Paracapillaria (Ophidiocapillaria) siamensis sp. nov. (Nematoda: Trichuroidea): a new nematode in Naja kaouthia from Thailand

A comprehensive investigation, incorporating both morphological and molecular analyses, has unveiled the existence of a hitherto unknown nematode species, Paracapillaria (Ophidiocapillaria) siamensis sp. nov., residing in the intestine of the monocled cobra, Naja kaouthia, in the central region of Thailand. This study integrates morphological characteristics, morphometric examination, scanning electron microscopy and molecular phylogenetic analysis (COI, 18S rRNA and ITS1 genes). The findings place the newly described species within the subgenus Ophidiocapillaria, elucidating its distinctive characteristics, including a frame-like proximal spicule shape, approximate lengths of 19 000 and 22 500 μm with approximate widths of 90 and 130 μm for males and females, 39‒45 stichocytes, elevated lips without protrusion, a dorsal bacillary band stripe with an irregular pattern of bacillary cells and evidence of intestinal infection. These features serve to differentiate it from other species within the same subgenus, notably Paracapillaria (Ophidiocapillaria) najae De, , a species coexisting P. siamensis sp. nov. in the monocled cobra from the same locality. This study addresses the co-infection of the novel species and P. najae within the same snake host, marking the second documented instance of a paracapillariid species in the monocled cobra within the family Elapidae. The genetic characterization supports the formal recognition of P. siamensis sp. nov. as a distinct species, thereby underscoring its taxonomic differentiation within the Capillariidae family. This research identifies and characterizes the new nematode species, contributing valuable insights into the taxonomy of this nematode.

Between 2000 and 2023, authors of this research conducted parasitological surveys in wildlife (Unpublished data), leading to the discovery of 2 distinct paracapillariid species residing within various digestive organs of the monocled cobras, Naja kaouthia, in central regions of Thailand.Additionally, other studies have reported finding eggs from these worms within the aforementioned snake host and location.However, precise identification has posed a considerable challenge, resulting in a taxonomic classification limited to the genus level, denoted as Capillaria sp.(Chaiyabutr and Chanhome, 2002;Vasaruchapong et al., 2017).The morphological similarity between these 2 paracapillariid species, without genetic investigation, caused confusion and led to the erroneous assumption in the survey that they were the same species.One of these species, P. najae, located in the cobra's oesophagus, was initially described by De in 1998 and subsequently redescribed by Charoennitiwat et al. (2023).Conversely, the other species, infesting the cobra's intestine, exhibited discernible variations in both morphology and genetic sequences when compared to the data documented for P. najae, as delineated by Charoennitiwat et al. (2023).This comprehensive examination ultimately resulted in the formal recognition of these as distinct new species and the revelation of the co-infection of this novel species alongside P. najae in the same snake host species.
This led to the inception of the present study.A substantial number of Paracapillaria samples retrieved from the monocled cobra's intestine underwent detailed molecular phylogenetic analysis and thorough morphological examination.The scrutiny of external morphological characteristics aimed to distinguish the new nematode species within its taxonomic context, aligning with insights derived from genetic data.The exploration of phylogenetic relationships involved the reconstruction utilizing genes, including cytochrome c oxidase subunit 1 (COI), internal transcribed spacer1 (ITS1) and small subunit nuclear ribosomal RNA (18S rRNA).Our findings conclusively validate the presence of the newly described Paracapillaria (Ophidiocapillaria) siamensis sp.nov., reinforcing previous reports of co-infection incidents involving P. najae in the same hosts organisms.

Specimen preparation
Five carcasses of wild monocled cobras were provided by the Snake Farm at Queen Saovabha Memorial Institute, Thai Red Cross Society in Bangkok, Thailand.These cobras underwent dissection to investigate the presence of parasites in their intestine, following the protocols outlined by Toland and Dehne (1960) as well as Terrell and Stacy (2007).Following the dissection, the organs were placed in Petri dishes filled with 0.85% normal saline and were subsequently examined under stereo-microscopes (Olympus, SZ30 and SZ51, Japan).Micro-dissecting needles were employed to observe and isolate the parasites, focusing on the paracapillariid nematodes.These isolated parasites were counted and preserved in 70% ethanol at −20 °C until required.Additionally, 4 specimens were separated and preserved in glutaraldehyde for scanning electron microscopic imaging.

