Steinernema pui sp. n. (Rhabditida, Steinernematidae), a new entomopathogenic nematode from Yunnan, China Author Qiu, Lihong Author Zhao, Jingxiu Author Wu, Zhongdao Author Lv, Zhiyue Author Pang, Yi text Zootaxa 2011 2767 1 13 journal article 10.5281/zenodo.206861 b0b8b516-e294-4680-af46-462f316273c9 1175-5326 206861 Steinernema pui sp. n. description Measurements. Morphometrics of the holotype (the first generation male), allotype (the first generation female) and paratypes of IJs and the first- and second- generation males and females are listed in Table 1 . FIGURE 1. Steinernema pui sp. n. in lateral view. A, D and E: the whole body (A), Spicule (D) and tail (E) of first generation male; B, F and G: the anterior region (B), epiptygma (F) and tail (G) of the first generation female; C and F: the anterior region (C) and tail (F) of the infective juvenile. Scale bar (in µm): A = 320; B = 100; C = 32; D = 32; E = 65; F = 78; G = 28 and H = 90. First-generation males: Body of heat-relaxed specimens C-shaped and curved posteriorly. Head truncated and slightly swollen anteriorly. Six lips fused at base, each with a papilla. Four cephalic papillae present. Two amphidial apertures distinct, located behind lateral labial papillae ( Fig. 2 A). Stoma shallow, circular anteriorly and triradiate internally. Pharynx extends nearly to mouth opening. Both cheilorhabdions and prorhabdions small but distinct. FIGURE 2. SEM micrographs of Steinernema pui sp. n. A: face view of a first generation male showing 6 labial papillae (l), 4 cephalic papillae (c) and 2 amphids (a); B: the tail of a first generation female showing a mucron (arrow) and anus; C: the tail of a first generation male showing the shape (conoid, short without mucron) and the distribution of genital papillae (arrows); D: the head of an ensheathed infective juvenile (IJ) showing 4 cephalic papillae (c) and an amphid (a); E: the middle portion of an IJ showing lateral field pattern; F: the tail of an IJ showing lateral field pattern and phasmids (p). Scale bar (in µm): A = 11; B = 30; C = 25; D = 7; E and F = 8. Pharynx muscular, with a cylindrical procorpus, metacorpus somewhat swollen, isthmus well defined, basal bulb slightly enlarged. The maximum diameter of metacorpus is equal to that of basal bulb. Nerve ring surrounds anterior portion of basal bulb. The excretory pore opening located slightly anterior to nerve ring. Lateral fields and phasmids not observed. Gonad single, reflexed. Spicules paired with a light brown color, moderately curved. The spicule length/width of the mature male is about 5.2 ( Fig. 3 A and B). Spicule head longer than wide with length/ width ratio of about 1.7 (1.5–2.0). Lateral and dorsal limb distinct, starting from head and extending to the spicule tip. Ventral limb starts from head and end well before the spicule tip. The tip of spicule blunt, bearing an aperture located on the tip with a circular shape and a slit-like concave located on the ventral side of the blade closed to the tip ( Fig. 3 D). This character was observed clearly in all of 4 specimen in posterior and ventral view (the only position that enables this character to be observed) among about 20 dissected spicules examined under SEM. Velum moderately developed, starting from anterior portion of ventral limb and ending at the tip of the ventral limb. Gubernaculum is boat-shaped in lateral view. In ventral view, it tapers anteriorly and ends at a slightly enlarged, ventrally bent head ( Fig. 3 E and F). Cuneus is needle-shaped and short. Spicules of the young males ( Fig. 3 C) are slightly different from those of the mature ones ( Fig. 3 A and B). Twelve pairs and a single precloacal genital papillae distributed in a pattern showed in Fig. 1 A and E and Fig. 2 C, including seven pairs preanal papillae, one pair adanal, one pair dorsolateral and three pair postanal (two being subterminal and one subdorsal). Tail conoid and short, without mucron. FIGURE 3. SEM micrographs of spicules and gubernacula of Steinernema pui sp. n. A and B: spicules of mature males; C: spicule of a young male; D: spicule tip showing an aperture and an irregular shaped concave; E and F: gubernacula in ventral (E) and lateral view (F). Scale bar (in µm): A = 15; B = 13, C = 12; D = 2.6; E and F = 12. TABLE 1. Morphometrics of Steinernema pui sp. n. Character1 First generation Second generation Infective 1EP, NR and ES: distance from anterior end to excretory pore, nerve ring and the base of pharynx, respectively; H%: hyaline tail length in % of total tail length. 2Mean ± SD and range in µm.

