Morphological, morphometrical, and molecular characterization of Metarhabditis amsactae (Ali, Pervez, Andrabi, Sharma and Verma, 2011) Sudhaus, 2011 (Rhabditida, Rhabditidae) from India and proposal of Metarhabditis longicaudata as a junior synonym of M. amsactae

Abstract A new population of Metarhabditis amsactae from India is morphologically, morphometrically, and molecularly characterized. This material is characterized by having 0.65 to 1.14 mm length, lips rounded, and grouped in pairs, stoma with metastegostoma bearing setose denticles, pharynx with metacorpus slightly swollen and fusiform, nerve ring, and excretory pore located at isthmus level, female reproductive system didelphic-amphidelphic with vulva equatorial, female tail conical-elongate with acute tip, male tail conical with large and robust posterior filiform part, spicules free with hooked manubrium slightly bent ventrad, gubernaculum with narrow corpus, bursa open leptoderan with eight genital papillae and phasmids posterior to the GP8. Molecular studies based on 18S and 28S rDNA genes are provided for the first time for the species. In addition, integrated morphological, morphometrical, and molecular characters are compared with other previous records of the species. According to our analysis, Metarhabditis longicaudata and other material described as different species are proposed as new junior synonyms of M. amsactae.


Nematode sampling
A survey to obtain the nematodes was conducted in soils of the district Shamli (29.6189° N, 77.4329° E; 280 m altitude), Uttar Pradesh, India. This location has a semiarid and moderate-to-tropical monsoon (humid subtropical) predominant climate. The type of soil is sandy loam and loamy and the pH of soil samples ranged from 6.5 to 8.4. A total of eighty-nine soil samples were taken from meadows, pastures, agricultural fields, open fields, and orchards.
Each soil sample consisted of 1 kg of soil that was a mixture of five soil subsamples collected at 15 to 20 cm depth in five locations within each field (one sample from each corner of the field, and one from the center of the field). The soil was first made fine to remove any debris (i.e., rocks, pieces of wood or bark, leaves, etc.) and then moistened with distilled water using a spray bottle to facilitate the movement of nematodes. To recover insectassociated nematodes from these soil samples, the 'Galleria mellonella baiting' method and the White (1927) trap method modified (Bedding and Akhurst, 1975) were used. Seven 4th instar Galleria mellonella larvae were buried in 250 ml of autoclaved plastic containers filled with the collected soil up to the brim. The plastic containers were then covered with tissue paper and muslin cloth. The containers were inverted upside-down and stored in the dark in an incubator at 27 ± 2°C for 7 days. The plastic containers were checked daily to recover dead insect larvae. Insect cadavers were rinsed with double-distilled water (ddH 2 O) to remove soil particles and disinfected with 0.1% sodium hypochlorite before being placed on the modified White traps to obtain emerging infective juveniles. The White traps were incubated at 27 ± 2°C in an incubator and checked daily for the emergence of third-stage juveniles from the cadavers. Emerged third-stage juveniles migrate after 5 to 7 days to water surrounding the Petri dish and nematodes were collected regularly until nematode emergence ceased after 10 to 20 days (Bhat et al., 2018. Emerged IJs were sterilized with 0.1% sodium hypochlorite and washed with ddH 2 O, and finally stored in tissue culture flasks at 15°C. Thirdstage juveniles were used within seven days after emergence Bhat et al., 2020a).

Nematode morphology and morphometry
Nematode third-stage juveniles were surfacesterilized with 1% NaOCl. Then, greater wax moth (Galleria mellonella) larvae were injected with 100 juvenile nematodes in sterile Petri plates using a 1 ml of insulin syringe. The male, female, and juvenile (third-stage) nematode generation were recovered from White traps as described above. All nematode generations were heat-killed in Ringer's solution and fixed in triethanolamine formalin (Courtney et al., 1955). Nematodes were infiltrated in glycerol by the Seinhorst method (Seinhorst, 1959) and processed further as described by Bhat et al. (2017). Briefly, the nematodes were kept in pure glycerol. Three females, specimens of five males, and 10 infective juvenile nematodes were mounted separately in a drop of glycerol on a clean glass slide. Paraffin wax was used to seal and to prevent the flattening of nematode specimens (Bhat et al. 2020b;Kajol et al., 2020). The morphology and morphometric analysis of the specimens was conducted using light compound microscope (Magnus MLX) and phase-contrast microscope (Nikon Eclipse 50i). Twenty specimens of adults (male and female) and 20 of juveniles were analyzed. Morphometric analyses were carried out with the aid of in-built software of the phase-contrast microscope (Nikon DS-L1). The best-preserved specimens were also photographed using a Nikon Eclipse 80i (Nikon, Tokyo, Japan) light microscope provided with differential interference contrast optics (DIC) and a Nikon Digital Sight DS-U1 camera. Micrographs were edited using Adobe® Photoshop® CS. Nematode species were identified based on morphological and morphometric characters using the key provided by Abolafia and Peña-Santiago (2019a). Demanian indices (de Man, 1881) and other ratios were calculated. The terminology used for the morphology of the stoma and spicules/gubernaculum follows the proposals by De Ley et al. (1995) and Abolafia and Peña-Santiago (2017), respectively.