Morphological study
For the morphological studies, 21 male and 21 female intact worm specimens were selected from the preserved 70% ethanol stock and subjected to comprehensive examination.This examination was conducted using an inverted microscope (Zeiss, Primovert, Germany) equipped with a Zeiss Axiocam and ZEN2 blue edition software.The measurements in micrometers (μm) of these morphological characters strictly adhered to the methodologies outlined by Biserkov et al. (1994), De (1998) and Charoennitiwat et al. (2023).Illustrations depicting selected morphological traits of both male and female worms were crafted under a light microscope with a camera lucida (Leitz, Wetzlar, Germany).Out of the total number of specimens, after the morphological investigations, 26 specimens (13 males and 13 females) were initially mounted in glycerine jelly and subsequently double mounted again with permount to establish permanent slides of type specimens.The remainder of the specimens (8 specimens for each sex) were designated for genetic analysis.

Scanning electron microscopy study
For a scanning electron microscope (SEM) analysis, 2 intact male and 2 intact female specimens were carefully selected.Initially, these specimens were immersed in a solution containing 2.5% glutaraldehyde in a 0.1 M sucrose phosphate buffer (SPB) for primary fixation.Subsequently, a secondary fixation step was performed using a 1% osmium tetroxide solution in the same 0.1 M SPB.The specimens were then dehydrated with ethanol and subsequently dried using a critical point drying device (CPD300 auto, Leica, Wetzlar, Germany).A fine coating of material was applied using a coat-sputter (Q150R PLUS, Quorum, East Sussex, England).Finally, these prepared specimens were examined under the SEM (JSM-6610LV, JEOL, Tokyo, Japan).This SEM analysis was conducted at the Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University.

Molecular study
For DNA extraction, 8 male and 8 female specimens were homogenized and processed using the DNeasy Blood & Tissue Kit (Qiagen, Germany) following the manufacturer's instructions.The extracted genomic DNA was eluted with 30 μL of nucleasefree water and quantified using spectrophotometry.
The PCR reactions were conducted using a T100 thermocycler (Bio-Rad, California, the United States of America).The PCR reaction mixture contained a final volume of 30 μL, including 15 μL of 2X i-Taq master mix (Biotechnology, Gyeonggi, South
Korea), 10 μM of each primer and 1 ng/μL of DNA.For the COI primers, the thermocycling profile was as follows: an initial denaturation step at 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 52 °C for 1 min and 72 °C for 45 s.The reaction concluded with a final extension step at 72°C for 5 min, as described by Charoennitiwat et al. (2023).For the ITS1 primers, the thermocycling profile was as follows: an initial denaturation step at 95 °C for 5 min, followed by 30 cycles of 94 °C for 40 s, 60 °C for 1 min and 72 °C for 2 min.The reaction concluded with a final extension step at 72 °C for 5 min.Regarding the 18S rRNA primers, the thermocycling profile consisted of an initial denaturation at 94 °C for 5 min, followed by 5 cycles of 94 °C for 30 s, 45 °C for 30 s and 72 °C for 70 s, and 35 cycles of 94 °C for 30 s, 54 °C for 30 s and 72 °C for 70 s, as described by Holterman et al. (2006).
For the visualization of the PCR amplicons, a 1% agarose gel stained with SYBR safe (Thermo Fisher Scientific, Waltham, the United States of America) was employed.Following this, the PCR products from the 3 most exemplary sample sequences underwent Barcode Taq sequencing, a method that does not necessitate primer walking (Celemics, Seoul, South Korea).The nucleotide sequences derived from the parasite specimens in this study have been submitted to the NCBI database and allocated the respective accession numbers: OR839870-72 for the COI, OR840538-40 for the 18S rRNA and OR840543-45 for the ITS1.
The partial sequences of the 3 target genes were verified through manual inspection of electropherograms.The complementary strands were compared and adjusted using BioEdit version 7.2.5 (Hall, 1999).For the analysis of phylogenetic relationships, a phylogenetic tree was constructed using the obtained sequences and additional sequences from parasites of Capillariidae, Trichuridae and/or Trichinellidae retrieved as outgroups from GenBank.The alignment of the data matrix was performed using ClustalX 2.1 (Hall, 1999;Thompson et al., 2002), and the aligned sequences were visually analysed using BioEdit.The aligned sequences were verified before conducting the phylogenetic analysis using maximum likelihood (ML) in MEGA-X (Chan et al., 2020).The best-fit nucleotide substitution model was determined by the Bayesian Information Criterion (BIC), and the analysis was performed with 1000 bootstrap replicates (Tamura et al., 2013).A bootstrap value exceeding 70% generally provides strong support (Hillis and Bull, 1993).The phylogenetic trees were constructed using the ML method, employing the Hasegawa-Kishino-Yano (HKY) model for the ITS1, Kimura 2-parameter (K2) with a gamma distribution for the 18S rRNA and the General Time Reversible (GTR) model with a gamma distribution for the COI.

