Morphological and molecular characterization of Acrobeloides saeedi Siddiqi, De Ley and Khan, 1992 (Rhabditida, Cephalobidae) from India and comments on its status

Abstract Two cultured populations of Acrobeloides saeedi are described from India. Morphologically and morphometrically this material agrees with other species of the Maximus-group (A. bodenheimeri, A. longiuterus, and A. maximus), especially with A. longiuterus. However, molecular studies based on 18 S, 28 S and ITS rDNA confirmed the Indian material is well differentiated from all of these species. According to this, A. saeedi is considered a valid taxon distinguished mainly from A. bodenheimeri by having dextral female reproductive system (vs sinistral), from A. longiuterus by having larger females (1.03-1.57 vs 0.57-0.88 mm) and from A. maximus by having seta-like labial processes (vs absent) and males as frequent as females (vs males very infrequent). Molecular and phylogenetic studies revealed the present specimens to be conspecific to undescribed Acrobeloides sp. population from Iran, and hence, both regarded to be conspecific to each other. In addition, other similar species are revised: Acrobeloides ishraqi is considered new junior synonym of A. saeedi, Acrobeloides mushtaqi is considered new junior synonym of A. bodenheimeri, while Acrobeloides gossypia is also considered junior synonym of A. saeedi.

Acrobeloides saeedi was described by Siddiqi et al. (1992) to erect the material previously described as Cephalobus litoralis (Akhtar, 1962;Andrássy, 1984) from Pakistan by Saeed et al. (1988). This last material, based only in two females was observed having morphology and morphometry somewhat different (Siddiqi et al., op. cit.) with respect to the type material of Paracephalobus litoralis described by Akhtar (1962) from Pakistan. Later, Khan and Hussain (1997) proposed the new genus Rafiqius to include A. saeedi and other morphological related species as A. bodenheimeri (Steiner, 1936;Thorne, 1937). This newly proposed genus was differentiated from Acrobeloides (Cobb, 1924) according to the morphology of the lip region, having seta-like processes at labial primary axils. However, the creation of this new genus was considered unjustified by De Ley et al. (1999). Unfortunately, none of these studies provided molecular study.
With respect to the isolation of soil nematodes using the Galleria soil baiting technique of Bedding and Akhurst (1975), the insect associate nature of some Acrobeloides species has been previously reported (Azizoglu et al., 2016). Besides their insect associate nature, their infestation has also been observed with some mollusks, arthropods, and annelids (Grewal et al., 2003). Kraglund and Ekelund (2002) reported infestation of A. nanus (de Man, 1880;Anderson, 1968) in earthworm cocoons. Baquiran et al. (2013) studied the association of these nematodes with microbes and repeatedly observed the presence of three bacterial species in association with A. maximus (Thorne, 1925(Thorne, , 1937. Later, Thiruchchelvan et al. (2018) found a free-living nematode similar to A. longiuterus (Rashid and Heyns, 1990;Siddiqi et al., 1992) in Sri Lanka infecting crop pests. Additionally, Suman et al. (2020) collected other rhabditid species, Distolabrellus veechi Anderson, 1983, from soil samples using the insect baiting technique. Their involvement in soil nutrient cycle and soil mineralization is well evident and during these processes, they interact with many arthropods and other invertebrate species, which may be phoretic to pathogenic, thus may be important for their use in biological control programs.
During a survey of soil nematodes in Meerut, Uttar Pradesh, India, two isolates of Acrobeloides were obtained and were labeled as KMW and DH1. Study of the specimens of these two populations showed that they were conspecific to A. saeedi (Siddiqi et al., 1992). Detailed redescription of this species based in morphological and morphometrical data is provided. We also provided a high quality photographic documentation of important morpho logical characters of A. saeedi through light micro scopy (LM) and scanning electron microscopy (SEM). Additionally, molecular data of this species based in the D2-D3 region of the 28 S rDNA, 18 S rDNA, and internal transcribed spacer (ITS) regions of rDNA genes are included to support the morpho-taxometrical studies. This is the first molecular study of this species and its first valid report from India.