Scanning electron microscopy (SEM)
For the SEM, male and female specimens preserved in glycerin were selected for observation and processed according to the  protocol. Thus, they were hydrated in distilled water, dehydrated in a graded mixture of ethanol-acetone series, critical point-dried with liquid carbon dioxide, and coated with gold. The mounts were examined with a Zeiss Merlin microscope (5 kV).

Sequence alignment and phylogenetic analyses
The sequences were edited and compared with those already present in GenBank using the Basic Local Alignment Search Tool (BLASTN) of the National Center for Biotechnology Information (NCBI) (Altschul et al., 1990). The newly obtained ribosomal LSU (D2/ D3 rDNA), SSU (18S rDNA), and ITS (ITS1, 5.8S, ITS2) rDNA sequences were manually edited using BioEdit 7.2.6 (Hall, 1999) and aligned with other relevant segments of same rDNA gene sequences available in GenBank using Clustal W alignment in the program MEGA7 (Kumar et al., 2016). Poorly aligned regions were removed from the alignments using MEGA7. The base substitution model was evaluated using jModeltest2.1.10 (Darriba et al., 2012). Phylogenetic trees were elaborated using the Bayesian inference method as implemented in the program MrBayes 3.2.7 (Ronquist et al., 2012). For analysis in jModeltest, the HKY + I + G model was selected for the ITS tree, the GTR + I + G model was selected for the 18S tree, and the GTR + G was selected for the 28S tree. The selected models were initiated with a random starting tree and ran with the Markov chain Monte Carlo (MCMC) for 1 × 10 6 generations. The Bayesian tree was ultimately visualized using the FigTree program 1.4.4 (Rambaut, 2018). Heterorhabditis downesi (KU573061) was used as the outgroup and to root the trees for ITS1-5.8S-ITS2 rDNA tree, Myolaimus byersi (KU180676) for LSU rDNA tree, and M. byersi (KU180665) for SSU rDNA tree.
The details of all the nematode species used in the molecular and phylogenetic study, including their updated nomenclature, accession numbers of rDNA genes, isolation source, and origin of the sequences are given in Table 4.

Insect-associated nematode isolation
Nematodes of four genera: Metarhabditis, Steiner nema, Heterorhabditis, and Oscheius were recovered from the eighty-nine soil samples collected in this study. Two soil samples, taken around the rhizosphere of sugarcane (Saccharum officinarum L.) and groundnut (Arachis hypogaea L.), contained Metarhabditis nematodes. Five samples were found positive for the presence of Steinernema abbasi, two for the presence of Heterorhabditis indica, and two for the presence of Oscheius sp. The rest of the samples were found negative for the presence of insect-associated nematodes. In this study, we characterized Metarhabditis nematodes. Steinernema, Heterorhabditis, and Oscheius nematodes are characterized in other studies (Bhat et al., 2020c, Rana et al., 2020b.

