Zoosyst. Evol. 94 (2) 2018, 425-460 | DOI! 10.3897/zse.94.28793 Zieh Breas eS ieODS BRT AVS ____ap__. D> PENSOFT. an Nitee A NATURKUNDE BERLIN Exploring the evolutionary potential of parasites: Larval stages of pathogen digenic trematodes 1n their thiarid snail host Zarebia granifera in Thailand Nuanpan Veeravechsukij', Suluck Namchote!, Marco T. Neiber*, Matthias Glaubrecht?, Duangduen Krailas! 1 Parasitology and Medical Malacology Research Unit, Department of Biology, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand 2 Center for Natural History (CeNak), Zoological Museum, Universitat Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany http://zoobank. org/54F23E BE-F115-4F 12-8 D82-B86973CC3C6B Corresponding author: Matthias Glaubrecht (matthias.glaubrecht@uni-hamburg.de) Abstract Received | August 2018 Minute intestinal flukes from several distinct families of endoparasitic platyhelminths Accepted 27 September 2018 are a medically important group of foodborne trematodes prevalent throughout South- Published 8 November 2018 east Asia and Australasia. Their lifecycle is complex, with freshwater snails as prima- ry intermediate hosts, with infecting multiple species of arthropods and fish as second Academic editor: intermediate hosts, and with birds and mammals including humans as definitive hosts. Andreas Schmidt-Rhaesa 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 7arebia Key Words 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 Trematoda during 2014—2016 when snails from the same localities were collected and new localities Cerithioidea found. From ninety locations a total of 15,076 T. granifera were collected and examined Thiaridae for trematode infections. With 1,577 infected snails the infection rate was found to be human health 10.46 %. The cercariae were categorized into fifteen species from eight morphological- cercariae ly distinguishable types representing several distinct families, viz. (1) virgulate xiphid- intermediate hosts iocercariae (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 tridac- tyla); (iv) pleurophocercous cercariae (Centrocestus formosanus), (v) megarulous cer- cariae (Philophthalmus gralli), (vi) furcocercous cercariae (Cardicola alseae, Alaria mustelae and Transversotrema laruei); as well as (vii) echinostome-type cercariae, and (vill) gymnocephalous-type cercariae. In addition, a phylogenetic marker (internal tran- scribed spacers 2, ITS2) was employed in generic and infrageneric level classifications of these trematodes, using sequences obtained from shed cercariae isolated from 7. gran- ifera 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 spe- cies 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. Copyright Nuanpan Veeravechsukij et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 426 Introduction Trematodes (or flatworms) are endoparasitic platyhel- minths that not only infect fishes, birds and other wildlife worldwide but also mammals as well as humans. As food- borne 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., Parag- onimus spp. and the minute intestinal trematodes such as Fasciolopsis buski, Heterophyes spp. and Metagonimus spp., are estimated to be 2.02 million worldwide (Torg- erson 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 con- taminated 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 tai- chui 1s a possible agent of irritable bowel syndrome-like symptoms, and Centrocestus formosanus may cause epi- gastric pain and indigestion accompanied by occasion- al diarrhea (Watthanakulpanich et al. 2010, Sripa et al. 2010, Chai et al. 2013). The prevalence of human trem- atode 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 mil- lion people are infected with the liver fluke, O. viverrini (Shin et al. 2010). As Thailand has the highest incidence of cholangiocarcinoma associated with O. viverrini (Sri- pa 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 trema- todes 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 in- volving 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, 1.e. the miracidium, hatches from the egg in a suit- able medium (usually water) being adapted for actively recognizing and penetrating the first intermediate host; zse.pensoft.net Veeravechsukij, N. et al.: Trematodes in Thai Jarebia granifera or the miracidium remains embryonated within the egg and infects the first intermediate host through passive up- take 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, 1.e. the cercariae, then de- velops 