Host and parasite specimen preparation

Rainbow water snakes (E. enhydris) were sampled from Nakhon Si Thammarat and neighbouring provinces in southern Thailand, with the assistance of local villagers and wildlife rescuers. The collected specimens were subsequently transported to the Snake Farm at the Queen Saovabha Memorial Institute in Bangkok. Between 2021 and 2025, a total of 119 snakes that died during quarantine at the facility were dissected, and their remains were transferred to the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok, for parasitological examination. Prior to dissection, the snakes were stored at −20 °C.

Before dissection, each specimen underwent external morphological examination and measurements following the criteria outlined by Cox et al. (Reference Cox, Hoover, Chanhome and Thirakhupt2012) to confirm species identity and record host information. Dissections were conducted using the methods described by Ratnarathorn and Kongrit (Reference Ratnarathorn and Kongrit2025). As the gallbladder was the primary site of infection, it was carefully removed, placed in Petri dishes with water to prevent desiccation and subsequently opened and examined under a stereomicroscope (Olympus SZ51, Japan) for the presence of Gogatea spp. When detected, parasites were gently isolated and transferred to Petri dishes for further examination.

Among the recovered trematodes, 2 distinct morphological groups were identified: (1) individuals possessing fully developed gonads and (2) individuals lacking gonads. Four well-preserved specimens with developed gonads were fixed in 2.5% glutaraldehyde in 0.1 m sucrose phosphate buffer for scanning electron microscopy. The remaining parasites were preserved in 70% ethanol. In addition, 6 immature trematodes were recovered from the small intestine of 1 snake; they were preserved in 70% ethanol for subsequent molecular analysis to verify their conspecificity with the specimens derived from the gallbladder.

Morphological study

For the specimens with gonads – which were the majority of the parasites found – 18 complete individuals from 4 out of 25 infected snakes were selected from the 70% ethanol stock for morphological study and permanent slide preparation. The remaining specimens were unsuitable for measurement due to deformation or incompleteness, with a few retained for genetic analysis. An additional 3 individuals without gonads were also prepared for permanent slide mounting. Each selected specimen was fixed in 10% formalin overnight, rinsed in distilled water for 3 min and stained with acetocarmine for 48 h. Destaining was performed using a 1% HCl solution for 10 s, followed by a graded ethanol dehydration series (70%, 80%, 90%, 95% and 100%), with each step lasting 12 min – except for the 95% ethanol and absolute ethanol steps, which lasted 20 min and 1 h, respectively, to ensure complete dehydration. The specimens were then immersed in a 1:1 solution of 100% ethanol and xylene for 5 min, briefly placed in pure xylene and subsequently mounted in Permount™ on glass slides with coverslips. The mounted slides were left at room temperature for several days to ensure complete drying, and later photographed using the same light microscope (see Figure 1 for illustrations).

Figure 1. Permanent slides (acetocarmine dye) of Gogatea serpentum: (A) entire body of a mature specimen with complete-gonad (white-dashed lines show 2 testes), ventral view; (B) anterior region, ventral view; (C) entire body of an immature specimen, ventral view; (D) entire body of a mature specimen with incomplete gonad (white-dashed line shows 1 testis), ventral view; and (E) entire body of a mature specimen without gonad, ventral view. Abbreviations: ATT, anterior testis; CB, caeca bifurcation; CE, caecum; CS, cirrus sac; E, eggs; GA, genital atrium; HO, holdfast organ; M, metraterm; OPS, ophistosoma; ORS, oral sucker; OS, oesophagus; OV, ovary; PH, pharynx; PTT, posterior testis; PS, prosoma; TT, testis; VF, vitelline follicle; VS, ventral sucker.