Description
Adult: Body 0.72 to 1.14 mm long in females and 0.65 to 1.00 mm long in males, mostly straight rarely arcuate upon gentle heat killing with tapering to the anterior and posterior ends, more tapering toward the posterior end. The cuticle striated with scarcely prominent annuli 1.0 to 1.5 µm wide varying with body regions. Lateral fields were indistinct under light microscopy; however, four longitudinal lines are visible under scanning electron microscopy. The Lip region was almost continuous from contiguous body. Lips rounded and swollen, organized in doublets forming three pairs (one dorsal and two subventral) around the triradial oral orifice. Amphids small, oval, positioned at the base of lateral lips. Stoma rhabditoid type, 1.5 to 3.4 times the lip region width in length, with stomatal tube (gymno-promesostegostom) well developed. Cheilostom short with poorly refringent rhabdia; gymnostom tubular with cuticularized rhabdia, shorter than promesostegostom, this later surrounded by a thin pharyngeal collar; metastegostom isomorphic and isotopic having glottoid apparatus with three valves bearing two setose denticles per valve; telostegostom with minute rounded rhabdia. Pharynx rhabditoid, differentiated into cylindershaped pharyngeal corpus, 0.9 to 2.0 times the isthmus length, metacorpus slightly swollen, fusiform, isthmus relatively thick, weakly narrowing until its junction with the basal bulb, this more or less rounded, occasionally pyriform, with a weak to moderately developed valvular apparatus and faintly double-chambered haustrulum. Nerve ring surrounding the pharynx at the level of isthmus, 83 to 89% of neck length. Secretory-excretory pore at 79 to 86% of neck length, variable in position ranging from middle of isthmus to closely anterior to basal bulb. Deirids and hemizonid poorly visible, posterior to excretory pore, at 84 to 96% of neck length, at level of isthmus. Cardia small, conoid, surrounded by intestinal tissue. Intestinal lumen wider and dilated posterior to the basal bulb. Female: Reproductive system didelphic-amphidelphic, the anterior and the posterior branches in sinistral and dextral position to intestine, respectively. Ovaries moderately developed, dorsally reflexed but with distal end not reaching to vulval level, anterior ovary slightly larger. Usually one or two small rounded pseudocoelomocytes observed in close proximity to the proximal end of ovaries. Ovi ducts proximally enlarged, connected to ovoid spermatheca frequently filled with sperm. Uteri well developed, differentiated into long glandular and muscular regions, filled with sperm and one to ten intrauterine eggs, 40−49 × 22−23 µm, in different stages of embryonation. Vagina thick-walled, often cuticularized, at right angle to the longitudinal body axis, with length equal to about one-third of the vulval body diameter. Vulva a wide transverse slit, with protruding lips, unremarkable or weak epiptygma but distinct cuticular flap. Rectum short, 1.0 to 1.8 times anal body diameter, allied with rectal glands at its junction with prerectum. Prerectum distinguishable from intestine in lacking prominent cell nuclei. Tail elongate conoid, gradually tapering to a fine terminus. Phasmids short tubular, located posterior to anus, about 38 to 42% of tail length.
Male: Similar to female in general morphology except for smaller size, posterior body curvature prominent and cuticular striations relatively fine. Reproductive system monorchic, with single testis reflexed ventrad anteriorly on the right side of the  intestine. Vas deferens a broad tube, packed with sperm without delineation of seminal vesicles. Ejaculatory glands not observed. Spicules paired and symmetrical, ventrally arcuate, free with slightly bent ventrad manubrium, ventrally hooked, calamus short conoid and slightly ventrally curved lamina with ventrally bent finely rounded tip in lateral view. Gubernaculum well-developed, slightly ventrad cur ved with long manubrium and narrow corpus, 50 to 60% of spicule length. Three small gland-like cells are distinguishable around the anterior end of the cloaca. Tail conoid with posterior two-thirds abruptly tapering and reduced. Bursa anteriorly open, narrow, leptoderan, not enclosing large tail spike, having smooth margins and eight pairs (1 + 1 + 1/3 + 2 + ph) of genital papillae, with GP1 and GP2 spaced, precloacal, GP3 slightly posterior to cloaca in most specimens, pairs GP4 to GP6 located at conoid part of tail and GP7 to GP8 located at posterior part of the bursa, dorsally directed. Phasmid small, tubular, located posterior to the GP8, at 45 to 50% of tail length.
Comparing with other sequences (unpublished) of the species available from GenBank, the 18S rDNA fragment, from a common aligned fragments with 794 bp, the present populations from India shows one (0.1%) change from the other sequence available from India (NM453373), 1 (0.1%) and 16 (2.0%) changes from the sequences submitted from Philippines (MT012150 and MT043860), respectively. For the 28S rDNA fragment, there are no other available sequences to compare. The ITS rDNA sequences, from a common aligned fragments with 645 bp, show 3 (0.5%) or 6 (0.9%) changes from other sequences submitted from India (KP834432/KP834433/ KY083045 and MH392568), respectively; 1 (0.2%) or 2 (0.3%) changes with respect to two sequences from Philippines (MT422254 and MT576957), while other two sequences (MT452472 and MT576957) deposited from Philippines show too much changes (51 and 64, respectively); the sequence submitted from Pakistan (MK973071) show 24 (3.7%) changes, the most of them consistent in two long contiguous deletions in the Pakistani sequence (10 and 12 gaps, respectively, after aligning sequences), which must be considered as M. amsactae.

Voucher material
Twenty females and twenty males of each isolate were deposited at the museum of the Department of Zoology, Chaudhary Charan Singh University Meerut, India. Ten females and ten males were deposited at the nematode collection of the Department of Animal Biology, Plant Biology and Ecology of the University of Jaén, Spain.