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 metacer- cariae. Through predation metacercariae are taken up by the definitive host and then develop into the adult trem- atode 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 over- view, see Galaktionov and Dobrovolskij 2003). The occurrence of trematodes depends on the pres- ence of first and second intermediate host species, as well as the eating habit of local people (Radomyos et al. 1998). In Thailand, medically-important freshwa- ter snails have been investigated since 1980 for trem- atode infections (Upatham et al. 1980, Nithiuthai et al. 2002, Krailas et al. 2003, 2008, 2014, Sri-aroon et al. 2005, Dechruksa et al. 2007, 2013, 2017, Ukong et al. 2007). For example, the liver fluke Opisthorchis viver- rini (Family: Opisthorchiidae) is found in Thailand in freshwater snails Bithynia funiculata, B. siamensis go- niomphalos and B. siamensis siamensis (Bithyniidae). However, despite the importance of the snail interme- diate host(s) to the lifecycle of trematodes, the faunistic and biosystematic knowledge of these limnic molluscs is scarce in general. In particular, among the Cerithioidea which is ecologically and phylogenetically a highly im- portant caenogastropod group of molluscs (Glaubrecht 1996, 2009, 2011; Strong et al. 2011), several freshwater gastropods are known especially in the Thiaridae Gill, 1871 to be important first intermediate hosts of trema- todes. For example, species of the intestinal lung fluke Paragonimus have been identified in paludomids and/ or thiarids, such as e.g. species of Paludomus as well as in Melanoides tuberculata and Tarebia granifera. Pinto and de Melo (2011) list 37 species of cercariae and an- other 81 trematode larval forms for Melanoides tubercu- lata Miller, 1774. For Thailand Brandt (1974) lists five snail species, viz. Melanoides tuberculata, M. jugicostis Hanley & Theobald, 1876, Sermyla riqueti Grateloup, 1840, Neoradina prasongi Brandt, 1974 and Tarebia granifera Lamarck, 1816 (see Lamarck 1816), that are currently assigned to the Thiaridae (Glaubrecht 1996, 1999, 2011, Lydeard et al. 2002, Glaubrecht et al. 2009, Strong et al. 2011). Most recently, Krailas et al. (2014) and Dechruksa et al. (2017) investigated the cercarial fauna of M. tuberculata and M. jugicostis populations from Thailand in detail, reporting 18 different cercariae from the former and four from the latter; among them C. Zoosyst. Evol. 94 (2) 2018, 425-460 formosanus, H. taichui, Haplorchis pumilio Looss, 1896 and Stictodora tridactyla Martin & Kuntz, 1955 that are known to be human pathogen (Watson 1960, Malek and Cheng 1974, Upatham et al. 1995, Pointier and Jourdane 2000, Dechruksa et al. 2007, Ukong et al. 2007). In the present study, the cercarial fauna of 7arebia granifera populations from Thailand is investigated. This thiarid species is widespread in the Oriental re- gion, 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 Caribbe- an 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, Point- ier 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 par- allel study on Jarebia granifera (also published in this journal; see Veeravechsukiy et al. 2018) shows this spe- cies to be widely distributed throughout Thailand, with several named and described congeneric constituent populations, as is revealed by respective collections car- ried out in the North, Northeast, South, East, and Cen- tral 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 (cox/) 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; 1.e. sequencing parts of the nucle- ar 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 po- tential within the complex snail-host relationship. Materials and methods 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 427 were unavailable, coordinates were determined as ac- curately 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 col- lected 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 addi- tional 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 ac- cording to their shell morphology , following essentially Brandt (1974), and subsequently examined for trema- tode infections. Cercarial study Collected snails were investigated for trematode infec- tions by using shedding and crushing methods. Descrip- tions of their morphology were based on living cercariae that had escaped from the snails. The emerged cercari- ae 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 speci- mens fixed in 10% formalin. Some cercariae (c. 20 spec- imens from each