Materials and methods
Nematode isolation, culture, and processing Soil samples were collected from agricultural farmlands in Mawana, Meerut (28°9´N, 77°71´E, and elevation of 225 m), India, and were tested for the presence of nematodes. Nematode specimens were isolated from two soil samples by Galleria soil baiting technique and were designated as DH1 and KMW. The cadavers were transferred to white trap (White, 1927) after proper washing with double distilled water and sterilization with 1% NaOCl. The nematodes that emerge in white trap were harvested, and stored in 250 ml tissue culture flasks in incubator at 15°C as described by Bhat et al. (2019). For observations and morphometrics, thirdstage juveniles (200) were injected to larvae of Galleria mellonella by Insulin Syringe 1 ml and larvae were killed within 36 hr at 27°C. The dead larvae were then transferred to white trap. The adult generations and third-stage juveniles were collected from white trap which emerge into water within six to seven days. These specimens were then killed with hot water, transferred to TAF (2% triethanolamine and 7% formaldehyde) for fixation. The fixed nematodes were processed to dehydrated glycerine as described by Seinhorst (1959) and mounted in pure glycerine on permanent glassslides (Siddiqi, 1964).

Light microscopy (LM)
Nematode specimens were observed for morphological characters under phase contrast microscope (Nikon Eclipse 50i) and light microscope (Magnus MLX) while morphometric characters were measured with built-in software of the Nikon Eclipse 50i (Nikon DS-L1). Demanian indices (de Man, 1880) and other morphometrical ratios were calculated. Line drawings were made with the help of drawing tube attached to the Nikon microscope provided with differential interference contrast (DIC) optics. Images were taken with the Nikon microscope that was provided with DIC optics and Nikon Digital Sight DS-U1 camera. Micrographs were edited using Adobe® Photoshop® CS. The terminology used for the morphology of stoma and spicules follows the proposals by De Ley et al. (1995) and Abolafia and Peña-Santiago (2017a), respectively.

Scanning electron microscopy (SEM)
For the SEM, male and female generations were first fixed in TAF and then preserved in glycerine. Glycerine preserved specimens were used for SEM observations according to the Abolafia's (2015) proctocol. They were hydrated in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried with liquid CO 2 , mounted on SEM stubs and finally coated with gold. The mounts were examined with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany).

Phylogenetic analyses
The sequences were edited and compared with those already present in GenBank using the basic local alignment search tool (BLAST) of the National Centre for Biotechnology Information (NCBI) (Altschul et al., 1990). An alignment of nematode samples together with sequences of related cephalobid species was produced for the LSU (D2-D3 rDNA), SSU, and ITS rDNA sequences using default Clustal W parameters in MEGA 6.0 (Kumar et al., 2016) and optimized manually in BioEdit (Hall, 1999). Pairwise distances were computed using MEGA 6.0 (Kumar et al., 2016). All characters were treated as equally weighted and gaps as missing data. Drilocephalobus sp. (AY284679) for the 18 S tree and Teratolobus sp. (KJ652552) for the 28 S tree were used as the out-group taxa and to root the trees. ITS tree was not included because too few sequences are available in the GenBank database for their comparisons. The base substitution model was evaluated using jModeltest 0.1.1 (Posada, 2008). Phylogenetic trees were elaborated using the Bayesian inference method as implemented in the program MrBayes 3.2.7 (Ronquist et al., 2012). The HKY + Γ (gamma distribution of rate variation with a proportion of invariable sites) model was selected. The selected model was initiated with a random starting tree and run with the Markov Chain Monte Carlo for 10 6 generations. The Bayesian tree was ultimately visualized using the FigTree program 1.4.3 (Rambaut, 2018).

