Exploring the evolutionary potential of parasites : Larval stages of pathogen digenic trematodes in their thiarid snail host Tarebia granifera in Thailand

Minute intestinal flukes from several distinct families of endoparasitic platyhelminths are a medically important group of foodborne trematodes prevalent throughout Southeast Asia and Australasia. Their lifecycle is complex, with freshwater snails as primary intermediate hosts, with infecting multiple species of arthropods and fish as second intermediate hosts, and with birds and mammals including humans as definitive hosts. In Southeast Asian countries, the diversity of snail species of the Thiaridae which are frequently parasitized by trematode species is extremely high. Here, the thiarid Tarebia granifera in Thailand was studied for variation of trematode infections, by collecting the snails every two months for one year from each locality during the years 2004–2009, and during 2014–2016 when snails from the same localities were collected and new localities found. From ninety locations a total of 15,076 T. granifera were collected and examined for trematode infections. With 1,577 infected snails the infection rate was found to be 10.46 %. The cercariae were categorized into fifteen species from eight morphologically distinguishable types representing several distinct families, viz. (i) virgulate xiphidiocercariae (Loxogenoides bicolor, Loxogenes liberum and Acanthatrium histaense), (ii) armatae xiphidiocercariae cercariae (Maritreminoides caridinae and M. obstipus); (iii) parapleurophocercous cercariae (Haplorchis pumilio, H. taichui and Stictodora tridactyla); (iv) pleurophocercous cercariae (Centrocestus formosanus); (v) megarulous cercariae (Philophthalmus gralli); (vi) furcocercous cercariae (Cardicola alseae, Alaria mustelae and Transversotrema laruei); as well as (vii) echinostome-type cercariae, and (viii) gymnocephalous-type cercariae. In addition, a phylogenetic marker (internal transcribed spacers 2, ITS2) was employed in generic and infrageneric level classifications of these trematodes, using sequences obtained from shed cercariae isolated from T. granifera specimens of the second study period collected in various regions in Thailand. We obtained ITS2 sequences of cercariae from nine species (of seven types): Loxogenoides bicolor, Loxogenes liberum, Maritreminoides obstipus, Haplorchis taichui, Stictodora tridactyla, Centrocestus formosanus, Philophthalmus gralli, as well as from one species each of echinostome cercariae and gymnocephalous cercariae. Thus, this analysis combines the parasites’ data on morphology and geographical occurrence with molecular phylogeny, aiming to provide the groundwork for future studies looking into more details of the parasite-snail evolutionary relationships.


