MOLECULAR AND MORPHOLOGICAL CHARACTERISATION OF FLATWORM LARVAE PARASITISING ON FISH IN CAT TIEN NATIONAL PARK, VIETNAM

Cat Tien National Park in southern Vietnam provides a unique opportunity to study the diversity of parasites associated with animals of the plains of tropical forests and lowland river basins in Southeast Asia. In this study we provide morphological description and phylogenetic analysis based on partial sequences of the 28S rRNA gene of metacercariae belonging to five species: Clinostomum sp., Posthodiplostomum sp. 1, Posthodiplostomum sp. 2, Crassiphialinae gen. sp. 1 and Crassiphialinae gen. sp. 2, collected in four species of freshwater fish (Rasbora paviana, Trichopodus trichopterus, Anabas testudineus and Channa striata). The digenean Clinostomum sp. was found to be phylogenetically close (possibly conspecific) to metacercaria of Clinostomum sp. recorded in Australia. There are no sequences in the GenBank database identical to any Posthodiplostomum spp. nor two other crassiphialine metacercariae found by us. Phylogenetic analysis supports the sister position of Crassiphialinae gen. sp. 1 to the Uvulifer spp. + Crassiphiala spp. group of crassiphialine trematodes. At the same time, phylogenetic relationships of Crassiphialinae gen. sp. 2 were poorly resolved. We also provide morphological description, cox1 and 28S rRNA genes-based reconstruction of phylogeny for the plerocercoid of solenophorid cestode Scyphocephalus sp. ex liver of Trichopodus trichopterus. Phylogenetic analyses unite plerocercoid of Scyphocephalus sp. in one group with solenophorid Duthiersia expansa, while the type species of the genus Scyphocephalus – S. bisulcatus– appears as a sister to Duthiersia fimbriata on the tree. Thus, phylogenetic data cast doubt on both the monotypy and the monophyly of the genus Scyphocephalus. This is the second record of plerocercoids of solenophorids and the first one of fish as second intermediate host of this cestode family.


Introduction
National parks and wildlife sanctuaries are essential for the preservation of native biodiversity of relevant natural communities. Untouched nature in the Cat Tien National Park in southern Vietnam provides a unique opportunity to study the diversity of parasites associated with animals of the plains of tropical forests and lowland river basins in Southeast Asia. Most of the Cat Tien National Park's territory is covered by rain forest with a few large permanent water bodies (Tordoff et al., 2004). However, temporal streams and ponds that occur in the forest during the wet season are of no less interest in terms of metazoan parasites' life cycle investigation. Temporary water bodies are the habitat of truly aquatic animals, and due to their shallow depth, are accessible to a wide range of terrestrial, near-water and amphibiotic hosts, which makes them an ideal environment for parasitic larvae distribution and the successful development of a fish-borne zoonosis.
The fauna of trematodes and cestodes parasitising in the metacercarial and metacestoid phases on fish in the oriental zoogeographical region is still poorly resolved, despite certain achievements in this field (e.g. Pandey & Agrawal, 2013;Gupta, 2016;Choudhary et al., 2017;Patarwut et al., 2020). Many species of metacercariae that parasitise on fish in this region are able to infect humans (Rim et al., 2008;De & Le, 2011;Hung et al., 2015), which also gives the study of their fauna practical significance.
The species identification of the larval stages of parasitic worms is a problem that all parasitologists encounter. Adequate identification of larvaelike forms of helminths is possible only with a combination of morphological and molecular genetic approaches (Galazzo et al., 2002;Faltýnková et al., 2014). In this paper, we provide morphological and molecular data on metacercariae and plerocercoids collected from fish that inhabit both permanent and temporal water bodies in the Cat Tien National Park.

Study area
Cat Tien National .57° E) is located in District Tân Phú, Dong Nai Province in South Vietnam (Tordoff et al., 2004). The topography of Cat Tien National Park varies greatly among the three sectors: Cat Loc, Nam Cat Tien, and Tay Cat Tien. The first one is rather hilly, although altitudes only reach 659 m a.s.l., the hills are relatively steep. The other two sectors (Nam Cat Tien and Tay Cat Tien) are situated in the lowlands. The topography of these sectors is characterised by low, gentle hills. The River Dong Nai, the second largest river in southern Vietnam, flows through the national park (Tordoff et al., 2004).
Cat Tien National Park is a well-developed protected area with a long history of efforts to preserve the pristine rainforest (Nguyen & Yen, 2013). The uniqueness of the ecosystem and the status of the protected area attract scientists who are actively studying the flora (Blanc et al., 2000), soil formation (Chernov et al., 2019), climate (Deshcherevskaya et al., 2013) and other aspects of tropical forest life.

