Morphological and Molecular note on the identity of Mylonchulus sigmaturus Cobb, 1917 (Nematoda: Mylonchulidae) from Pakistan

Nematodes were extracted from soil samples by Cobb’s wet sieving and decanting methods (Cobb, 1918), followed by the modified Baermann funnel technique (Baermann, 1917). Specimens were fixed with hot 4 % formaldehyde solution and processed to anhydrous glycerin by the method of Seinhorst’s method (Seinhorst, 1959). Preserved specimens were observed under different magnifications of a compound microscope. Measurements were taken directly using an ocular micrometer. De Man’s formula was used for denoting the dimensions of the nematodes. Line drawing was made with the aid of a camera lucida attached to the microscope and photomicrographs were taken by Nikon DS L2 camera. Ahmad and Jairajpuri (2010) was followed for taxonomic descriptions.

The principal component analysis was used to study the variation among the populations of M. sigmaturus and M. arenicolus. We have included M. arenicolus, because of being placed together with M. sigmaturus through molecular analysis of 18S rDNA. A total of fourteen populations were analyzed for this study. Twenty-two morphometric characters, viz. body length (L), a, b, c, c’, V, G1, G2, lip region width (LW), buccal cavity length (BCL), buccal cavity width (BCW), dorsal tooth apex % (DTA%), amphids opening to anterior end, amphid diameter, nerve ring to anterior end (NR), neck length, neck base diameter (NBD), mid-body diameter (MBD), anal body diameter (ABD), rectum, tail length (TL), and tail length % of body length (TL%BL) were used for analysis. Measurements for the PCA were taken from original descriptions. Data on the morphometric measurements of the species were analyzed using XLSTAT (Addinsoft, 2007). The morphometric measurements for the PCA analysis were taken from the original descriptions. The measures were normalized using XLSTAT software before their analysis (Addinsoft, 2007). The score values were determined for each species based on each of the principal components. The scores for the first two components were used to form a two-dimensional plot (F1 and F2) of each isolate based on the eigenvalues given by the software XLSTAT.

DNA extraction was done using the Chelex method (Shokoohi, 2021). Two specimens of each species were hand-picked with a fine tip needle and transferred to a 1.5 ml microcentrifuge tube containing 5 μl double distilled water. The nematodes in the tube were crushed with the tip of a fine needle and vortexed. Thirty microliters of 5 % Chelex® 50 and 2 μl of proteinase K were added to each of the microcentrifuge tubes that contained the crushed nematodes and mixed. These separate microcentrifuge tubes with the nematode lysate were incubated at 56 °C for two hours and then incubated at 95 °C for 10 minutes to deactivate the protein-ase K and finally spun for 2 min at 16000 rpm (Shokoohi and Abolafia, 2021). The supernatant was then extracted from each of the tubes and stored at −20 °C. Following this step, the forward and reverse primers, SSU F04 (5′-GCTTGTCTCAAAGATTAAGCC–3′) and SSU R26 (5′-CATTCTTGGCAAATGCTTTCG–3′) (Blaxter et al., 1998); D2A (5′-ACAAGTACCGTGAGGGAAAGTTG–3′), D3B (5′-TCGGAAGGAACCAGCTACTA–3′) (De Ley et al., 1999), were used in the PCR reactions for partial amplification of the 18S rDNA, and 28S rDNA regions. PCR was conducted with 5 μl of the DNA template, 12.5 μl of 2X PCR Master Mix Red (Promega, USA) for the Pakistani specimens, 1 μl of each primer (10 pmol μl⁻¹), and ddH2O for a final volume of 30 μl. The amplification was processed using an Eppendorf master cycler gradient (Eppendorf, Hamburg, Germany), with the following program: initial denaturation for 3 min at 94 °C, 37 cycles of denaturation for 45 s at 94°C; 54 °C, and 56 °C annealing temperatures for 18S, and 28S rDNA, respectively; extension for 45 s to 1 min at 72 °C, and finally an extension step of 6 min at 72 °C followed by a temperature on hold at 4 °C. After DNA amplification, 5 μl of product from each tube was loaded on a 1 % agarose gel in TBE buffer (40 mM Tris, 40 mM boric acid, and 1 mM EDTA) for evaluation of the DNA bands. The bands were stained with GelRed® and visualized and photographed on a UV transilluminator. The amplicons of each gene were stored at −20 °C. Finally, the PCR products were purified for sequencing. Available sequences for other Mononchida were obtained from NCBI GenBank for comparison. Also, Mermis nigrescens Dujardin, 1842 (KF583882; AF036641) and (KF886018) were used as outgroups for 18S and 28S rDNA, respectively. Outgroups were selected based on Van Megen et al. (2009). The ribosomal DNA sequences were analyzed and edited with BioEdit (Hall, 1999) and aligned using CLUSTAL W (Thompson et al., 1994). The length of the alignments was 1015 and 850 bps for 18S, and 28S rDNA, respectively. Phylogenetic trees were generated using the Bayesian inference method as implemented in the program Mr Bayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The GTR model was selected using jModeltest 2.1.10 (Guindon & Gascuel, 2003; Darriba et al., 2012). Then, the selected model was initiated with a random starting tree and ran with the Markov chain Monte Carlo (MCMC) for 106 generations. The 18S sequence alignments were also used to construct phylogenetic Median Joining Networks using PopART software (http://popart.otago.ac.nz) (Bandelt et al., 1999). The partial rDNA sequences of M. sigmaturus were deposited in GenBank under the accession numbers: OM278259–OM278262 for 18S rDNA, and OM317560–OM317561 for 28S rDNA.