Introduction
Trematodes (or flatworms) are endoparasitic platyhelminths that not only infect fishes, birds and other wildlife worldwide but also mammals as well as humans.As foodborne parasites they are of medical importance resulting in significant morbidities and mortalities worldwide.For example, the disability adjusted life years (also known as DALYs) for the foodborne trematodiases including Fasciola spp., Clonorchis spp., Opisthorchis spp., Paragonimus spp.and the minute intestinal trematodes such as Fasciolopsis buski, Heterophyes spp.and Metagonimus spp., are estimated to be 2.02 million worldwide (Torgerson et al. 2015).
Especially as liver flukes and intestinal flukes human infecting parasites are highly prevalent in Southeast Asian countries (Wongratanacheewin et al. 2001, Chai et al. 2005, 2013, Krailas et al. 2014).Infections caused by these flukes have a major public health impact and are also of economic importance in veterinary medicine.Humans or domestic animals become infected when they eat raw, salted, pickled or smoked fish containing the infective metacercariae (e.g.Opisthorchis spp.,) or contaminated to raw or uncooked vegetables (e.g.Fasciola spp.) (see e.g.Krailas et al. 2011, Krailas et al. 2014).
Examples include the liver fluke Opisthorchis viverrini, which can cause cholangiocarcinoma, a kind of cancer in the bile ducts.The intestinal fluke Haplorchis taichui is a possible agent of irritable bowel syndrome-like symptoms, and Centrocestus formosanus may cause epigastric pain and indigestion accompanied by occasional diarrhea (Watthanakulpanich et al. 2010, Sripa et al. 2010, Chai et al. 2013).The prevalence of human trematode infections of the mentioned species was found to be the highest in the northern and northeastern regions of Thailand (Srisawangwong et al. 1997, Pungpak et al. 1998, Radomyos et al. 1998, Sukontason et al. 1999).In Northeast Thailand alone, for example, about six million people are infected with the liver fluke, O. viverrini (Shin et al. 2010).As Thailand has the highest incidence of cholangiocar cinoma associated with O. viverrini (Sripa et al. 2007), opisthorchiasis received greater attention for research than infection with the minute intestinal flukes, such as Haplorchis taichui, for which no such associations have been documented.Nevertheless, Thai people have considerably underestimated these trematodes in the past by continually eating traditional Thai food prepared from raw freshwater fish (Chuboon et al. 2005).Hence, the prevalence of trematodes in Thailand remains a continuous problem (Krailas et al. 2014).
Trematodes often have very complex life cycles involving at least one, sometimes two or four, but usually three different hosts, of which the first is almost always a mollusc (Galaktionov and Dobrovolskij 2003).Eggs are released by the definitive host and either the first larval stage, i.e. the miracidium, hatches from the egg in a suitable medium (usually water) being adapted for actively recognizing and penetrating the first intermediate host; or the miracidium remains embryonated within the egg and infects the first intermediate host through passive uptake and subsequent hatching and penetration within the host.The miracidium develops directly into a (mother) sporocyst that may produce daughter sporocysts or rediae (sometimes rediae also produce a second generation of rediae).Another larval form, i.e. the cercariae, then develops either within the sporocyst or within the redia in the first intermediate host and is typically released into the environment where it either actively searches and penetrates the host or is passively taken up.Within the second host cercariae encyst and develop into metacercariae.Through predation metacercariae are taken up by the definitive host and then develop into the adult trematode completing the life cycle.Deviations from this typical life cycle occur either in the number of different life cycle stages that actually develop or in the number of hosts involved in the development (for a detailed overview, see Galaktionov and Dobrovolskij 2003).
In the present study, the cercarial fauna of Tarebia granifera populations from Thailand is investigated.This thiarid species is widespread in the Oriental region, with an autochthonous range including South and Southeast Asia, South China and numerous islands of the Western Pacific (Brandt 1974, Glaubrecht 1996).The species has been introduced to Africa, the Near East, North and Central America as well as to the Caribbean region and is considered to be invasive there (Abbott 1952, Chaniotis et al. 1980, Prentice 1983, Vargas et al. 1991, Férnandez et al. 1992, Gutierrez et al. 1997, Pointier et al. 1998, Appleton 2002, Mukaratirwa et al. 2005, Facon and David 2006, Appleton et al. 2009, Miranda et al. 2010, 2011, Miranda and Perissinotto 2012).A parallel study on Tarebia granifera (also published in this journal; see Veeravechsukij et al. 2018) shows this species to be widely distributed throughout Thailand, with several named and described congeneric constituent populations, as is revealed by respective collections carried out in the North, Northeast, South, East, and Central region, and morphological documentation conducted detailing the biometrical parameters of the adult shells.In addition, molecular phylogenies using fragments of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and 16 S rRNA genes have been constructed, as well as the reproductive strategy documented (i.e. the various stages of embryos and juveniles in the brood pouch) and analysed as to the effect of cercariae infection in female snails.
Here we apply, aside from traditional morphological methods, molecular genetic techniques in order to delimit species of cercariae; i.e. sequencing parts of the nuclear ribosomal RNA gene cluster that have been shown to be efficient for the identification of species of trematodes from their distinct life stages (Skov et al. 2009, Prasad et al. 2011, Davies et al. 2015, Anucherngchai et al. 2016, 2017).With this combination of molecular phylogeny and the parasites' data on morphology and geographical occurrence, we attempt to provide the groundwork for future studies determining the parasite's evolutionary potential within the complex snail-host relationship.

Sampling
Specimens of Tarebia granifera were collected in streams, ponds, rivers, brooks, trenches and mountain creeks in all major regions of Thailand (North, South, East, Central and Northeast).Geographic coordinates (WGS84 datum) of sampling sites were determined with the global positioning system (GPS) (Garmin PLUS III, Taiwan).Where GPS data for sampling sites were unavailable, coordinates were determined as accurately as possible from a map.Sampling sites were mapped on a dot-by-dot basis on a public domain map (ArcGIS, Esri, Redlands, California, USA) and then compiled using Photoshop CS6 (Adobe Systems, San Jose, California, USA).

Collection methods and determination of snails
Snail collections were done during two periods.In the first period, from 2004 to 2009, the snails were collected every two months for one year from each of all the locations.During the second period, from 2014 to 2016, the same localities were visited again, but additional samples were also taken at several new localities, this time collected once only from each location.The snails were collected using the counts per unit of time sampling method (Olivier and Schneiderman 1956).Five researchers collected samples by handpicking and scooping every 10 minutes at each sampling site.The snails were transferred and studied in the laboratory of the Parasitology and Medical Malacology Research Unit, Silpakorn University, Nakhon Pathom, Thailand (PaMaSU: code SUT).The snails were identified according to their shell morphology , following essentially Brandt (1974), and subsequently examined for trematode infections.

