reference Spiridonov, Subbotin, Hunt and Nguyen2016) within the glaseri-group (sensu Spiridonov et al. Reference Spiridonov, Reid, Podrucka, Subbotin and Moens2004). This clade comprises species known exclusively from South and South-East Asia, namely S. adamsi; S. guangdongense Qiu, Fang, Zhou, Pang & Nguyen, 2004; S. hermaphroditum Stock, Griffin & Chaerani, 2004; S. keralense Anes, Patil, Babu, Aarthi, Gotyal, Josephrajkumar, Mhatre, Sajan, Gowda & Půža, Reference Anes, Patil, Babu, Aarthi, Gotyal, Josephrajkumar, Mhatre, Sajan, Gowda, Sushil and Půža2025; S. lamjungense Khatri-Chhetri, Waeyenberge, Spiridonov, Manandhar & Moens, 2011; S. longicaudum Shen & Wang, 1992; S. pui Qiu, Zhao, Wu, Lv & Pang, 2011; and S. taiwanensis Tseng, Hou & Tang, 2018. The new species can be distinguished from other members of the longicaudum clade by a combination of morphological and morphometric characters of first-generation males (Table 2) and IJs (Table 3).
First-generation males of S. chainatense n. sp. differ from those of S. adamsi in having longer spicules (88 (80–95) μm vs 62 (44–69) μm) and gubernaculum (60 (57–64) μm vs 37 (28–59) μm), a greater maximum body diameter (113 (102–156) μm vs 70 (56–90) μm), a higher SW% (178 (143–215) vs 127 (93–143)), and a higher number of genital papillae (25 vs 23). IJs of S. chainatense n. sp. can be distinguished from those of S. adamsi by a longer pharynx (132 (121–138) μm vs 103 (90–112) μm), a lower b ratio (6.6 (6.0–7.1) vs 8 (8–10)), and a lower D% (54 (48–60) vs 68 (61–78)).
Males of S. chainatense n. sp. can be distinguished from those of S. guangdongense by having a higher number of genital papillae (25 vs 23). IJs of S. chainatense n. sp. differ from those of S. guangdongense in having a shorter body (871 (798–939) μm vs 1055 (987–1145) μm), a smaller maximum body diameter (27 (24–30) μm vs 42 (30–48) μm), and a lower b ratio (6.6 (6.0–7.1) vs 7.9 (7.3–8.5)).
Males of S. chainatense n. sp. differ from those of S. hermaphroditum in having longer spicules (88 (80–95) μm vs 68 (65–70) μm), a longer gubernaculum (60 (57–64) μm vs 48 (47–50) μm), a higher D% (82 (75–88) vs 48 (44–50)), and a higher number of genital papillae (25 vs 21).
Males of S. chainatense n. sp. differ from those of S. keralense in having longer spicules (88 (80–95) μm vs 68 (60–72) μm) and a lower GS% (68 (63–76) vs 82 (72–92)). IJs of S. chainatense n. sp. differ from those of S. keralense in having a thinner body, with a smaller maximum body diameter (27 (24–30) μm vs 39 (30–43) μm).
Steinernema chainatense n. sp. can be distinguished from S. lamjungense by a smaller mean maximum body diameter in first-generation males (113 (102–156) μm vs 154 (116–205) μm) and a higher c ratio in IJs (11 (10–13) vs 9.6 (7.7–11)). In addition, first-generation females of S. chainatense n. sp. have a more posteriorly located excretory pore relative to the pharynx, as indicated by a considerably higher D% (71 (64–79) vs 33 (21–47)).
Males of S. chainatense n. sp. can be distinguished from those of S. longicaudum by a higher number of genital papillae (25 vs 23). IJs of S. chainatense n. sp. differ from those of S. longicaudum in having a smaller maximum body diameter (27 (24–30) μm vs 37 (34–40) μm).
IJs of S. chainatense n. sp. differ from those of S. pui in having a smaller maximum body diameter (27 (24–30) μm vs 36 (33–40) μm), a shorter distance from the anterior end to the excretory pore (72 (64–81) μm vs 85 (80–95) μm), a lower c ratio (11 (10–13) vs 15 (13–16)), and a lower E% (93 (78–105) vs 125 (109–142)).
Males of S. chainatense n. sp. can be distinguished from those of S. taiwanensis by a higher D% (82 (75–88) vs 70 (66–75)), a lower SW% (178 (143–215) vs 213 (210–214)), and a higher number of genital papillae (25 vs 23). IJs of S. chainatense n. sp. differ from those of S. taiwanensis in having a shorter body (871 (798–939) μm vs 1012 (983–1045) μm), a smaller maximum body diameter (27 (24–30) μm vs 35 (33–37) μm), a shorter distance from the anterior end to the excretory pore (72 (64–81) μm vs 82 (80–84) μm), a shorter distance from the anterior end to the nerve ring (100 (86–111) μm vs 124 (120–127) μm), and a shorter pharynx (132 (121–138) μm vs 147 (143–150) μm).
Financial support
This work was supported by the Faculty of Agriculture and Technology, University of South Bohemia, the Czech Science Foundation (Grant No. 23-06457S), and the Royal Golden Jubilee Ph.D. Program (Grant No. N41A640215), through the National Research Council of Thailand.
Competing interests
The authors declare none.