Holotype	Male	Allotype	Female	Male	Female	Juvenile
n L Greatest body diam EP	2025 130 140	20 2059±124 2 (1800–2350) 137±15 (118–180) 152±13	5200 238 133	20 6081±1151 (4850–8875) 228±32 (155–275) 147±17	10 1425±67 (1300–1500) 84±7 (70–90) 138±10	10 2495±278 (2070–2950) 138±10 (125–150) 126±6.1	20 1004±75 (900–1120) 36±2 (33–40) 85±4
NR ES Tail length (T) Anal body diam. (ABD) Spicule length (SP) Gubernaculum	150 193 34 55 80 65	(130–180) 157±10 (143–188) 196±12 (175–228) 32 ± 2 (29–38) 55±3 (48–63) 84±4 (78–88) 62±2	150 225 55 90	(125–188) 166±17 (150–218) 236±21 (213–288) 57±10 (43–75) 98±13 (85–125)	(120–150) 110±12 (93–125) 141±13 (120–162) 27±3.7 (21–30) 38±4 (35–48) 65±4.3 (58–70) 44±4.4	(120–137) 137±9.3 (125–152) 183±16 (162–207) 60±3.7 (53–63) 56±5 (50–65)	80–95 117±7 (100–125) 144±8 (130–150) 69±5 (60–80) 22±1 (20–25)
length (GU) a b c	15 10 60	(58–65) 15±1.3 (14 –16) 11± 1.4 (9.5–12) 64±6	22 23 94	27±5 (20–35) 26±3 (22–31) 107± 14	(35–50) 17±3 (14–20) 10.1±0.2 (9.6–11) 53±4	18± 4 (14–22) 14±3 (11–18) 42±3	28±1.7 (26–31) 7.03±0.74 (6.00–8.15) 15±0.8
c’ H% D% =EP/ES x 100	0.62 73	(58–70) 0.58±0.06 (0.53–0.61) 77±5.8	0.61 59	(96–118) 0.58±0.07 (0.53–0.61) 62.3±4.5	(48–66) 0.71±0.06 (0.53–0.61) 98±12	(37–46) 1.07±0.1 (0.53–0.61) 68.9±6.7	(13–16) 3.1±0.2 (2.9–3.3) 45±3.7 (40–50) 59.5±3.04
E% = EP/T x 100 SW=SP/ABD	412 1.45	(70–91) 465±48 (403–512) 1.52±0.10	241	(54–71) 258±35 (201–312)	(77–114) 511±48 (389–621) 1.71±0.20	(57–81) 210±45 (139–272)	(55.00–66.04) 125±8.0 (109–142)
GS=GU/SP	0.81	(1.40–1.84) 0.74±0.04	(1.32–1.93) 0.68±0.08
V	(0.69–0.81)	51	50±5 (37–55)	(0.52–0.78)	52±2.3 (50–57)

Second-generation males: Similar to that of the first generation male except that the excretory pore is located much more posterior and most morphometrics, such as spicule and gubernaculum length, are smaller. Tail conoid without mucron. First generation female: Body C-shaped when killed by gentle heat. Cuticle smooth or with faint annules. Head rounded and slightly truncated anteriorly ( Fig. 1 B). The morphology of the anterior part and pharynx is similar to those of males. The excretory pore opening is anterior to nerve ring. Lateral fields and phasmids not observed. Gonads didelphic, reflexed. Vagina muscular and short. Vulva a transverse slit, slightly protruding from body surface and with a short double flapped epiptygma. Tail conoid, pointed, with a short projection ( Fig. 1 G; Fig. 2 B). Second-generation female: Similar to the first generation female but smaller. Vulva asymmetric, protruding from body surface. Short double flapped epiptygma present in the first generation female indistinct or absent in the second generation females. Tail longer than anal body width, tapering to a pointed end. Infective juveniles: Body slender. Mouth and anus closed. Anterior end rounded, slightly truncated and continuous with body contour ( Fig. 1 C, Fig. 2 D). Labial region smooth, papillae not seen. Amphidial apertures distinct, slit –shaped. Four cephalic papillae prominent. Pharynx long and narrow, isthmus distinct and surrounded by nerve ring, basal bulb slightly elongated, cardia indistinct ( Fig. 1 C). Excretory pore located anterior to nerve ring, in slightly posterior position of pharynx. Bacterial pouch located in the anterior portion of the intestine. Cuticle with prominent transverse striations. Lateral field begins anteriorly with one line and increases to 9 posterior to the base of pharynx, making a total of 8 ridges ( Fig. 2 E). The nine-line pattern extends posteriorly to a position closed to anus where the number of ridges reduced to five followed by two and then disappeared ( Fig. 2 