Sample collection and morphological observations
All fish were caught between 16 November 2017 and 14 December 2017 during an expedition carried out within the framework of the Russian-Vietnamese Tropical Centre (Gordeev et al., 2018;Sokolov & Gordeev, 2019). Fish specimens in temporal water bodies (Nui Tuong ponds, the brook Da Brout and an unnamed pond) were caught using hand nets and fish net traps. Specimens from the Bao Sao (Crocodile) Lake were provided by employees of the Cat Tien National Park. Immediately after capture, specimens were dissected using standard methods (Bykhovskaya-Pavlovskaya, 1985;Klimpel et al., 2019). At all, 57 fi sh specimens were examined in this re-57 fish specimens were examined in this research: one Rasbora paviana Tirant, 1885 (total length (TL) 7.4 cm; weight 3.91 g) of the Cy-cm; weight 3.91 g) of the Cy-cm; weight 3.91 g) of the Cy-g) of the Cy-g) of the Cy-Cyprinidae, 49 Trichopodus trichopterus (Pallas, 1770) (TL 4.0-10.5 cm; weight 1.17-16.44 g) of the Osphronemidae, six Anabas testudineus (Bloch, 1792) (TL 6.4-10.6 cm; 5.84-37.46 g) of the Anabantidae, and one Channa striata (Bloch, 1793) (TL 14.9 cm; weight 30.55 g) of the Channidae. Worms collected for morphological study were taken out of the cysts using dissecting needles and fixed in hot 70% ethanol, stained with acetocarmine and mounted in Canadian balsam.
All the measurements were made in micrometres based on one specimen. Ecological terms followed Bush et al. (1997). Host species were identified by Dr Ekaterina Vasilyeva (Zoological Museum of Lomonosov Moscow State University, Russia). Specimens destined for molecular analysis were fixed in 96% ethanol and stored at -18°C. The whole mounts were deposited in the Museum of Helminthological Collections at the Centre of Parasitology of the A.N. Severtsov Institute of Ecology and Evolution of RAS (IPEE RAS) in Moscow, Russia.

DNA extraction, amplification and sequencing
The total DNA was extracted from 96% ethanol-fixed metacercariae using a «hot shot» technique (Truett, 2006). The nuclear 28S rRNA gene of trematode larvae was amplified using the polymerase chain reaction (PCR) with the primers DIG12 (5′-AAGCATATCAC-TAAGCGG-3′) and 1500R (5′-GCTATCCT-GAGGGAAACTTCG-3′), which were described earlier . The initial PCR was performed in a total volume of 20 µl that contained 0.25 mM of each primer pair, 1 µl DNA in water, 1× Taq buffer, 1.25 mM dinucleotide triphosphates (dNTPs), 1.5 mM MgCl2 and 1 unit of Taq polymerase. The amplification was carried out in a GeneAmp 9700 (Applied Biosystems) with a 3-min. denaturation hold at 94°C, 40 cycles of 30 s at 94°C, 30 s at 55°C and 2 min. at 72°C, and a 7-min. extension hold at 72°C. Negative and positive controls were amplified using both primers. The PCR products were directly sequenced using the ABI Big Dye Terminator v.3.1 Cycle Sequencing Kit, as recommended by the manufacturer, with the internal sequencing primers . The PCR products were analysed using an Applied Biosystems 3130xl Genetic Analyser at the Russian Federal Research Institute of Fisheries and Oceanography. Unfortunately, an attempt to obtain sequences of ITS1-5.8S-ITS2 r DNA locus for all studied metacercariae using universal primers BD1 (5'-GTCGTAACAAGGTTTCCGTA-3') and BD2 (5'-TATGCTTAARTTCAGCGGGT-3') (Luton et al., 1992) was unsuccessful.

