Description of Oscheius indicus n. sp. (Rhabditidae: Nematoda) from India Author Kumar, Puneet Author Jamal, Wajih Author Somvanshi, Vishal S. Author Chauhan, Khushbu Author Mumtaz, Sabia text Journal of Nematology 2019 2019-04-02 51 1 1 11 http://dx.doi.org/10.21307/jofnem-2019-004 journal article 299040 10.21307/jofnem-2019-004 84540dba-696f-41d2-b1fb-647832784d14 2640-396X PMC6929651 31115203 11644501 Oscheius indicus n. sp. ( Figs 1 : A–H, 2: A–I, and 3: A–D) Measurements: given in Table 1 . Female The female body is straight, slender, tapering toward extremities but more toward posterior end. Cuticle is finely annulated, more prominent at the mid-body region, with faint punctations all over the body. Lateral fields distinct with four lateral lines, 4–5µm wide. Lips six, not fused, each bearing one bristle-like labial sensilla. Lip region continuous with body contour, 11 to 15µm wide. Amphidial apertures elliptical, at the base of lateral lips. Stoma tubular, 13 to 18µm long, 3 to 4 times longer than wide, with a thin pharyngeal collar reaching up to the 70% of stoma length. Chielostom with indistinct cheilorhabdion; metastegostmal walls usually isomorphic, with three small warts. Telostegostom narrow, funnel-shaped. Pharynx 194 µm (181–210) long; corpus cylindrical, strongly developed, 117µm (108–127) long. Medium bulb not set off. Isthmus 41µm (37–47) long, distinct, forming 20–22% of pharynx length. Basal bulb spherical, 36µm (33–41) long, 28µm (27–30) wide. Valvular apparatus prominent, haustrulum medium sized. Nerve ring located in posterior half of isthmus at 65 to 77% of pharyngeal length. Excretory pore at 85 to 95% of pharyngeal length. Deirids not observed and hemizonid obscure. Cardia 4 to 5µm long, protruding into intestine. Phasmid conspicuous, pore like. Reproductive system is amphidelphic. Anterior branch situated on right side and posterior branch on left side of intestine, with dorsally reflexed gonad, often extending beyond the vulva. Spermatheca consisting large sperm cells. Double-flapped epiptygma is present on the vulval opening. Uterus large, with 8 to 15 eggs . Vulva transverse slit situated at the mid-body, vulval lips protruding. Mating plug present in inseminated female. Vagina strong, thick walled about one-third to one half body width long, provided with oblique muscle. Rectum dilated, anus slit like, with protruding lips. Tail elongate, gradually tapering to fine point, 5 to 6 times as long as anal body diameter. Male Male body is similar to females in general morphology, but smaller in size and with posterior region strongly curved. Testis single, reflexed ventrally on right side of intestine. Bursa leptoderan, anteriorly open, margin smooth leaving a short part of tail protruding beyond bursa. Genital papillae nine pairs, three pairs pre-cloacal, and six pairs post cloacal. Arrangement of GP 1, GP 2, and GP 3 are either equally spaced with the length of spicule or GP 1 and GP 2 more spaced than GP 2 and GP 3. GP 4 and GP 5 sub-ventral and GP 6 lateral in position. All are equally spaced. GP 8 sub-dorsal, evenly spaced with GP 7 and GP 9. GP 7 and GP 9 sub-ventral in position. Spicules paired, separate, pointed terminus, and dagger-shaped. Gubernaculum thin, arcuate, boat shaped 42 to 44% of spicule length. Tail-1.1–1.4 anal body diameter long, protruded slightly outside the bursa. Table 1. Morphometric data of OSCHeiUS indiCUS sp. n. Measurements are in µm and in the form: mean ± standard deviation (range).

