Steinernema texanum * n

Steinernema texanum

Nguyen, = Stuart, Andalo, Gozel & Rogers, 2007

Measurements

Description

Male, first-generation male: (Fig.1) Body C-shaped to strongly curved p= osteriorly 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 papilla= e. Labial pap= illae longer than cephalic papillae. Stoma shallow and narrow, usually with pronounced cheilorhabdions, posterior part funnel-shaped, well cuticularise= d. Excretory pore anterior of nerve r= ing, anterior part of excretory duct well cuticularised, excretory cells not observed. Pharynx with cylindrical procorpus, metacorpus slightly swollen, isthmus present. Nerve ring surrounding isthmus, ba= sal bulb distinct. Pharyngo-intes= tinal valve present. Gonad monorchi= d, 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 el= ongate (spicule head length/width =3D 1.20-1.40); shaft (calomus) very short or ab= sent; rostrum present; blade (lamina) thick, tapering slightly posteriorly; blade terminus blunt; velum prominent. Each spicul= e 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 papill= a and eleven pairs of genital papillae arranged in normal position for Steiner= nema (i.e., six or seven pairs precloacal subventral, one pair adcloacal, one pa= ir 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 variab= le. 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 mo= re 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 w= ith one ridge. Tail terminus with a mucron, usually subterminal subventral in position.

<= span lang=3DEN-ZA style=3D'font-size:12.0pt;color:teal'>Females, firs generation= : (= Fig. 3) Body C-shaped or strongly spiraled= when heat killed and fixed with 4% formalin.&nb= sp; Head rounded and continuous with body. Cuticle smooth or with faint annules. Lateral fields with = one line and present on all females observed.&= nbsp; 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, valvat= e. Nerve ring surrounding isthmus, ju= st anterior to basal bulb. Phary= ngo-intestinal valve prominent. Excretory po= re position variable, near mid-pharynx. Pharyngo-intestinal valve present.&= nbsp; Vulva a transverse slit on a protruding area, a small epiptygma present. Tail with bluntly po= inted tip, one or two projections present. Postanal swelling present in most fema= les, more pronounced in fully mature females.&n= bsp; 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.

<= span lang=3DEN-ZA style=3D'font-size:12.0pt;color:green'>Females, secondgenerati= on: Body an open C when heat killed an= d 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 elonga= te, almost straight or slightly curved. Sheath (second-stage cuticle) present immediately after harvesting, but many infective juveniles losing sheath in storage. Exsheathed juvenile with fo= ur cephalic papillae. Labial reg= ion smooth, continuous with body. Amphids prominent. Cut= icle 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, n= umber 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 othe= rs in middle part of body. Near = anus all ridges become smaller, only ridges 1, 3, 5, 7 are raised and seen clear= ly (Fig. 4D), others disappear gradually.&nbs= p; 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. Phary= nx 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 poste= rior 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. Hya= line portion occupying 58% (54-61) of tail length.

=

Type host and locality

Steinernema te= xanum was isolated using the Galleria<= /i>-baiting technique from soil samples t= aken 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), Beltsvill= e, Maryland. Allotype (female,= first generation): Same data as hol= otype, deposited in the USDANC, Belts= ville, Maryland. Paratypes: Same data as holotype. Many males and females of the first generation and several third-stage infective juveniles in 4% formalin depos= ited in USDANC, Beltsville, Maryland. Several males, females and infecti= ve 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 7= 56 μm, distance from anterior e= nd to the excretory pore 59 μm, tail 73 μm, ratio a =3D 25, H% =3D 59, and E% = =3D 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 t= he spicule and the gubernaculum lengths and shapes, position of the excretory pore, D% =3D 67, and GS% =3D 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 IT= S 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. hebeiens= e, S. jollieti, S. kraussei, S. kushi= dai, 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<= span lang=3DEN-ZA style=3D'mso-ansi-language:EN-ZA'>. E% of the new species is similar t= o that of S. kraussei but smaller than= that of S. feltiae and S. oregonen= se. The new species has similar body length with S. jollieti and S. = weiseri but body diam. is differen= t in both means and ranges. Consequently, ratio a value is different, 25 compare= d to 30.5 and 29, respectively. Additionally, E% of the three species are differ= ent. The new is unique in feltiae gr= oup in having seven ridges in lateral fields compared to six in S. akhursti and S. joll= ieti 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 spic= ules and gubernaculum. D% and GS% are also different (Table 3). The second-generation male differe= s 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

Crossbreedi= ng 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.&nbs= p;

Molecular characterisati= on

The sequence of ITS regi= ons of S. texanum n. sp., flanked by primers 18S and 26S is characterised = by its sequence length 956 base pairs (bp), ITS1 =3D 263 bp, ITS2 =3D 286 bp a= nd its composition. The sequence len= gth 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 interspecif= ic relationships, pairwise dista= nces 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 specie= s, 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 differe= nce (see the original description).

For D2/D3 regions, the sequence of S. texanum n. sp. is 855 bp, composition, A =3D 0.24912, C =3D 0.19= 532, G =3D 0.30292, T =3D 0.25263. Pairwise distances show that the new species differ= s from its closest taxon S .feltiae<= /span> 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; Tab= le 7).

Phylogenetic analysis

For ITS regions, maximum parsimony analysis shows that the alignment resulted in 1247 charac= ters in which 152 in ambiguous reg= ions 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 spec= ies of Steinernema are presented in Figure 6 (for MP, tree length =3D 3622, CI =3D 0.4268, RI = =3D 0.4616, RC =3D 0.1970, HI =3D 0.4616). In this consensus tree, 13 species o= f 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 a= ny 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 St= einernema are presented in Fig. 5 (tree length =3D 1125, CI =3D 0.6009, RI =3D 0.6999= , RC =3D 0.4205, HI =3D 0.3991). The s= ix species, S. texanum n. sp., <= /span>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 <= /i> and S. kushidai stand separa= tely. 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. Ne= matology 9, 379-396.

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