Steinernema poinari, Mráček & Půža & Nermuť, 2014

Mráček, Zdeněk, Půža, Vladimír & Nermuť, Jiří, 2014, Steinernema poinari sp. n. (Nematoda: Steinernematidae) a new entomopathogenic nematode from the Czech Republic, Zootaxa 3760 (3), pp. 336-350 : 338-349

Steinernema poinari 1* sp. n.

( Figs 1–3 )

Measurements. See Table 1 .

Description. Infective juvenile: Body elongate, on average 768 µm long, bow-shaped when heat killed, usually exsheathed from the second-stage cuticle. Head region continuous, slightly flattened, not offset from body contour, with four cephalic papillae and a pair of pore-like amphidial apertures laterally. Cuticular striations (distinct under SEM) ca 1.5 µm wide at mid-body, but lip region and tail terminus smooth. Lateral fields consisting of six, not equally spaced and developed ridges in mid-body region (i.e., seven lines).

Only submarginal ridges prominent. Lateral field beginning with irregular cuticular depressions (lines) close to eight annule from anterior end; a short distance posteriorly, two ridges appearing and changing to five (six lines) at about excretory pore level; more posteriorly still central ridge dividing into two, making a total of six ridges. In posterior part of body, number of ridges reducing gradually. Anterior to anus, two middle ridges merging to leave five ridges in lateral field. At phasmid opening, lateral field reduced to two and at tail dorsal constriction to one ridge, extending almost to tail tip. With above arrangement, formula of lateral field is 2, 5, 6, 5, 2, 1. Deirids not observed. Hemizonid visible as a 4–5 µm wide cuticular depression usually located between anterior of basal bulb and nerve ring. Pharynx collapsed, not well seen, corpus slender, cylindrical, isthmus indistinct, surrounded by nerve ring. Excretory pore located at level of mid-pharynx (D% = 46). Cardia ca 5–9 µm long, bacterial vesicle usually not well seen, ellipsoid, approximately 30x 10 µm. Rectum ca 75% of anal body diam. long. Tail conoid with characteristic dorsal constriction that is situated slightly before proximal portion of the hyaline layer that occupies approximately one half of tail length (50%), terminus sharply pointed. Anus wide, sickle-shaped. Phasmids located at ca 45–50% of tail length posterior to anus, with aperture near point where dorsal constriction begins.

Adults: Head rounded, almost continuous with body contour. Head morphology characterized by six lips fused at their base, each lip with a labial papilla. Four cephalic papillae forming an outer circle. Amphidial aperture distinct, located laterally between labial and cephalic circle of papillae. Cuticle with distinct transverse striation, lateral fields absent. Excretory pore located at the level of mid procorpus. Stoma wide, shallow, cheilorhabdions distinct, sclerotized, followed by sclerotized smaller prorhabdions. Pharynx muscular with procorpus extending into slightly swollen non-valvate metacorpus, narrower isthmus region surrounded by nerve ring, followed by rounded basal bulb. Cardia rather long, intestine generally with a wide lumen. Distinct, papilliform deirid situated slightly anterior to nerve ring.

First generation male: Body characteristically J or C-shaped in heat-relaxed specimens, tapering toward head region, tail curved ventrally. Stoma ca 3–4 µm long and 5–8 µm wide. Excretory pore located posterior to midpharynx but anterior to nerve ring (42–64% of distance from anterior body end to the base of pharynx). Spicules paired, symmetrical, finely curved, robust, colorless, ca 62–78 µm long, spicule tip pointed, spicule manubrium slightly variable, finely rounded, with an average length/width ratio approximate of 1:1. Manubrium forming slightly less than 25% of spicule length, calomus and rostrum distinct. Lamina robust with two ribs, finely curved. Dorso-proximal portion of lamina often with a distinct hump. Velum bow-like, distinct, extending from rostrum to 2/3 of lamina length. Rostrum developed or fused with calomus. In lateral view, gubernaculum boat-shaped, short proximal part straight, never bent like in many steinernematids, approximately from 3/4 its length strongly attenuated to distal point. In ventral view, cuneus small, needle-shaped, or absent, corpus with two wings. Tail bluntly conoid, concave on ventral surface with papillated protuberance on its tip, filamentous mucron absent. Genital papillae totaling 23, comprising 11 pairs and a single midventral papilla located just anterior to cloacal opening. Of the paired papillae, six pairs located precloacal (five pairs subventral, one pair lateral). Two pairs adcloacal subventrally. Three pairs on the tail tip and consistently near tail terminus with one pair subdorsal and two at tail tip.