Results and discussion
The morphological and morphometrical studies and molecular (D2-D3, 18 S and ITS rDNA) analyses confirmed the present strains KMW and DH1 as conspecific to A. saeedi (Siddiqi et al., 1992) and hence, described as the same. This is the first report of this species from Indian subcontinent.
Measurements: see Tables 1 and 2. Female: Body is larger, 1.31 to 1.57 mm long, in the KMW population and smaller, 1.06 to 1.45 mm, in the DH1 population, more or less fusiform with a sudden narrowing behind the vulva, tapering anteriorly from mid-pharynx to lip region, fusiform, slightly arcuated ventrally and becomes open C shaped upon heat killing. Cuticle with annuli separated from each other by a narrow groove. Lateral fields with four alae limited by five longitudinal incisures ending at tail tip terminus, showing only three incisures after the phasmids. Lip region bears six inner labial papillae and four outer cephalic papillae. Lips are in pairs, with smooth margin; primary axils are "U"-shaped, usually with acute tip; secondary axils are "V"-shaped; guard processes are absent. Labial probolae is low, triangular in section, connected by tangential ridges. Amphidial apertures are pore like, oval. Oral opening is triangular leading into a narrow cephaloboid stoma bearing well-developed refringent rhabdia, cheilostom is short with bar-shaped cheilorhabdia, gymnostom is very short and stegostom is elongated with robust rhabdia. Pharynx is cephaloboid, divided in three regions: pharyngeal corpus is slightly fusiform, 2.7 to 3.1 times the isthmus length in KMW population while 3.7 to 5.4 times in case of DH1; isthmus is robust and basal bulb is spheroid with well-developed valvular apparatus. Excretory pore is located at isthmus level, at 60 to 89% of neck length, at 53 annuli; renette cells are just behind pharyngeal bulb. Hemizonid is present just anterior to the excretory pore. Deirids are present at basal bulb level, at 70 to 92% of neck length, at 48 annuli. Nerve ring surrounds the isthmus at metacorpus-isthmus junction or slightly posterior.
Intestine with anterior end with thinner walls. Reproductive system is monodelphic, prodelphic: ovary well developed, with several lines of oocytes, with or without a double flexure at postvulval region; oviduct short; spermatheca well developed, 0.4 to 0.5 times longer than the body width; uterus is very long, divided in two parts only observed in young females, one distal tubular part and other proximal swollen part with thinner walls; in old females all length usually swollen containing 16 to 30 uterine eggs, 41 to 55 µm long and 24 to 35 µm wide; post-vulval uterine sac 0.7 to 0.9 times the body width; vagina is straight or slightly arcuate, 21 to 31% of body width; vulva ventral. Rectum is distinct, shorter than anal body width with three unicellular glands at its junction with the intestine. Anus is large, directed posteriorly. Tail is straight, conoid, truncated to slightly rounded terminus with 15 to 20 annuli ventrally. Phasmids are distinct pore like and located at 59 to 62% of tail length.
Male: Body is 0.81 to 1.16 mm long in the KMW population, and 0.80 to 1.14 mm long in the DH1 population, "J" shaped after heat killing with general morphology similar to female. Reproductive system is monorchic with testis ventrally reflexed anteriorly. Two deep latero-subventral grooves are extended from the sides of the cloacal apparatus approximately to the first preanal pair of the papillae. Genital papillae are in eight pairs, three pairs are pre-cloacal and five pairs are post-cloacal (two at mid tail length, one lateral at lateral field and one subventral, and three terminal, two subventral and one subdorsal), and one midventral papillae. Phasmids are well observed, located posterior to the anterior lateral papillae, at 67 to 70% of tail length. Spicules are long, broad and arcuate, larger than gubernaculum, with manubrium reduced, ventrally bent, rounded-elongate, calamus is conoid and lamina is slightly ventral curved with angular dorsal hump, long ventral velum and very thin  Lip region width 8.9 ± 0.8 (8-11) 6.3 ± 0.7 (5-8) 5.0 ± 0.5 (4-5) Stoma length 12.9 ± 2.2 (9-15) 14.6 ± 1.9 (11-17) 12 ± 1.6 (7-14) Pharyngeal corpus length 108 ± 8.  Notes: All measurements are in µm (except n, ratio, and percentage) and in the form: mean ± SD (range). -= character absent, ? = character not observed.

Diagnosis (of Indian populations)
The material examined of A. saeedi from India is characterized by having 1.06 to 1.57 mm in females and 0.80 to 1.16 mm in males, lateral field with five longitudinal incisures, lip region with six paired lips, smooth, primary and secondary axils lacking guard processes, labial probolae low, triangular in section and frontally flattened, stoma cephaloboid with rounded cheilorhabdia, pharynx cephaloboid with slightly swollen metacorpus, female reproductive system monodelphic-prodelphic, dextral, with spermatheca well developed and postvulval uterine sac slightly shorter than the body diam., female rectum shorter than anal body diam., female tail conoid with truncate to slightly rounded terminus
From A. maximus, Indian strains (KMW and DH1) can be distinguished by having lips lacking seta-like processes (vs bearing seta-like process at primary axils), pharyngeal metacorpus slightly fusiform (vs fusiform in Thorne (1925) but not well appreciated in Steiner (1936)), lateral field with five incisures (vs three according Smythe and Nadler (2006), being unknown in Thorne (1925) and Steiner (1936)), males as frequent as females (vs male rare or absent, presumably parthenogenetic females ), female tail terminus truncate (vs finely rounded). Although the size of the females of the Indian populations of A. saeedi are similar to A. maximus (1.31-1.57 (1.2-1.4) vs 1.2 mm) but they differed in Demanian indices.