Cercarial study
Collected snails were investigated for trematode infections by using shedding and crushing methods.Descriptions of their morphology were based on living cercariae that had escaped from the snails.The emerged cercariae were studied unstained or vitally stained with 0.5% neutral red.Details of the cercariae were drawn using a camera lucida and identified according to Schell (1970), Yamaguti (1975), Ito (1980) and Krailas et al. (2014).Sample measurements (average size) in micrometers were taken, using an ocular micrometer, from 10 specimens fixed in 10% formalin.Some cercariae (c.20 specimens from each location) belonging to identified trematode species were then preserved in 95% ethanol for further DNA analysis.

Molecular study of cercariae
The preserved cercariae were processed for molecular identification at the Department of Animal Diversity, Zoological Museum of the Center for Natural History (CeNak), Universität Hamburg, Germany.Genomic DNA from the cercariae was extracted using the DNeasy blood and animal tissue kit (QIAGEN, Venlo, The Netherlands).Amplification by polymerase chain reaction (PCR) of the nuclear internal transcribed spacer 2 (ITS2) region were performed with the following primers ITS2-F (5'-CTT GAACGC ACA TTG CGG CCA TGG G-3') and ITS2-R: (5'-GCG GGT AAT CACGTC TGA GCC GAG G-3') (Sato et al. 2009).
Reactions were set up in 20 μl volumes containing 1.0 µl dNTPs (2 mM each), 2.0 µl 10× mM DreamTaq Green buffer (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.3 µl GreenTaq DNA polymerase (5 U/µl, Thermos Fisher Scientific), 1.0 µl of each primer (10 μM) and 14.7 µl ddH 2 O.The DNA samples were initially denatured at 94 °C for 4 min followed by 35 cycles (denaturation at 94 °C for 1 min, annealing at 60 °C for 30 s, and elongation at 72 °C for 2 min; see Sato et al. 2009) and a final elongation step at 72 °C for 7 min.The PCR products were purified according to the protocol for enzymatic PCR product clean-up with exonuclease I (20 U/µl, Thermo Fisher Scientific) and FastAP thermosensitive alkaline phosphatase (1 U/µL, Thermo Fisher Scientific).Purified PCR products were sequenced at Macrogen Europe Lab.(Amsterdam, The Netherlands).Alignments of forward and reverse strands were conducted using Geneious 10. 1.3 (Biomatters Ltd.,Auckland,New Zealand).The ITS2 consensus sequences were aligned in MEGA 7 (Kumar et al. 2016) using MUSCLE (Edgar 2004) under default settings.A Neighbor joining (NJ) analysis was performed based on p-distances with 1,000 bootstrap replicates.For details on sequences used for this study, see Table 1.

Geographical origin of collected snails
Specimens of Tarebia granifera were found at 90 sampling sites in five regions of Thailand (Fig. 1).During the first sampling period (2004)(2005)(2006)(2007)(2008)(2009), infected snails were reported from 18 sampling sites.In the second period (2014)(2015)(2016), infected snails were reported from 51 sampling sites.At a total of 58 localities in four regions of Thailand snails with cercarial infections were found.For information on sampling sites including geographic coordinates and the number of infected snails, see Table 2.

Occurrence of trematodes obtained from Tarebia granifera in Thailand
The various trematode cercariae (distinguished and described in more detail below) exhibit a certain geographical pattern within the various water bodies in Thailand.Only two among the fifteen trematode species found in the thiarid T. granifera, viz.Loxogenoides bicolor and Stictodora tridactyla, were recorded in the present study from almost all major river systems in Thailand (Fig. 2).
In contrast, several species exhibit a more restricted distribution.For example, Haplorchis taichui was only detected in T. granifera samples from the Nan River (Chao Phraya river system) and the Loei River (Mekong river system), whereas Philophthalmus gralli and gymnocephalous cercaria were only detected in the Phachi River (Mae Klong river system).Echinostome cercaria were only present in the T. granifera population from the Khek River (Chao Phraya river system).
Cercariae of Loxogenes liberum, Centrocestus formosanus and Maritreminoides obstipus had again a somewhat wider distribution in Thai T. granifera populations, being present in several rivers of the Chao Phraya, Mae Klong and Gulf of Thailand drainages (Fig. 2).
In this study, neither double trematode infections nor triple trematode infections of collected Tarebia granifera were found.