1. * The new species is named after Prof. G.O. Poinar Jr, the leader in EPN recovery and description.

Second generation male: Similar to first-generation, but smaller in body and internal organs size (see Table 1 ). Mucron short, either 2–3 µm filamentous or papillated. Spicules slightly variable in shape, similar to the first generation male.

First generation female: Body variable in length, usually C-shaped on heat relaxation. Head bluntly rounded, slightly tapering anteriorly, not offset from body contour. Stoma ca 4–7 µm long and 10–14 µm wide. Genital tract amphidelphic with reflexed ovaries. Vulva distinct, mostly symmetrical, rarely slightly asymmetrical and in form of a transverse slit, not or only slightly (3–4 µm), protruding from body contour. Vulval opening always posterior to mid-body, on average 55%. Vulva slightly protruding (ca 12 µm), epiptygmata not developed. Vagina short, leading into paired uteri. Rectum narrow, anal opening distinct. Tail of mature females obese, bearing two minute protuberances. Postanal swelling slightly (2–8 µm) or not developed in mature females. Pigmy forms observed. Endotokia matricida rarely seen.

Second generation female Similar to first generation in general morphology, but smaller ( Table 1 ). Vulval opening slightly posterior to mid-body, slightly to moderately protruding. Tail conical, sharply pointed, longer than anal body diam., extending to form a conical/filamentous mucron (6 to 10 µm long). Postanal swelling absent or slightly developed. Endotokia matricida observed.

Type host and locality. The type isolate, CZ 1152, was recovered from brown clay soil at an elevation of 450 meters, from a bank of unnamed creek covered by willow and alder bush in west Bohemia near Plzeň city, Czech Republic (49°50'54.162"N, 13°8'8.154"E). Insect hosts unknown .

Other localities. Same species, isolate CZ 1160 was recovered from beneath the canopy of willow in Novohradské Hory Mts. (48°38'55.221"N, 14°41'23.277"E, 825 m asl). Other strain CZ 1093 was isolated in birch and aspen stand on the edge of the maize field in South Bohemia (48°59'22.723"N, 14°19'47.778"E, 441 m asl). Genetically and morphologicaly identical isolate from the vicinity of Tomsk city, Siberia, Russia was received from Dr. Sergei Spiridonov .

Type material. Holotype 1st generation male, paratype males (19 slides with 51 specimens), paratype females (10 slides with 24 specimens) and third-stage juveniles, (6 slides with 87 specimens) were deposited at the EPN collection in the Laboratory of Entomopathogenic nematodes, Institute of Entomology , Biological Centre of the AS CR, Česke Budějovice , Czech Republic. Three slides with paratype IJ, males and females deposited at the USDA Nematode Collection, Beltsville, MD, USA.

Diagnosis and relationships. Morphological and morphometric data of S. poinari sp. n. were compared with the original description of S. intermedium in Poinar (1985) and published information by Nguyen et al. (2007). The new species is characterized morphologically by a combination of features of the IJ and adults. For IJ, the medium body length of 768 (687–848) µm, distance from anterior end to excretory pore of 64 (56–70) µm, anterior end to the end of pharynx of 141 (119–151) µm; tail length of 77 (68–87) µm, D% = 46 (40–55), E% = 84 (76–95), H% = 50 (43–56) and lateral field formula 2, 5, 6, 5, 2, 1 are characteristic. For first generation males, the diagnostic characters include the spicule length of 70 (62–78) µm; D% = 54 (42–64); E% = 207 (167–246); SW% = 109 (98– 123) and GS% = 70 (58–87). The spicule tip is pointed, manubrium length/width ratio approximate of 1:1. Females have a slightly protruding symmetrical, rarely slightly asymmetrical vulva, V% = 55 (52–59). The tail of mature first generation females is obese with two indistinct minute protuberance and a postanal swelling is slight or not developed. Second generation females with slight or without postanal swelling and with distinct conical/ filamentous mucron.

