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Steinernema innovationi n. sp. (Panagrolaimomorpha: Steinernematidae), a new entomopathogenic nematode species from South Africa

Published online by Cambridge University Press:  03 April 2014

H. Çimen,
M.-M. Lee,
J. Hatting,
S. Hazir and
S.P. Stock
H. Çimen
Affiliation:
Adnan Menderes University, Faculty of Arts and Science, Department of Biology, 09010Aydin, Turkey
M.-M. Lee
Affiliation:
Department of Entomology, University of Arizona, 1140 E. South Campus Dr., Tucson, AZ 85721-0036, Arizona, USA
J. Hatting
Affiliation:
South African Agricultural Research Council, Small Grain Institute, Private Bag X29, Bethlehem, 9701, South Africa
S. Hazir
Affiliation:
Adnan Menderes University, Faculty of Arts and Science, Department of Biology, 09010Aydin, Turkey
S.P. Stock*
Affiliation:
Department of Entomology, University of Arizona, 1140 E. South Campus Dr., Tucson, AZ 85721-0036, Arizona, USA
*
*Fax: +1-520-621-1150 E-mail: spstock@email.arizona.edu
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Abstract

Morphological and molecular sequence data were combined with cross-hybridization studies and used to identify a new Steinernema sp. from Free State, South Africa. Molecular and morphological data indicate that the new species belongs to the ‘glaseri-group’ of Steinernema spp. Key morphological diagnostic characters for S. innovationi n. sp. include the morphometric features of the third-stage infective juveniles: total body length = 1054 (1000–1103) μm, tail length = 108 (97–117) μm, location of the excretory pore = 88 (82–91) μm, and D% = 58 (54–63), E% = 115 (104–137) and H% = 43 (37–46). Additionally, the morphology of the spicules and gubernaculum of the first-generation males are considered key diagnostic traits. Steinernema innovationi n. sp. was also characterized by analysis of both rDNA and mitochondrial gene sequence data, which further indicate the uniqueness of this Steinernema species.

Information

Type
Research Papers
Information
Journal of Helminthology , Volume 89 , Issue 4 , July 2015, pp. 415 - 427
Copyright
Copyright © Cambridge University Press 2014 

Introduction

Several surveys have been conducted to document the diversity of entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) in South Africa (Malan et al., Reference Malan, Nguyen and Addison2006, Reference Malan, Knoetze and Moore2011, Reference Malan, Knoetze and Tiedt2012; Hatting et al., Reference Hatting, Stock and Hazir2009). The rationale for such work was driven by the need to find locally adapted species and/or isolates that could be considered for control of native insect pests. The first EPN species found in this country was Heterorhabditis bacteriophora, which was isolated from an infected maize beetle, Heteronychus arator (Spaull, Reference Spaull1988). Subsequently, focused surveys were conducted in the provinces of KwaZulu-Natal (Spaull, Reference Spaull1990, Reference Spaull1991) and Western Cape (Malan et al., Reference Malan, Nguyen and Addison2006, Reference Malan, Nguyen, De Waal and Tiedt2008; Nguyen et al., Reference Nguyen, Malan and Gozel2006). As a result, several uncharacterized novel Steinernema and Heterorhabditis species were isolated, including S. khoisanae Nguyen, Malan and Goezel, 2006; Heterorhabditis safricana Malan, Nguyen de Waal and Tiedt, 2008; Steinernema citrae Stokwe, Malan, Nguyen, Knoetze and Tiedt, 2011; and Heterorhabditis noenieputensis Malan, Knoetze & Tiedt, 2013. Currently, many of these endemic species are being implemented in successful pest management programmes (Malan & Manrakhan, Reference Malan and Manrakhan2009; De Waal et al., Reference De Waal, Malan, Levings and Addison2010; Van Niekerk & Malan, Reference Van Niekerk and Malan2012).

The survey conducted by Hatting et al. (Reference Hatting, Stock and Hazir2009) was the first systematic sampling of indigenous EPN species in South Africa. This study encompassed all seven geographic regions of this country and also considered sampling of diverse habitats (natural and human-impacted) as well as different soil types. Five per cent of all samples taken in this survey yielded EPNs. Specifically, four Steinernema species, including S. khoisanae and three undescribed species, and one Heterorhabditis species, H. bacteriophora, were collected (Hatting et al., Reference Hatting, Stock and Hazir2009).