Taxonomy
Phylum Site of infection: Small intestine Parasite intensity: 16-82 (on average 47) nematodes in the fivehost examined ZooBank LSID: 7072F98E-09EC-4D3C-BC10-B86D654A7C60 Etymology: The specific epithet 'siamensis' indicates that the nematode species is found in Thailand or belongs to Thailand.'Siam' is an original and informal name for 'Thailand,' and the suffix '-ensis' in Latin is commonly used to create adjectives that signify 'of' or 'belonging to' specific places or localities.

Type materials
Holotype: mature male was deposited at the Mahidol University Museum of Natural History (Voucher no.: MUMNH-NEM0001; specimen code: SN62PMI05) was collected by Vachirapong Charoennitiwat and his team, on July 18, 2023, in the small intestine of a monocled cobra, Naja kaouthia (IDs: SN062 for this project; AAS089 [EL-Nk-025] for the Applied Animal Science laboratory's catalogue), at the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University.
Allotype: gravid female was deposited at the Mahidol University Museum of Natural History (Voucher no.: MUMNH-NEM0002; specimen code: SN62PFI07) was collected by Vachirapong Charoennitiwat and his team, on July 18, 2023, in the small intestine of a monocled cobra, Naja kaouthia (IDs: SN062 for this project; AAS089 [EL-Nk-025] for the Applied Animal Science laboratory's catalogue), at the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University.
Description of allotype: body length 19 720; maximum width 102.Whole oesophagus 7986 long; muscular oesophagus and stichosome 327 and 7630, respectively.Stichosome with a single row of 41 stichocytes.Anterior: posterior body ratio, 1:1.47.Tail 23 long.Vulva with no elevated lips situated at 71 from oesophagus end; vagina directed posteriorly from vulva and uterus containing 135 eggs.Eggs elongate with slightly protruding polar plugs and uncleaved content, 60 long and 30 wide; egg wall thick with 2 layers: an inner hyaline layer and a thicker outer layer with fine longitudinal sculpture.

Diagnosis
As specified (see Table S1), Paracapillaria (Ophidiocapillaria) siamensis sp.nov.can be distinguished from other Paracapillaria species by diagnostic characteristics: body length 7150-18 098 in males and 11 816-25 160 in females.Maximum width 53-86 in males and 54-112 in females.Stichosome with a single row of 39-45 stichocytes.Specifically, this nematode species was found in the snake intestine, frequently in the small intestine.Anterior: posterior body ratio in males, usually 1:<1 with average 1:0.9.The number of eggs in uterus is usually <199 with average 99.The eggs arrange in 1-2 rows in uterus.Proximal end of spicule of male frame-like shape (Fig. 1I); spicule length 749-1815.The cephalic region displayed elevated lips without protrusion (Figs 2A-B).The body's midsection featured transverse striations on the ventral side (Fig. 2C).A dorsal stripe of bacillary band extending from the nearly beginning of the body to the cloaca (Figs 2D-E).This bacillary band consists of irregular pattern of bacillary cells (Fig. 2D).P. siamensis sp.nov.primarily infected the small intestine of the snake host.