Alignment and phylogenetic analysis
Partial sequence of the 28S rRNA and cox1 gene, used in our study to evaluate the phylogenetic relationships, were assembled using the Geneious ver. 10.0.5 software and aligned with sequences retrieved from the Genbank database (Electronic Supplement 1; Electronic Supplement 2) using the ClustalW DNA weight matrix within the MEGA 10.0.5 software alignment explorer (Kumar et al., 2018) selected using the BLAST search and Phylogenetic analysis of the nucleotide sequences was undertaken using the maximum likelihood (ML) and Bayesian (BI) methods. Phylogenetic trees using ML and BI methods were reconstructed using the MEGA 10.0.5 (Kumar et al., 2018) and MrBayes v. 3.6.2 software (Ronquist & Huelsenbeck, 2003), respectively. Best nucleotide substitution model for the dataset was estimated using jModelTest version 0.1.1 software (Posada, 2008). In both methods, the general time-reversible model GTR+G+I was used based on the Aikake Information Criteria (AIC). A Bayesian algorithm was performed using the Markov chain Monte Carlo (MCMC) option with ngen = 10 000 000, nruns = 4, nchains = 4 and samplefreq = 1000. The burn-in values were 2 500 000 for the «sump» and «sumt» options. The robustness of the phylogenetic relationship was estimated using bootstrap analysis with 1000 replications (Felsenstein, 1985) for ML and with posterior probabilities for BI (Ronquist & Huelsenbeck, 2003). The values of p-distance for solenophorid cestodes that are given in Table 1 were calculated using MEGA 10.0.5 (Kumar et al., 2018).
Prevalence and intensity: one of one host; two worms/host specimen.

Phylogenetic data
A partial sequence of our specimen's 28S rRNA (1313 bp) is identical to metacercaria of Clinostomum sp. (AY222175) ex Hypseleotris galii (Ogilby, 1898), Moggil Creek, Queensland, Australia. Unfortunately, no morphological information on this sample is available . On the phylogenetic tree (Fig. 2), both metacercariae are members of the Clinostomum spp. group that also includes Clinostomum complanatum (Rudolphi, 1814), C. cutaneum Paperna, 1964, andC. phalacrocoracis Dubois, 1931. This group is weakly supported by BI analysis and moderately supported by ML analysis, but it was poorly resolved internally.   Prevalence and intensity: one of 25 hosts; one worm/host specimen (T. trichopterus) and one of six hosts; 26 worms/host specimen (A. testudineus).
Representative DNA sequences: partial 28S rRNA gene sequence was deposited in Genbank (NCBI) as MT394045.
Representative DNA sequences: partial 28S rRNA gene sequence was deposited in Genbank (NCBI) as MT394053.
Representative DNA sequences: partial 28S rRNA gene sequence was deposited in Genbank (NCBI) as MT394052.
Representative DNA sequences: two partial 28S rRNA and cox1 genes sequences deposited in Genbank (NCBI) as MT408587 and MT375386, respectively.