The genus Mylonchulus is characterized by goblet or funnel shaped stoma, dorsal wall thicker than ventral. Dorsal tooth large directed forward with pointed apex in anterior half of stoma. Subventral walls bearing several small rasp like denticles arranged in rather regular transverse rows. Often ventro-sublateral denticles opposite to the base of the dorsal tooth. Pharyngo-intestinal junction non-tuberculate.

Female genital system didelphic, amphidelphic. Spicules slender, arcuate, gubernaculum simple or bidentate with or without lateral accessory pieces. Tails similar in both sexes, short or slightly elongated with usually conspicuous glands and terminal or subterminal spinneret.

Mylonchulus sigmaturus was originally described by Cobb, 1916 from various parts of the United States and Mexico. This species is recognized by the outlandish ventrally bent tail which is similar in both males and females. Tail of the male bears a series of 13 to 15 low mammliform accessary organs which have distinct connections with the inner body tissues. Spicule slightly arcuate, tapering and subacute and about as long as the anal body diameter. The wedge shaped accessory pieces taper at both ends. Besides the accessory pieces, there are furcate lateral guiding pieces. The caudal glands lie tandem in the tail (Thorne, 1924).

Four populations of a reported species of predatory nematode, M. sigmaturus Cobb, 1917 have been isolated from the different geographical locations of Pakistan viz., P1-Thatta (Sindh), P2-Matiari (Sindh), P3-Lasbella (Balochistan), P4-Khanewal (Punjab), from the soil around plantation of banana (Musa paradisiaca L.) (Table S1; Table 1; aggregated).

M. sigmaturus is a cosmopolitan species described or reported from various parts of the world (Koohkan et al., 2014). This species was first reported in Pakistan by Khan & Saeed (1987). In the present study, the specimens were collected from around the roots of banana plants (Musa paradisiaca L.) in different localities of Pakistan.

In comparison with the material studied by Koohkan et al., (2014) the present specimens have a slightly wider buccal cavity (15 – 17 vs 14 – 15 μm), having slightly anterior position of amphid aperture (8 – 10 vs 12 μm) and more anterior nerve ring (78 – 88 vs 89 – 117 μm). Compared with the specimens studied by Coetzee (1966) present specimens have a wider lip region (25 – 27 vs 19 μm), longer buccal cavity (25 – 27vs 22 μm) and longer tail (30 – 40 vs 26.5 μm). Compared with the material examined by de Bruin & Heyns (1992), the present population differs in lower c’ value (1.0 – 1.4 vs 1.5) and smaller buccal cavity width (15 – 17 vs 13 μm). The specimens described by Khan & Saeed (1987) differ from the present population in higher ‘a’ and ‘c’ ratios (a=25.8 – 33.7 vs 23; c=26 – 40 vs17.9) and in slightly posteriorly located vulva (V=60 – 70 vs 58). The present specimens examined fits well with description of Ahmad et al., (2010), but differs in anteriorly located nerve ring (78 – 88 vs 102 – 114 μm), in shorter neck length (318 – 372 vs 370 – 410 μm) and in shorter G₂ ratio (5 – 14 vs 23 – 27). In comparison with the specimens examined by Farahmand et al., (2009), the present specimens have a longer buccal cavity (25 – 27 vs 21 – 25 μm) and nerve ring located more anteriorly (78 – 88 vs 87.5 – 122.5 μm). In the specimens described by Chaves (1990), advulval papillae were observed, but this character was not observed in the Pakistani specimens. The present specimens have a longer neck and long buccal cavity (318 – 372 vs 279 – 309 and 25 – 27 vs 20 – 25 μm), respectively.