The morphological characters of the mounted specimens were measured using ZEN 2 Blue Edition software connected to an inverted microscope (Zeiss Primovert, Germany) equipped with a Zeiss Axiocam, while the illustrations (Figure 2) were created using a light microscope with a camera lucida (Leitz, Wetzlar, Germany). A total of 23 morphological characters were recorded, including body length, forebody length (i.e. distance from the oral sucker to the ventral sucker), prosoma length, prosoma width (i.e. maximum body width), pharynx length, pharynx width, oesophagus length, oral sucker length, oral sucker width, ventral sucker length, ventral sucker width, holdfast organ length, holdfast organ width, cirrus sac length, cirrus sac width, ovary length, ovary width, length and width of each testis, egg length and egg width (Table 1; Supplementary Tables S1–S3). Three of these characters – forebody length, oesophagus length and cirrus sac length – were also calculated as percentages of the total body length. All measurements were expressed in micrometres (μm) (Table 1).

Figure 2. Camera lucida drawing illustrations of Gogatea serpentum: (A) entire body of a mature specimen with complete gonads, ventral view; (B) entire body of a mature specimen lacking gonads, ventral view; (C) holdfast organ, ventral view; and (D) posterior region of a mature specimen with complete gonads, ventral view. Abbreviations: ATT, anterior testis; CS, cirrus sac; E, eggs; M, metraterm; ORS, oral sucker; OS, oesophagus; OV, ovary; PH, pharynx; PTT, posterior testis; VF, vitelline follicle; VS, ventral sucker.

Table 1. Morphological features and measurement data of Gogatea serpentum examined in this study (including both gonad-bearing and gonad-less specimens), compared with those from previous studies and Gogatea burmanicus syn. n. All measurements are in micrometres (µm) and are presented as ranges with the mean ± standard deviation in parentheses

Scanning electron microscopy (SEM) analysis was conducted on 4 specimens that were initially preserved and fixed in 2.5% glutaraldehyde solution to assess tegument spine characteristics, including shape, size and distribution pattern. Specimen preparation was carried out at the Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University. Each specimen underwent secondary fixation in 1% osmium tetroxide in 0.1 m SPB, followed by ethanol dehydration and drying using a critical point dryer (CPD300 auto, Leica, Wetzlar, Germany). The dehydrated specimens were then coated with gold using a sputter coater (Q150R PLUS, Quorum, East Sussex, UK). The samples were subsequently transferred to the Central Instrument Facility, Faculty of Science (Phaya Thai Campus), Mahidol University, where SEM imaging was performed (Hitachi SU8010, Hitachi High-Tech, Japan) (see Figure 3 for illustrations).

Figure 3. Scanning electron micrographs of Gogatea serpentum: (A) entire body with a very large protruded holdfast organ, ventral view; (B) anterior region with densely distributed dot-like tegumental spines, dorsal view; (C) oral sucker, anterior region, ventral view; (D) large protruded holdfast organ with small ventral sucker anteriorly (white arrow), ventral view; (E) genital pore at apical end, ventral view; (F) densely distributed tegumental spines, anterior body; (G) sparsely distributed tegumental spines, posterior body; and (H) magnified view of tegumental spine.

Principal component analysis (PCA) was conducted to analyse the multivariate data matrix of 16 morphometric characters using PAST version 4.06b (Hammer et al., Reference Hammer, Harper and Ryan2001). To illustrate morphological variation among the specimens (Figure 4), the correlation model was used to generate 2-dimensional scatter plots with the corresponding percentage variances. PCA was performed on all 21 mounted individuals, including the 3 parasites lacking gonads. Ventral sucker, ovary and egg measurements were excluded from the analysis due to being unobservable in some specimens; likewise, all gonad-related measurements were omitted, as these structures were absent in the gonad-less individuals (Supplementary Tables S2 and S3).

Figure 4. Principal component analysis (PCA) of Gogatea serpentum was performed using 16 morphological characters, accounting for 67.323% of the total variance. Black dots and red triangles represent specimens with and without gonads, respectively.

