Steinernema sandneri n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Poland

Body straight or slightly abdominally curved when heat-relaxed, tapering gradually from the base of esophagus to the anterior end and from anus to the distal end. Second-stage cuticle present shortly after leaving the host body, with six labial and four cephalic papillae, but lost in storage after a few days/weeks (depending on the temperature). Cephalic region continuous with body smooth, truncate-conical, with four cephalic papillae and prominent amphidal apertures (Figs 1A and 2B). Mouth and anus closed (Fig. 2B,F). Cuticle with prominent striation along almost the whole body (Fig. 2B,D,F). Lateral fields beginning as a single line close to the anterior end, increasing to eight ridges, posteriorly gradually reduced to four (anus level) and two (phasmid level) (Fig. 2D,F). Deirids not visible. Esophagus with narrow corpus, slightly swollen metacorpus, isthmus surrounded by nerve ring (Fig. 2A,C). Excretory pore in the middle between anterior end and basal bulb (Figs 1A and 2A). Hemizonid distinct, between nerve ring and esophagus base. Cardia present. Bacterial vesicle well developed, with visible rod-shaped bacteria (Fig. 2C). Tail conical, tapering gradually. Phasmids distinct, located 40% of tail length, posterior to anus. Hyaline portions comprising ca. 1/3 of tail length (Figs 1E and 2E,F).

Figure 1:

Figure 2:

Body C- or J-shaped when heat-relaxed. Cuticle with faint transverse striation visible in SEM (Fig. 3A–D). Lateral fields not observed. Cephalic region smooth, rounded, with four cephalic and six smaller labial papillae and slit-like amphid openings (Fig. 3A,B). Stoma shallow, funnel-shaped, cheilorhabdions prominent. Esophagus with cylindrical procorpus, slightly swollen metacorpus, and narrower isthmus surrounded by nerve ring located anteriorly to basal bulb. Excretory pore anterior to nerve ring, close to metacorpus (Figs 1B and 4A). Cardia prominent. Anterior deirids similar to genital papillae in shape and size (Fig. 3A). Posterior deirids usually located anteriorly, just before the first pair of genital papillae. Testis monorchic, reflexed. Spicule with two ribs and velum not reaching spicule tip (Figs 1F,G, 4E). Gubernaculum boat-shaped in lateral view, with ventrally curved manubrium (Figs 1H and 4F,G). Typically 23 genital papillae present, comprising 11 pairs and 1 single precloacal midventral. Additional papillae – if occur – usually before posterior deirids. Phasmid openings between ventral last pair of genital papillae. Tail terminus with mucron (Figs 1F, 3D and 4B).

Figure 3.

Figure 4:

Similar to first-generation male but shorter and more slender. Excretory pore located more posteriorly. Tail relatively longer, with prominent mucron (Table 1; Fig. 4C,D).

Body C-shaped when heat-relaxed and fixed. Cuticle smooth when observed in a light microscope, with faint striation in SEM (Fig. 5B,D,F). Lateral fields not observed. Deirids inconspicuous, difficult to observe even under SEM. Labial region rounded, continuous with the body. Six labial and four cephalic papillae (Fig. 5B). Slit-like amphidial apertures. Cheliorhabdions large, sclerotized. Stoma prominent. Esophagus with cylindrical procorpus, swollen metacorpus, and distinct isthmus. Excretory pore in mid-esophagus region. Nerve ring just anterior to basal bulb (Fig. 5A). Cardia prominent. Gonads amphidelphic, reflexed. Vulva in the form of transverse slit located slightly posterior to mid-body (Table 1). Vulval lips slightly protruding, asymmetrical, with larger posterior lip (Figs 1D and 5C,D). Tail length shorter than body anal diameter, with slight post-anal swelling. Tail terminus with mucron (Figs 1C and 5E,F).

Figure 5:

Similar to first-generation female but smaller. Vulva more protruding, with distinct asymmetry between lips. Tail with mucron, but without pronounced post-anal swelling (Table 1).

Steinernema sandneri n. sp. can be successfully reared on G. mellonella or Tenebrio molitor (Coleoptera: Tenebrionidae) larvae at a temperature in the range of 15–20°C. The life cycle of S. sandneri n. sp. is similar to that of other Steinernema species. G. mellonella larvae exposed to 50–100 IJs die within 3–4 days. Adults of the second generation can be found in insect cadaver 8–12 days after infection. Pre-infective juveniles migrate from the host body, mature for a few days, and migrate to water trap after 18–21 days.