The new species belongs to the affine /intermedium- group ( Spiridonov et al. 2004). Phylogenetic analysis, spicule shape, position of excretory pore of IJ, number and arrangement of lateral fields, place it within this group. Affine/ intermedium group includes: S. affine , S. intermedium , S. beddingi , S. sichuanense and S. arasbaranense . Of these, two species, S. beddingi and S. sichuanense , are Palearctic, whereas S. intermedium , S. affine and S. arasbaranense are from the Holoarctic zoogeographical subregion (Mráček et al. 2005).

Infective juveniles of S. poinari sp. n. differ from S. affine by lacking a refractile spine in its tail terminus. The tail of the first generation females bearing papilla-like protuberances should play an important role in the steinernematid taxonomy. First generation females of S. poinari sp. n. possess a rounded terminus with two minute protuberances on the tail tip whereas S. affine and S. intermedium possess a wedge-like terminus with 2–4 protuberances, S. sichuanense four protuberances in position 3+1, while the wedge-like terminus of S. beddingi carries two protuberances and S. arasbaranense possess a terminal peg. S. poinari sp. n. can be differentiated from other species of the affine / intermedium group by several morphometric characters ( Tables 2–3 ). The mean body length of S. poinari sp. n. IJ is less than 800 µm, the latter being a major character to distinguish species of the affine / intermedium group from other Steinernematids. The IJ c´ ratio is greater than in S. affine and S. arasbaranense , at 4.1 (3.3–4.6) vs 3.6 (3.3–4.2) and 3.6 (3–5), respectively. The D% of S. poinari sp. n. IJs is lower than in S. beddingi and S. intermedium at 46 (40–55) vs 57 (52–64) and 54, respectively, whilst E% of S. poinari sp. n. differs from S. affine , S. arasbaranense , S. beddingi , S. intermedium at 84 (76–95) vs 94 (74–108), 67 (53– 75), 92 (84–103) and 100, respectively, and H% also differs from S. affine at 50 (43–56) vs 40 (36–45), respectively. Six ridges in the lateral field of S. poinari sp. n. should distinguish this species from S. arasbaranense with eight ridges. However, according to Fig. 5 in Nikdel et al. (2011), the number of ridges was wrongly calculated and S. arasbaranense has the same formula as all other species of affine / intermedium group.

*data from Nguyen et al. 2007.

*data from Nguyen et al. 2007

** recalculated from the original description ( Qiu et al. 2005)

The spicule tip of S. poinari sp. n. is pointed while blunt in S. affine , S. beddingi , S. intermedium and S. sichuanense . The SW% value of the first generation male of S. poinari sp. n. is much lower than that of S. affine , S. arasbaranense , S. intermedium and S. sichuanense at 109 (98–123) vs 117, 187 (174–213), 121, 130 (120–140), respectively, whilst the GS% value also differs from S. arasbaranense , S. beddingi and S. sichuanense at 70 (58– 87) vs 63 (52–70), 61 (55–66) and 80 (60–80), respectively. The spicule shape of S. poinari sp. n. differs significantly from the S. arasbaranense spicule. The former is finely curved, robust, colorless, with rostrum, manubrium L/W – 1/1 whilst the latter (according to Fig. 2 C, D in Nikdel at al. 2011)) is slightly curved, brownish with oblongate manubrium and without rostrum.

A major support for separation of species is the cross breeding with related species, in this case with S. affine and S. intermedium . There were no offspring between S. poinari sp. n. and these species.

Molecular characterization and phylogenetic analysis. Gel electrophoresis revealed a PCR-product of 1052 bp for ITS-region ( Table 4 ), comprising the transcribed spacer regions ITS1 and ITS2, the 5.8S rRNA gene and flanking regions of the 18S and 28S rRNA gene. From its closest relatives S. affine , S. intermedium , S. sichuanense and S. beddingi , S. poinari sp. n. is separated by 37–52 bp ( Table 4 ).