Molecular characterization and its taxonomical implications
A. saeedi strains DH1 and KMW were molecularly characterized by ITS rDNA (901 bp, 938 bp), 18 S rDNA (894 bp, 895 bp) and flanking regions D2-D3 of rDNA (984 bp, 997 bp), respectively. The NBlast analysis of D2-D3, 18 S and ITS rDNA sequences of present specimens showed 100% similarity with D2-D3 (KY914573), 18 S (KY090631) and ITS (KY090632) rDNA sequences of Acrobeloides sp. ES-2017 isolate SMF3 from Iran. 18 S sequences of the present two strains do not show any nucleotide difference with each other and with Acrobeloides sp. ES-2017 present in the GenBank. ITS and D2-D3 sequences of DH1 do not show any nucleotide difference with Acrobeloides sp. ES-2017 (KY090632), however, together these regions show two and one nucleotide differences with KMW, respectively. According to this, the Acrobeloides material from Iran could be considered conspecific with A. saeedi.
On the other hand, A. saeedi was considered a probable junior synonym of A. maximus by De Ley et al. (1999) based on morphological data. However, the 18 S sequence alignment of present strains DH1 and KMW showed 21 bp differences with A. maximus (JQ237850), while 28 S sequence alignment showed 51 bp differences and three gaps with A. maximus (AF147067). ITS sequences of A. maximus are lacking. This shows that both species are not conspecific.
On the other hand also, A. saeedi displays some similar morphology with A. longiuterus, two almost undistinguished taxa. However, molecularly both Table 3. Comparative morphometrics of females from populations of Acrobeloides maximus -group (all measurements in µm except L in mm).
Distance matrix analyses with other closely related populations of several Acrobeloides species were also carried out using above three genes studies. Thus, the 18 S rDNA sequences of DH1 and KMW are separated from those of other closely related species of Acrobeloides by 9 to 89 bp ( Table 5). The D2-D3 segment of 28 S rDNA gene in the Indian isolates differed in 5 to 76 bp from other closely related species of Acrobeloides ( Table 6).
All of these data showed that A. saeedi is molecularly different with respect to its more similar species, A. bodenheimeri, A. longiuterus, and A. maximus, and hence, it should be considered as valid species.

Phylogenetic analysis
The phylogenetic analyses of the present stains based on 18 S rDNA and flanking region D2-D3 segment of 28 S rDNA gene also supported the molecular data. Phylogenetic analyses based on 18 S rDNA sequences (Fig. 5) showed a clear monophyly Table 6. Pairwise distances of the D2D3 regions of 28 S rDNA regions between present strains of Acrobeloides and already described species.   of the group formed by the isolates DH1 and KMW and other undescribed Acrobeloides species ES-2017 from Iran, probably conspecific isolates within a highly supported (100%) clade and together formed a sister clade with other species of "maximus" group from different geographical regions, namely A. maximus and A. bodenheimeri. In D2-D3 rDNA tree (Fig. 6), present two strains DH1 and KMW formed a monophyletic group with Acrobeloides sp. ES-2017, and together formed sister clad with A. longiuterus (including A. camberenensis, its junior synonym (Abolafia and Peña-Santiago, 2017a, b) from USA. Here also, this pair was sister to the other two species of "maximus" group from different geographical regions, namely A. maximus and A. bodenheimeri. For the ITS rDNA region, there were not enough sequences within Acrobeloides genus to construct any useful phylogenetic tree or use it for comparisons. However, both resulting sequences were added to GenBank with accession numbers of KU721840 (KMW) and KU721841 (DH1) for future comparisons.

Taxonomical remarks
Acrobeloides strains DH1 and KMW obtained during the present study were conspecific to A. saeedi from Pakistan. Although they shared morphological similarities with A. longiuterus, A. maximus and A. bodenheimeri but some divergences were also found and displayed morphometrical differences (Tables 3 and 4). This is the first molecular study of this species and first valid report from India. ITS, 18 S, and D2-D3 rDNA studies confirm it to be different from morphologically closely related species of Acrobeloides. Molecular and phylogenetic studies based on the above three genes revealed the specimens studied now and the Acrobeloides population examined from Iran, could be conspecific.