Morphology of infecting cercariae
The cercariae were categorized by their morphology and organ characters, using as reference previous morphological descriptions (e.g.Schell 1970, Yamaguti 1975, Frandsen and Christensen 1984, Krailas et al. 2014).They are described as follows for the eight distinct morphological cercarial types known and found to date, attributable to at least seven distinct trematode families.Body oval; throughout with granules.Oral sucker bigger than ventral sucker; globular in shape and with stylet.Virgulate organ in the anterior part of the body.Pharynx small; an esophagus was not observed.Three pairs of penetration glands present located at about two thirds of the body, two anterior pairs with fine granules and a posterior pair with rather coarse, dark granules.Genital primordial C-shaped; excretory bladder U-shaped.Tail shorter than body; spinose at its tip.The infection rate was 0.05% (7/15,076) (Table 3).Size range and average size (in micrometers, calculated from 10 cercariae):   The cercarial body is pear-shaped.It has a circular oral sucker that is located near the proximal end of the body.
The mouth is equipped with transverse rows of spines.
The small ventral sucker is located approximately at two-thirds of the body length measured from the front.The small pharynx is situated in the anterior part of the body just distal of the oral sucker between the two distinct eyespots; an esophagus is absent.There are seven pairs of penetration glands, which are arranged laterally in two longitudinal rows in the posterior two thirds of the body.The excretory bladder has an oval shape and is dark pigmented.A genital primordium is present, located between the ventral sucker and the excretory bladder.
The tail is longer than the body and rather slender, and is equipped with lateral finfolds proximally and a dorsoventral finfold along the longer distal portion.
The cercariae develop within rediae.
The infection rate was 0.72% (108/15,076) (Table 3).Size range and average size (in micrometers, calculated from 10 cercariae):   Body is oval in shape.The oral sucker is located at the anterior of body.The mouth aperture is equipped with transverse rows of spines.A pair of pigmented eyespots and pharynx are present.Seven pairs of penetration glands extend from the pharynx to the posterior end of the body.Cystogenous cells are arranged in lateral fields from the level of the pharynx to the posterior end of the body.The excretory bladder is saccular and thick-walled.The tail is longer than the body.There are lateral finfolds at one-third of tail tunk and a dorso-ventral finfold at the distal portion.
The cercariae develop within rediae.
The infection rate was 0.09% (14/15,076) (Table 3) Size range and average size (in micrometers, calculated from 10 cercariae): Body 43-83 µm (mean: 61 µm) × 105-140 µm (mean: 120 µm) four groups of 3:4:4:3 are present that are situated between the pharynx and the excretory bladder.A pair of pigmented eyespots and a pharynx are present.The ventral sucker is poorly developed.The excretory bladder is V-shaped and thick-walled.The tail is longer than the body.There is a bilateral finfold and a dorso-ventral finfold on the tail.
The cercariae develop within rediae.
The infection rate was 2.92% (440/15,076) (Table 3).The body is oval in shape.The oral sucker has oral spines or rostellar hooks like a tapeworm on the dorsal wall of the mouth aperture.A pair of eyespots is located above the prenetration glands at the same level as the pharynx.There are seven pairs of penetration glands.The genital primordial is elongated-triangular and located between the ventral sucker and the excretory bladder.The excretory bladder has dark granules and is thin-walled.The tail is slender and longer than the body.It is equipped with very narrow finfolds.
The cercariae develop within rediae.
The infection rate was 1.14% (172/15,076) (Table 3).The body is elongate pear-shaped and distinctly granulose.Eyespots are absent.The pharynx is large and extends into an esophagus that is bifurcating (Y-shape) into two blind ending intestinal caeca that almost reach the posterior end of the body.The ventral sucker is bigger than the oral sucker.The excretory bladder is rather small.The tail is about as long as the body and relatively slender.There is an adhesive gland present at its tip.The cercariae encyst rapidly after developing within rediae.
The infection rate was 0.02% (3/15,076) ( (Fig. 13) The body is elongate-oval, slightly bent.Eyespots, a pharynx, an esophagus, intestinal caeca and a ventral sucker are absent.There is a narrow dorsal finfold in the middle part of the body.The penetration gland is located in the anterior part of the body.The excretory bladder is small and thin-walled, located at the posterior end of the body.The tail is forked.The stem of the tail is rather thick and longer than the furcae.Finfolds are present along the margins of the furcae.The cercariae develop within sporocysts.
The infection rate was 0.05% (7/15,076) (  The body is elongate-oval in shape.A pair of unpigmented eyespots is present.A prepharynx is present but rather short.The pharynx is small and roundish in shape.The esophagus is long, bifurcating into two intestinal caeca that are shorter than half the length of the esophagus.The oral sucker is larger than the ventral sucker.There are two pairs of penetration glands, filled with dark granules that are located around the ventral sucker.There is a Y-shaped excretory bladder located medially close to the posterior end of the body.The tail is longer than the body and divided into two furcae.The tail stem is slender and about as long as the furcae.
The cercariae develop within sporocysts.
The infection rate was 0.15% (23/15,076) (  The body is of a bowl-like shape.The surface of the body is covered with spines that have the appearance of fish scales.
The genital pore of the seminal vesicle is located in the anterior part of the body.Eyespots are present.The mouth is located near the ventral sucker.The esophagus is narrow and the intestinal caeca form a ring.There is one pair of testes present, and an ovary is located anterolateral to the left of the testes.The excretory bladder is small and short, and is situated close to the posterior end of the body.The tail is longer than the body and possesses spatulate furcae.At the base of the tail a pair of bilaterally symmetrical appendages is present, each equipped with an adhesive pad at its distal end.
The cercariae develop within rediae.
The infection rate was 0.05% (8/15,076) (Table 3).Type 7. Echinostome cercariae (Fig. 16) The body is elongate pear-shaped.Eyespots are absent.The oral sucker is circular in shape and is equipped with collar spines.The prepharynx is long.The esophagus is shorter than the prepharynx, bifurcating into two intestinal caeca that almost reach to the posterior end of the body.The relatively large ventral sucker is located approximately at two-thirds of the body length measured from the front.Penetration glands are absent.The excretory bladder is small and triangular in shape, its two main collecting tubes beginning at the level of the esophagus.The tail is slender and almost of the same length as the body.
The cercariae develop within rediae.
The infection rate was 0.07% (10/15,076) (Table 3).The body is oval and covered with spines.The terminal oral sucker is equipped with minute spines.Eyespots are absent.
The prepharynx is long and thin.The pharynx is rather large and of a round shape.The esophagus is short but rather wide, bifurcating into two intestinal caeca that extend towards the posterior part of the body.There are 4-5 penetration glands present that are located laterally of the caeca between the level of the pharynx and the ventral sucker.The ventral sucker is of about the same size as the oral sucker.The excretory bladder is roundish, with a thin wall and located medially near the posterior end of the body.Two thin, undulating excretory tubules that begin just anterior of the pharynx insert into the excretory bladder.The tail is longer than the body, with the opening duct of the excretory bladder located at its end.There are groups of of 3-5 distinct pigment granules present in the tail but flame cells could not observed.
The cercariae develop within rediae.