F). Phasmid distinct, located on the posterior end of the first ridge from ventral side. Tail short, attenuate and tapering evenly ( Fig. 1 F). Hyaline portion occupying 46% of the tail length. Type locality and host. The soil sample was collected from a rubber plantation at Xiao-jie town, Jing-hong city, Xi-shuang-ban-na district ( 22.01o N , 100.47 o E ), Yunnan province, People’s Republic of China . The type host of this nematode in nature is unknown as it was recovered from soil using Galleria larvae as bait. Type specimens and etymology. The slides of holotype (first generation male), allotype (first generation female) and paratypes of about 60 infective juveniles on three slides, 10 each of the first and second generation males and females on 20 slides (one male and one female from the same generation on each slide) of S. pui sp. n. were deposited in the State Key Lab for Biocontrol ( SKLB ), School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China . Additional paratypes (one each of the first and second generation male and female and about 20 IJs on three slides) will be deposited in the Nematode Collection of the Department of Agriculture, USA . Living infective juveniles are also preserved in liquid nitrogen in the nematode collection of SKLB , Zhongshan University. This species is named after the late Professor Zhelong Pu, a distinguished scientist in the area of biological control and the founder and first director of SKLB , Sun Yat-sen University. Cross-breeding. Normal offspring were observed on the majority of slides on which two inoculated IJs developed into opposite sex adults in the self-cross controls of CWL05, GDc339 and YNd339. However, no offspring were observed from any slide in the cross-breeding treatments of YNd339 x CWL05 and YNd339 x GDc339. Molecular characterization. The lengths of ITS and partial 28S rDNA of S. pui sp. n. amplified using the above mentioned primers were 977bp (235A, 255G, 291T and 195C) and 800bp (193A, 247G, 196T and 164C), respectively. The phylogenetic tree of the partial 28S rDNA ( Fig. 4 ) shows that S. pui sp. n. forms a monophyletic group with S. khoisanae , S. longicaudum , S. guangdongense , S. scarabaei and S. hermaphroditum . This is a subgroup of the S. glaseri group that comprises EPNs with long IJ body and adopted a cruiser host finding strategy ( Campbell et al. , 2003 ). The sequence alignment of the S. pui sp. n. group ( Fig. 5 ) shows that it has 22 diagnostic characters (= number of base pairs, in the same column of the alignment, present in one sequence but not in others) in the group and differs from its closest nematode, S. khoisanae by 33 characters while there are only 11 different characters between S. guangdongense and S. longicaudum . Pairwise distances ( Table 2 ) clearly differentiate it from all other nematodes in the group. The phylogenetic tree of the rDNA ITS regions ( Fig. 6 ) support the relationship of S. pui sp. n. with other Steinernema species revealed by the partial 28S rDNA sequences. The pairwise distances of the ITS sequences ( Table 3 ) can also be used to differentiate the new species from other nematodes in the group. FIGURE 4. Phylogenetic relationship of Steinernema pui sp. n. with other Steinernema species based on the D2D3 domain of 28S rDNA sequence. FIGURE 5. Alignment of the D2D3 domain of 28S rDNA sequence of Steinernema pui sp. n. with those of closely related Steinernema species. TABLE 2. Percentage of similarity (upper triangle) and genetic distance (lower triangle) on the sequences of D2D3 domain of 28S rDNA of Steinernema pui sp. n. with other closely related Steinernema species.