Specific diagnosis
Among these Ophidiocapillaria, the newly described Thai species, Paracapillaria siamensis sp.nov.can be distinctly differentiated from Italian Paracapillaria mingazzinii (Rizzo, 1902), American Paracapillaria colubra (Pence, 1970) 1934) due to differences in vulva lip characteristics.P. siamensis sp.nov. is characterized by the absence of elevated vulva lips, distinguishing it from the mentioned species.Furthermore, P. siamensis sp.nov.differs from these species in several measured characteristics, including longer spicule length (in P. colubra, P. heterodontis, and P. viperae), shorter spicule length (in P. cesarpintoi), a larger distance between the vulva opening and the end of the oesophagus (in P. colubra and P. heterodontis), and a shorter distance between the vulva opening and the end of the oesophagus (in P. cesarpintoi).Importantly, P. siamensis sp.nov.was recorded in Thailand, Asia, while all the other species were discovered on different continents.
In addition, P. colubra, P. heterodontis and P. murinae were found in the oviduct, rectum and stomach of snakes, unlike P. siamensis sp.nov., which specifically inhabits the small intestine.Based on Moravec (1986), P. siamensis sp.nov.does not exhibit the same levels of characteristic variation as Paracapillaria sonsinoi (Parona, 1897), which includes body length of both genders, spicule length, the ratio of the anterior and posterior ends, egg length and importantly, its recorded locality in Europe and America.

Variation
The number of P. siamensis sp.nov.individuals, ranging from ∼16 to 82 worms, displays significant morphological variation among the cobra specimens (see Table S2).Key characters that vary among the cobras include relative-to-body size and oesophagus characteristics of both male and female worms, encompassing body length and width, oesophagus lengths and other distance characters.Notably, cobra ID: SN013 exhibited relatively small parasites (average 7946.35,n = 4), whereas other cobra samples displayed nearly double the size in body length (14 171.67-15 788.92, n = 3-7).The spicule length stands out as another example of character with high variation; the worms in cobra ID: SN059 had an average spicule length of 2480.26 (n = 3), surpassing those in cobra IDs: SN018 and SN013, which measured 1265.00 (n = 3) and 969.60 (n = 4), respectively.This trend is also observable in other morphological characteristics.Despite the pronounced morphological diversity among P. siamensis sp.nov.individuals, specimens subjected to genetic analysis revealed minimal sequence differences (Fig. 2).Sexual dimorphism, reflected in traits such as larger size in females (average 19 630.29 vs 13 672.54 in males), distinct vulva lips in females (Fig. 1E) and specific features of the spicular sheath in males , is observed in this novel species (Table S2).

Genetic characterization and phylogenetic position
The molecular characterization of the newly identified species, P. siamensis sp.nov., involved the amplification and sequencing of the nuclear 18S rDNA and ITS1 genes, as well as the mitochondrial COI gene.The outcomes of the phylogenetic analysis clearly indicated that P. siamensis sp.nov. is a distinct species within the taxonomic classification of Capillariidae.The species available for comparison included Baruscapillaria sp., Pseudacapillaria sp., Pearsonema sp., Aonchotheca sp., Capillaria spp., Eucoleus spp., Paratrichosoma sp. and other Paracapillaria sp.Specifically, the COI strongly suggested that P. siamensis sp.nov.forms a sister clade with P. najae (Fig. 3A), even though the 2 species infect the same snake host species.This illustrates the co-infection between both paracapillariid species.Both nuclear 18S rRNA and ITS1 also supported the differentiation of P. siamensis sp.Nov. from P. najae and other capillariid sequences available in GenBank, similar to the mtDNA result (Figs 3B-C).The genetic variation between P. siamensis sp.Nov. and other capillariids ranged from 14 to 25% for the COI, 2-14% for the 18S and 41-63% for the ITS1.The closest genetic distances were observed between P. siamensis sp.nov.and members of P. najae, with 14% for the COI, 2% for the 18S rRNA and 41% for the ITS1.