Molecular analysis
In the present study, ITS2 sequences from nine distinct cercarial types (collected during the second period of this study) of a total of fifteen trematode species found in Thai populations of Tarebia granifera could be amplified by PCR and sequenced.The ITS2 sequences of the virgulate xiphidiocercariae and the armatae xiphidiocercariae had a length of approximately 320 bp, while the ITS2 sequences of the parapleurophocercous cercariae and the pleurophocercous cercariae had a length of approximately 380 bp.The ITS2 sequences of the remaining cercarial types, i.e. megarulous cercariae, echinostome cercariae and gymnocephalous cercariae, had a length of approximately 500 bp.
The phylogenetic tree obtained from the neighbor-joining analysis (Fig. 18) was rooted with the nematode Angiostrongylus cantonensis (Chen, 1935) (GenBank accession number: HQ540551.1).All trematode species from Thai populations of T. granifera that were distinguished on the basis of cercarial morphology and for which more than one sequence was obtained, formed well supported groups in the phylogenetic analysis.These are highlighted in the following: -Specimens of S. tridactyla, C.  sa et al. 2007, Ukong et al. 2007, Krailas et al. 2011, 2014).Furthermore, the phenotypically highly diverse and, thus, systematically problematic thiarid snails are widely distributed in Southeast Asia and Australasia (e.g.Glaubrecht 1996, 2009, 2011, Glaubrecht et al. 2009).
This not only renders them most suitable objects for various systematic, biogeographical and evolutionary studies but also brings them into special focus from a parasitological perspective.
The present study aimed at bringing together the classical parasitological approach of the morphological characterization of the cercariae stages of trematodes obtained from their snail host, with a molecular parasitology approach, presenting a phylogenetic analyses of the minute intestinal flukes identified from their thiarids host, exemplified here for the first time with Tarebia granifera from Thailand.This particular snails host is common in many Thai freshwater systems, inhabiting rivers, lakes, streams and ponds (Hyslop 2003).Pillay and Perissinotto (2008) recorded that T. granifera was also able to colonize moderately saline habitats (brackish water).Without doubt, therefore, this thiarid is well established as an intermediate host for several species of trematodes.
We here focussed on the larval trematode infections found in this snail collected in various regions in Thailand during two periods of field work.When we started the research in the first period (2004)(2005)(2006)(2007)(2008)(2009), T. granifera was found in 18 sampling sites.In the second period (2014-2016), we not only went back to the same sampling sites but also added samples from new locations.Thus, snails from a total of 72 locations could be analysed from all over Thailand, covering for the first time most of the distributional range of the snail host T. granifera in this country.In more than two-thirds of these locations, i.e. in populations at 51 sampling sites, infected snails were found, indicating the wide prevalence of these trematodes in Thailand.
As we mentioned above, only three species of trematodes, viz.L. bicolor, S. tridactyla, C. formosanus, were found to commonly occur in Tarebia granifera from most river systems and regions in Thailand.They were also found during all seasons, thus independent of the time of the year the snails were collected.By re-visiting during the years 2014 to 2016 the same locations of the first collecting period five to ten years earlier, and recording infected snails in 18 of these sampling sites, we also found that these trematode infections are apparently long-lasting, in the sense of a permanent phenomenon of these snail host populations, despite seasonal variation in the abundances of plants and animals in general (Shimadzu et al. 2013).Among the total of 15 species from 8 types of cercariae recorded in our study, we found only half of them (i.e. 8 species from 4 types) during the first period; whereas 11 species from 7 types were found in the second period.Thus, with the new study period and with collecting at various other and thus new locations all over Thailand we were able to expand our knowledge with respect to the taxonomical and geographical aspects of this analysis.
In the following we discuss in more details various aspects for the distinct trematode species found in their Thai thiarid snail host Tarebia granifera: Parapleurophocercous cercariae and pleurophocercous cercariae were reported to be commonly found also in other freshwater snails in Thailand, such as e.g.Melanoides tuberculata (Krailas et al. 2014).In this study, three species of parapleurophocercous cercariae and one species of pleurophocercous cercariae were found in T. granifera.
Various reports have indicated the presence of parapleurophocercous cercariae and some species of pleurophocercous cercariae of the intestinal trematodes Heterophyidae, such as H. taichui, H. pumilio, S. tridactyla and C. formosanus (e.g.Chontananarth andWongsawad 2013, Waikagul andThaekham 2014).These parasites have an aquatic life cycle, using freshwater snails as the first and cyprinid fish as the second intermediate host, with definitive hosts being fish-eating mammals and humans (Nithikathkul and Wongsawad 2008, Krailas et al. 2011, 2014, 2016).