1	2	3	4	5	6	7	8	9	10	11	12	13
1. S. boemarei	***	95.0	92.1	89.2	92.4	93.1	92.4	94.0	92.7	90.2	90.7	90.8	89.4
2. S. arenarium	2.7	***	91.7	89.4	93.0	92.2	92.6	91.9	91.4	90.1	90.0	90.2	89.9
3. S. glaseri	6.0	5.2	***	97.1	92.1	92.0	92.0	90.5	90.9	88.2	88.9	89.9	88.0
4. S. cubanum	6.6	5.7	0.9	***	91.7	91.0	91.1	89.1	89.7	87.5	88.3	89.0	87.6
5. S. puertoricense	5.1	4.0	6.3	6.4	***	97.6	95.9	94.2	95.7	91.5	93.6	94.7	91.9
6. S. diaprepesi	5.0	4.7	6.4	6.9	2.2	***	96.2	94.7	96.2	92.9	93.6	94.6	91.0
7. S. khoisanae	4.8	4.8	6.3	6.7	3.2	2.8	***	95.5	96.1	95.4	94.4	95.4	93.5
8. S. longicaudum	4.9	5.4	7.8	8.3	5.1	4.7	3.9	***	98.0	94.2	94.0	95.2	92.2
9. S. guangdongense	5.5	5.8	7.6	8.0	4.1	3.7	3.2	1.4	***	94.5	94.5	95.7	93.0
10. S. pui sp. n.	6.9	7.4	9.7	10.1	7.0	6.2	4.5	4.7	4.7	***	92.8	93.6	92.1
11. S. hermaphroditum	5.9	5.9	8.0	8.4	4.7	4.4	3.4	3.5	3.9	4.9	***	99.2	91.6
12. S. scarabaei	6.1	5.9	7.9	8.3	4.6	4.4	3.3	3.4	3.6	4.8	0.0	***	91.8
13. S. karii	7.6	7.8	9.4	9.5	6.7	7.2	5.8	6.8	6.2	7.2	5.7	6.5	***

Diagnosis. S. pui sp. n. is characterized by its unique nucleotide sequences of either the partial 28S rDNA or the rDNA ITS regions. Morphologically, it is characterized by the combination of the features of various developmental stages of the nematode. For IJs, the combination of the following characters: body length (1004 ± 75 μm); distance from anterior end to excretory pore (85 ± 4 μm), to the base of pharynx (114 ± 8 μm); tail length (69 ± 5 μm); E% (77 ± 4.5); lateral field with eight evenly distributed and identical ridges at the middle body portion; and tail short and attenuate with a hyaline portion occupying 40–50% of the tail length can be used to differentiate the new species from other Steinernema species. For the first generation males, the new species can be recognized by spicule length (84 ± 4 μm, measured along the arch); GS (0.74 ± 0.04); SW (1.52 ± 0.10); D (77 ± 5.8); spicule possesses an aperture on the tip and a pit on the ventral side of the lamina close to the tip; gubernaculum with a short needle-shaped cuneus; tail conoid and short without mucron. For female, S. pui sp. n. is recognized by a conoid and pointed tail with a short mucron on the tip and a slightly protruding and symmetrical vulva with a short double flapped epiptygma. FIGURE 6. Phylogenetic relationship of Steinernema pui sp. n. with other Steinernema species based on rDNA sequence of the internal transcribed spacer regions. TABLE 3. Percentage of similarity (upper triangle) and genetic distance (lower triangle) on the rDNA internal transcribed spacer region sequences of Steinernema pui sp. n. with other closely related Steinernema species.