Principal component analysis using morphometric features of females of M. sigmaturus showed a close relationship to these populations.

An accumulated variability of 55.61 % was observed in female-based PCA, 40.02 % in the F1, and 15.69 % in the F2.

The PCA indicated that P1-(Thatta) Sindh and P4 (Khanewal) Punjab are more similar to the South African and Iranian populations. In addition, P2-(Matiari) Sindh and P3-(Lasbella) Balochistan are similar to each other. Overall, the populations of M. sigmaturus showed a low morphometric variation based on the female’s morphometrics (Fig. 3). The Pakistani populations of M. sigmaturus were categorized into two groups on the based on their morphometrics. Furthermore, a population P4 (Khanewal) Punjab from Pakistan stands separately from other M. sigmaturus in the present work, possibly belonging to another species than M. sigmaturus. The results indicated that M. arenicolus stands separate from the M. sigmaturus populations, differentiating them into two valid species.

Fig. 3.

The sequence flanked by the two primers SSU F04 and SSU R26 of the 18S regions of M. sigmaturus isolates is 800 bp long. The blastn search revealed that this population has 5 nucleotides differences with M. sigmaturus from Japan (acc. no. AB361446; AB361447) with 99 % similarity. Estimates of genetic distance using the maximum composite likelihood method among the 18S rDNA region of M. sigmaturus ranges from 0.007 to 0.014. The result showed that Pakistani M. sigmaturus (OM278261) has 0.016 genetic distance from other populations in Pakistan, which is the highest variation. In addition, M. arenicolus Clark, 1961 (AF036596) showed 0.002 genetic distance from M. sigmaturus (AY284755; The Netherlands).

The consensus tree inferred using our 18S rDNA marker (Fig. 4) indicated that the mononchids included in the molecular study were grouped into six clades, including I) Bathyodontus mirus Andrássy, 1956, and Cryptonchus tristis (Ditlevsen, 1911) Filipjev, 1934 and unidentified Cryptonchus; II) Mylonchulus spp. (e.g., M. sigmaturus (Cobb, 1917) Altherr, 1953; M. oceanicus Andrássy, 1986; M. brachyuris (Bütschli, 1873) Cobb, 1917; and M. rotuni-caudatus Skwarra, 1921; and M. arenicolus Clark, 1961, III) Actus salvadoricus Baqri & Jairajpuri, 1974, IV) Iotonchus species and Anatonchus tridentatus (de Man, 1876), De Coninck, 1939, V) Mononchus aquaticus Coetzee, 1968; M. truncatus Bastian, 1865, and M. tunbridgensis Bastian, 1865 and Coomansus gerlachei (de Man, 1904) Jairajpuri, 1970, and VI) Clarkus papillatus (Bastian, 1865) Jairajpuri, 1970; Prionchulus punctatus (Cobb, 1917) Andrássy, 1958; P. muscorum (Du Jardin, 1845) Wu and Hoeppli, 1929; Parkellus zschokkei (Menzel, 1913) Ahmad & Jairajpuri, 2010 and unidentified Parkellus.

Fig. 4.

The consensus tree inferred using our 28S rDNA marker (Fig. 5) indicated that the mononchids included in the molecular study are grouped into four clades, including I) Prionchulus punctatus, Clarkus papillatus, and Mylonchulus sigmaturus; II) Coomansus parvus, Parkellus zschokkei and unidentified Parkellus, III) Anatonchus tridentatus and Iotonchus sp., IV) Mononchus aquaticus, M. truncates, M. tunbridgensis, M. maduei Schneider, 1925 and unidentified Mononchus.

Fig. 5.

A phylogenetic median-joining network showing the relationships between populations of M. sigmaturus, is given in Fig. 6. The populations studied could be divided into two groups, including (I) OM278259–OM278262, AY284755, AB361446, and AB361447, and (II) AY284756 and AY284757. The Pakistani M. sigmaturus (OM278259 and OM278262) differed from Dutch M. sigmaturus (AY284755) and Japanese M. sigmaturus (AB361446; AB361447) in 2 nucleotides. Besides, Pakistani M. sigmaturus (OM278259 and OM278262) in the first group was represented as an identical sequence (Fig. 6). Nucleotide differences of 18S rRNA sequences of 27 populations of Mylonchulus was estimated as Л = 0.338214.

Fig. 6.