The PCR-product of part of the 28S rRNA gene containing the D2D3 expansion fragments had a length of 873 bp and from its closest relatives it is separated by 6–21 bp ( Table 4 ).

The phylogenetic relationships between species of Steinernema are presented in Fig 4 and Fig 5 . For Bayesian analysis of the ITS sequence, 1428 characters were included (tree length = 5527, CI = 0.363, RI = 0.590, RCI = 0.214 and HI = 0.637) for D2D3 region, 933 characters were included (tree length = 1333, CI = 0.464, RI = 0.659, RCI = 0.306 and HI = 0.536).

Both trees support S. poinari sp. n. as a new species belonging to the affine / intermedium group. In agreement with some previous studies (e.g. Spiridonov et al. 2004) the ITS tree further shows a basal position of the affine / intermedium group within the genus Steinernema . Morphological and molecular characteristics presented above clearly show S. poinari sp. n. as a new species of affine / intermedium group.

Distribution. Three recent findings of S. poinari sp. n. show that the species can be quite abundant in the Czech Republic. Its omission in previous surveys ( Mráček et al. 1999, Mráček et al. 2005) could be due to misidentification with morphologically similar S. intermedium . Another strain of S. poinari sp. n. “Tomsk” originates from a distant area of the Tomsk region, Siberia. A Genbank search of the ITS region of S. poinari revealed close similarity (99–100%) to several steinernematid strains referred to as “ Steinernema sp. 6 ” ( Spiridonov et al. 2004) that were isolated in UK, Belgium, Estonia, and Moscow and Altai regions of Russia. These strains are most likely conspecific with S. poinari sp. n. and thus the geographic distribution of S. poinari sp. n. seems to be quite wide, probably covering a large area of the Palearctic region.

Figures

FIGURE 1. Steinernema poinari sp. n. (A–C) First generation female. (A) Anterior portion with pharynx and excretory pore. (B) Vulval region. (C) Tail with minute protuberances. (D–E) Second generation female. (D) Tail with mucron. (E) Vulval region. (F–H) Second generation male. (F) Tail with an arrangement of genital papillae, laterally. (G) Tail with an arrangement of genital papillae (ventrally). (H) Spicules (laterally) and gubernaculum (ventrally). (I–J) Infective juvenile. (I) Anus and hyaline portion. (J) Lateral field with ridges (numbered). (K) Arrangement of labial and cephalic papillae, and amphids. Scales (in µm): A – D = 100, E = 130, F, H = 30, G = 35, J = 38, L = 25.

FIGURE 2. Steinernema poinari sp. n. SEM of infective juvenile, male and female. (A–D) Infective juvenile. (A) Head region with four papillae and amphid openings. (B) Lateral field in neck region. (C) Lateral field in mid–body. (D) Lateral field in tail region with phasmid opening. (E–G) First generation male. (E) Tail with genital papillae. (F) Posterior region of a male tail showing 7 pairs of precloacal genital, and one pair adcloacal papillae. (G) Higher magnification of posterior part of a male showing pairs 7–11. (H) First generation female with four minute terminal protuberances.

FIGURE 3. Steinernema poinari sp. n. LM of infective juvenile, male and female and LM of Steinernema intermedium spicule. (A–B) First generation female. (A) Vulval region. (B) Tail. (C–D) Second generation female. (C) Tail with spine–like mucron. (D) Vulval region. (E–G) First generation male. (E) Tail with spicules and gubernaculum. (F) Spicule and gubernaculum. (G) S. intermedium spicule. (H–I) Infective juvenile. (H) Anterior portion—rounded head and bacterial vesicle (arrows point to excretory pore and bacterial vesicle); (I) Tail with anus and hyaline layer. Scales (in µm): A, B, D = 140, C = 120, E, F, G, H, I = 50.

FIGURE 4. Phylogenetic relationships of 67 species of Steinernema based on analysis of ITS rDNA regions. Caenorhabditis elegans was used as outgroup. Bayesian posterior probability values are given next to the nodes.

FIGURE 5. Phylogenetic relationships between 47 species of Steinernema based on analysis of D2D3 rDNA regions. Caenorhabditis elegans was used as outgroup. Bayesian posterior probability values are given next to the nodes.