Natural history
Paracapillaria siamensis sp.nov., appears to be a prevalent parasite in the monocled cobra, given its discovery in all 5 snake samples examined.This species, exclusively found in the small intestine of the cobras, has been documented to co-occur with P. najae, which predominantly resides in the oesophagus.This co-occurrence was observed consistently across all snake samples examined in the study.As a common behaviour, the worms tend to aggregate through ball-coiling within the intestinal mucus, extending throughout the entire intestinal tract and occasionally reaching into the proximal large intestine area.Importantly, visual inspection revealed no discernible lesions on the target organ.
Although the precise geographic coordinates of the sample collection sites were not recorded, the cobras were captured in suburban areas surrounding Bangkok, directly linked to their prey choices.Within the gut contents, a conspicuous presence of small conical structures resembling the teeth of lizards or rodent claws was observed under the stereomicroscope.This observation raises the possibility of intermediate hosts for the new paracapillariid species within the ecosystem.

Discussion
Paracapillaria siamensis sp.nov., as per Moravec and Justine (2020), belongs to the subgenus Ophidiocapillaria, as it resides in the snake species, the monocled cobra, Naja kaouthia, found in Thailand.The key diagnostic morphology of this new species aligns with that of other Paracapillaria spp., including the presence of a bursa supported by lateral lobes and a non-spiny spicular sheath at the male caudal end (e.g.Moravec, 1986;Moravec et al., 1995;Moravec and Justine, 2020).Furthermore, the phylogenetic results from both mitochondrial and nuclear genes in this study strongly support the morphological findings, indicating that this new paracapillariid species falls within the capillariid lineage.
The newly discovered Ophidiocapillaria species exhibits distinct morphological differences from other species within the same subgenus, characterized by variations in morphological measurements and proportions.Key features used for classifying this new paracapillariid species include body length, body width, oesophagus length, distance from the nerve ring to the anterior end, stichosome and spicule length, among others (e.g.Moravec, 1986;Moravec and Gibson, 1986;Moravec et al., 1995;De, 1998;Moravec and Justine, 2020;Sakaguchi et al., 2020;Charoennitiwat et al., 2023).Several characteristics, such as the target organ of infection, type of vulva lips, shape of the proximal spicule and locality, can serve as alternatives for species identification (e.g.Moravec, 2001;Timi et al., 2007;Moravec andJustine, 2010 and, 2020;Melnychuk et al., 2020;Sakaguchi et al., 2020;Carvalho et al., 2023).Despite the distinctiveness of many species based on those characteristics, a few original papers on Paracapillaria lacked morphological details, resulting in difficulties in the identification of species, particularly Paracapillaria ptyasi found in the southern part of China.This species exhibited minimal morphological differences from P. siamensis sp.nov., except for a few measurements i.e. nerve-ring position, stichocytes numbers, anterior part length, female body length and importantly, the spicule shape, a key factor in Paracapillaria's identification (sensu Moravec and Justine, 2020;Carvalho et al., 2023).The spicule shape was markedly different between these 2 speciesround for P. ptyasi and frame-like for P. siamensis sp.nov.
Additional evidence for the differentiation of these 2 worm species lies in the isolation of their hosts and the host distribution (the monocled cobra, Naja kaouthia, vs the Oriental rat snake, Ptyas mucosa).Moreover, there are reports of geographical distribution separation in the rat snake (genus Ptyas) between China and Thailand (Gernot and Sjon, 2013).This separation is attributed to the high elevation of the Himalayan Mountain ranges, suggesting that parasites restricted to their hosts might not cross the border.Similar isolation of reptiles is observed in other Colubrid species (Bain and Hurley, 2011), such as the keelback snake, Rhabdophis subminiatus (Liu et al., 2021) and Dendrelaphis sp.(Biakzuala et al., 2022).
However, species infected within the same snake host, like P. siamensis sp.nov.and P. najae are distinguished by the described morphological measurements and ratios in Table 1, as well as descriptive characteristics such as proximal spicule shape (frame-like for P. siamensis sp.nov.vs spatula-like for P. najae, sensu Charoennitiwat et al., 2023) and the organ of infection (the intestine for P. siamensis sp.nov.vs the oesophagus for P. najae).Nevertheless, these characteristics, which closely resemble each other between the 2 species, pose challenges in species identification when the parasites are isolated from the host body and in low numbers.Therefore, microscopic structure and molecular genetic studies play a crucial role in species identification for paracapillariid worms, given their high morphological variation (Charoennitiwat et al., 2023), potentially leading to species misidentification.The microscopic surface images clearly display variation in the position of the bacillary band between the 2 species.The genetic information derived from the mt COI, as well as the nuclear 18S rDNA and ITS1 (slowly evolving nuclear genes, Mallatt et al., 2004) markers, provides valuable insights into the identification, confirming the genetic distinctiveness of P. siamensis sp.nov.from P. najae.This emphasizes the importance of genetic data for the species identification of Paracapillaria, although the available data in GenBank on Paracapillaria species is still limited.