Herein, we describe one of these new Steinernema isolates, originally labelled as ‘species 1’ (Hatting et al., Reference Hatting, Stock and Hazir2009). We utilized differential interference contrast optics (DIC) and scanning electron microscopy (SEM) for morphological observation and morphometric analysis, in addition to DNA sequence analysis and cross-hybridization methods, to fully describe and illustrate this novel Steinernema species.

Materials and methods

Nematode isolation and rearing

Steinernema isolate SGI-60 was recovered from a grain field at the Small Grain Institute in Bethlehem, Free State province (Hatting et al., Reference Hatting, Stock and Hazir2009). Nematodes were recovered directly from soil samples using the insect-baiting method of Bedding & Akhurst (Reference Bedding and Akhurst1975). Cadavers with positive signs of nematode infection were placed in modified White traps (Kaya & Stock, Reference Kaya, Stock and Lacey1997) to recover infective juvenile (IJ) progeny. A 30% bleach solution was used to surface sterilize the nematodes for 15 min. Surface sterilized nematodes were used to infect fifth-instar Galleria mellonella larvae (100 IJs per insect) to confirm Koch's postulates for pathogenicity (Kaya & Stock, Reference Kaya, Stock and Lacey1997). Emerging IJ progeny were stored in 250-ml tissue-culture flasks for subsequent identification and establishment of cultures, following the procedures described by Stock & Goodrich-Blair (Reference Stock, Goodrich-Blair and Lacey2012).

Morphological characterization

Morphometrics

Third-stage infective juveniles and adult stages (first and second generation) were randomly collected from White traps and infected cadavers, respectively. Twenty-five randomly selected specimens of each nematode stage were examined after heat killing and relaxation in M9 buffer in a water bath heated to 60°C. Heat-killed specimens were fixed in formaldehyde–acetic acid solution (FA 4:10) (Franklin & Goodey, Reference Franklin and Goodey1949), slowly dehydrated and processed to anhydrous glycerin (Seinhorst, Reference Seinhorst1959). Specimens were mounted on glass slides using Pliobond® industrial contact cement to both seal and provide cover glass support (Lee et al., Reference Lee, Sicard, Skeie and Stock2009). An Olympus BX51 microscope equipped with differential interference contrast optics and Olympus Microsuite software (Soft Imaging System Corp., Lakewood, Colorado, USA) was used to obtain morphometric data of each nematode specimen. Morphological characters measured were based on the recommendations of Hominick et al. (Reference Hominick, Briscoe, del Pino, Heng, Hunt, Kozodoy, Mráček, Nguyen, Reid, Spiridonov, Stock, Sturhan, Waturu and Yoshida1997). Line drawings were prepared from digitized camera lucida and/or from video images.

Scanning electron microscopy (SEM)

Male and IJ specimens were heat-killed in M9 buffer and then fixed in 8% glutaraldehyde buffered in cacodylate at pH 7.30 overnight. Fixed nematodes were rinsed in distilled water three times, post-fixed in osmium tetroxide for 1 h, rinsed again in distilled water before being subjected to a serial dehydration process in ethanol (McClure & Stowell, 1978). Specimens were critical point dried in liquid carbon dioxide, mounted on SEM stubs and coated twice with gold. Observations and image recordings were made at 5 kV accelerating voltage on a Hitachi S-4800 Type II series microscope equipped with a digital camera (Hitachi, Clarksburg, Maryland, USA).

Molecular characterization

Nematodes were molecularly characterized using two rDNA genes: the internal transcribed spacer region (ITS) and the 28S large subunit region, inclusive of the D2D3 domain. Total genomic DNA isolation, polymerase chain reaction (PCR) amplification (reaction, cycling conditions and primers) followed protocols described by Stock et al. (Reference Stock, Campbell and Nadler2001a) and Nguyen et al. (Reference Nguyen, Maruniak and Adams2001). Sequence data from rDNA and mitochondrial genes were compared to an existing library of more than 60 Steinernema spp. (P. Stock's laboratory, University of Arizona, USA) and available sequences found in GenBank.