Characters	Holotype female	Paratype females	Paratype males
n	1	14	14
L	1,320	1,272.3 ± 111.7 ( 1,072 –1,480 )	1,062.7 ± 104.5 (886–1,209)
a	20.3	18.9 ± 1.1 (16.8–20.6)	19.6 ± 1.6 (17.4–22.4)
b	6.5	6.5 ± 0.4 (5.7–7.1)	6.2 ± 0.6 (4.9–6.9)
c	9.9	8.9 ± 0.9 (7.5–10.4)	31.2 ± 2.8 (26.1–36.2)
c’	5.3	5.8 ± 0.4 (5.0–6.6)	1.3 ± 0.1 (1.2–1.4)
V	50.2	48.2 ± 1.8 (45.4–50.5)	–
Maximum body diam.	65	67.8 ± 20.2 (59–82)	54.4 ± 16.3 (46–64)
Lip width	12	12.6 ± 1.1 (11–15)	11.6 ± 0.7 (11–13)
Length of stoma	16	15.4 ± 1.7 (13–18)	15.6 ± 0.9 (14–17)
Corpus	123	117.7 ± 6.1 (108–127)	105.4 ± 4.0 (101–114)
Isthmus	42	40.7 ± 3.4 (37–47)	35.4 ± 3.5 (31–41)
Basal bulb length	37	36.4 ± 2.2 (33–41)	29.9 ± 1.4 (27–32)
Basal bulb width	29	27.6 ± 1.8 (24–30)	23.8 ± 0.9 (22–25)
Pharynx	202	194.8 ± 9.8 (181–210)	172.4 ± 6.0 (159–181)
Excretory pore from anterior end	201	179.9 ± 15.1 (154–201)	173.6 ± 9.7 (158–187)
Nerve ring from anterior end	162	142.0 ± 14.1 (118–162)	136.7 ± 7.6 (122–146)
Anterior gonad Posterior gonad Vulva body diam. Vulva-anus distance Rectum Tail length Anal body diameter Pharynx gonad distance Testis Spicule length along axis Gubernaculum	524 352 64 525 72 133 25 175 _ _ _	558.2 ± 43.2 (501–640) 504.4 ± 90.3 (324–614) 66.4 ± 5.5 (58–80) 512.1 ± 40.2 (447–576) 67.9 ± 5.8 (58–76) 142.6 ± 8.9 (131–156) 24.8 ± 1.4 (23–28) 118.0 ± 31.6 (89–175) – – –	– – – – 54.3 ± 4.3 (46–61) 34.2 ± 2.3 (32–39) 25.6 ± 1.2 (24–28) 82.8 ± 17.2 (61–116) 883.0 ± 82.1 (760–1,035) 60.0 ± 4.2 (54–66) 25.1 ± 1.2 (24–28)

Figure 4: Molecular phylogenetic relationship of Oscheius indicus n. sp. (highlighted in bold) inferred using 28S D2/D3 extension region of 28 rDNA gene. The evolutionary history was inferred by the Bayesian analysis using the General Time Reversible substitution model with gamma-distributed rate variation across sites and a proportion of invariable sites (GTR+G+I). The posterior probability values exceeding 50% are indicated at appropriate clades. The scale bar indicates expected changes per site. Orange and green shaded boxes indicate insectivora and dolichura group, respectively. The 18s rDNA sequences failed to amplify after repeated attempts. The ITS and 28S D2/D3 sequences were deposited to the GenBank with accession nos. MF441252 and MF441494. The blast search analysis of ITS identified Oscheius sp. ( JQ 002565.1) and Oscheius chongmingensis ( EF 503690, EU273598) ITS sequences as the closest matches with 92 to 91% sequence identity, 99% query coverage and E-value = 0. The blast search analysis of 28S D2/ D3 sequence showed Oscheius chongmingensis (EU273599), Rhabditis sp. Hy CM-2010 ( HM 474859) and Oscheius insectivora (EU195968) as highest matching sequences at 96, 92, and 92% sequence similarity, respectively, at 93 to 100% query coverage and E-value = 0. The phylogenetic analysis using ITS and 28S D2/D3 expansion region sequences of Oscheius indicus sp. n. indicated that it is a member of insectivora group ( Figs 4 and 5 ). Both the markers showed O. chongmingensis to be the closest relative. The ITS sequence-based tree also grouped O. rugaonensis along with O. indicus and O. chongmingensis . In summary, the sequences of ITS and 28S D2/D3 expansion segment of the 28s rDNA gene of O. indicus were sufficiently divergent from any other Oscheius species for which these marker sequences are available, and suggest that our species might be a new species within insectivora group ( Figs 4 and 5 ). Diagnosis and Relationships Oscheius indicus n. sp. is characterized by the