In this study, we found human trematodes, viz.H. taichui, H. pumilio, S. tridactyla and C. formosanus.Especially the snail infections by the minute intestinal fluke of S. tridactyla (2.92%) and C. formosanus (1.14%) showed a high level of prevalence in Thailand.In addition, H. taichui is important for public health, as was shown in several studies.For example, Kumchoo et al. (2005) reported high prevalence of fish as being the second intermediate host (91.4%) of H. taichui from Mae Taeng district of Chiang Mai province.Also, in the PDR Laos many patients have been infected by H. taichui, as cases were reported with mucosal ulceration, chronic inflammation and fibrosis of submucosa (Sukontason et al. 2005, Sohn et al. 2014).Chai et al. (2013) reported for seven patients who were infected by C. formosanus in Laos that they had abdominal pain, indigestion and diarrhea.Chung et al. (2011) reported the first case in Korea for patients being infected by H. pumilio.This heterophyid trematode is an important and continuing public health problem in many countries, as there are case reports not only from Southeast Asia but also from other Asian countries.
Therefore, it is from this perspective that for the epidemiology of zoonosis in general we recommend the study of snail intermediate hosts of human and animal trematode infections.It would be interesting to study whether there are geographically related higher or lower incidenc-es of human infections, perhaps also correlated to infected fishes in these areas.
In contrast, known as parasites to animals only, xiphidiocercariae can be distinguished by their stylet organ in the mouth part of the cercariae.They can be divided into two morphological types, the first type being the virgulate xiphidiocercariae, and the second type the armatae xiphidiocercariae (see e.g.Frandsen and Christensen 1984).The virgulate xiphidiocercariae have a virgular organ present in the region of the oral sucker.For this group, the present study reported three species of parasites from the Lecithodendriidae, viz.L. bicolor, L. liberum and A. histaense, for which the hosts are amphibians (Brooks et al. 1985).It should be noted that we found L. bicolor to have the highest prevalence, with an infection rate of 3.84 %, and to be distributed in every water body, river system and region of Thailand.In the second group, i.e. the armatae xiphidiocercariae, the cercariae do not possess a virgular organ.For this group, we here reported two species, viz.M. caridinae and M. obstipus of the Microphallidae, which are parasites in birds as definite hosts.For the time being, we refrain from speculating on what might cause these differences before more detailed studies will be done.
Megarulous cercariae have been morphologically characterized as belonging to Philophthalmus.This parasite is commonly known as the oriental avian eyefluke and it had been reported in connection with human accidental infections (Waikagul et al. 2006, Derraik 2008).Nollen and Murray (1978) reported that P. gralli parasitized the conjunctival sac of various galliform and anseriform birds.This fluke was also found in ostriches, causing conjunctivitis.In earlier studies the cercariae of this trematode were found in the thiarid snail Melanoides tuberculata as intermediate host (Kalatan et al. 1997, Pinto and de Melo 2010, Krailas et al. 2014).In this study, we found P. gralli now also in the thiarid Tarebia granifera from the Phachi River in western Thailand.This river system originates in the Tenasserim mountain range and tributes to the Mae Klong river system to the east of it.
Furcocercous cercariae are generally from trematodes of the Sanguinicolidae; they develop to cercariae in brackish-water and freshwater snails, while the definitive hosts were found in fishes.In this study, we found cercariae of three species, viz.C. alseae, A. mustelae and T. laruei, to parasitize Tarebia granifera as intermediate host.Cercariae of all three trematode species were also found in other thiarid snails, as they were reported in Melanoides tuberculata (Krailas et al. 2014, Anucherngchai et al. 2017).
Echinostome cercariae are distributed throughout Southeast Asia (Chai 2009).Most species mainly parasitize avian hosts, such as migratory birds, but sometimes also infect mammals including humans.The echinostome trematodes are associated with the ingestion of raw snails and amphibians that transmit metacercariae as the infective stage (Esteban and Antoli 2009).In the present study, echinostome cercariae were found in Tarebia gran-ifera populations from the north of Thailand only; which corroborates the report by Nithikathkul and Wongsawad (2008) that echinostomiasis cases have been commonly found in the north and northeast of Thailand.
Gymnocephalous cercariae are small larval stages of trematodes, in general attributed to the Fasciolidae (e.g.Schell 1970).In this study, we found only one snail infection with cercariae that morphologically are obviously attributable to Fasciola cercariae.However, the molecular identification showed that these cercariae were actually neither F. gigantica nor F. hepatica.Instead, the phylogentic analyses indicate a closer affinity of these sequences to those from cercariae with echinostoma type.By morphology the echinostome cercariae are clearly distinguishable by being elongated spinose with a reniform collar, armed with a single or double row of spines surrounding the dorsal and lateral margins of the oral sucker (Anucherngchai et al. 2016, Ayoub et al. 2017).Thus, our study here revealed one case of obvious conflict between the morphologically based identification and the molecular indication of affinity, which clearly is in need to be studied further.We cannot exclude the possibility of a simple laboratory mix-up, but should also keep in mind e.g.hybrid effects.
In a previous report, the gymnocephalous cercariae were produced by trematodes of the Fasciolidae.They were found in Biomphalaria sp., Bulinus sp., Ceratophallus sp., Gabbiella sp., Gyraulus sp., Lymnaea sp., and in Melanoides sp.(Frandsen and Christensen 1984).However, thiarid snails were never reported with fasciolid trematode infections in Thailand.Even though, the morphology of gymnocephalous cercariae was obviously to be Fasciola cercariae.The sequence of DNA was shown to match with that from the group of echinostome cercariae.