Phylogenetic analysis

SeqEdit software (DNA Star Inc., Madison, Wisconsin, USA) was used to perform contig assembly and sequence ambiguity resolution. Sequences were aligned using ClustalW v2.1 (Larkin et al., Reference Larkin, Blackshields, Brown, Chenna, McGettigan, McWilliam, Valentin, Wallace, Wilm, Lopez, Thompson, Gibson and Higgins2007) under default alignment parameters, and alignment inconsistencies were corrected by hand in Mesquite v2.75 (Maddison & Maddison, Reference Maddison and Maddison2011). Caenorhabditis elegans was used as the outgroup taxon for all analyses, according to criteria described by Nadler et al. (Reference Nadler, Bolotin and Stock2006). Ribosomal and mitochondrial DNA sequences for the new Steinernema species were deposited in GenBank (accession numbers KJ578793 and KJ578794 for ITS and 28S rDNA genes, respectively). ITS and 28S sequence data were analysed separately. Each dataset was analysed by unweighted maximum parsimony (MP). MP methods were performed in PAUP* v.4.0b10 (Swofford, 2002) following standards described by Stock et al. (Reference Stock, Campbell and Nadler2001a) and Nadler et al. (Reference Nadler, Bolotin and Stock2006).

Cross-hybridization

Reproductive compatibility of the new species was tested using the modified hanging-blood assay described by Kaya & Stock (Reference Kaya, Stock and Lacey1997). Two morphologically similar and close relatives of S. innovationi n. sp., S. khoisanae Nguyen et al., Reference Nguyen, Malan and Gozel2006 and Steinernema glaseri (Steiner, Reference Steiner1929), were crossed for assessing reproductive compatibility of this new species. Additionally, another South African isolate recovered from the survey conducted by Hatting et al. (Reference Hatting, Stock and Hazir2009), ROOI-352 (which represents another novel undescribed Steinernema sp.), was used for the cross-breeding experiments. Controls consisted of crosses with male and single female nematodes of the same species, as well as a single female only. There were ten replicates per cross and tests were repeated twice.

Results and discussion

Description of Steinernema innovationi n. sp

First-generation male

Body slender, ventrally curved posteriorly, J-shaped when heat-killed (figs 1A, 2A). First-generation male larger (average 1896 μm) than second-generation male (average 1322 μm). Cuticle smooth under light microscopy. Lateral field and phasmids inconspicuous under light microscopy. Head truncate to slightly round, continuous with body (figs 1B, 2B). Six lips amalgamated but tips distinct, with one labial papilla each. Four conspicuous cephalic papillae (fig. 2B). Amphidial apertures small, located posterior to lateral labial papillae (fig. 2B). Stoma reduced (cheilo-, gymno- and stegostom vestigial), short and wide, with inconspicuous sclerotized walls. Pharynx muscular, with a cylindrical procorpus and a metacorpus slightly swollen and non-valvate. Isthmus indistinct followed by pyriform basal bulb with reduced valve. Nerve-ring usually located about mid-isthmus level or on the anterior part of the basal bulb (fig.1A). Excretory pore opening circular, located anterior to nerve ring at anterior third of metacorpus (fig. 2C). Testis monorchic, ventrally reflexed (figs 1A, 2A). Spicules paired, symmetrical, curved, with ochre–brown colouration (figs 1D, 2D). Manubrium rhomboidal (fig. 1D). Shaft distinct. Blade with rostrum or retinaculum and two internal ribs. Velum present, narrow (fig. 1D). Blade terminus blunt (figs 1D, 2D). Gubernaculum boat-shaped or arcuate, c. 3/4 length of spicules. Manubrium of gubernaculum curved ventrally (figs 1E, 2E). Tail conoid and non-mucronate, with no bursa (figs 1C, 2F). There are 23 genital papillae (11 pairs and one single) arranged as follows: six precloacal pairs, of which five pairs are subventral and one pair is lateral, and one single ventral papilla, and five pairs of postcloacal, of which one pair is subventral, two pairs are subdorsal and two pairs are terminal (figs 1C, 2G).

Fig. 1

Steinernema innovationi n. sp., line drawings. First-generation male (A–E), first-generation female (F–I), second-generation female (J) and third-stage infective juvenile (K, L). First-generation male: (A) full body, lateral view; (B) anterior end (lateral view), showing stoma region; (C) tail (lateral view), showing genital papillae, spicules and gubernaculums; (D) spicule, lateral view; (E) gubernaculum, lateral view. First-generation females: (F) full body, lateral view; (G) anterior end (lateral view), showing pharyngeal region and excretory canal; (H) vulva, lateral view; (I) tail, lateral view. Second-generation female: (J) tail, lateral view. Third-stage infective juvenile: (K) anterior end (lateral view), showing pharyngeal region, nerve ring and excretory canal; (L) tail, lateral view. Scale bars: A = 2 μm; B = 3 μm; C, G = 50 μm; D, E = 30 μm; F = 190 μm; H = 70 μm; I = 55 μm; J, K, L = 40 μm.