Molecular and phylogenetic study

The molecular analysis was performed to compare whether the individual group with gonads and without gonads are conspecific. DNA was extracted from 2 specimens of individuals with gonads, and one parasite from the individuals without gonads using the Genomic DNA Mini Kit (Geneaid Biotech Ltd., New Taipei City, Taiwan) according to the manufacturer’s protocol. Additionally, the DNA was also extracted from 2 immature parasites found in the small intestine. The DNA amplification was conducted on 3 regions, including the nuclear 28S ribosomal RNA (28S rRNA), the nuclear ITS2 and the mitochondrial cytochrome COI. The selected genes are those that are widely utilized in molecular identification and genetic diversity studies in trematodes (Thaenkham et al., Reference Thaenkham, Chaisiri and Chan2022). The obtained sequences were subsequently concatenated using the aligned sequences from all 3 genetic markers. These targeted genes were amplified by polymerase chain reaction (PCR) using a T100™ thermocycler (Bio-Rad) in a 30 µL mixture containing 15 µL of 2× i-Taq master mix (iNtRON Biotechnology, Gyeonggi, South Korea), 10 µm of each primer and 1 ng µL⁻¹ of DNA. The PCR details for each of the targeted regions including the primers used, expected amplicon lengths and the thermocycling profile are provided in Supplementary Table S4 (Curran et al., Reference Curran, Tkach and Overstreet2011; Routtu et al., Reference Routtu, Grunberg, Izhar, Dagan, Guttel, Ucko and Ben-Ami2014; Van Steenkiste et al., Reference Van Steenkiste, Locke, Castelin, Marcogliese and Abbott2015; Besprozvannykh et al., Reference Besprozvannykh, Tatonova and Shumenko2018). The PCR amplicons were visualized on a 1% agarose gel stained with SYBR Safe (Thermo Fisher Scientific, USA). The products that were successfully amplified, as indicated by clear bands on the agarose gel, were sent for sequencing using Fast Next-Generation Sequencing (Tsingke, Beijing, China). The obtained nucleotide sequences were submitted to the NCBI database under the accession numbers PV988016–18 for 28S rRNA, PV988019–21 for COI and PV988350–52 for ITS2.

After sequencing, the partial sequences of each marker were manually inspected and edited using BioEdit version 7.2.5. Multiple sequence alignment was performed by ClustalX 2.1 with reference sequences of other Gogatea species. Phylogenetic analysis was conducted using maximum likelihood (ML) in MEGA-11. Best-fit nucleotide substitution models were determined for each marker: the Hasegawa–Kishino–Yano model with an additional parameter accounting for a proportion of invariable sites (+I) for 28S rRNA and COI, the Jukes–Cantor with an additional parameter accounting for a proportion of invariable sites (+I) for ITS2 and the general time reversible model with a discrete gamma distribution (+G) for concatenated sequences from all 3 markers. Phylogenetic trees were constructed using 1000 bootstrap replicates to ensure analytical robustness (Hall, Reference Hall1999; Thompson et al., Reference Thompson, Gibson and Higgins2002; Tamura et al., Reference Tamura, Stecher, Peterson, Filipski and Kumar2013). The COI, 28S rRNA and ITS2 analyses included sequences from all available Gogatea species, with Cyathocotyle prussica used as the outgroup for each marker. All sequences were obtained from GenBank (Figure 5; Supplementary Table S5).

Figure 5. Phylogenetic analyses were conducted using the maximum likelihood method in MEGA-12 based on the 28S rRNA (A), ITS2 (B), COI (C), and concatenated sequences of all 3 markers (D) from available species within the genus Gogatea. Branch length scale bars indicate the number of substitutions per site, and node values represent bootstrap support. Specimens of Gogatea serpentum with and without gonads (the latter corresponding to Gogatea burmanicus syn. n.) Identified in this study are highlighted in dark red and light red font/boxes, respectively.