Discussion
Both Clinostomum Leidy, 1856 and Posthodiplostomum Dubois, 1936 are widespread genera of trematodes, the adult stages of which mainly parasitise the piscivorous birds (Dubois, 1968;Locke et al., 2015). In regions with poorly studied fauna, species diagnostics of Clinostomum and Posthodiplostomum metacercariae, based on morphological characters, does not give an adequate and useful result, in view of cryptic diversity in these genera (e.g., Locke et al., 2015;Pérez-Ponce de León et al., 2016;Boone et al., 2018). This is the first study of the genera Clinostomum and Posthodiplostomum metacercariae parasitising on fish in Vietnam, carried out using molecular methods. Previously, only four species of this genera were recorded in fish in Vietnam, namely Clinostomum complanatum, C. piscidium Southwell & Prashad, 1918, Posthodiplostomum cuticula (Nordmann, 1832), and P. grayi (Verma, 1936 (see Arthur & Te, 2006;Lysenko, 2013;Guseva et al., 2014). Our Clinostomum sp. was found to be phylogenetically close (possibly conspecific) to metacercaria of Clinostomum sp. recorded in Aus-tralia. Presently there are no sequences in the Gen-Bank database identical to any of Posthodiplostomum sp. found by us. Also Posthodiplostomum sp. 1 and Posthodiplostomum sp. 2 differ from each other in body size, suckers ratio and utilisation of host tissues. Current phylogenetic analysis, like the data of our predecessors (e.g. Locke et al., 2010;López-Hernández et al., 2018), does not support the monophyly of the genus Posthodiplostomum.
Crassiphialinae gen. sp. 1 closely fits the morphology of Subuvulifer sabahensis (Fischthal & Kuntz, 1973), originally described in kingfisher Pelargopsis capensis javana (Boddaert, 1783) in North Borneo, namely forebody shape, morphology of pseudo-suckers and holdfast organ (compare with Fischthal & Kuntz, 1973). However, the absence of molecular data on the genus Subuvulifer Dubois, 1952 and its life cycle does not allow us to verify this assumption. Crassiphialinae gen. sp. 2 differs from Crassiphialinae gen. sp. 1 by a set of significant morphological features, e.g. lack of pseudo-suckers, shape and position of holdfast organ that are a clear sign of a different generic affiliation. The position of Crassiphialinae gen. sp. 2 on the tree (Fig. 3) suggests that this trematode belongs to a separate and unknown genus.
The morphology of the studied plerocercoids' scolexes is indicative of belonging to the genus Scyphocephalus (see Riggenbach, 1899;Waeschenbach et al., 2017). According to the current concept of the genus Scyphocephalus, it is a monotype taxon, and previously described nominal species S. secundus Tubangui, 1938 andS. longus Sawada &Kugi, 1973 are conspecific to the type species S. bisulcatus (see Schmidt & Kuntz, 1974;Vlnová, 2014). The values of p-distance in the 28S rRNA and cox1 genes in pairs Scyphocephalus sp./S. bisulcatus and Scyphocephalus sp./D. expansa ranged from 1.4-3.6% and 2.7-12.2%, respectively ( Table 1). These values correspond to at least the interspecific level of differences in cestodes (e.g. Agustí et al., 2005;Zhang et al., 2014). Together with the topology of the trees (Fig. 5, Fig. 6) these data allow us to consider genera Duthiersia and Scyphocephalus as polyphyletic taxa. The question of the generic affiliation of cestodes included in the Duthiersia spp. + Scyphocephalus spp. clade has no clear solution yet. The taxonomic interpretation of the clade requires the union of all species into one genus or consideration as independent genera of Scyphocephalus bisulcatus, Scyphocephalus sp., Duthiersia fimbriata and all «D. expansa». Thus, the molecular data we obtained cast doubt on not only the monotypy, but also the monophyly of Scyphocephalus.
This study is the second record of solenophorids' plerocercoid and the first on a fish as the second intermediate host of this cestode family. The plerocercoid stage was described earlier only in D. expansa. Pandey & Rajvanshi (1984) found this metacestode in the amphibian Hoplobatrachus tigerinus Daudin in India.

Conclusions
The species richness of helminths in the tropics remains a topical question of biogeography, emphasising the importance of obtaining primary faunistic data from different hosts and different regions of this climate zone (Dobson et al., 2008;Poulin, 2010). The short-term collection of fish parasites that we carried out in November and December 2017 in only four of many temporal and permanent water bodies located in the Cat Tien National Park resulted in several years of work to identify the numerous larvae of parasitic flatworms. Following the establishment of Asaccotrema vietnamiense Sokolov & Gordeev, 2019 in this paper we provide a morphological description and molecular data on the 28S rRNA gene for five species of metacercariae: Clinostomum sp., Posthodiplostomum sp. 1, Posthodiplostomum sp. 2, Crassiphialinae gen. sp. 1 and Crassiphialinae gen. sp. 2, collected from four species of fish. Of these five, only Clinostomum sp. was found to have an identical sequence in GenBank. We also provide a morphological description as well as cox1 and 28S rRNA genes-based phylogeny reconstruction of solenophorid cestode Scyphocephalus sp. Phylogenetic analysis casts doubt on both the monotypy and monophyly of the genus Scyphocephalus. Fish were recorded as the second intermediate host of solenophorids for the first time.

Supporting Information
The dataset of 63 sequences of trematodes (Electronic Supplement 1: List of previously pub-List of previously published trematode sequences used in the phylogenetic analysis), and 31 sequences of cestodes (Electronic Supplement 2: List of previously pub-List of previously published cestode sequences used in the phylogenetic analysis) may be found in the Supporting Informa-) may be found in the Supporting Information here.