1	2	3	4	5	6	7	8	9	10	11	12	13	14
1. S. loci ***	82.7	58.8	62.5	61.3	47.9	50.2	52.1	56.9	58.5	54.1	55.0	58.7	55.0
2. S. leizhouense 4.3	***	63.8	63.8	54.3	52.3	59.3	55.0	53.9	58.2	56.3	59.8	64.6	54.2
3. S. thanhi 8.3	6.6	***	52.5	52.8	45.8	44.0	49.3	52.5	47.6	50.1	50.4	51.5	47.8
4. S. aciari 19.9	18.7	18.7	***	63.8	62.3	61.0	60.3	62.7	65.3	64.0	61.0	62.7	58.0
5. S. diaprepesi 20.3	19.1	21.1	18.3	***	57.0	63.1	62.0	58.1	59.2	68.9	55.4	58.9	61.6
6. S. glaseri 30.0	28.3	26.9	25.0	25.2	***	89.3	59.1	65.5	56.2	60.4	63.4	61.5	59.3
7. S. cubanum 29.5	28.4	27.5	27.4	27.5	4.4	***	60.1	63.3	61.8	61.0	62.2	61.8	59.0
8. S. boemarei 31.1	29.0	28.0	28.9	29.7	18.2	19.5	***	68.1	52.3	56.4	53.9	60.5	54.4
9. S. arenarium 28.1	26.0	24.2	27.3	25.8	16.6	17.3	12.6	***	59.2	60.2	58.2	63.7	58.3
10. S. longicaudum 24.5	23.0	21.6	21.1	17.5	22.6	24.1	25.6	22.0	***	84.0	77.2	67.1	65.2
11. S. guangdongense 23.7	22.9	21.5	21.5	15.7	22.3	24.0	23.9	21.6	4.3	***	80.0	65.3	63.5
12. S. pui sp. n. 23.5	22.3	21.9	23.3	18.0	22.1	24.5	23.9	21.8	8.9	7.2	***	63.2	62.9
13. S. khoisanae 27.5	27.1	27.6	26.0	22.3	29.5	31.1	27.5	26.4	17.5	15.4	17.3	***	60.4
14. S. karii 26.9	27.5	25.1	22.9	20.4	28.6	29.1	27.1	25.6	19.8	17.8	19.0	23.0	***

Relationships. Both molecular ( Figs. 4–6 ) and morphological characters ( Table 1 and 4 ) showed congruently that S. pui sp. n. belongs to the S. glaseri group. 28S rDNA sequence shows that the new species is most closely related to S. khoisanae . However, the genetic distance between them is 3.2 ( Table 2 ), greater than that of many closely related described species, such as S. cubanum and S. glaseri (0.9), S. longicaudum and S. guangdongense (1.4) and S. boemarei and S. arenarium (2.7). Several characters of various developmental stages can also separate the new species from S. khoisanae : IJ tail length of S. pui sp. n. is shorter (69 ± 5) vs (85 ± 5), the distance from anterior end to excretory pore is smaller (85 ± 4) vs (95 ± 5) and diameter greater (36 ± 2) vs (31 ± 2); vulva of the first generation females of S. pui sp. n. has a short double flapped epiptygma vs absence in S. khoisanae ; SW (the ratio of spicule length to tail width) of the first generation male of the new species is 1.52 ± 0.1, lower than that of S. khoisanae (2.03 ± 0.5); an aperture were seen on the spicules tip of the first generation S. pui sp. n. but this character is not reported in S. khoisanae . S. pui sp. n. can be distinguished from the rest of Steinernema species of the S. glaseri group, including S. khoisanae , S. guangdongense , S. longicaudum , S. scarabaei and S. karii by morphological characteristics of the IJs and the first generation males as listed in Table 4 .