Male (first generation) (Fig.1) (Fig. 2)
Body curved ventrally, posteriorly. Head rounded, usually slightly swollen. Anterior end with six labial papillae, two amphids, and four prominent cephalic papillae. Stoma shallow. Under light microscope, cheilorhabdions appearing as small and sclerotized structures at anterior end, sometimes indistinct. Under SEM, cheilorhabdions as a subtrianglar ring surrounding stoma. Excretory pore located between mid-pharynx and nerve ring. Anterior part of excretory duct well sclerotized. Pharynx with cylindrical procorpus, metacorpus slightly swollen, isthmus present, nerve ring around isthmus (just anterior to basal bulb), basal bulb distinct. Esophago-intestinal valve present. Gonad monorchic, reflexed. Distance from base of pharynx to anterior end of testis variable. Spicules paired, brown in color. Head (manubrium) of spicules enlarged anteriorly (Fig. 3), with rounded anterior end, longer than wide (length/width = 1.5-2.0). Shaft (calomus) very short, mostly absent. Blade (lamina) thin, slightly curved, tapering gradually posteriorly, blade terminus blunt, velum very small or absent. In general, spicules of the new species similar to those of S. feltiae but a little more curved and anterior end mostly bluntly rounded. Each spicule with two internal sclerotized ribs. Gubernaculum boat-shaped in lateral view, tapering gradually anteriorly. Anterior end usually ventrally curved. Eleven pairs and one single precloacal genital papillae distributed as in Fig. 2E, F. Tail conoid, curved ventrally (Fig. 4); tail terminus rounded without a mucron.
Male
(second-generation)
Similar to that of the first-generation except body, spicule and gubernaculum shorter and thinner, posterior end spiral shaped (Fig. 4).
Female (first-generation) (Fig. 2)
Body cuticle smooth or with faint annules. Lateral fields and phasmids not observed. Head rounded or flattened anteriorly, continuous with body; six labial and four cephalic papillae. Lips indistinct. Amphids usually inconspicuous even under SEM (Fig. 2). Stoma shallow. Cheilorhabdions well sclerotized but small. A smaller sclerotized structure posterior to cheilorhabdions (presumably the prorhabdions), observed in other species, indistinct in this species. Pharynx with procorpus cylindrical, muscular; metacorpus somewhat swollen; isthmus distinct; basal bulb valvate as in other steinernematids (Fig. 5). Nerve ring surrounding isthmus, variable in position. Esophago-intestinal valve present. Excretory pore located anterior to mid-pharynx, anterior to nerve ring, excretory duct sclerotized anteriorly. Gonads amphidelphic, reflexed, often containing many eggs. Vulva a transverse slit; mostly protruding; small double-flapped epiptygma present occasionally; posterior lip larger than anterior one (75%). Body diameter right posterior to vulva usually larger than that anterior to vulva. Vagina sclerotized. Tail shape variable, nipple-shaped, with blunt terminus, occasionally (about 10%) a mucron-like structure present on dorsal side of tail terminus (seen more often on young females). Ventral postanal swelling present but small; tail shorter than anal body width. Lateral fields and phasmid not observed.
Similar to first-generation female but smaller. Vulva less protruding, epiptygma absent. Tail tapering to a pointed end, longer than anal body width; ventral postanal swelling present.
Body elongate. Sheath (second-stage cuticle) present immediately after harvesting, but many infective juveniles will lose their sheath in storage. Labial region not striated, rounded or slightly angular anteriorly, continuous with body (Fig. 6A). Labial papillae not seen; four cephalic papillae prominent. Amphids slit-shaped, prominent under SEM. Cuticle marked with prominent transverse striations. Both hemizonid (anterior to excretory pore) and hemizonion (posterior to excretory pore) present. Deireid not observed under either light microscope or SEM. Lateral field begins anteriorly with one line at the first annule. Two additional lines appear at annules 7-8 to form two ridges (Fig. 6B). Near excretory pore level, the number of ridges in lateral fields increases from two to six . Near the end of pharynx, one marginal line appears, and the central ridge divides into two (Fig. 6C), making a total of eight ridges, the maximum number in the lateral field. The portion with eight ridges is the longest part (compared to portions with 2, 6, 4 ridges) of the lateral field. Near anus the number of ridges reduced to four. Some annules anterior to phasmid, the 4 ridges in lateral field become two ridges. With the above description, the formula of the lateral field is 2, 6, 8, 4, 2.
Pharynx with thin corpus, basal bulb more or less elongate with visible valve. Tail attenuate, tapering abruptly to form a typical tail. Phasmid present near mid-tail, close to ventral marginal line of lateral field. Hyaline portion short, occupies 38% (33-45) of tail length.
Steinernema leizhouense can be recognized
by large IJ body diameter 38 (35-43) μm, distance from anterior end to
nerve ring 101 (95-108) μm; pharynx length 133 (125-143) μm and a =
28 (23-38). Lateral field pattern variable, the formula for the arrangement of
ridges from head to tail is 2, 6, 8, 4, 2 (Fig. 6). The hyaline portion
occupies 38% (33-45) of tail length. The new species can be recognized further
by males with spicule shape, spicule length averaging 82 (75-93) μm,
spicule head longer than wide, spicule blade thin, velum present but narrow (Fig.
3) and the ratios SW = 210 and GS = 50 (Table1). Female of the new species is characterized by
nipple-shaped tail terminus (Fig. 2).
