Steinernema ashiuense*, Phan, Takemoto & Futai, 2006

Steinernema ashiuense

Phan, Takemoto & Futai, 2006

Me= asurements

See Table 1.

De= scription

Males, first generation: Body curved ventrally; C- or J-shaped. Cuticle appearing smooth under LM, lateral field not observed. Head rounded, slightly offset from bo= dy. Six pointed labial papillae and four cephalic papillae visible under SEM. Amphids inconspicuous. Buccal cavity funnel- or cup-shaped. Stoma shallow. Pharynx muscular; procorpus cylindrical; metacorpus slightly swollen, non-v= alvate; isthmus distinct; basal bulb pyriform, valve distinct. Nerve ring located j= ust anterior to basal bulb. Cardia prominent and protruding into intestinal lum= en. Excretory pore located at middle of pharynx. Excretory duct cuticularised; excretory gland swollen and elongated. Testis monorchic, reflexed. Spicules paired, slightly yellowish in colour, strongly curved; length / width ratio= =3D 4 (3.5-4.4); head (manubrium) as long as wide. Blade arcuate with straight tip and three well-defined lobes; dorsal lobe well curved, terminating at spicu= le tip; lateral lobe prominent, usually greater in width anteriorly, terminati= ng at spicule tip; ventral lobe enlarged anteriorly at dorsal and ventral side= to form prominent apex and rostrum, terminating posterior to rounded spicule t= ip. Velum large, not covering spicule tip. Gubernaculum 40-70% of spicule lengt= h, boat-shaped in lateral view, swollen at middle, with or without slightly ventrally curved knob at proximal end; in ventral view, cuneus long, needle-shaped, pointed posteriorly. Wing of corpus expanding laterally. Gen= ital papillae disposed as single midventral precloacal papilla and ten pairs; la= tter arranged as follows: five pairs subventral pre-cloacal, one pair lateral pre-cloacal; one pair adcloacal; two pairs subventral postcloacal and one p= air subdorsal postcloacal. Tail conoid with small mucron usually present. Phasm= ids inconspicuous.

Males, second generation: Similar to first generation but slender, b= ody diam. ca half that of first generation. Testis flexure extending more posteriorly. Mucron more distinct= , 2.5-7.5 µm long.

Females, first generation: = Body robust, C-shaped. Cuticle appearing smooth under LM, late= ral field not observed. Head broadly rounded. Six pointed labial papillae and f= our cephalic papillae visible with SEM. Amphids inconspicuous. Buccal cavity funnel- or cup-shaped. Stoma shallow. Pharynx with cylindrical procorpus; metacorpus slightly swollen and non-valvate; isthmus distinct; basal bulb pyriform, valve visible. Nerve ring located just anterior to basal bulb. Excretory pore usually located at mid-pharynx level. Excretory duct cuticularised and excretory gland swollen. Cardia prominent protruding into intestinal lumen. Genital system didelphic, amphidelphic, reflexed; filled with eggs. Vulva a median transverse slit, protruding from body, no epiptygma. Vagina short, oblique with muscular wal= ls. Tail dome-shaped, shorter than anal body diam. with terminal peg.

Females, second generation: Similar to first generation but smaller, body length about half that of first generation and= one third of body diam. Eggs arranged in one row. Tail conoid without mucron. Postanal region slightly swollen.

Infective juvenile: Body slender, mod= erately ventrally curved when heat-relaxed, <= /b>often enclosed in cuticle of second-stage, tapering regularly from base of pharyn= x to anterior end and from anus to terminus. Mouth and anus closed. Head broad, offset from body; labial papillae not observed; transverse slit-like amphid= ial apertures situated posterior to labial disc but anterior to cephalic papill= ae. Pharynx long, narrow; isthmus distinct, surrounded by nerve ring; basal bulb elongate with valve. Cardia prominent. Excretory pore located at mid-pharynx level. Hemizonid distinct, located anterior to base of basal bulb. Bacterial vesicle elongate (21-34 µm long × 7-10 µm wide). Genital primordium clear, about 1= 20-160 µm long. Lateral field at mid-body with five equally developed ridges= (six lines or incisures). Tail conical, straight, often bent towards dorsal side; terminus finely rounded. Hyaline portion distinct, about 36% of tail length. Phasmids distinct, located in anterior half of tail.

