The study was conducted in the Khun Thale Swamp, Surat Thani, Southern Thailand (9°4′ 3″N 99°19′52″E). The swamp is a part of the Tapi River’s catchment area (Supplementary Fig. S1).

Freshwater fish belonging to the family Cyprinidae (15 species) were collected from a local fisherman at the Khun Thale Swamp, Surat Thani, Thailand. In 2020, 577 fish were collected for 6 months during the summer (February–April) and rainy (September–November) seasons. The species of each fish were identified and then examined for trematode metacercariae and adults.

The scales and fins of the fish were removed, placed in a Petri dish containing 0.85% saline solution, and examined under a stereomicroscope. The gill filaments were dissected and placed in 0.85% saline for the parasites infecting gills. The fish’s body was examined after dissecting the internal organs. Briefly, scissors were used to cut open the fish, from the anus to the posterior part of the operculum. Internal organs, such as the liver, gall bladder, stomach, and intestine, were dissected and placed in separate Petri dishes containing 0.85% saline and then examined using a stereomicroscope. Caudal fin muscle tissues were minced to detect metacercariae, as reported previously [14,19]. The metacercarial stage was isolated using 1% acid pepsin solution for 2 h at 36°C, and the digested material was passed through a sieve tube and rinsed with 0.85% saline [14,16,20]. The intestines of the fish were examined for adult flukes. The collected parasite specimens were rinsed in 0.85% saline, fixed in 4% formalin, and set between a slide and coverslip with light pressure. The adult specimens were stained by acetocarmine or hematoxylin, dehydrated in an alcohol series, cleared in xylene, and mounted in Permount [14]. The parasite species were morphologically identified under a compound microscope following the related literature [1,2,7,14,20,21]. The prevalence and the mean intensity of parasite species were calculated following the method of Margolis et al. [22]. An Olympus CX31 microscope (Olympus, Tokyo, Japan) with a drawing tube (UDA60) was used to make morphometric data and illustrations. Measurements are given in millimeters as the means with ranges in parentheses.

Out of 15 species of cyprinoid fish examined, 8 were found infected with larval or adult trematodes. The detected metacercariae included 3 species belonging to the Heterophyidae family, i.e., Haplorchis taichui, Haplorchoides mehrai, and Centrocestus formosanus (syn. Centrocestus caninus). The adult trematodes included 2 species, Aspidogastrea (Rohdella siamensis) and Digenea (Helostomatis cyprinorum) (Fig. 1A–F, Fig. 2A–D).

The overall prevalence of larval or adult trematodes was 32.4% (187/577), generally (regardless of trematode species) higher in the rainy season (av. 41.4%; 5.0–91.7% by fish species) than in the summer season (average: 23.6%; 15.0–75.0% by fish species) (detailed data not shown). The metacercariae of H. taichui and H. mehrai were found in the muscle and fin and the muscle, fin, and scale of the fish, respectively. The metacercariae of C. formosanus was found in the gills of the fish. The species of adult trematodes (R. siamensis and H. cyprinorum) were found in the intestine of the fish.

H. taichui metacercariae were detected in 7 species of fish (Table 1), with its highest prevalence in B. gonionotus of 36.4% (summer: 31.3% and rainy: 38.5%) with a mean intensity of 25.6 and 28.9 metacercariae/fish in the summer and rainy seasons, respectively. The overall prevalence was 20.1% (summer: 12.6% and rainy: 28.3%), with 1,179 detected metacercariae in 76 infected fish (average: 15.5 metacercariae per fish; 1–93 by individual fish) (Table 1). H. mehrai metacercariae were found in 7 species of fish (Table 2), with its highest prevalence in L. siamensis at 62.5% (summer: 47.5% and rainy: 77.5%), with a mean intensity of 105.4 and 119.3 metacercariae/fish in summer and rainy seasons, respectively. The overall prevalence was 37.9% (summer: 31.7% and rainy: 43.6%), with 10,800 detected metacercariae in 159 fish (average: 67.9 metacercariae/fish; 1–321 by individual fish) (Table 2). C. formosanus metacercariae were found only in R. toneri fish with a prevalence of 11.7% (summer: 15.0% and rainy: 5.0%), and mean intensity of 3.2 and 5.0 metacercariae/fish in the summer and rainy seasons, respectively (Table 3).