The new species is characterized genetically by sequence length and composition of ITS regions (1032 bp, ITS1 = 300 bp, ITS2 = 325 bp and A = 25.2%, C = 19.6%, G = 25% and T = 30.1%) (accession # AY170340), and a partial 28S rDNA sequence (446 bp, A = 23.8%, C =18.9%, G = 31.8%, T = 25.5%) ( AY169557). The phylogenetic tree of ITS regions shows that the nematodes S. leizhouense , and S. loci form a monophyletic group well supported by bootstrap analysis. Additionally, the alignment of ITS regions (not shown) of seven nematode species of the same clade (S. aciari, S. diaprepesi, S. glaseri, S. guangdongense, S. leizhouense, S. loci, and S. longicaudum) shows that S. leizhouense has 28 diagnostic character states (=number of characters, in the same column of the alignment, present in one sequence but not in others) and differs from its sister taxa, S. loci at 155, and S. aciari at 136 total characters of the ITS sequence. Tracing process from McClade using a phylogenetic tree of ITS regions of S. glaseri group with S. intermedium as an outgroup, shows that the number of autapomorphies that changed unambiguously on branches is seven for S. leizhouense.
The new species can also be differentiated from other closely related nematodes by characteristics of its partial 28S sequence. The phylogenetic tree of 28S the partial sequence shows that S. leizhouense and S. aciari comprise a monophyletic group, a sister group of the S. glaseri group. This clade is well supported by bootstrap analysis (Nguyen et al., 2006).
Relationships
Steinernema leizhouense can be distinguished from the closest species S. loci and S. aciari by characteristics of infective juveniles, males and females. Compared to S. loci, both nematodes have almost the same length but IJs of the new species have a shorter pharynx, 133 (125-143) µm in the new species compared to 141 (126-155) µm in S. loci, shorter tail, 66 (58-75) µm compared to 75 (66-83) µm, and higher E%, 120 (105-140) compared to 107 (94-120). Males of the new species have a different shape (Fig. 2, 3 compared to Fig. 2C in Phan et al., 2001) and longer spicule, 82 (75-93) µm compared to 71 (60-80) µm, longer gubernaculums, 55 (50-60) µm compared to 46 (40-52) µm, and higher SW%, 213 (177-251) compared to 190 (170-210) (Table 1). Comparing to S. aciari, IJs of the new species have a shorter body length, 983 (900-1050) µm compared to 1113 (975-1250) µm. Because of the shorter body length, all other measurements are shorter, especially tail length, 66 (58-75) µm compared to 78 (68-88) µm. Males of the new species have a different shape of spicule (Fig. 2 compared to Fig. 3 in Qiu et al., 2004), and higher SW%, 213 (177-251) compared to 204 (180-240).
Type host and locality
The type host of this nematode in
nature is unknown as it was recovered from soil using Galleria larvae as
bait. The soil sample was collected in an eucalypt plantation in Tangjia town
(20.8oN, 109.8oE),
Type
material
Holotype male, allotype female, 10 paratype
first generation males, 5 paratype females, 15 paratype infective juveniles and
other population slides deposited in the State Key Lab for Biocontrol, College
of Life Sciences, Zhongshan University, Guangzhou 510275, China. Living
infective juveniles are also preserved in liquid nitrogen in the nematode
collection of SKLB,
Distribution
Steinernema
leizhouense was found only in
Reference
Nguyen, K.B., Qiu, L., Zhou, Y. & Pang, Y.
(2006). Steinernema leizhouense sp. n.
(Nematoda: Steinernematidae), a new entomopathogenic
nematode from southern
Legends
for figures
Fig. 1. Steinernema
leizhouense males and infective juvenile. A: anterior region of a
first-generation male; B,C: variations of first-generation male tails and
spicules and gubernacula; D,E: variation of second-generation male tails,
spicules and gubernacula;
F,G: anterior and posterior region
of an infective juvenile. Scale bar = 50 µm. After Nguyen et al. 2006, courtesy
of Russian Journal of Nematology.
Fig. 2. SEM of
Steinernema leizhouense first-generation female and male. A: anterior region
of a female; B,C: first-generation female tail; D: face of a first generation
male; E,F: first-generation male tails showing distribution of genital
papillae. Scale bars: A: 12 µm; B: 16.7 µm; C: 60.0 µm; D: 6.0 µm; E: 37.5 µm; F: 16.7 µm. After Nguyen et al.
2006.
Fig. 3. SEM of
Steinernema leizhouense spicule and gubernaculum of the first-generation
males. A-D: variations of spicule shape; E,F: gubernacula showing ventral and
dorsal views. Scale bars: A, B: 23.1 µm; C, D: 20.0 µm; E, F: 15.0 µm. After
Nguyen et al. 2006.
Fig. 4. Steinernema
leizhouense male tails. A: first-generation male tail; B: second- generation
male tail. Scale bar (in A): A: 20 µm; B: 18.5 µm.
Fig. 5. Steinernema leizhouense females. A, B, C: first-generation female anterior region and variations in tail shape; D, E: variations of vulva shape. F, G: variation of tail shape of second-generation females. Scale bar = 50 µm.
Fig. 6. SEM of lateral field pattern of Steinernema leizhouense juveniles. A: anterior region showing smooth head, amphid, and one line in lateral field; B: lateral field showing the number of ridges change from two to six; C: lateral field showing a new line appears (arrow), and the middle ridge divides into two, making the lateral field become eight; D: lateral field with eight ridges. E: Lateral field near anus showing four ridges and phasmid (p); F: posterior region showing tail shape, lateral field with two ridges and phasmid. Scale bars: A: 6.0 µm; B, C, E, F: 6.67µm; D : 7.50 µm.