The prevalence, mean intensity, and mean abundance of metacercariae found in the present study are shown in Table 1. Both fish species were found to be infected with metacercaria of Clinostomum. Of six metacercariae fed to the birds, five adults were recovered after 8 days (success rate of 83%). For all three parasite species, six metacercaria were fed to the birds but only 5 adults were recovered after eight days (success rate of 83%). The presence of eggs confirmed the maturity of the worms collected from the birds. It was observed that after feeding the birds with metacercariae, they were not visible in the buccal cavity, assuming they entered the gut of the birds. The birds’ buccal cavities were regularly checked by opening their beaks. Worms first appeared after 7 days but were collected after 8 days.

The parasites were identified to species level based on the morphology of the adult worms collected from the birds, and then the sequences of both adults and metacercaria were obtained. Metacercariae were identified based on the matching sequence with adult specimens.

A total of three species were found in the present study, C. giganticum, C. piscidium, and Euclinostomum heterostomum (Fig. 1). Metacercariae of C. giganticum and E. heterostomum were found in Channa punctatus whereas C. piscidium metacercaria was found in Trichogaster fasciata. No mixed infection was observed in the individual of Channa punctatus examined in the present study. Euclinostome metacercaria were found beneath the operculum within the branchial chamber of the host, whereas clinostome metacercariae were found in the excysted form in the body cavity of the host attached to visceral organs.

Metacercaria and adult C. giganticum (a and b, respectively), metacercaria and adult C. piscidium (c and d, respectively), and metacercaria and adult E. heterostomum (e and f, respectively). OS oral sucker, ES esophagus, VS ventral sucker, T1 anterior testis, T2 posterior testis, OV ovary, CS cirrus sac, VF vitelline follicles, EG eggs

Metacercaria and adult C. giganticum both had a spinose body, a subterminal oral sucker smaller than the ventral sucker, a short tubular esophagus, and intestinal caecae up to the hind end of body, forming shoulders. There were two testes with a crenated margin in the metacercaria, but smooth in adult C. giganticum. The cirrus sac in both metacercaria and adults was elongated to oval, opening into the genital atrium. Other characteristics for both developmental stages included a tubular vesicula seminalis, a small ovary between the two testes, vitellaria consisting of small vitelline follicles distributed from the level of the posterior border of the ventral sucker and extending to the posterior extremity of the body (adult) and a V-shaped excretory bladder.

Metacercaria and adult C. piscidium had a linguiform body with spines only in the anterior half, a small subterminal oral sucker, smaller than the ventral sucker in metacercaria but almost equal in adults, a pseudo-pharynx, smooth intestinal caecae, forming shoulders in the adult, gonads in the middle third of the body, two testes, deeply lobed with a crenated margin in the metacercaria but with a smooth border in the adults, cirrus sac, ovoidal ovary between the two testes, large uterus in the middle line, extending anterior to the lower margin of the ventral sucker, opening posteriorly into the genital atrium, a genital pore at the middle level of the anterior testis, an excretory U-shaped bladder and excretory pore at the posterior end.

Metacercaria and adult E. heterostomum had aspinous adult but thin, spinous, fibrous, round to oval shape metacercaria with a subterminal oral sucker smaller than the ventral sucker. There was a short esophagus in both metacercaria and adults with ten to eleven lateral diverticles in the latter form, lobed testes (anterior curved and U-shaped; posterior Y and triangular shaped in metacercaria and adult forms, respectively), an elongated-tubular cirrus sac between two limbs of the anterior testis, a saccular, vesicula seminalis, a globular ovary between the two testes, and a subterminal excretory pore.

Measurements of taxonomically important features are provided in Table 2.

Clinostomum giganticum and C. pisicidium can be morphologically differentiated based on the distance between the ventral sucker and oral sucker and the width of the ventral sucker, as well as deep, multilobed testes with a crenated margin in the metacercaria of the latter species, whereas almost triangular testes with less digitation in the metacercaria of the former species. However, as adults, in both species, the testes have a smooth border. The position of the genital pore also varies in both species. In C. giganticum, the genital pore opens at the left external margin of the anterior testis, both in metacercaria and adult forms, whereas in adult C. pisicidium, the genital pore opens at the right side, slightly away from the external margin of the anterior testis, and in metacercariae, it opens close to the external margin to anterior testis in the middle of the body. The shape of the excretory bladder also varies; it is V- or Y-shaped in C. giganticum; however, it is U-shaped in C. piscidium. The extension of intestinal caeca may be better visualized in both the metacercariae, which can be concealed by vitellaria in adults.

The 28S, ITS1, and ITS2 sequences of both metacercariae and adults of Clinostomum species (C. giganticum and C. piscidium) and E. heterostomum were consistent and identical with their respective adults. The phylogenetic relationship between parasite taxa in the present study and closely related taxa is shown in the Supplementary Figure. Panels b and c in the Supplementary Figure, which are trees built based on the ITS regions, show that taxa included in the tree are resolved from one another, whereas in panel a, a tree built based on 28S sequences, adults, and metacercaria of C. giganticum are grouped together along with metacercaria and adults of C piscidium, which suggests ITS-1 and ITS-2 regions may be more reliable for interspecific differentiation of Clinostomum spp.