The adult flukes of R. siamensis were found only in B. gonionotus fish with a prevalence of 7.3% (6.3% in summer and 7.7% in rainy seasons), and a mean intensity of 1.0 and 1.3 parasites/fish in the summer and rainy seasons, respectively (Table 3). The adults of H. cyprinorum were found in 2 fish species: L. siamensis and O. vittatus, with an overall prevalence of 11.9% (summer: 12.5% and rainy: 11.3% in seasons) and a mean intensity of 2.7 and 2.4 parasites/fish in the summer and rainy seasons, respectively (Table 3).

Haplorchis taichui (metacercaria; Fig. 1A, B): The encysted metacercaria is oval-shaped and is 0.18 (0.18–0.22) mm in length and 0.13 (0.12–0.18) mm in width (n=10). The excysted metacercaria is elongated and oval and is 3.52 (0.32–0.55) mm in length and 1.50 (0.08–0.20) mm in width (n=10). The ventrogenital sac is located ventrally in the middle region of the body and armed with 16 long, chitinous, and crescentic spines, which is a specific characteristic. A single testis is observed.

Haplorchoides mehrai (metacercaria; Fig. 1C, D): The encysted metacercaria is oval-shaped and is 0.17 (0.16–0.20) mm in length, and 0.12 (0.12–0.16) mm in width (n=10). The excysted metacercaria is elongated and is 0.40 (0.35–0.50) mm in length and 0.13 (0.12–0.18) mm in width (n=10). The number of acetabular spines is 19–27 in total, including 5–7 in the anterior, 7–10 in the median, and 7–10 in the posterior groups, which is a specific characteristic of this species. The testis is single.

Centrocestus formosanus (metacercaria; Fig. 1E, F): The encysted metacercaria is oval-shaped and is 0.21 (0.18–0.22) mm in length, and 0.14 (0.12–0.16) mm in width (n=10). The excysted metacercariae are pyriform in shape and measured 0.45 (0.40–0.48) mm in length and 0.14 (0.13–0.15) mm in width (n=10). The oral sucker is surrounded by 32 circumoral spines in 2 rows. The excretory bladder is characteristically X-shaped, situated in the posterior third of the body between the 2 testes.

Rohdella siamensis (adult; Fig. 2A, B): The body is elongate-oval and measured 3.75 (3.10–4.13) mm in length, and 1.63 (1.35–1.88) mm in width (n=4). The holdfast organ at the ventral surface is composed of 23 (22–23) rows of transverse alveoli divided by 3 longitudinal septa, arranged in a marginal ring of 46–48 and 22–23 medial alveoli, which is one of the specific characteristics of this species. The genital pore is sinistro-lateral near the forebody and overlaps the holdfast organ. A single testis is post-ovarian and globular to oval. Eggs are ovoid and measured 0.09 (0.08–0.10) mm in length and 0.04 (0.03–0.05) mm in width.

Helostomatis cyprinorum (adult; Fig. 2C, D): The body is oval, with its posterior part slightly broader than the anterior one and measured 2.80 (1.35–3.90) mm in length and 0.91 (0.68–1.28) mm in width (n=10). Two cecae extend to the posterior 1/5 of the body, having a 3-shape configuration, which is one of its specific characteristics. Two oval-lobulate testes are located pre-ovarian, and the ovary is ovoid, situated between the testes and the ventral sucker. Eggs are ovoid, embryonated, and measured 0.11 (0.08–0.15) mm in length and 0.70 (0.05–0.10) mm in width.