Fig. 2

Steinernema innovationi n. sp., light and scanning electron microscope (SEM) photographs. First-generation male: (A) full body, lateral view; (B) SEM of anterior end, face view (lp, labial papillae; cp, cephalic papillae; a, amphid); (C) anterior end, lateral view showing location of excretory canal (arrow); (D) spicule, lateral view; (E) gubernaculum, lateral view; (F) tail, lateral view; (G) tail, SEM, ventrolateral view, showing arrangement of genital papillae (pr, precloacal; po, postcloacal; v, ventral; t, terminal). Scale bars: for light microscopy, the scale bar is given in (A), and for SEM images, in (G). A = 150 μm; B = 5 μm; C = 35 μm; D, E, F = 40 μm; G = 100 μm.

Second-generation male

General morphology similar to that of first-generation males, but smaller in size. Tail with or without mucron. Spicules with manubrium morphology similar to those of first-generation male. Gubernaculum more slender and longer than that of first-generation male.

First-generation female

Lip region, stoma and pharyngeal region as in male (figs 1F, 3A). Body C-shaped when heat-relaxed. Cuticle smooth under light microscope, with slight annulations under SEM. First-generation females larger (average 4070 μm) than second-generation females (average 2063 μm). Excretory pore located about mid-procorpus level or surrounding isthmus (fig. 1G). Genital system didelphic, amphidelphic. Ovaries opposed, reflexed in dorsal position; oviduct well developed; glandular spermatheca and uterus in ventral position. Vagina short, with muscular walls. Vulva located near middle of body with slightly protruding lips and mostly symmetrical (figs 1H, 3B). First-generation female tail blunt, conoid and with a digitated tip (figs 1I, 3C). Postanal lips non-protruding or slightly protruding (figs 1H, 3C).

Second-generation female

Body open C-shaped when heat-killed. Similar to first-generation female but smaller. Vulva shape and lips similar to first-generation female. Tail conoid, without postanal swelling (fig. 1J).

Third-stage infective juvenile

Body of heat-relaxed specimens almost straight, slender, gradually tapering posteriorly. Cuticle with fine transverse striae. Head region continuous with body, slightly truncate (figs 1K, 3D). Six lips each bearing one small labial papilla. Four conspicuous cephalic papillae. Amphidial apertures pore-like (fig. 3G). Lip region smooth, continuous; stoma closed (fig. 3G). Lateral field begins anteriorly with one line at fourth or fifth annule and splits posteriorly into two additional lines forming two ridges. More posteriorly, the number of ridges increases to six. At the mid-body region there are nine distinct lines ( = eight ridges) evenly spaced and developed (fig. 3H). Ridges reduced to six near the anus and to two near the phasmid region. Lateral line formula is 2, 6, 8, 6, 2. Pharynx long, narrow, with slightly expanded procorpus, narrower isthmus and pyriform basal bulb with valve (figs 1K, 3D). Nerve-ring located at isthmus level (figs 1K, 3D). Excretory pore located about mid-corpus (figs 1K, 3D). Anterior portion of intestine with small bacterial receptacle (fig. 3E). Intestine filled with numerous fat globules, lumen of intestine narrow. Rectum long, straight; anus distinct (figs 1L, 3F). Genital primordium evident. Tail conoid with pointed terminus. Hyaline portion occupying c. 32% of tail length (figs 1L, 3F).

Fig. 3

Steinernema innovationi n. sp., light and scanning electron microscope (SEM) photographs. First-generation female (A–C) and third-stage infective juvenile (D–H). First-generation female: (A) anterior end (lateral view), showing procorpus region of pharynx; (B) vulva, lateral view; (C) tail (lateral view), showing digitate mucron (arrow). Third-stage infective juvenile: (D) anterior end (lateral view), showing pharynx and excretory canal (arrow); (E) close-up of basal bulb and bacterial receptacle (br); (F) tail (lateral view), showing rectum (arrow) and hyaline portion; (G) anterior end (face view), showing position of labial (lp) and cephalic papillae (cp); (H) lateral field pattern in mid-body region. Scale bars (based on the scale bar in (A), except for SEM images): A = 40 μm; B = 54 μm; C, E = 20 μm; D = 25 μm; G = 5 μm; F, H = 10 μm.