Remarks
The material examined in this study agrees well with the type material described by Ali et al. (2011)  Morphologically, the present material does not show important morphological differences with previous described populations. With respect to other populations described from different geographical regions of India (Shaheen et al., 2011;Pervez et al., 2012 as Oscheius ciceri and O. hussaini;Asif et al., 2013 as Oscheius gingeri), the material examined now shows close similitude to each other with only variations in body length, pharyngeal corpus, and isthmus length in adult generations (see Tables 2 and  3). The variation in morphometry in the present Indian population compared with the other populations can be attributed to differences in their geographical origin.
Recently, Tabassum et al. (2019) described a new species, Metarhabditis longicaudata Tabassum, Salma and Nasir, 2019 from Pakistan. According to its morphology, especially males with posterior filiform part well developed, robust, and bursa posteriorly appearing parallel along it at its proximal part (unfortunately, the LM Fig. 2C, D provided by these authors seems to be strongly stretched making the stoma too much long and narrow, and not agreeing with the line drawing Fig. 1C, D provided by these same authors), and morphometric characteristics (Tables 1 and 2), the specimens described are highly similar to M. amsactae. Given these considerations, we propose that Metarhabditis longicaudata is a junior synonym of Metarhabditis amsactae. Moreover, the specimens described as M. amsactae in the same study by these Pakistani authors do not present characteristics of this species, especially because the males lack posterior filiform part of the tail, spicules lack ventral bent or hooked manubrium, and females have a long rectum. In addition, the nematode population described as M. rainai (Carta and Osbrink, 2005) Sudhaus, 2011 in the same study by Tabassum et al. (2019) are morphologically very similar to M. amsactae nematodes (Tables 1 and 2) and, hence they were misidentified. ITS-phylogenetic trees support these conclusions as the sequences submitted to GenBank by these authors, Metarhabditis sp. (MK973071), cluster together with other M. amsactae (Fig. 4).
According to this, the updated list of synonyms of Metarhabditis amsactae is as follows: Metarhabditis amsactae (

Phylogenetic relationships
The phylogenetic relationships as inferred from the Bayesian Inference analysis between Metarhabditis amsactae and other closely related are provided based on ITS- (Fig. 4), 18S- (Fig. 5), and 28S- (Fig. 6) rDNA fragments. Based on the three phylogenetic trees, M. blumi (Sudhaus, 1974) Sudhaus, 2011and M. rainai (Carta and Osbrink, 2005 Sudhaus, 2011 are sister species of M. amsactae. The phylogenetic tree inferred using 18S rDNA gene sequences, shows three clusters that contain sequences of nematodes that have been suggested to belong to Metarhabditis (Fig. 5). One cluster is composed of M. rainai (AF083008, JQ237848, MT012133, MT012135 and MT012153) and one nematode isolate that might have been misidentified as Rhabditis sp (MN082353) but could correspond to M. rainai. A second cluster composed of M. amsactae (MT872504, MT872503) and three nematode isolates that might have been misidentified as M. blumi (MT043860, MT012150, MN453373). A third cluster composed of M. blumi (MF989442, U13935), and Rhabditis sp. (MN082355). As M. amsactae isolates that correspond to accession numbers MT872504 and MT872503, and the M. blumi isolate that correspond to accession numbers U13935 have been morphologically and molecularly characterized, we conclude that nematodes isolates with NCBI accessions MT043860, MT012150 and MN453373 Finally, analyzing ITS rRNA gene sequences, we arrive to the same conclusions derived from the analysis of 28S-and 18S rDNA gene sequences.
ITS-based phylogenetic tree show a clear cluster that separates M. amsactae and M. blumi (Fig. 4). Unfortunately, there are no available M. rainai sequences. Sequence comparisons show that a common ITS rDNA fragment of 802 bp in length of M. amsactae (MT873043-4) and of M. blumi (DQ121436) differ in 496 changes, which again support the status of the nematode isolates of this study as M. amsactae (Table 4).   Dr. Aashaq Hussain Bhat is thankful to the Department of Science and Technology (DST), New Delhi, India, for providing financial assistance through a DST INSPIRE Fellowship/2014/76. The authors thank the University of Jaén, Spain, for financial support received for the Research Support Plan 'PAIUJA 2019/2020: EI_RNM02_2019'. SEM pictures were obtained with the assistance of technical staff (Amparo Martínez-Morales) and equipment of the 'Centro de Instrumentación Científico-Técnica (CICT)' at the University of Jaén. The work of RARM is supported by the Swiss National Science Foundation (PZ00P3_186094). Aasha Rana is thankful to DST, New Delhi India for providing financial assistance through DST WOS-A (SR/WOS-a/LS-1083/2014).