Mating attempts were observed between S. sandneri n. sp. and S. kraussei and S. silvaticum, but no fertile offspring was produced in any of the crosses. Hybridization tests with S. kraussei, S. silvaticum, S. feltiae, S. oregonense, S. ichnusae, S. weiseri, S. jollieti, and S. cholashanense showed that S. sandneri n. sp. was reproductively isolated. The positive control always yielded a progeny.

The new species was characterized by analysis of the morphology and morphometrics of IJs and adults (Table 1). IJs have a body length of 843 μm (708–965), a body diameter of 27 (23–32) μm, and a tail length of 75 (64–86) μm. The distance from the anterior end to the excretory pore is 56 (44–64) μm and to the esophagus base is 138 (123–151) μm, D% = 40 (36–45), E% = 74 (63–86). The lateral field in the mid-body region has 8 ridges, and the hyaline part of the tail occupies ~1/3 of its length. The first-generation male is characterized by a spicule length of 60 (53–65) μm and by a gubernaculum length of 44 (39–50) μm. The spicule manubrium is almost as long as it is wide, the shaft is short, and the velum expands from the calomus to the end of the ventral rib. The tail of both generation males is mucronated. The first- and second-generation females of S. sandneri n. sp. have a slightly protruding vulva and a mucron at the posterior end.

The species belongs to the feltiae-kraussei group of Steinernema, which comprises over 20 species. The length of S. sandneri n. sp. IJs 843 μm (708–965) is typical for this group, identical with the length S. cholashanense 843 μm (727–909), and similar to that of S. feltiae 849 μm (766–928), S. silvaticum 860 μm (670–975), S. xueshanense 860 μm (768–929), and S. ichnusae 866 μm (767–969). The IJs of S. sandneri n. sp. can be distinguished from these species by the relatively thicker body a = 6.1 (5.5–6.9) vs > 6.3 (5.6–7.7), the more anteriorly located excretory pore 56 μm (44–64) vs >62 μm (51–73), and the lower D% value 40 (36–45) vs > 46 (42–53). The position of the IJ nerve ring 103 μm (83–118) is more anterior than in S. feltiae 113 μm (108–117). The esophagus of S. sandneri n. sp. IJs is longer 138 μm (123–151) than in most of the clade species except for S. ichnusae 138 μm (119–148) (Table 2).

First-generation males of S. sandneri n. sp. can be distinguished from other feltiae-kraussei clade members by shorter spicules 60 μm (53–65), except of S. hebeiense 57 μm (51–63), S. xinbinense 56 μm (49–62), S. silvaticum 51 μm (42–64), and S. kraussei 49 μm (42–53). The SW% value of S. sandneri n. sp. males 111 (97–127) is similar only to that of S. feltiae 113 (99–130), S. kraussei 110 (range: data not available), and S. cholashanense 115 (92–144). The relative length of the gubernaculum is high GS% = 79 (61–83) and comparable only with S. oregonense GS% = 79 and S. weiseri GS% = 80 (70–85). The position of the excretory pore in S. sandneri n. sp. males D% = 51 (42–59) is more anterior than in most clade species, with the exception of S. hebeiense D% = 51 (48–59), S. nguyeni D% = 48 (38–57), S. weiseri D% = 49 (39–60), S. sangi D% = 49 (42–63), and S. kraussei D% = 53 (range: data not available). The mucron in first-generation males distinguishes S. sandneri n. sp. from European S. weiseri, S. ichnusae, and S. silvaticum (Table 3).

Natural host unknown. The nematode isolate S17-050 was obtained from sandy-loamy soil samples collected in eastern Poland (51°46’55”N 22°42’35”, 147 m a.s.l.) in 2017. The soil samples were collected in a mixed forest from 0 to 20 cm depth. Nematodes were isolated using a modified live trap method (Bedding and Akhurst, 1975) with the use of G. mellonella larvae as a bait. Detailed studies were performed on a straight line of nematodes (offspring of 2 IJs) reproducing successfully in G. mellonella and maintained in our laboratory.

Holotype male, paratype males, paratype infective juveniles, paratype females, and second-generation paratype males and females were deposited in the nematode collection of the Museum and Institute of Zoology, Polish Academy of Sciences, Wilcza 64, Warsaw, Poland (see Table S2 for deposition numbers).