Morphological remarks

The trematode G. serpentum (Gogate, 1932) was found infecting 25 of 119 examined E. enhydris, indicating a moderate prevalence of 21.0%, with intensities ranging from 1 to 33 parasites per snake (average of 5 parasites per infected snake). In total, 131 parasites were recovered from the gallbladder of the snakes. Of these, 14 parasites recovered from 3 different infected snakes (with intensities of 2, 4 and 8) lacked gonads, while the rest had both ovaries and testes present. Additionally, 6 immature parasites – identified by their small size and the absence of a visible uterus and eggs – were collected from the small intestine of one of the infected snakes.

Morphological identification confirmed that the parasites obtained from E. enhydris in this study are G. serpentum. The specimens possess a large holdfast organ (i.e. adhesive apparatus) with a vitellarium consisting of large vitelline follicles confined within it, which is a key characteristic of the genus Gogatea. The morphometric measurements of the species are mostly similar in size to those reported from other populations of G. serpentum previously recorded in the Southeast Asian region (Table 1; Supplementary Table S1). The presence of a small ventral sucker rules out G. anacetabulata (Achatz et al., Reference Achatz, Von Holten, Binh and Tkach2024), which is the only other species reported from Southeast Asia but lacks a ventral sucker.

Based on 18 parasitic adult specimens with complete gonads, the description of the parasite is as follows (Table 1; Supplementary Tables S1 and S2) (all measurements in μm): body elongated, linguiform, with a total length of 1258–2087 (Figures 1A, 2A and 3A). Forebody (oral to ventral sucker) 316–748 (Figure 1B). Prosoma oval (Figure 1A), 729–1432 × 494–965. Opisthosoma tail-like, elongated, cylindrical, 291–705 × 215–531. Oral sucker well-developed, 59–143 × 82–154 (Figures 2A,B and 3C). Ventral sucker small, round, overlapping with a large holdfast organ, 51–109 × 39–136 (Figures 2A,B and 3D). Ratio of ventral sucker to oral sucker: length 1:1, width 2:1. Holdfast organ well-developed, very large, round, or oval with longitudinal opening, 395–747 × 350–812 (Figures 1A, 2C and 3D). Prepharynx short or absent. Pharynx small, 57–85 × 56–101. Oesophagus short, 82–212 (E = 8%) (Figures 1B and 2A,B). Tegumental spines minute, sharply pointed; most numerous and densely arranged on forebody, progressively sparser towards posterior end (Figure 3B, F–H).

Testes round, tandem, situated posterior to ventral sucker (Figures 1A and 2A,D). Anterior testis 165–263 × 128–211; posterior testis slightly larger, 148–342 × 127–259. Cirrus sac elongate, narrow, 343–827 × 57–126, extending from level of anterior testis to genital pore (CS = 36%). Metraterm thick, joined with cirrus sac at genital atrium (Figures 1A and 2D). Genital pore terminal (Figure 3E). Ovary round or oval, intertesticular or at the same level as anterior testis (Figure 2A,D). Vitellarium composed of large follicles confined to holdfast organ (Figure 2C). Eggs oval, large, 119–151 × 69–100, with 0–15 eggs per individual.

Morphological variation

The morphology of 3 other individuals without gonads showed measurements similar to those with gonads and an identical overall appearance, except for the absence of testes and ovary, and a slightly thicker wall of the cirrus sac, which fits well with the description of G. burmanicus (Chatterji, 1940) (see full measurements in Table 1; Supplementary Tables S1 and S3; Figures 1E and 2B). Interestingly, there was also one individual who had only one testis present (Figure 1D). In addition, the individuals from the small intestine appeared to be immature or underdeveloped, as indicated by their noticeably smaller size (700–900 μm) and the lack of developed internal organs (Figure 1C).