Molecular analyses of cercaria and their host correlations
In general, morphological as well as molecular studies of cercariae were able to confirm the specific identity and prevalence of various infectious trematodes in Thai freshwater snails of Tarebia granifera.In this study, we found that the ITS2 marker allowed to distinguish a total of nine trematode species, with the cercariae attributable to seven of the morphologically distinguishable types, viz. the parapleurophocercous cercariae, pleurophocercous cercariae, the virgulate xiphidiocercariae and armatae xiphidiocercariae, megarulous cercariae, as well as echinostome cercariae and gymnocephalous cercariae; only the furcocercous cercariae were not available for molecular studies.And in one case only we found a conflict insofar, as the sequence data revealed that cercariae of the morphologically distinguished gymnocephalous type grouped closely with those of the echinostome type.
We also used the ITS2 marker for a phylogenetic reconstruction (Fig. 18), in which two characteristics are most noteworthy: First, while the relationships of species within molecular clades found could not be resolved robustly, our analyses revealed, second, two well-supported major molecular clusters or groups.These clusters can be well interpreted in context of their respective zoonotic parasites and human pathogens.
The first group with parapleurophocercous and pleurophocercous cercariae, respectively (marked f and g in Fig. 18), i.e. S. tridactyla, C. formosanus, Centrocestus sp., H. taichui, H. pumilio, O. viverrini, O. felineus, all have cyprinoid fish as second intermediate host, while birds and mammals, including in particular humans, are the definite host.Note that the latter two trematode species have a bithyniid instead a thiarid snail as first intermediate host.
In a second group cluster trematode species with virgulate xiphidiocercariae and armatae xiphidiocercariae, respectively (marked a and b in Fig. 18), i.e.L. bicolor, L. liberum, Lecithodendrium spathulatum, L. linstowi and M. obstipus), which all have arthropods (Insecta or Crustacea) as second intermediate hosts while amphibians, birds and mammals, but with the exclusion of humans, are the definite hosts.
In addition, also the sequences of trematode species with echinostome cercaria and the gymnocephalous cercaria obtained from T. granifera grouped together with relatively high support.However, no clear picture as to a correlation with their second intermediate hosts and definitive hosts is visible to date, as we lack knowledge on the latter in particular for the gymnocephalous cercaria.Nevertheless, the latter two form a well-supported clade together with P. gralli, F. hepatica and F. gigantica, which all have gymnocephalous, echinostome or megarulous cercariae (Fig. 18, c,d,e).Note that the latter two trematode species are known to have an eupulmonate instead of a thiarid snail host.Interestingly, in this latter monophyletic clade, formed by P. gralli together with F. hepatica and F. gigantica, only those trematodes are known to be human pathogens (as definite hosts) when the cercaria are encysting in the open instead of parasitizing a second intermediate host; see Fig. 18.
We anticipate that more detailed studies, based on molecular phylogenetic analyses, looking into these and other correlations of intermediate hosts, their regional occurrences and ecological specifics will shed more light on the evolutionary potential of trematode parasites from thiarid snails.

