Overall information on the sampling and survey data
G. pervia samples were collected from 54 sites (as shown in Fig. 1G) with about 38-214 snails in each site. Trematodes were found in 17 sites investigated following dissection, including Tianbao Reservoir and Hede village in Nanning city, Liushan Town, Liutang Village, Guangrong village, and Cha Village in Liuzhou City, and Maling Town in Guilin City. Various stages in the life history of this trematode (redia, cercaria and metacercariae) were found during dissection procedures.
Morphological characters and molecular identification of trematodes larvae
The rediae of echinostomes were cylindrical, blunt at both ends, slightly pointed at the head and more pointed at the tail. The body was curved to the ventral surface with muscular feet, and the movement was slow. The tail of the cercariae is not forked. The head of H. conoideum cercariae shows prominent spines, as well as well-developed ventral suckers, pharynx, and intestines (Fig. 2D-F). The metacercaria were round and have two transparent walls (the outer wall was thicker than the inner wall). Abdominal suckers and refractive granules of larvae could be seen inside the cyst. Due to the movement of the larvae inside the sac, the small spines around its head were not easily observed. The rediae of Australapatemon sp. forms a distinct bulge at the head. The cercaria larvae had a forked tail, which was obviously longer than the body length. The cercaria of P. cordatum also had visible forked-tail, oral sucker and pharynx (Fig. 2J-L).
Figure 2
Based on morphologically available keys, the species of isolated trematodes were identified by amplification of ITS2 region and verified through BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi) with the highest identity after sequencing. Finally, we identified five different species of trematodes including Australapatemon sp., Echinostoma sp., E. revolutum, H. conoideum, and P. cordatum. The lengths of ITS2 were 292 bp, 429bp, 430bp, 432 bp and 294 bp, respectively. The nucleotide sequences obtained in the present study have been deposited in GenBank database under the accession numbers KM980466-KM980471 (Australapatemon sp.), KJ848453-KJ848455 (Echinostoma sp.), KM980474 and KM980476-KM980477 (E. revolutum), KJ944311-KJ944313 (H. conoideum) and OL870492 (P. cordatum).
The prevalence of trematodes in G. pervia
The overall trematodes infection rate was 22.0% (1818/8258). Echinostoma revolutum were detected in the snails from 11 sampling sites, with an infection rate of 12.9% (1069/8258); Hypoderaum conoideum infection was detected in the snails from two sampling sites, with an average infection rate of 3.8% (315/8258). Infection of Australapatemon sp. was detected in the snails form 2 sampling sites, with an infection rate of 2.5% (206/8258); Infection of Pharyngostomum cordatum and Echinostoma sp. were detected at 1 sampling site with an infection rate of 0.4% (34/112) and 2.3% (194/8258), respectively.
Phylogenetic analyses
In total, 15 representative high-quality ITS2 sequence data was obtained. Figure 2 shows an NJ tree based on the submitted sequences and relevant GenBank sequences. The ITS2 sequences of Echinostoma sp. constituted a monophyletic clade (Fig. 3A shaded pink area), distinct from the clade formed by E. robustum, E. friedi and E. miyagawai. The sequences of E. revolutum and H. conoideum constituted a monophyletic group together with E. revolutum (AY168930) and H. conoideum (AJ564385) references (Fig. 3A shaded blue area). The ITS2 sequences of Australapatemon sp. formed a group with A. burti (KU950451) at 99% bootstrap value but formed a unique clade at 75% bootstrap value. Figure 3B showed that the ITS2 sequences of P. cordatum were identical to the reference sequences of P. cordatum (KJ137231).
Figure 3
Laboratory infection experiment with Echinostoma sp., E. revolutum, and H. conoideum
Because there was no suitable second intermediate host for P. cordatum and definitive host for Australapatemon sp., we conducted an infection experiment for isolated three kinds of trematode to evaluate rates of parasite establishment in ducklings. Ducklings were individually exposed to Echinostoma sp., E. revolutum, and H. conoideum larvae and all were successfully infected. Subsequent observation on Ducklings (17 dpi) fed with Echinostoma sp. infected G. pervia, we detected eggs (195.8×143.8 µm) in the feces, and the morphological characteristics of adult Echinostoma sp. were presented as measures: body length 9.8 mm, width 1.2 mm, oral suker 638.9×399.2 µm, acetabulum 1591.2×1338.2 µm, pharynx 492.6×331.7 µm, anterior testis 1120.5×707.4 µm, posterior testis 1274.9×880.4 µm, ovary 818.9×527.9 µm. In contrast, we found H. conoideum eggs in ducklings fed with infected G. pervia from three sites from a median of 12 dpi (range: 9 dpi to 14 dpi). The morphological characteristics of adult H. conoideum were: body length 1.05 mm, width 1.5 mm, oral suker 424.2×293 µm, acetabulum 1610.2×1594.6 µm, pharynx 379.8×253.6 µm, anterior testis 1902.6×875.3 µm, posterior testis 2045.2×898.2 µm, ovary 751.7×553.9 µm, and also characterized by the possession of 50 spines. E. revolutum eggs (104.1×63.1 µm) were found on 10 dpi. The morphology of adult E. revolutum was characterized by: body length 8.5 mm, width 2.2 mm, oral suker 260×180.1 µm, acetabulum 741.6×598.3 µm, pharynx 193.1×150.6 µm, anterior testis 628×459.4 µm, posterior testis 725.5×557.9 µm, ovary 411.8×311.2 µm, and presence of a head collar with 37 spines.
The developmental characteristics of E. revolutum in duckling host from juvenile to adult
As there were not sufficient metacercariae of other trematodes, experiments were only designed to gain insight into how E. revolutum developed in duckling hosts. The developmental characteristics of E. revolutum was recorded by dissecting infected ducklings from 1 dpi to 10 dpi (eggs in the feces were first detected). E. revolutum could be obtained in the small intestine from 1 to 7 dpi and then migrate and reside in the cecum and colon around 8-10 dpi. The body length developed from 490 µm to 8500.5 µm (a dramatic 17-fold increase). At 1 dpi, juveniles presented a circumoral collar bearing 37 spines in a double circle and characterized by clearly visible oral suckers, acetabulum, pharynx, esophagus, and cecum. At 1 dpi, the tiny structure of the testis appeared. By 4 dpi, the ovaries were beginning to organize and develop, and the seminal receptacle began to form. The tubular-shaped uterus loomed at 4 dpi, and maturation of the reproductive and digestive organs occurred around 6~8 dpi. The vitelline glands were the last to appear, and several eggs deposited in the uterus could be observed at 9 dpi. E. revolutum larvae matured at 10 dpi and excreted eggs (Fig. 4). The daily measurement of E. revolutum development was recorded in detail, as shown in Table S2.
Figure 4