Steinernema texanum

Nguyen, Stuart, Andalo, Gozel & Rogers, 2007

Measurements

See Table 1.

Description

 

Male, first-generation male:  (Fig.1)  Body C-shaped to strongly curved posteriorly when heat killed. Cuticle smooth under a light microscope but with striation under SEM. Lateral field not observed. Head rounded, gently tapering anteriorly, slightly swollen and flattened anteriorly in some specimens.  Face view with six labial papillae, two amphids and four cephalic papillae. Labial papillae longer than cephalic papillae. Stoma shallow and narrow, usually with pronounced cheilorhabdions, posterior part funnel-shaped, well cuticularised.  Excretory pore anterior of nerve ring, anterior part of excretory duct well cuticularised, excretory cells not observed.  Pharynx with cylindrical procorpus, metacorpus slightly swollen, isthmus present.  Nerve ring surrounding isthmus, basal bulb distinct.  Pharyngo-intestinal valve present.  Gonad monorchid, reflexed. Testis with ventral reflexion, germinal zone, growth zone and vas deferens.  Distance from base of pharynx to anterior end of testis variable, longer than pharynx length. Spicules paired, dark brown in color.  Head (manubrium) of spicules (Fig. 2), somewhat elongate (spicule head length/width = 1.20-1.40); shaft (calomus) very short or absent; rostrum present; blade (lamina) thick, tapering slightly posteriorly; blade terminus blunt; velum prominent.  Each spicule with two internal ribs.  Under SEM, spicule with three lobes: dorsal lobe prominent, well curved anteriorly; lateral lobe well defined on dorsal edge and divided into two posteriorly; ventral lobe short. Gubernaculum boat-shaped in lateral view, cuneus needle-shaped (Fig. 2E), anterior end curved ventrally.  Usually a single precloacal papilla and eleven pairs of genital papillae arranged in normal position for Steinernema (i.e., six or seven pairs precloacal subventral, one pair adcloacal, one pair lateral, two pairs subterminal and one pair subdorsal).  The number of caudal papillae is constant (3 pairs), but the number of precloacal subventral pairs is variable.  Tail conoid without bursa, without phasmid; tail terminus without mucron.

 

Male, Second generation: Second-generation male similar to that of the first-generation male except body shorter and body diameter less and the following characteristics: Head usually somewhat swollen. Excretory pore more anterior, in posterior part of corpus. Posterior part of body with a single precloacal papilla and 13-14 pairs of genital papillae in which four pairs are caudal compared to three in the first generation. Lateral field prominent with one ridge. Tail terminus with a mucron, usually subterminal subventral in position.

 

Females, firs generation:  (Fig. 3)  Body C-shaped or strongly spiraled when heat killed and fixed with 4% formalin.  Head rounded and continuous with body.  Cuticle smooth or with faint annules.  Lateral fields with one line and present on all females observed.  Phasmids inconspicuous. Cheilorhabdions prominent, well sclerotized.  Another smaller sclerotized structure present, posterior to cheilorhabdions, presumably the prorhabdions, posterior part funnel-shaped.  Pharynx with procorpus cylindrical, muscular; metacorpus swollen; isthmus distinct; basal bulb enlarged, valvate.  Nerve ring surrounding isthmus, just anterior to basal bulb.  Pharyngo-intestinal valve prominent.  Excretory pore position variable, near mid-pharynx.  Pharyngo-intestinal valve present.  Vulva a transverse slit on a protruding area, a small epiptygma present.  Tail with bluntly pointed tip, one or two projections present. Postanal swelling present in most females, more pronounced in fully mature females.  Tail shorter than anal body width, tapering to a blunt end.  Two projections usually present at tail end. Two wart-like structures anterior to tail tip always present on ventral side.

 

Females, secondgeneration: Body an open C when heat killed and fixed with 4% formalin.  Similar to first-generation female but smaller.  Body diameter greater anterior to vulva than posterior to vulva.  Vulva on asymmetrical protuberance and situated at midbody; epiptygma not observed.  Postanal swelling present.  Tail mostly longer than anal body diameter, tapering gently to a sharp point. For some large females (about 5%), tail shorter than anal body diameter.