Conclusion and outlook
To date, studies on freshwater snails and their interactions with parasitic trematodes are under-represented worldwide (Adema et al. 2012).There is an urgent need for collaboration bringing together deeper understanding on the basic biology, biodiversity, and evolutionary associations of parasitic trematodes on the one hand and their snail hosts on the other, i.e. those studying parasitology and malacology, taking advantage of their respective expertise in host-parasite interactions and evolutionary systematics.
Accordingly, the aims of this approach presented here were to establish reliable and reproducible data for the morphological identification as well as the methodology for the extraction of high quality DNA from preserved trematode cercariae in specifically known populations of their thiarid snails hosts (including museum samples collected several years ago).It was also the aim to conduct a phylogenetic analysis of the minute intestinal flukes.In addition, the present paper adds to a more in-depth evolutionary systematic analysis with data on reproductive biology, geographical distribution, morphology and molecular phylogenies of T. granifera.
Using this combinational approach, it will eventually be possible to identify in more details the host-parasite relationships of thiarid snails as first intermediate host populations not only in Thailand, and also to determine the role of parasitic infections in these gastropods and as human pathogens.
The body is oval in shape.The oral sucker is located at the anterior end of the body.There are three transverse rows of oral spines present.Seven pairs of penetration glands in 3

Figure 18 .
Figure 18.Neighbor-joining tree on the basis of ITS2 sequences of cercarial species obtained from Thai populations of Tarebia granifera (Lamarck, 1816) and several published sequences obtained from GenBank.Nodes are annotated with bootstrap support values ≥ 50.Taxon names and voucher or GenBank accession numbers are provided at the tips of the tree (see also Table 1).First and second intermediate hosts and definitive hosts are indicated (see legend).Abbreviations -DH: definitive host: IH1: first intermediate host; IH2: second intermediate host.Cercarial types -a: virgulate xiphidiocercariae; b: armatae xiphidiocercariae; c: gymnocephalous cercariae; d: echinostome cercariae; e: megarulous cercariae; f: parapleurophocercous cercariae; g: pleurophocercous cercariae.

Table 1 .
List of ITS2 sequences used for the phylogenetic analysis.For SUT numbers, see the material lists in the main part of the text.

of cercariae Type of cercariae Locality GenBank accession number Reference
Body oval.Oral sucker at the anterior end of body, with stylet.Virgulate organ present.Ventral sucker roundish, smaller than oral sucker.Pharynx very small, a prepharynx, an esophagus and ceca were not observed.Four pairs of penetration glands present, located near the middle of the body; the two anterior pairs with fine granules and the two posterior pairs with coarse granules.Excretory bladder V-shaped.Tail shorter than body, rather slender and spinose at its tip.The cercariae develop within sporocysts.

Table 3 )
. Size range and average size (in micrometers, calculated from 10 cercariae):

Table 3 )
. Size range and average size (in micrometers, calculated from 10 cercariae):

Table 3 )
. Size range and average size (in micrometers, calculated from 10 cercariae):

Table 3 )
. Size range and average size (in micrometers, calculated from 10 cercariae):
intestinal and hepatic systems not only in some domestic animals but also in humans.As outlined in the Introduction, this represents a serious threat to public health.For example, thiarid snails such as Melanoides tuberculata, T. granifera, Mieniplotia scabra and Sermyla riqueti have been reported as the intermediate hosts of a wide array of diverse trematodes, such as Haplorchis pumilio, H.