Type material

Holotype male, first generation; five paratype males, first generation; five paratype females, first generation; five paratype third-stage infective juveniles deposited in the USDA Nematode Collection, Maryland.

Five paratype males, first generation; five paratype females, first generation; five paratype third-stage infective juveniles deposited at the University of California Davis Nematode Collection, Davis, California, USA.

Dimensions of holotype and paratype specimens are provided in table 1.

Table 1

Morphometric characters of male, female and third-stage infective juvenile of Steinernema innovationi n. sp.; n=number of examined specimens; measurements in μm include mean values ± standard deviation and ranges.

ABD, anal or cloacal body diameter; D% = (EP/PH) × 100; E% = (EP/TL) × 100; EP = distance from anterior end to excretory pore; GS% = (GuL/SpL) × 100; GuL = gubernaculum length; H = length of hyaline portion of tail; H% = H as % of TL; MBW = maximum body width; NR = distance from anterior end to nerve ring; PH = distance from anterior end to base of pharynx; SpL = spicule length (measured in situ, along the curvature in a line along the centre of the spicule); StL = stoma length; StD = stoma diameter; SW% = (SpL/ABD) × 100; TBL = total body length; TL = tail length; V = distance from anterior end to vulva; a = TBL/MBW; b = TBL/PH.

Type host. The natural host of this novel species is unknown.

Type locality. Isolate SGI-60 was collected from a grain field at the Agricultural Research Center, Small Grain Institute in Bethlehem, South Africa.

Etymology. This species is dedicated to the ‘Innovation Foundation’ that provided financial support for the survey conducted in 2009 to isolate entomopathogenic nematode species in South Africa.

Cross-hybridization results

Cross-hybridization assays between males and females of S. innovationi n. sp. with S. khoisane, S. glaseri and another South African Steinernema sp. isolate ROOI-352 yielded no progeny. No progeny were observed in the single female control plates.

Diagnosis and relationships

Based on morphological and morphometric traits, the new species is considered a member of the ‘glaseri-group’. Nematodes in this group are characterized by having the largest third-stage infective juveniles (average total body length (TBL) ≥ 1000 μm) and by the lateral field in infective juveniles, which possess eight ridges in the mid-body region.

Phylogenetic analyses of the ITS and 28S genes dataset also confirmed affiliation of S. innovationi n. sp. in clade V (as depicted by Spiridinov et al., Reference Spiridinov, Reid, Podrucka, Subbotin and Moens2004) which encompasses taxa of the ‘glaseri-group’. Within this clade, the new species is closely related to the four described Steinernema species: S. khoisanae Nguyen et al., Reference Nguyen, Malan and Gozel2006, from South Africa and three Asian species: S. longicaudum Shen & Wang, 1992, S. lamjungense Khatri-Chhetri et al., Reference Khatri-Chhetri, Waeyenberge, Spiridonov, Manandhar and Moens2011 and S. guangdongense Qiu et al., Reference Qiu, Fang, Zhou, Pang and Nguyen2004. However, the novel species can also be distinguished from these taxa by several morphological and morphometric characters, which are listed below.

Infective juveniles of S. innovationi n. sp. are wider (average 37 μm versus 33 μm) and slightly shorter than those of S. khoisanae (average 1054 μm versus 1075 μm). The excretory pore in the new species is more anteriorly located (average 88 μm versus 93 μm), and the tail is usually shorter than that of S. khoisanae (average 76 μm versus 85 μm). Additionally, differences exist in the values of D% and E% (table 2). Males of S. innovationi n. sp. can be distinguished from S. khoisanae by the D% (average: 83 versus 88) and SW ratio (average: 1.4 versus 1.9). In S. khoisanae the number of pairs of genital papillae may differ between 11–12 pairs plus one single papilla; however, in the new species a consistent number of 11 pairs plus one single papilla was observed in all specimens examined. Moreover, the value of SW% between these two species is quite different (table 2). Females of S. innovationi n. sp. have a digitated tail, similar to those of S. khoisanae. However, females of the new species lacked the postanal swelling or, if present, this was slightly protruding when compared to S. khoisanae.

Table 2

Comparative morphometric traits of third-stage infective juveniles and males of Steinernema innovationi n. sp. with other members of the S. glaseri-group (clade V); measurements in μm include mean values with ranges; see table 1 for abbreviations.