S. sandneri n. sp. was characterized genetically by the sequences of the ITS rDNA, D2D3 of 28 S rDNA, and the mitochondrial cox1 gene. No variation in the sequences of these genes was found between the analyzed individuals. The D2D3, ITS, and cox1 sequences of S. sandneri n. sp. were deposited in the GenBank with accession numbers MW078535, MW078536, and MW078544, respectively.

As it is known that the molecular diversity in the group of nematodes assigned as S. kraussei is relatively high, we included multiple S. kraussei sequences in the molecular analysis, also these of S. kraussei from the Lublin region, which are sympatric to the new species (Table S1). Compared to other species of the genus Steinernema with ITS sequences available in the GenBank, S. sandneri S17-050 showed the highest ITS sequence identity with S. kraussei strains, i.e. 96.0–97.7%, corresponding to 16–28 nucleotide substitutions (Table S3). It was also noted that the GeneBank sequence AY171250, attributed to S. kraussei from Belgium, displayed 99.7% identity and 2 bp difference from this of S17-050 isolate, which implies that this nematode is a conspecific to S. sandneri n. sp and should be considered as misidentification. Among the other Steinernema species, the most similar sequence of the ITS region with S. sandneri n. sp. was displayed by S. silvaticum (94.5–95.0% identity, 34–37 substitutions) and S. xinbinense (94.5% identity, 35 nucleotide substitutions). The ITS sequences of the other species of the Steinernema genus were more divergent from that of S. sandneri n. sp., showing identity ≤ 94% and at least 41 nucleotide substitutions (Tables 4 and S3).

The highest sequence identity of the D2D3 region of the new species was 98.2%, corresponding to 15 nucleotide substitution, in respect to the analyzed S. kraussei strains. The new species differs from other species from the feltiae-kraussei group by at least 21 bp, showing ≤ 97.5% nucleotide identity (Table 5).

The analysis of the cox1 gene sequences showed 92.9–93.8% identity to the sequences of S. kraussei isolates (36–40 bp difference) and ≤ 87.7% identity (minimum 70 bp difference) to other sequences of Steinernema spp. (Table 6).

The alignment of the analyzed ITS sequences resulted in 870 positions, in which 266 positions were conserved, while 573 positions were variable, including 426 parsimony-informative and 136 singleton ones. The phylogenetic tree based on the ITS sequences shows that S. sandneri n. sp., S. kraussei, and S. silvaticum form a monophyletic cluster with 95% bootstrap support (BS) within the feltiae-kraussei group. It is also noted that S. sandneri n. sp. clusters with S. kraussei isolates as a sister group with 94% BS (Fig. 6). The ITS rDNA of S. sandneri n. sp. differs from that of the other species of the feltiae-kraussei group by four unique traits (present in the sequence alignment only in the new species but not in the others from the group) in the following positions: 530, 608, 656, and 713. In addition, S. sandneri n. sp. contains a unique stretch of eight adenine nucleotides in position 480–487 of the ITS sequence.

Figure 6:

In the case of the D2D3 region sequences, the alignment resulted in 899 positions, in which 611 positions were constant, while 247 positions were variable, including 130 parsimony-informative and 117 singleton ones. The phylogenetic tree based on D2D3 sequences shows that S. sandneri n. sp. and S. kraussei isolates form a monophyletic group with BS 56%, which is a part of the clade comprising S. silvaticum, S. xinbiense, S. cholashanense, S. tielingense, and S. oregonense with BS 68% (Fig. 7). S. sandneri n. sp. differs from the other species of the feltiae-kraussei group in the D2D3 region by six diagnostic traits in the following sequence positions: 52, 155, 374, 444, 456, and 463.

Figure 7:

The alignment in the cox1 gene sequences resulted in 567 positions, in which 376 positions were conserved, while 191 positions were variable, including 109 parsimony-informative and 82 singleton ones. The analysis involved only nine steinernematid nematode species, as the number of cox1 sequences available in the GeneBank is still limited. The phylogram based on cox1 gene sequences shows a clade separating S. sandneri n. sp., S. kraussei, and S. silvaticum with BS 88% (Fig. 8). In this clade, the new species and S. kraussei strains form a sister branch with 100% BS. Steinernema sandneri n. sp. differs from the other species of the feltiae-kraussei group in the cox1 gene by five diagnostic traits in positions 21, 138, 165, 225, and 263.

Figure 8: