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Introduction
Siddiqi (1969) proposed the genus Laimydorus to accommodate those species of MesodorylaimusAndrássy, 1959 which possesses ‘double’ guiding ring, longitudinal vulva, numerous ventromedian supplements and prerectum in males extending well beyond the range of supplements and designated Laimydorus prolificus (=Mesodorylaimus prolificus (Thorne & Swanger, 1936) Goodey, 1963) as its type species. Andrássy (1988) noted that LaimydorusSiddiqi, 1969 bears a strong resemblance to Dorylaimus Dujardin, 1845 in its general appearance, with the primary distinction being the absence of longitudinal ridges on the cuticle. Andrássy (1988) identified several distinguishing features of Laimydorus, including a double guiding ring, a notably long prerectum in males originating well before the supplements, contiguous ventromedian supplements, and a plump, short, rounded tail in males. He also suggested that Laimydorus needs revision. Loof (1985) reassigned all monosexual species of Laimydorus to ProdorylaimusAndrássy, 1959, while Jairajpuri and Ahmad (1992) argued for retaining these species within Laimydorus. Currently, Laimydorus comprises approximately 55 recognized species along with 5 species inquirendae (Peña-Santiago, 2021).
Documentation on Laimydorus species from India is sparse. However, several species have been described by Baqri and Jana (1982), Dey and Baqri (1986), Baqri (1991), and Baniyamuddin and Ahmad (2006), Mushataq and Ahmad (2006) from several parts of India. Ahmad and Ahmad (2002) provide a detailed description with fine line drawings of L. siddiqii based on specimens collected from several localities (Uttarakhand, Kerala, Goa and Andhra Pradesh) in India. This species has been described from West Bengal (Baqri & Jana, 1982). All the previous descriptions of this species lack molecular data. The lack of this molecular data for L. siddiqii presents a significant gap in the literature, hindering phylogenetic studies and accurate species identification.
Nematodes of the genus Laimydorus play a critical role in soil ecosystems, contributing to nutrient cycling and soil health (Tu et al., 2024). However, accurate identification and understanding of these species are essential for ecological studies and biodiversity conservation (Baerwald et al., 2020). The lack of molecular data for many species within this genus, including L. siddiqii, has led to their taxonomy and phylogeny challenges. Molecular approaches have become increasingly vital for resolving taxonomic ambiguities and exploring evolutionary relationships within nematode taxa (Nisa et al., 2022). Addressing these gaps is crucial for advancing our understanding of nematode diversity and their ecological roles, especially in regions like India, where documentation is limited.
This study aims to redescribe Laimydorus siddiqii isolated from Uttarakhand, India, using both morphological and morphometric analysis and provides the first molecular data for this species. By addressing these gaps, this research contributes to the taxonomy and phylogenetics of Laimydorus, facilitating a better understanding of nematode diversity and its ecological implications.
Materials and Methods
Nematode isolation
Soil samples were collected from sugarcane crop fields in the district of Haridwar Uttarakhand, India (29°91′ N, 78°16′ E and elevation of 314 m above sea level). The samples were collected from soils around the sugarcane rhizosphere and collected in well-labeled polythene bags. Nematodes were isolated from soil samples using sieving and decantation (Cobb, 1918), followed by a modified Baermann funnel technique (Baermann, 1917). The extracted nematodes were then collected in a Petri dish for further studies.
Morphology and morphometry
Following Cobb’s (1918) sieving and decantation, the nematodes were extracted from soil samples and modified Baermann’s funnel techniques. The extracted nematodes were fixed in hot triethanolamine-glycerol fixative, dehydrated by the slow evaporation method (Seinhorst, 1959), and mounted in anhydrous glycerine. Permanent mounts were prepared using the paraffin wax ring method (de Maeseneer & d’Herde, 1963). The measurements were taken using an ocular micrometer, and the position of pharyngeal gland nuclei and their orifices were calculated according to Loof and Coomans (1970). Line drawings were prepared using a drawing tube, and photographs were taken with a Nikon DS digital Camera attached to the Nikon Eclipse 80i microscope. Raw photographs were edited using Adobe® Photoshop®.
DNA extraction, amplification and sequencing
Genomic DNA was extracted from five single females using the Qiagen DNeasy® Blood and Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. The D2-D3 regions of 28S rRNA and the 18S rRNA fragments were amplified using specific primers (Delay et al., 1999; Floyd et al., 2005). PCR conditions included initial denaturation at 95°C for 3 min, 40 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min, and a final extension at 72°C for 15 min (Bhat et al., 2023). Amplified products were confirmed on agarose gel electrophoresis (Bhat et al., 2018), and sequencing was performed using an ABI 3730 (48 capillary) electrophoresis instrument (Bioserve Pvt. Ltd, Hyderabad, India). Identical sample sequences were submitted to GenBank (accession numbers OP532315 and OP532812).
Phylogenetic analyses
Phylogenetic analyses were performed by editing and comparing sequences with those in GenBank using BLAST (Altschul et al., 1990). Alignments were generated for nematode samples and related species, including 18S and 28S rRNA genes, using Clustal W within MEGA 11.0 (Tamura et al., 2021). Phylogenetic relationships were reconstructed using the Maximum Likelihood method, with model selection based on best-fit nucleotide substitution analyses in MEGA 11 (Kimura, 1980; Hasegawa et al., 1985; Nei & Kumar, 2000; Tamura et al., 2021). MUSCLE (v3.8.31) was used for sequence alignment (Edgar, 2004), and trees were generated using Neighbor-Joining and BioNJ algorithms with MCL-based pairwise distance matrices. The tree topology with the highest log likelihood was selected. Discrete Gamma (+G) and invariant sites (+I) were included where applicable. Phylogenetic trees were visualized and edited using Interactive Tree of Life (v3.5.1) (Chevenet et al., 2006; Letunic & Bork, 2016).
Morphometry of Laimydorus siddiqiiBaqri and Jana, 1982 from Uttarakhand, India. All the measurements are in μm (except n, ratio and percentage) and are in the form of mean ± standard deviation (range).
Characters
Females
Males
N
10
6
Total body length (L)
2913 ± 162 (2601 – 3100)
2710 ± 312 (2393 – 3108)
a (L/BD)
38 ± 4.1 (31 – 42)
35 ± 2.4 (33 – 39)
b (L/NL)
5.1 ± 0.2 (4.8 – 5.2)
4.6 ± 0.4 (4.2 – 5.2)
c (L/TL)
12.1 ± 0.8 (11 – 14)
110.14 ± 16.6 (94 – 141)
c' (T/ABD)
7.9 ± 0.6 (6.5 – 8.9)
0.8 ± 0.1 (0.7 – 1)
V % (AV/L) ×100
48 ± 1.9 (45 – 52)
–
Lip region width
15.7 ± 1.2 (14 – 18)
15.2 ± 0.4 (15 – 16)
Lip region height
5.7 ± 0.4 (5.1 – 6)
5.9 ± 05 (5 – 5.6)
Amphid aperture width
8.2 ± 1.3 (7 – 10)
6.6 ± 0.57 (6 – 7)
G1((AUL + AOVL+ AOL)/L×100)
22.4 ± 1.5 (20.6 – 25.6)
–
G2 ((PUL+ POVL+ POL/L) × 100)
21.4 ± 1.9 (19.3 – 23.6)
–
Cuticle at anterior region
1.85 ± 0.2 (1.5 – 2)
1.9 ± 0.2 (1.4 – 2)
Cuticle at mid region
2 ± 0.35 (1.5 – 2.5)
1.2 ± 0.2 (1 – 1.5)
Cuticle at tail region
2.52 ± 0.4 (2 – 3)
2.9 ± 0.2 (2.5 – 3)
Odontostyle length
31.5 ± 0.8 (30 – 33)
31.9 ± 0.6 (31 – 33)
Odontostyle length-dorsal side
21.5 ± 2.0 (19 – 26)
21.7 ± 1 (20 – 23)
Odontostyle length-ventral side
30.7 ± 1.4 (29 – 34)
30.6 ± 0.8 (30 – 32)
Odontostyle width
3.6 ± 0.6 (3.1 – 5.2)
3.7 ± 0.5 (3 – 4)
Odontostyle aperture length
10.5 ± 0.9 (9 – 12)
10.2 ± 1.1 (9 – 12)
Odontophore length
35.8 ± 2.2 (33 – 40)
36.4 ± 0.8 (35 – 37)
Distance guiding ring-anterior end
19.3 ± 0.7 (18 – 20)
18.2 ± 0.7 (17 – 19)
Distance nerve ring-anterior end
170.6 ± 9.6 (155 – 185)
182.3 ± 27.5 (155 – 210)
Neck length (NL)
583 ± 31.7 (532 – 652)
581.6 ± 12.9 (563 – 598)
Pharyngeal expansion length
283 ± 21.2 (244 – 306)
282 ± 14.8 (255 – 299)
Pharyngeal expansion width
38 ± 2.8 (35 – 44)
46.5 ± 3.1 (44 – 51)
Body diameter at neck base
77 ± 8.2 (65.2 – 86)
67.6 ± 2.6 (63 – 70)
Cardia length
24.9 ± 3.1 (19 – 30)
24.1 ± 3.1 (21 – 30)
Cardia width
19.4 ± 1.9 (18 – 24)
16.3 ± 1.6 (14 – 18)
Body diameter at vulva
78.1 ± 8.1 (65 – 86)
–
Anterior uterus length (AUL)
272.3 ± 11.5 (250 – 290)
–
Anterior oviduct length (AOVL)
190.4 ± 0.5 (190 – 191)
–
Anterior ovary length (AOL
187.8 ± 3.5 (183 – 192)
–
Posterior uterus length (PUL)
261.6 ± 18 (225 – 292)
–
Posterior oviduct length (POVL)
173.5 ± 7.7 (160 – 182)
–
Posterior ovary length (POL
188.7 ± 5.3 (180 – 198)
–
Vagina length
21.5 ± 1.9 (20 – 26)
–
Distance vulva-anterior end (AV)
1407 ± 93.2 (1241 – 1558)
–
Prerectum length
188 ± 16.9 (167 – 210)
333 ± 61.9 (290 – 404)
Rectum length
42.5 ± 5.2 (34 – 48)
65.2 ± 10.10 (53 – 80)
Anal body diameter (ABD)
30.4 ± 1.3 (28 – 32)
31.5 ± 2.6 (28 – 35)
Tail length (TL)
213 ± 27.8 (183 – 268)
26.5 ± 1.3 (25 – 28)
Vulva to anus
1272 ± 99.2 (1124 – 1410)
–
Body diameter (BD)
78.1 ± 8.1 (65 – 86)
72.1 ± 5.1 (63 – 78)
Spicule length
–
59.5 ± 2.8 (56 – 63)
Max. width of spicule
–
10.0 ± 0.8 (9.0 – 11.0)
Distance of hump from anterior end of spicule
–
18.5 ± 0.7 (18 – 19)
Spicule width at posterior tip
–
5.2 ± 0.6 (4.7 – 6)
Ventromedian supplements
–
27.3 ± 0.8 (26 – 28)
Distance between supplements
–
1.6 ± 0.2 (1.5 – 2.0)
Description
Female: Moderately slender nematodes of large size, 2.6 – 3.1 mm long. Body tapering is done towards both extremities but more towards the posterior region, with the habitus slightly arcuated ventrally. Cuticle with fine striations, 1.5 – 2.0 μm thick in the anterior region, 1.5 – 2.5 μm thick at the mid-body and 2.0 – 3.0 μm on the tail. Lateral chords 18 – 20 μm wide at mid-body, occupying about 31 – 33 % of the corresponding body diameter. 2 – 3 ventral and two dorsal body pores are usually present at the odontostyle-odontophore junction, and lateral body pores are indistinct. The lip region truncates, set off from the adjoining body by slight depression, about 2.4 – 3.0 times as wide as high or about one-fourth diameter at the neck base. Lips amalgamated slightly projecting above the labial contour. Amphid aperture occupies slightly more than half of the lip region diameter. Odontostyle dorylaimoid is 7.8 – 10 times as long as wide, 1.9 – 2.3 times as long as lip region diameter and 1.2 – 1.4 % of body length; its aperture is 9 – 12 μm long or occupies 32 – 40 % of its length. Odontophore is linear, rod-like, and as long as the odontostyle length. The guiding ring is fixed, “double,” at about 1.1 – 1.3 times the lip region diameter from the anterior end. Nerve ring encircling the anterior slender part of the pharynx at about 28 – 38 % of the total neck length from the anterior end. The pharynx expands gradually, with basal expansion 6.5 – 7.6 times as long as broad or 4.1 – 4.6 times as long as the corresponding body diameter, occupying about 46 – 52 % of total neck length. Cardia elongate, conoid, about one-third to two-fifths of the corresponding body diameter long. Pharyngeal gland nuclei and their orifices are located as follows: DO = 52 – 56 %, DN = 53.6 – 58.3 %; DO–DN = 1.0 – 1.8; S1N1 = 71.6 – 73.8 %; S1N2 = 74.8 – 78.5 %; S2N = 85.7 – 89.66 %; S2O = 86.1 – 90.0 %. The genital system is amphidelphic; both sexual branches are almost equally developed. Ovaries reflexed, variably sized, usually surpassing the sphincter level, anterior 183 – 192 μm, posterior 180 – 198 μm long, with oocytes arranged in a single row except near its tip. The oviduct joins the ovary subterminally, measuring 190 – 191 μm or 2.9 – 3.2 (anterior) and 160 – 180 μm or 2.7 – 2.9 (posterior) times the corresponding body diameter long and consisting of a distal slender section made of prismatic cells and a distinct pars dilatata, with a distinct lumen (containing sperms). A well-developed sphincter separates the oviduct and uterus. The uterus is tripartite, i.e., consisting of a saccate distal portion with a distinct lumen, an intermediate coiled tube and a wider proximal part with a distinct lumen, measuring 250 – 290 μm or 4.0 – 4.8 (anterior) and 225 – 292 μm or 3.6 – 4.9 (posterior) times the corresponding body diameter long. Vagina with its three perceptible sections, pars proximalis 15 – 17 × 5 μm, with convex wall and surrounded with circular musculature; pars refringens vaginae measuring 10 × 4 – 5 μm with a combined width of 10 – 11 μm; pars distalis vaginae 3 – 4 μm. Vulva a longitudnal slit. Vagina extending inwards about 20 – 26 μm, occupying up to 43 % of body diameter. Prerectum 5.9 – 6.5 times and rectum 1.2 – 1.5 times anal body diameter long. Tail long filiform, 6.5 – 8.3 times anal body diameter long, hyaline part of tail 16 – 21 % of tail length. Caudal body pores have one pair on each side.