Entries in bold and with * are the most closely related species to S. innovation n. sp.

Third-stage infective juveniles of S. innovationi n. sp. differ from those of S. longicaudum by the location of the excretory pore (average 88 μm versus 82 μm), and by having a shorter tail (average 76 μm versus 94 μm). Males of S. innovationi n. sp. can be separated from those of S. longicaudum by the morphology of the spicules which are more robust and smaller than those of S. longicaudum (see table 2). Moreover, the lamina of the spicules in S. longicaudum is more slender than that of the new species. The gubernaculum in S. innovationi n. sp. is shorter than that of S. longicaudum. Differences also exit in the values of SW% and GS% (see table 2). Additionally, first-generation males of the new species have a longer tail than those of S. longicaudum (average 51 μm versus 30 μm).

Steinernema innovationi n. sp. can be distinguished from S. lamjungense by the size of the IJs, which are larger in the new species (table 2). Moreover, the excretory pore in S. lamjungense is more anteriorly located than that of S. innovationi n. sp. The tail of the IJs in the new species is slightly shorter than that of S. lamjungense. Additionally, differences exist in the values of D% and E% (table 2). Males of the novel species differ from S. lamjungense in the values of D% and SW%. Furthermore, the morphology of the spicules in S. innovationi n. sp. is different than that of S. lamjungense. In particular, the shape of the manubrium in S. lamjungense is usually round compared to that of S. innovationi n. sp. which is rhomboidal. The gubernaculum in the new species is boat-shaped to arcuate, whereas in S. lamjungense it varies from boat-shaped to almost straight, and with the anterior part slightly recurved. Differences also exit in the values of D%, SW% and GS% (table 2).

Infective juveniles of the new species can be distinguished from S. guangdongense by the location of the excretory pore which is more posteriorly located in the new species (average 80 versus 88 μm). Additionally, the tail of the IJs in the new species is shorter than that of S. guangdongense (average: 76 versus 91 μm). Males of S. innovationi n. sp. can be discriminated from those of S. guangdongense by the values of D%, SW% and GS% (table 2). Moreover, the gubernaculum in the new species is shorter than that of S. guangdongense. Also, first-generation males have a mucronated tail, whereas no mucron is present in S. innovationi n. sp.

Molecular characterization and phylogenetic analysis

Maximum parsimony (MP) analysis of the 28S dataset yielded 401 parsimony informative characters out of 935 characters. A heuristic search of 5000 random addition replicates produced 126 equally parsimonious trees with a tree length of 1215 steps. Bootstrap MP analysis of this dataset placed S. innovationi n. sp. as a member of clade V, and strongly supported (98%) its placement as sister to the undescribed Steinernema isolate ROOI-352 (fig. 4). The two South African isolates are closely related to S. khoisanae, with 89% branch support. This clade comprises Steinernema spp. known to have infective juveniles with exceptionally large body size (average ≥ 1000 μm). Clading of ‘glaseri-group’ individuals, including S. innovationi n. sp., was supported by bootstrap resampling (88%), for which a 50% majority rule consensus tree is given (fig. 4).

Fig. 4

Evidence of 28S rDNA (inclusive of D2D3 domain) lineage independence for S. innovationi n. sp. based on maximum parsimony, with bootstrap values above 50% shown.

A heuristic search of the ITS rDNA sequences yielded two most parsimonious trees with tree length of 4356 steps. Of 1244 characters, 683 were parsimony informative. The MP bootstrap analysis of ITS data also placed S. innovationi n. sp. as a member of clade V, and (strongly, 100%) sister to the undescribed Steinernema isolate ROOI–352. The two South African isolates were also found to be closely related to S. khoisanae in this dataset; however, support was lower (60%). Bootstrap resampling provided strong (99%) support for the clade V glaseri-group assemblage, as seen in the 50% majority rule consensus tree (fig. 5).

Fig. 5

Evidence of internal transcribed spacer region (ITS) rDNA lineage independence for S. innovationi n. sp. based on maximum parsimony analysis, with bootstrap values above 50% shown.

The 28S rDNA pairwise distances between S. innovationi n. sp and Steinernema isolate ROOI-352 and S. khoisanae, close sister taxa, are 4 and 10, respectively. The considerably more variable ITS region yielded pairwise distances between S. innovationi n. sp. and the same two species of 58 and 147, respectively. Results of these analyses provide further evidence for the distinctiveness of this species (tables 3 and 4).