 

Infective juvenile: (Fig. 4) Body elongate, almost straight or slightly curved.  Sheath (second-stage cuticle) present immediately after harvesting, but many infective juveniles losing sheath in storage.  Exsheathed juvenile with four cephalic papillae.  Labial region smooth, continuous with body.  Amphids prominent.  Cuticle marked with prominent transverse striations.  Excretory pore anterior to nerve ring.  Lateral field beginning anteriorly with one line at the fourth or fifth annule. A short distance posteriorly, two additional lines appear forming two ridges.  Near excretory pore level, number of ridges in lateral fields increasing from two to seven.  Seven ridges keeping unchanged from excretory pore to anus except the two submarginal ridges not raised as others in middle part of body.  Near anus all ridges become smaller, only ridges 1, 3, 5, 7 are raised and seen clearly (Fig. 4D), others disappear gradually.  Near phasmid, only two ridges (poorly separated) are observed in the lateral field.  With the above arrangement, the formula of the lateral field is 2, 7, 2.   Pharynx with thin corpus, metacorpus slightly swollen, isthmus present, nerve ring usually at middle of isthmus; basal bulb elongate with visible valve.  Cardia present.  Bacterial pouch located just posterior to cardia, 7-8.5 μm in diameter, 26-31 μm in length, containing bacterial cells, variable in shape, mostly fusilform or oval. Hemizonion and hemizonid not observed.  Tail four times as long as anal body diameter and attenuate.  Phasmid present near mid-tail, just ventral to lateral field.  Hyaline portion occupying 58% (54-61) of tail length.

 

Type host and locality 

Steinernema texanum  was isolated using the Galleria-baiting technique  from soil samples taken at a site along state road 77 south of Kingsville between the towns of Sarita and Armstrong in Kenedy County, Texas, in an area with sandy soil, weedy vegetation, and shaded by mature live oak trees (Quercus sp.). Natural host unknown.

Type material

Holotype (male, first generation):  Isolated from haemocoel of G. mellonella deposited in the United States Department of Agriculture Nematode Collection (USDANC), Beltsville, Maryland. Allotype (female, first generation):  Same data as holotype, deposited in the USDANC, Beltsville, Maryland. Paratypes:  Same data as holotype.  Many males and females of the first generation and several third-stage infective juveniles in 4% formalin deposited in USDANC, Beltsville, Maryland.  Several males, females and infective juveniles in formalin deposited in the Department of Entomology and Nematology, University of Florida, Gainesville, Florida.

Diagnosis and Relationships

Steinernema texanum n. sp. is characterised by morphometrics of the infective juvenile with body length 756 μm,  distance from anterior end to the excretory pore 59 μm, tail 73 μm, ratio a =  25,  H% = 59, and E% = 81.  Lateral pattern of the new species is 2, 7, 2, very typical for the species.  Male of the first generation can be recognised by the spicule and the gubernaculum lengths and shapes, position of the excretory pore, D% = 67, and GS% = 75.  Female can be recognised by the vulva with very low epiptygma, and two wart-like structures anterior to tail tip always present on ventral side.

Steinernema texanum  is characterised genetically by ITS and D2D3 regions as stated in the molecular characterisation.

Steinernema texanum is closely related to nematode species in the feltiae group, which include S. akhursti, S. feltiae, S. hebeiense, S. jollieti, S. kraussei, S. kushidai, S. litorale, S. monticolum, S. orogonense, S. sangi, S. silvaticum and S. weiseri.

The infective juvenile of S. texanum n. sp. differs from closerly related species, S. feltiae, S. kraussei, S. oregonense by its short body length, short pharynx length and lower a ratio value.  E% of the new species is similar to that of S. kraussei but smaller than that of S. feltiae and S. oregonense. The new species has similar body length with S. jollieti and S. weiseri but body diam. is different in both means and ranges. Consequently, ratio a value is different, 25 compared to 30.5 and 29, respectively. Additionally, E% of the three species are different. The new is unique in feltiae group in having seven ridges in lateral fields compared to six in S. akhursti and S. jollieti and eight in all others.  Steinernema texanum n. sp. can be distinguished from all other members of feltiae group by data in Table 2.