Male: Similar to female in general morphology except for the posterior region, more curved ventrally, 2.3 – 3.1 mm long. Genital system is diorchic, testes opposed, sperm spindle shaped. Supplements consisting of an ad-cloacal pair, situated at 7 – 10 μm from the cloacal aperture, and a series of 26 – 30 contiguous ventromedians, beginning (36 – 46 μm from the spicule head) beyond the spicular range, the anterior most of which is located at 86 – 96 μm from the ad-cloacal pair and posterior most at 187 – 210 μm from ad-cloacal pair. Spicules typical dorylaimid, slender curved ventrad, total length 56 – 63 μm along the arc, equal to that at the chord, 5.1 – 5.8 times its maximum width, 1.6 – 1.9 times the body diam. At the level of the cloacal aperture. The dorsal contour is regularly convex, and the ventral contour is very weakly concave, lacking a distinct hump and hollow. Curvature °. The head occupies 29.8 % of the total length of the spicule, and its dorsal contour is conspicuously curved and longer than the ventral, which is very short and straight. The median piece is 6.4 times as long as wide, occupying 63.8 % of the spicule maximum width, reaching the spicule terminal tip. Lateral guiding pieces 4.0 – 5.0 times as long as wide. Prerectums 9.0 – 12.7 times the cloacal body diameter long. Rectum 1.7 – 1.8 times the cloacal body diameter long. Tail short rounded, about 0.8 – 0.8 times the anal body diameter. Caudal body pores have one pair on each side
Locality and habitat
Nematodes were isolated from soil samples collected in sugarcane agricultural fields in the Haridwar district, Uttarakhand, India.
Voucher material
Ten females and six males were deposited in the Nematode Collection of Chaudhary Charan Singh University, Meerut, India. Also, three females and four males were deposited in the Nematode Biodiversity Research Laboratory, Department of Zoology, Aligarh Muslim University, Aligarh, India.
Molecular characterization
Two nuclear gene sequences were obtained for Laimydorus siddiqiiBaqri and Jana, 1982, specifically including D2 and D3 expansion segments of the 28S (large subunit, LSU) rRNA sequence (794 bp) and 18S (small subunit, SSU) rRNA sequence (878 bp). Analysis of the nucleotide composition revealed that the D2 and D3 expansion segments of the 28S (LSU) rRNA had a G+C content of 53.92 % and an A+T content of 46.08 %, while the 18S (SSU) rRNA exhibited a G+C content of 47.04 % and an A+T content of 52.96 %. BLASTn searches using the 18S rRNA sequences showed that L. siddiqii exhibits high similarity with Labronema ferox Thorne, 1939 (97.75 %; AY552972), Ecumenicus monohystera (de Man, 1880) Andrássy, 1959 (97.27 %; EU543179), and Amblydorylaimus isokaryon (Loof, 1975) Andrássy, 1998 (97.27 %; KM092519). It also showed similarity with Talanema ibericum (Khan & Araki, 2002) Andrássy, 2011 (97.15 %; OP793644), Aporcelaimellus obtusicaudatus (Bastian, 1865) Altherr, 1968 (97.15 %; KU662325), and Dorylaimoides sp. (97.15 %; KU662325). Similarly, BLASTn searches using the D2 and D3 expansion segments of 28S rRNA sequences found that L. siddiqii was closely related to Nevadanema nevadense Álvarez-Ortega and Peña-Santiago, 2012 (92.07 %; JN242245), Mesodorylaimus sp. (91.92 %; MG921257), Prodorylaimus sp. (91.73 %; EF207241), Laimydorus zehsazianiZahedi, Niknam and Vazifeh, 2023 (90.98 %; ON059339), Rhyssocolpus paradoxus (90.20 %; KM092522), and Laimydorus sp. (89.03 %; OR538326) as well as Opisthodorylaimus sylphoides (Williams, 1959) Carbonell and Coomans, 1986 (89.92 %; AY593010). The pairwise alignment of LSU rRNA shows that L. siddiqii shows 49 nucleotide base pair differences and 20 gaps with L. zehsaziani from Iran, which is the only known LSU sequence of the Laimydorus genus. These findings highlight the need for further molecular characterization of species within the family Dorylaimidae to understand the genus Laimydorus better.
