JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2019-004 e2019-04 | Vol. 51 Description of Oscheius indicus n. sp. (Rhabditidae: Nematoda) from India Puneet Kumar1,2, Wajih Jamal1, Vishal S. Somvanshi2*, Khushbu Chauhan2 and Sabia Mumtaz1 Abstract 1Department of Zoology, Aligarh A new amphimictic species Oscheius indicus n. sp. is described Muslim University, Aligarh, India. and illustrated with morphological and molecular data. The species 2 is characterized by a medium-sized and slender body (female: L = Division of Nematology, ICAR – 1.1 to 1.5 mm; a = 16.8 to 20.6; b = 5.7 to 7.1; c = 7.5 to 10.4; c’ = Indian Agricultural Research Insti- 5.0 to 7.6; V = 45 to 51%), presence of four incisures each in the tute, LBS Center, PUSA Campus, lateral fields with three minute warts, long rectum (2 to 3 anal body New Delhi, India. diameters), nine pairs of papillae arranged as 1+1+1/3+3 pattern, a *E-mail: [email protected]. prominent double-flapped epipytigma on vulval opening, presence of open leptoderan bursa and crochet needle-shaped spicules place This paper was edited by: Eyualem it in the insectivora group. Morphologically, O. indicus n. sp. closely Abebe. resembles O. carolinensis, O. chongmingensis, O. colombiana, and Received for publication September O. nadarajani. Molecular phylogenetic analysis carried out using ITS 3, 2018. and D2/D3 expansion region of 28S rDNA sequences suggests that O. indicus n. sp. is closer to O. chongmingensis and O. rugaonensis. In summary, the morphometrical data, morphological observations and molecular phylogenetic analysis suggested that O. indicus n. sp. is sufficiently different from any known species and is therefore proposed as a new species within the insectivora group. Key words Amphimictic, 28S D2/D3, ITS, New species, Oscheius, Taxonomy. Oscheius is a free-living bacteriophagous or ento- body plan into three groups, Pleiorhabditis, Synrhab- mopathogenic nematode found in saprobic biotypes ditis, and Anarhabditis. He positioned the genus Os- (detritus, dung), occasionally associated with beetles cheius under the group Synrhabditis which consisted (Lucanidae). Körner (1954) first described Rhabditis of 27 valid species, of which 13 species belonged to insectivora. Later, Andrássy (1976) erected the ge- dolichura group and 14 to insectivora group. Tabas- nus Oscheius with O. insectivorus as its type spe- sum et al. (2016) recognized 28 valid species under cies and considered Oscheius as the closest relative the genus Oscheius of insectivora group. With the of Rhabditis (Dujardin, 1845). Sudhaus (1976) placed recent descriptions of O. microvilli (Zhou et al., 2017) Oscheius under the family Rhabditidae and divid- and O. safricana (Serepa-Dlamini and Gray, 2018), at ed them into two main groups: dolichura group and present, the number of valid species under the insec- insectivora group. Diagnostic characters of both the tivora group of genus Oscheius is 30, whereas the groups are unique; species under the insectivora dolichura group comprises of 14 species. Six species group are characterized by leptoderan bursa, crochet of the insectivora group, namely, O. carolinensis (Ye needle-shaped spicules and normal rectum, whereas et al., 2010), O. niazii (Tabassum and Shahina, 2010), the dolichura group has peloderan bursa, probe head O. siddiqii (Tabassum and Shahina, 2010), O. ams- spicule tips and expandable rectum. On the basis of actae (Ali et al., 2011), O. microvilli (Zhou et al., 2017) morphological and molecular studies, Sudhaus (2011) and O. safricana (Serepa-Dlamini and Gray, 2018) and characterized the family Rhabditidae by generalized one species in the dolichura group O. onirici (Torrini © 2019 Authors. This is an Open Access article licensed under the Creative 1 Commons CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/ Description of Oscheius indicus n. sp. (Rhabditidae: Nematoda) from India et al., 2015) are considered as entomopathogenic. 800 µg/ml proteinase K) was added (Holterman et al., The genus Heterorhabditidoides was proposed as a 2006). The lysis was carried out in a thermocycler at separate genus (Zhang et al., 2008) but it was later 65°C for 3 hr with intermittent shaking followed by in- considered a junior synonym of Oscheius (Ye et al., cubation for 5 min at 100°C. Six µl of the lysate (diluted 2010). Another species, Heterorhabditidoides ru- 1: 10) was used as DNA template in PCR reaction, gaoensis, was also transferred to Oscheius (Darsouei and the surplus lysate was stored at −20°C. The ITS et al., 2014; Tabassum et al., 2016). Synonymizing region was amplified by primers TW81 (5′-GTTTCCG Heterorhabditoides with Oscheius has been widely TAGGTGAACCTGC-3′) (Joyce et al., 1994) and AB28 accepted in the field (Liu et al., 