Ty= pe host and locality

The type host of this species in nature is unknown as the nema= tode was collected from soil by baiting with Galleria mellonella. The soil sample was collected from a grassland habitat along the bank of the Yura river, near the Office of Ashiu Forest Research Statio= n of Kyoto University, Myama town, Kyoto prefecture, Japan (longitude 135º = 43' E, latitude 35º 18' N, altitude 356 m a.s.l., annual average of temperature 11.7ºC, precipitation 2353 mm/year, depth of snow during winter about 1 m, flora transit= ion zone from cool-temperate to warm-temperate). Soil type is dark grey brown s= andy clay silt.

Ty= pe material

Holotype male and paratype male, female and infective juveniles deposited in the nematode collection at the Herbarium and Insect Museum of = the National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki, Japan. Additional paratype slides at the Laboratory of Environmental Mycoscience, = Graduate School of Agriculture, Kyoto University, Japan and Institute of Ecology and Biological Resources, Vietnamese Academy of Sciences and = Technology, Vietnam= .

<= o:p>

Di= agnosis and relationships

Steinernema ashiuen= se is characterised by an IJ body length of 768 (720-800) μ= m, lateral field with five equally developed ridges (six lines or incisures), = head smooth, lacking horn-like structures, excretory pore located at level of mi= ddle of pharynx, hyaline portion short (about one-third of tail length). Males have broad, slightly yellowish, spicules 50-65 μm long; spicule length / spicule width =3D 4 (3.5-4.4); and two subve= ntral and one subdorsal pairs of postcloacal genital papillae.

The lateral field of this new species has five equally develop= ed ridges, a feature that distinguishes it from all species in the feltiae group: S. kraussei and S. oreg= onense have five equally developed ridges and two weak submarginal ones (total of seven ridges); S. feltiae, S. robustispiculum and S. sangi have eight ridges with the two submarginal and two central ridges less distinct;= S. akhursti and S. jollieti have six equally developed ridges; and S. litorale, S. silvaticum and S. we= iseri have eight equally developed ridges.

Steinernema ashiuen= se differs from S. monti= colum by the location of the excretory pore opening in IJ and male (located at mid-pharynx level vs at posteri= or one-third of pharynx), and some characters of the male such as a shorter spicule length of 59 (50-65) vs 70 (61-80) μm, a relatively broader spicule with spicule length / spicule width =3D 4 (3.5-4.4) vs 8.7 (8= -8.8) and spicule head (manubrium) elongate vs round.

Steinernema ashiuen= se has broad spicules as found in S. kushidai, a species that is also found in Japan, but can be separated f= rom the latter by longer IJ body length of 768 (720-800) vs 589 (524-662) μm, more posterior excretory pore of 55 (51-59) vs 46 (42-50) μm, = longer IJ tail length of 71 (66-76) vs 50 (44-59) μm, lateral lobe of spicule terminating at spicule tip vs not terminating at spicule tip,= and cuneus of the gubernacula long, needle-shaped and pointed posteriorly vs not well seen.

Mo= lecular characterisation

The ITS1 + 5.8S + ITS2 sequence length of S. ashiuense is 662 bp which is the shortest amongst eleven spe= cies in the f= eltiae group. Sequence divergence ranged from 3.0 to 20%. The sequence of S. ashiuense differed from those of other Steinernema species in 20= -135 substitutions. Maximum parsimony analysis of the alignment of Steinernema species in this group revealed 156 parsimony informative characters. In the single unrooted maximum parsimony tree, S. ashiuense clustered with S. robustispiculum + S. monticolum with high boot strap support.

Maximum parsimony analysis of the alignment of this group with= an out group taxon revealed 196 parsimony informative characters. The single M= P tree (tree length =3D 573) and M= L tree (DNA model =3D GTR + G, - ln likelihood =3D 3655.07) had a similar topology. Steinernema ashiuense clustered with S. robustispiculum + S. monticolum with high to moderate boot strap support (see the original d= escription).

The alignment of the ITS1 + 5.8S + ITS2 of S. ashiuense with two closely related species (S. robustispiculum and = S. monticolum) show that= the new species differs from these species by nine unique substitutions in the ITS1 and eight unique substitutions in the ITS2.

The ITS sequence is deposited in GenBank under accession number DQ354694.

Reference=

Phan, L.K., Takemoto, S. & Futai^,K. (2006). Steinernema ashiuense s= p. n. (Nematoda: Steinernematidae), a new entomopathogenic nematode from Japan. <= /span>Nematology 8, in press.