Phylogenetic relationships
This study marks the first documentation of the 28S and 18S rRNA sequences from a representative of the genus Laimydorus, except for one existing 28S rRNA sequence from Laimydorus zehsazianiZahedi, Niknam and Vazifeh, 2023, reported from Iran. The molecular analysis has not definitively clarified the phylogenetic relationships of Laimydorus siddiqii due to the limited number of sequenced genera within the Dorylaimidae family. Figures 3 and 4 illustrate the phylogenetic trees based on 28S and 18S rRNA, respectively.
Fig. 3.
Phylogenetic tree based on D2-D3 expansion segments of the 28S rRNA gene sequences of the nematodes isolated in this study and several related species. Phylogenetic relationships based on the nucleotide sequences of the D2-D3 expansion segments of the 28S rRNA gene were inferred by using the Maximum Likelihood method based on the General Time Reversible Model. The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. NCBI accession numbers of the sequences used for the analyses are shown.
Fig. 4.
Phylogenetic tree based on 18S rRNA sequences of the nematodes isolated in this study and several related species. Phylogenetic relationships based on 18S rRNA gene sequences were inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model. The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. NCBI accession numbers of the sequences used for the analyses are shown.
In the phylogenetic analyses utilizing the D2 and D3 expansion segments of the 28S rRNA, L. siddiqii was grouped into a clade with L. zehsaziani, which was described from Iran. This clade further formed a sister clade to an undescribed Laimydorus species from Iran (Fig. 3). Additional DNA sequences from other described Laimydorus species are necessary for a more precise reconstruction of the phylogenetic tree. Due to the absence of available 18S rRNA sequences from any known Laimydorus species, the phylogenetic analysis based on 18S rRNA sequences placed L. siddiqii within a clade that includes representatives of the genera Nevadanema, Dorylaimus, Labronema, Calcaridorylaimus, Mesodorylaimus, and Prodorylaimus (Fig. 4). More comprehensive sequencing efforts across the Laimydorus genus are essential to resolve the phylogenetic relationships within the Dorylaimidae family accurately.
Remarks
Laimydorus siddiqii was first collected and recorded from paddy fields in West Bengal, India, by Baqri and Jana (1982). Later, Ahmad and Ahmad (2002) redescribed it based on specimens collected from various states of India, including Andhra Pradesh, Goa, Kerala, and Uttarakhand. Our present population closely resembles the type population and its subsequent descriptions regarding general morphology and measurements (Baqri & Jana, 1982; Ahmad & Ahmad, 2002). This species is widely distributed across India, with all previously reported populations being found in paddy fields. This preference may be attributed to the wet habitat typical of paddy fields, which members of the Dorylaimidae family usually favor. However, in the present study, L. siddiqii was isolated from sugarcane fields in the Haridwar district of Uttarakhand, India. Unlike paddy fields, the habitat in sugarcane fields is not wet but does contain sufficient moisture suitable for the species’ survival. The original description of L. siddiqii was based on two females and four males. The populations of males and females observed in this study, although closely resembling the type specimens and other descriptions, provide a much broader range of measurements and variations. In the case of females, the rectum length was longer (47 – 53 vs. 30 – 46 μm), while the tail length was shorter (105 – 215 vs. 227 – 334 μm) compared to the previous description (Ahmad & Ahmad, 2002). For males, spicule length was longer (56 – 63 vs 47 – 54 μm). A comparison of morphometric characters of males and females of Laimydorus siddiqii species is given in supplementary tables S1 and S2. Additionally, this study offers the first molecular characterization of the species using LSU and SSU rRNA markers.
Since no sequencing results of 18S and 28S rRNA markers are available for previously described species of Laimydorus (except for 28S rRNA available for L. zehsaziani), more comprehensive sequencing efforts across the Laimydorus genus are essential. These efforts are crucial to accurately resolving the phylogenetic relationships within the Dorylaimidae family. By expanding our molecular understanding, we can better understand this group of nematodes’ evolutionary history and diversification.