2012; Campos-Her- (5′-ATATGCTTAAGTTCAGCGGGT-3′) (Howlett et al., rera et al., 2015; Torrini et al., 2015; Tabassum et al., 1992); and D2 F- 5′ ACAAGTACCGTGAGGGAAA 2016; Valizadeh et al., 2017). A total of 44 species of GTTG-3′ and D3 R- 5′-TCGGAAGGAACCAGCTAC Oscheius have been described till date, out of which TA-3′ were used for D2/D3 expansion region of 28s 16 are from the Indian sub-continent. Here we de- rDNA gene (Nunn, 1992). The amplification of near scribe the 45th species of the genus, Oscheius indi- full length 18s rDNA gene was attempted by prim- cus n. sp. ers SSU_F_07 (AAAGATTAAGCCATGCATG) and SSU_R_81 (TGATCCWKCYGCAGGTTCAC) (Gutiér- rez-Gutiérrez et al., 2012). Each PCR reaction com- Materials and methods prised of 1 unit of Platinum Taq polymerase (Invitro- The nematodes were extracted from soil samples gen Carlsbad, CA, USA), 1x Taq polymerase buffer, 0.2 mM dNTPs, 1.5 mM MgCl , 0.4 µM forward and collected from district Cachar, Assam, India by mod- 2 ified decanting and sieving and modified Baermann’s reverse primers and 6 µl of template DNA. The ther- funnel techniques (Flegg, 1967). Extracted nema- mocycling conditions for ITS were – Initial denatura- todes were cultured on standard NGM plates seeded tion at 95°C for 5 min, followed by 40 cycles of 95°C with Escherichia coli OP50 strain as a food source for 45 sec, 55°C for 30 sec, and 72°C for 1.30 min, as used for Caenorhabditis elegans cultures (Wood, and a final elongation at 72°C for 10 mins. For 28S 1988). Stock cultures were maintained from single D2/D3, initial denaturation was 95°C for 5 min, fol- gravid female. The life cycle was completed in six lowed by 40 cycles of 95°C for 30 sec, 60°C for days at 28°C. Nematodes were rinsed off the plates 45 sec, and 72°C for 45 sec and a final elongation of using distilled water. The nematode entomopatho- 72°C for 10 min. The 18s rDNA was amplified as per genicity toward the greater wax moth, Galleria mel- (Gutiérrez-Gutiérrez et al., 2012); one cycle of 94°C for lonella was evaluated by using fourth stage larvae of 2 min, followed by 35 cycles of 94°C for 30 s, anneal- the greater wax moth. For light microscopy, extracted ing temperature of 57°C for 45 s, 72°C for 3 min, and nematodes were killed and fixed in hot FG (Hooper, finally one cycle of 72°C for 10 min. The PCR ampli- 1970) for 24 hr and then transferred to glycerin-alco- fied products were checked on 1% agarose gel and hol (Seinhorst, 1959) for slow dehydration in a desic- eluted from the gel by using Qiagen MinElute Gel cator. Dehydrated specimens were mounted in anhy- extraction kit (Qiagen USA, Catalogue No. 28606). drous glycerin on glass slides using wax ring method The purified products were cloned using the pGEMT- (De Maeseneer and d’Herde, 1963). All observations, easy vector system I (Promega USA, Catalogue No. drawing, and photographs were made on an Olym- A1360), which was transformed into DH5α chemically pus BX 50 DIC microscope. For scanning electron competent cells using standard protocols. Positive microscopy, specimens were fixed in 3% glutaralde- colonies were screened by blue-white screening, hyde in 0.05 M phosphate buffer (Kiontke et al., 2001) confirmed by PCR amplification using gene-specific for 48 hr, washed in 0.05 M sodium phosphate buffer primers and sent for sequencing by using forward (pH = 6.9) several times, dehydrated in a graded etha- and reverse M13 primers. The forward and reverse nol series (30, 50, 70, 80, 90, 95, and 100%) for 20 to sequences were manually edited for quality, aligned 25 min each, and critical-point dried in carbon diox- using ClustalW, and a consensus sequence was gen- ide. Dried specimens were mounted on stubs, coated erated using BioEdit v.7.2.6 (Tom Hall, NCSU, USA). with 20 nm gold and observed in a scanning electron The phylogenetic analysis of the ITS and 28S D2/D3 microscope (Joel JSM-6510) at 15 KV. sequences were carried out as described (Ye et al., For characterization of the ITS and 28S D2/D3 2018). In brief, the new and previously published se- sequences for molecular taxonomy, a single nema- quences for each gene were aligned using ClustalW tode was taken in 25 µl of sterile water in a 0.2 ml PCR in MEGAX (Kumar et al., 2018) with default param- tube to which 25 µl of lysis buffer (0.2 M NaCl + 0.2 M eters. The alignments were subjected to Bayesian Tris-HCl (pH 8.0) + 1% (v/v) β -mercaptoethanol + Inference (BI) analysis using MrBayes 3.2.6 (Ronquist 2 JOURNAL OF NEMATOLOGY Figure 1: Oscheius indicus n. sp. (A) Entire male; (B) Entire female; (C) Pharyngeal region; (D) Anterior region; (E) Female reproductive system; (F) Male posterior region showing spicules and gubernaculum; (G) Male tail in dorso-ventral view (genital papillae and bursa); (H) Female posterior region.