Table 3

Pairwise distance matrix of 28S rDNA (inclusive of D2D3 domain) for representative nematodes in the S. glaseri-group (clade V).

Table 4

Pairwise distance matrix of ITS region for representative nematodes in the S. glaseri-group (clade V).

Financial support

This research was partially funded by the South African Agricultural Research Council (ARC) under Project GK05/14 to J.H. Training of H.Ç. in P. Stock's Laboratory was supported in part by NemaSym Research Coordination Network grant (PI P. Stock, NSF–IOS 0840932).

Conflict of interest

None.

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Figure 0

Fig. 1Steinernema innovationi n. sp., line drawings. First-generation male (A–E), first-generation female (F–I), second-generation female (J) and third-stage infective juvenile (K, L). First-generation male: (A) full body, lateral view; (B) anterior end (lateral view), showing stoma region; (C) tail (lateral view), showing genital papillae, spicules and gubernaculums; (D) spicule, lateral view; (E) gubernaculum, lateral view. First-generation females: (F) full body, lateral view; (G) anterior end (lateral view), showing pharyngeal region and excretory canal; (H) vulva, lateral view; (I) tail, lateral view. Second-generation female: (J) tail, lateral view. Third-stage infective juvenile: (K) anterior end (lateral view), showing pharyngeal region, nerve ring and excretory canal; (L) tail, lateral view. Scale bars: A = 2 μm; B = 3 μm; C, G = 50 μm; D, E = 30 μm; F = 190 μm; H = 70 μm; I = 55 μm; J, K, L = 40 μm.

Figure 1

Fig. 2Steinernema innovationi n. sp., light and scanning electron microscope (SEM) photographs. First-generation male: (A) full body, lateral view; (B) SEM of anterior end, face view (lp, labial papillae; cp, cephalic papillae; a, amphid); (C) anterior end, lateral view showing location of excretory canal (arrow); (D) spicule, lateral view; (E) gubernaculum, lateral view; (F) tail, lateral view; (G) tail, SEM, ventrolateral view, showing arrangement of genital papillae (pr, precloacal; po, postcloacal; v, ventral; t, terminal). Scale bars: for light microscopy, the scale bar is given in (A), and for SEM images, in (G). A = 150 μm; B = 5 μm; C = 35 μm; D, E, F = 40 μm; G = 100 μm.

Figure 2

Fig. 3Steinernema innovationi n. sp., light and scanning electron microscope (SEM) photographs. First-generation female (A–C) and third-stage infective juvenile (D–H). First-generation female: (A) anterior end (lateral view), showing procorpus region of pharynx; (B) vulva, lateral view; (C) tail (lateral view), showing digitate mucron (arrow). Third-stage infective juvenile: (D) anterior end (lateral view), showing pharynx and excretory canal (arrow); (E) close-up of basal bulb and bacterial receptacle (br); (F) tail (lateral view), showing rectum (arrow) and hyaline portion; (G) anterior end (face view), showing position of labial (lp) and cephalic papillae (cp); (H) lateral field pattern in mid-body region. Scale bars (based on the scale bar in (A), except for SEM images): A = 40 μm; B = 54 μm; C, E = 20 μm; D = 25 μm; G = 5 μm; F, H = 10 μm.

Figure 3

Table 1Morphometric characters of male, female and third-stage infective juvenile of Steinernema innovationi n. sp.; n=number of examined specimens; measurements in μm include mean values ± standard deviation and ranges.

Figure 4

Table 2Comparative morphometric traits of third-stage infective juveniles and males of Steinernema innovationi n. sp. with other members of the S. glaseri-group (clade V); measurements in μm include mean values with ranges; see table 1 for abbreviations.

Figure 5

Fig. 4Evidence of 28S rDNA (inclusive of D2D3 domain) lineage independence for S. innovationi n. sp. based on maximum parsimony, with bootstrap values above 50% shown.

Figure 6

Fig. 5Evidence of internal transcribed spacer region (ITS) rDNA lineage independence for S. innovationi n. sp. based on maximum parsimony analysis, with bootstrap values above 50% shown.

Figure 7

Table 3Pairwise distance matrix of 28S rDNA (inclusive of D2D3 domain) for representative nematodes in the S. glaseri-group (clade V).

Figure 8

Table 4Pairwise distance matrix of ITS region for representative nematodes in the S. glaseri-group (clade V).