 The first-generation male of the new species differs from all related species by  lengths and shapes of spicules and gubernaculum. D% and GS% are also different (Table 3).  The second-generation male differes from other species by the presence of 13-14 pairs of genital papillae.

The first generation female of S. texanum n. sp. differs from all other species by the presence of two wart-like structures on ventral side of tail. 

Cross hybridization tests

Crossbreeding between males and females of S. texanum n. sp. with S. feltiae and S. oregonense produced no progeny.  In the control, when all species were self crossed, males and females produced offspring normally. 

Molecular characterisation

The sequence of ITS regions of S. texanum n. sp., flanked by primers 18S and 26S is characterised by its sequence length 956 base pairs (bp), ITS1 = 263 bp, ITS2 = 286 bp and its composition.  The sequence length of ITS1 of the new species is longer than that of S. sangi but shorter than all other species in feltiae group. ITS2 is longer than that of S. monticolum but shorter than all others in Table 4. For more interspecific relationships,  pairwise distances show that the new species differs from its closest taxon S. jollieti at 67 bp and from the most divergent species S. monticolum at 141 bp. Among these species, the less divergent species are  S. litorale and S. weiseri with 27 bp difference and most divergent species are S. monticolum and S. kushidai with 150 bp difference (see the original description).

For D2/D3 regions, the sequence of S. texanum n. sp. is 855 bp, composition, A = 0.24912, C = 0.19532, G = 0.30292, T = 0.25263. Pairwise distances show that the new species differs from its closest taxon S .feltiae at 28 bp and from the most divergent species S. monticolum at 51 bp.  S. texanum n. sp. are different from closely related Steinernema species, from 28 to 37 bp. These results indicate that the new nematode is a good new species when comparing these distances with other species , the distances between some species are as low as zero bp (Nguyen et al., 2006; Table 7).

 

Phylogenetic analysis

For ITS regions, maximum parsimony analysis shows that the alignment resulted in 1247 characters in which  152 in ambiguous regions are excluded, 276 are constant, 261 variable characters are parsimony-uninformative and 558 characters (included) are parsimony-informative. Parsimony and distance based treebuilding approaches produce almost identical trees. The phylogenetic relationships  between 33 species of Steinernema are presented in Figure 6 (for MP, tree length = 3622, CI = 0.4268, RI = 0.4616, RC = 0.1970, HI = 0.4616). In this consensus tree, 13 species of feltiae group form a monophyletic group in which the new species, S. feltiae, S. hebeiense, S. jollieti, S. kraussei, S. litorale, .S. oregonense, S. sangi, S. silvaticum and S. weiseri form a monophyletic group. The new species and S. sangi do not group with any others. Bootstrap support in this clade is from low (50) to high (99). The reconstructed nucleotide character transformations show that the new species differs from other species of the S. feltiae group by 18 unambiguous, polarised autapomorphies.

For D2D3 regions, maximum parsimony analysis shows that the alignment resulted in 1032 characters in which 554 are constant, 128 variable characters are parsimony-uninformative and 350 characters are parsimony-informative. The phylogenetic relationships between 26 species of Steinernema are presented in Fig. 5 (tree length = 1125, CI = 0.6009, RI = 0.6999, RC = 0.4205, HI = 0.3991).  The six species, S. texanum n. sp.,  S. feltiae, S. kraussei, S. kushidai, S. monticolum and S. oregonense form a monophylectic group well supported by bootstrap proportion  (99).  The two species S. monticolum  and S. kushidai stand separately. Additionally, the reconstructed nucleotide character transformations show that S. texanum  differs from species of S. feltiae group by 15 unambiguous, polarised autapomorphies (see the original description).

 

REFERENCES

 

Nguyen, K.B., Stuart, R, Andallo, V., Gozel, U. & Roger, M.E. (2007). Steinernema texanum n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode fron Texas, USA. Nematology 9, 379-396.

Nguyen, K.B., Malan, A.P. & Gozel, U. (2006). Steinernema khoisanae n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from South Africa. Nematology 8, 157-175.