Parasitology
Despite the considerable morphological variation observed in P. siamensis sp.nov., the genetic sequence exhibited minimal differences.This suggests that the observed morphological differentiation may arise from other factors, such as environmental influences (e.g.Thompson and Lymbery, 1990;Benesh and Kalbe, 2016).Therefore, considering alternative sources of information, in addition to morphological characteristics, for species identification may be advisable.
P. siamensis sp.nov., appearing in the adult stage, infects the monocled cobra, a generalist species predominantly found in the central lowland region of Thailand (Cox et al., 2012;Ratnarathorn et al., 2019Ratnarathorn et al., and, 2023)).It is plausible that transmission of the parasite species occurs through various prey items consumed by the cobra, such as rodents, chicks, amphibians or other reptiles (Cox et al., 2012;Kalki et al., 2022).Given the capability of paracapillariid species to infect multiple hosts (Charoennitiwat et al., 2023), a further investigation involving their prey and other generalist snake species, such as rat snakes (Ptyas spp.) and the Siamese spitting cobra (Naja siamensis), which share similar prey preferences with the monocled cobra (Cox et al., 2012), may contribute to a more comprehensive understanding of the natural history aspects, including the life cycle of this newly described species.

Figure 2 .
Figure 2. Scanning electron micrograph of Paracapillaria (Ophidiocapillaria) siamensis sp.nov.: (A) lateral view and (B) apical view of head region of male, elevated oral lip (white arrow) surrounded by papillae (black arrows); (C) lateral view of middle body with transverse striations; (D) dorsal view of middle body with a bacillary band and (E) button-like bacillary cells (black arrow); (F) lateral view of posterior end of male with two post-cloacal papillae (white arrows); (G) spicular sheath without spines.

Figure 3 .
Figure 3. Phylogenetic analysis of capillariids based on different genetic markers: (A) COI gene, (B) 18S rRNA and (C) ITS1.The analyses were conducted using MEGAX with the maximum likelihood method.Branch length scale bars indicate the number of substitutions per site.Coloured lines/fonts represent genetic data from various capillariid genera sourced from GenBank, with the red line/font specifically highlighting the genus Paracapillaria.The blue box indicates the specimens of Paracapillaria siamensis sp.nov.used in the present study.Synonym remarks include Capillaria hepatica = Calodium hepaticum, Capillaria gastrica = Eucoleus gastricus, Capillaria putorii = Aonchotheca putorii, Capillaria plica = Pearsonema plica, and Capillaria suis = Aonchotheca suis.