presence of four incisures in the lateral fields, three minute warts in metastegostom, long rectum (2–3 anal body diameters), open leptoderan bursa, nine pairs of papillae arranged as 1+1+1/3+3 pattern, a prominent double-flapped epipytigma. Oscheius indicus n. sp. closely resembles O. carolinensis ( Ye et al., 2010 ) , O. chongmingensis ( Zhang et al., 2008 ) , O. colombiana ( Stock et al., 2005 ) and O. nandarajani (Ali et al., 2011) . Figure 5: Molecular phylogenetic relationship of Oscheius indicus n. sp. (highlighted in bold) inferred using ITS region. The evolutionary history was inferred by the Bayesian analysis using the General Time Reversible substitution model with gamma-distributed rate variation across sites and a proportion of invariable sites (GTR+G+I). The posterior probability values exceeding 50% are indicated at appropriate clades. The scale bar indicates expected changes per site. Orange and green shaded boxes indicate insectivora and dolichura group, respectively. The new species is differentiated from Oscheius carolinensis in having smaller stoma (13–18µm vs 18–27µm), shorter pharynx (181–210µm vs 228–264µm) and anal body diameter (24–28 µm vs 28–43µm). In male shorter stoma (14–17 µm vs 18–23µm), longer pharyngeal collar extension to stoma (70 vs 50%), arrangement of GP 1, GP 2, and GP 3 (either equally or unequally spaced vs equally spaced) and GP 5 (sub-ventral vs dorsal). The new species is distinguished from Oscheius chongmingensis by longer stoma in female (13– 18 µm vs 8.9–10.3µm), longer tail (131–156µm vs 67– 102 µm), number of metastegostomal warts (3 vs 5), double-flapped epiptygma (present vs absent), and in males longer pharynx (181–210 µm vs 113–186 µm). The new species can be differentiated from Oscheius colombiana by smaller stoma in females (13–18µm vs 21–28µm) and in males (14–17µm vs 19–24µm). Males have a shorter tail (32–39µm vs 51–71µm), smaller c’ ratio (1.2–1.4 vs 2.1–3.5), higher c ratio (26.1–36.2 vs 13–16), arrangement of GP 5 (sub-ventral vs dorsal), and GP 8 (sub-dorsal vs dorsal). Oscheius indicus n. sp. different from O. nadarajani by a smaller stoma in females (13–18µm vs 21– 28µm), shorter pharynx (181–210 µm vs 256–267µm), longer tail (131–156µm vs 121–123µm) and smaller c ratio (7.5–10.4 vs 11–13). Males have a shorter stoma (14–17µm vs 19.4µm), shorter pharynx length (159– 181µm vs 243–257µm), narrow body or smaller a ratio (17.4–22.4 vs 24–28), and in number of pre-cloacal papillae (3 pairs vs 4 pairs). Due to the presence of leptoderan bursa, crochet needle-shaped spicules and ITS and 28S D2/D3 molecular marker sequences showing sufficient difference to the closely related genera, we propose the position of the new species under insectivora group. The nematode did not show any entomopathogenicity against Galleria mellonella . Any invertebrate associate for this species is not known. Type habitat and locality Collected from soil samples of fallow fields from district Cachar, Assam , India . Type specimens Holotype female on slide Oscheius indicus n. sp. / 1, and 10 paratype females and 10 paratype males on slides Oscheius indicus n. sp. /2 to 10 are deposited in the nematode collection of Department of Zoology , Aligarh Muslim University , Aligarh , India and 4 paratypes females and 4 paratype males slides of Oscheius indicus n. sp. /11 to 14 are deposited in the National Nematode Collection of India housed at the Division of Nematology , ICAR – Indian Agriculture Research Institute , New Delhi , India .