Thirty individual specimens of 4 species of freshwater fish, including D. pusillus, Henicorhynchus siamensis, Puntius brevis, and Cyclocheilichthys armatus, were subjected to analyses by surveying the metacercarial infections of heterophyid trematodes. Each individual fish was digested using an acid pepsin solution (conc. HCl 1 ml: pepsin 1 g: 0.85% sodium chloride solution 99 ml) for 3 hr at 37˚C. The digested material was then rinsed with 0.85% sodium chloride solution and examined for metacercariae under a light microscope. Some metacercarial specimens were excysted and fixed in 4% formalin under the pressure of a cover slip. Specimens were then stained with hematoxylin, dehydrated in alcohol series, cleared with xylene, and mounted in Permount^®. The metacercariae were confirmed in terms of species, based on Pearson and Ow-Yang [2] and Chai and Sohn [3].

The half-beaks (D. pusillus) were collected from Chiang Mai province, Thailand and were examined and isolated for the metacercarial stage of S. falcatus infection. The fish were digested by 1% acid pepsin solution at 37˚C for 2 hr. The digested material was passed through graded sieves, rinsed in 0.85% saline solution, and then collected for metacercariae under a light microscope. Two hundred metacercariae of S. falcatus were orally infected to 34 chicks, Gallus gallus domesticus. The exposed hosts were killed daily and their small intestines, which were roughly divided into the duodenum, jejunum, and ileum, were opened in 0.85% saline solution. Worms were collected in a modified Baermann’s apparatus, and then examined and observed under a stereomicroscope. The specimens of S. falcatus were fixed in 4% formalin under the cover slip pressure, stained with hematoxylin, dehydrated in alcohol series, cleared with xylene, and mounted in permount. The specimens were measured and drawn under a compound microscope for morphological studies.

The metacercariae of S. falcatus, H. taichui, and C. formosanus were collected from different fish hosts; D. pusillus, H. siamensis, and P. brevis, respectively. Meanwhile, the adult specimens of Haplorchoides sp. were gathered from the intestines of the Yellow cat fish, Hemibragus filamentus, and the adult stages of other trematodes, namely the giant liver fluke, Fasciola gigantica and the rumen fluke, Orthocoelium streptocoelium, were recovered from cows (Bos taurus).

The genomic DNA of all parasites was extracted and purified from the adult worms using a DNeasy Tissue Kit (Vivantis, Oceanside, California, USA) according to the instructions of the manufacturer. All extracted genomic DNA were diluted to a working concentration of 30 ng/μl and stored at -20˚C until used.

The PCR amplification of partial ITS2 fragments was done using a pair of primers as has been described in the previous report [20]. This consisted of (ITS3) 5´GCA TCG ATG AAG AACGCA GC 3´ as a forward primer and (ITS4) 5´TCC TCCGCT TAT TGA TAT GC 3´ as a reverse primer. The reaction was carried out at a final volume of 20 μl containing 1× PCR buffer, 2 mM MgCl₂, 10 μM of each dNTP, 1 μM of each primer, and 1 U of Vivantis Tag DNA polymerase. The analysis was then performed in a MyCycler^TM Thermocycler (BioRAD, Hercules, California, USA). Recommended PCR protocols were as follows; 1 cycle of 95˚C for 2 min, 35 cycles of 94˚C for 45 sec, 50˚C for 45 sec, 72˚C for 1 min, and 1 cycle of final extension at 72˚C for 7 min. Specific PCR products were separated on 1.4% TBE agarose gel electrophoresis, stained with ethidium bromide, and photographed using a Kodak digital camera Gel Logic 100 and direct sequenced by the dideoxy terminator method (First Base Company). The sequence was checked using the BLAST program in the NCBI (National Center for Biotechnology Information, Bethesda, Maryland, USA) database, to confirm the PCR target. The eletropherograms of each sequence were examined for sequence accuracy using a Sequence Scanner version 1.0 and Bioedit version 7.1. All sequences were aligned automatically using Clustal X version 2.0. The phylogenetic relationships of 11 trematodes were constructed using MEGA version 5.0. All molecular data were analyzed by UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method (Table 1). The reliability of the internal branches was assessed using the bootstrap method per 1,000 replicates.

The prevalence of infection in 4 heterophyid trematodes; S. falcatus, H. taichui, Haplorchoides sp., and C. formosanus, were recorded individually from their fish hosts which revealed the prevalence of 90.0%, 73.3%, 33.3%, and 33.3%, respectively, while only D. pusillus was infected by S. falcatus with the highest value of intensity (919 metacercariae/fish) (Table 2).

Worms were recovered in 33 (97.1%) of 34 chicks that were experimentally infected with 200 S. falcatus metacercariae each. The average worm recovery rate was 43.0%. Measurements of worms recovered from the chick are summarized in Fig. 1. The body length and width were remarkably increased from days 1-2 post-infection (PI), and then gradually increased until day 14 PI (Fig. 1A). The oral sucker, pharynx, esophagus, and ventrogenital sucker also showed a similar developmental pattern to the body size over days 1-2 PI, before stabilizing at a plateaued level (Fig. 1B). The left testis was a little bit larger than the right one in terms of both length and width. The size of 2 testes was dramatically increased from day 1 to day 2 PI, and gradually increased until day 11 PI. The ovary and the expulsor developed at an equivalent level, as was also observed in the testes (Fig. 1C). Uterine eggs were recognized in 87.5% of the specimens at day 2 PI. Eggs in the uterine duct were constant in size during days 2-14 PI, with a length of 20.0-22.5 μm and a width of 7.5-10.5 μm.

On day 1 PI, the worms differed morphologically from the metacercariae particularly in terms of their body size. The body measured 240.0-375.0 μm in length and 130.0-160.0 μm in width. The ovary was 30.0-37.50 μm in length and 27.5-45.0 μm in width. The right testis was found to be 42.5-90.0 μm long and 20.0-42.5 μm wide, while the left testis was 37.5-70.0 μm long and 22.5-30.0 μm wide. On day 2 PI, mature worms were observed. The body measured 360.0-510.0 μm in length and 130.0-210.0 μm in width. The ovary enlarged to 30.0-62.5 μm long and 27.5-50.0 μm wide. The right testis was 55.0-87.5 μm long and 32.5-52.5 μm wide, while the left testis was 47.5-80.0 μm long and 30.0-52.5 μm wide. The expulsor was oval and relatively large, with a length of 32.5-82.5 μm and a width of 25.0-37.5 μm. The ventrogenital sucker was slightly submedian to the right side and contained a ventral sucker. The ventral sucker armed with 2 dense lateral groups of small spines on the lip. The ovary was submedian and located slightly to the right side between the ventral sucker and right testis and was 30.0-62.5 μm long and 27.5-50.0 μm wide. The vitellaria extended dorsally into the ovariotesticular zone (Fig. 1D).

The partial sequence data of the ITS2 region were used to understand their phylogenetic relationships. The length of this fragment was 410-590 bp. The trematodes in the family Heterophyidae appeared to be in a monophyletic clade. The heterophyids were separated into 2 sister groups including S. falcatus group and the other heterophyid group (Fig. 2).