PCA conducted on both complete-gonad (n = 18) and incomplete-gonad (n = 3) individuals revealed no distinct separation or morphological clustering, as shown in the 2-dimensional PC1–PC2 plots (Figure 4). The analysis accounted for 67.323% of the total variance, supporting the morphological homogeneity of the G. serpentum samples. Interestingly, the 3 gonad-less individuals did not cluster together but were instead dispersed throughout the plot, further indicating no consistent morphological difference between gonadal and non-gonadal specimens. Based on these findings, the synonymization of G. burmanicus as a new synonym of G. serpentum is proposed.

Genetic characterization and phylogenetic position

Despite exhibiting different morphologies, phylogenetic analyses using the 3 markers (ITS2, 28S rRNA and COI) showed that all specimens were 100% identical, with no genetic variation observed (Figure 5A–C). Molecular evidence thus further reinforces morphometric analyses, supporting our proposal that the Gogatea specimens, which were initially thought to be G. burmanicus due to the lack of gonads, are identical to G. serpentum. All 4 phylogenies revealed that G. serpentum is genetically closest to G. mehri, forming a monophyletic clade. The genetic distance obtained between these 2 species were 0.7% for both 28S rRNA and ITS2 markers, and 14.2% for COI. Gogatea bijirrii, G. acrochordi and G. anacetabulata formed another monophyletic clade, with G. bijirrii and G. acrochordi sharing a closer genetic relationship than G. anacetabulata. Additionally, G. bijirrii and G. acrochordi, recently described from Australia, were genetically indistinguishable in both the nuclear 28S rRNA and ITS2 analyses (Figure 5A,B), while their COI sequences were clearly distinct, differentiating them as separate species (Figure 5C).

Nomenclatural note

The species was originally described as Prohemistomum serpentum by Gogate (Reference Gogate1932). Subsequent authors have variably referred to it as Gogatea serpentium or Gogatea serpantium (Lutz, Reference Lutz1935; Frank, Reference Frank1966; Gupta, Reference Gupta1967), resulting in some inconsistency in the literature. However, according to the International Code of Zoological Nomenclature (ICZN, 1999: Articles 32–33.3), the original spelling serpentum should be retained unless demonstrably incorrect. As there is no evidence that Gogate’s use of serpentum was inadvertent, this spelling is regarded as the correct and valid one. In his monograph on trematodes, Yamaguti (Reference Yamaguti1958) also employs the term serpentum, citing the original description by Gogate (Reference Gogate1932) and the subsequent work of Lutz (Reference Lutz1935), and thereby retained the original nomenclature. In addition, recent taxonomic consensus (Chatterji, Reference Chatterji1940; Achatz et al., Reference Achatz, Von Holten, Binh and Tkach2024) also follows serpentum in accordance with the original description, further supporting its validity. The alternative spellings serpentium and serpantium are considered incorrect subsequent spellings under ICZN Article 33.3 and are treated here as such.

Taxonomic summary

Phylum: Platyhelminthes Minot, 1876

Class: Trematoda Rudolphi, 1808

Order: Plagiorchiida La Rue, 1957

Family: Cyathocotylidae Mühling, 1896

Genus: Gogatea Lutz, 1935

Species: Gogatea serpentum (Gogate, 1932)

Host: Enhydris enhydris (Schneider, 1799)

Locality: Aquatic areas such as lakes, ponds and swamps in Nakhon Si Thammarat (e.g. Pak Phraek [8°14′56.04″ N, 100°13′18.96″ E], Thung Song [8°17′18.6″ N, 99°29′42.6″ E] District) and neighbouring provinces in southern Thailand (e.g. Songkhla Lake [7°12′34.2″ N, 100°27′43.56″E]) served as collection sites. However, the exact coordinates of individual hosts were not documented.

Collection date: 1 January 2021 to 7 February 2025

Site of infection: gallbladder and small intestine (duodenum)

Parasite intensity: 1–33 worms, mean approximately 5

Parasite prevalence: 0.21 (25 out of 119 hosts infected)

Specimens deposited: Department of Helminthology, Faculty of Tropical Medicine and Department of Biology, Faculty of Science, Mahidol University