location) belonging to identified trem- atode 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 Histo- ry (CeNak), Universitat Hamburg, Germany. Genom- ic 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 spac- er 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 ul volumes containing 1.0 ul dNTPs (2 mM each), 2.0 ul 10x mM DreamTaq zse.pensoft.net 428 Green buffer (Thermo Fisher Scientific, Waltham, Mas- sachusetts, USA), 0.3 ul GreenTaq DNA polymerase (5 U/ul, Thermos Fisher Scientific), 1.0 ul of each primer (10 uM) and 14.7 wl ddH,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/ul, Thermo Fisher Scientif- ic) and FastAP thermosensitive alkaline phosphatase (1 U/uL, Thermo Fisher Scientific). Purified PCR prod- ucts were sequenced at Macrogen Europe Lab. (Am- sterdam, 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 (Ku- mar 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. Results Geographical origin of collected snails Specimens of Zarebia granifera were found at 90 sam- pling sites in five regions of Thailand (Fig. 1). During the first sampling period (2004—2009), infected snails were reported from 18 sampling sites. In the second pe- riod (2014—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 co- ordinates 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 geo- graphical pattern within the various water bodies in Thailand. Only two among the fifteen trematode spe- cies found in the thiarid 7! granifera, viz. Loxogenoi- des 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 7’ granifera samples from the Nan River (Chao Phraya river system) and the Loei River (Mekong river system), whereas Philophthalmus gralli and gym- nocephalous cercaria were only detected in the Phachi River (Mae Klong river system). Echinostome cercaria were only present in the 7’ granifera population from the Khek River (Chao Phraya river system). zse.pensoft.net Veeravechsukij, N. et al.: Trematodes in Thai Jarebia granifera Cercariae of Loxogenes liberum, Centrocestus formo- sanus and Maritreminoides obstipus had again a some- what wider distribution in Thai 7? granifera populations, being present in several rivers of the Chao Phraya, Mae Klong and Gulf of Thailand drainages (Fig. 2). Cercarial diversity and infection rates A total of 15,076 snails of 7! granifera were collected and examined for trematode infections. With 1,577 par- asitized snails the overall infection rate was found to be 10.46 %. The obtained cercariae were classified into a total of fifteen species from eight morphologically dis- tinguishable types representing at least seven distinct trematode families, viz. (1) virgulate xiphidiocercariae (Loxogenoides bicolor, Loxogenes liberum and Acan- thatrium histaense), (11) armatae xiphidiocercariae (Mar- itreminoides caridinae and Maritreminoides obstipus), (ii1) parapleurophocercous cercariae (Haplorchis pum- ilio, Haplorchis taichui and Stictodora tridactyla), (iv) pleurophocercous cercariae (Centrocestus formosanus), (v) megarulous cercariae (Philophthalmus gralli), (v1) furcocercous cercariae (Cardicola alseae, Alaria muste- lae and Transversotrema laruei), as well as (vil) echinos- tome cercariae, and (vil) gymnocephalous cercariae. The virgulate xiphidiocercariae were the dominant cercarial type infecting snails (5.10%), while infections with oth- er cercarial types were found at rates of (i1) 0.15%, (111) 3.73%, (iv) 1.14%, (v) 0.02%, (vi) 0.25%, (vil) 0.07%, (vili) 0.01%, respectively; see Table 3 for details. In this study, neither double trematode infections nor triple trematode infections of collected TZarebia granifera were found. Morphology of infecting cercariae The cercariae were categorized by their morphology and organ characters, using as reference previous morpholog- ical descriptions (e.g. Schell 1970, Yamaguti 1975, Frand- sen 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. Type 1. Virgulate xiphidiocercariae cercariae Lecithodendriidae Liihe, 1901 (sensu Odhner 1910) 1.1 Loxogenoides bicolor (Krull, 1933) (sensu Kaw 1945) (Fig. 3) 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. Phar- ynx 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. Zoosyst. Evol. 94 (2) 2018, 425-460 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. GenBank accession 429 Species of cercariae ALIERBes Reference Angiostrongylus cantonensis —— HQ540551 C. Y. Liu (unpubl.) Lecithodendrium spathulatum JF784192 Lord et al. (2012) Lecithodendrium linstowi Xiphidiocercariae - KJ934792 Kudlai et al. (2015) SUT 0515066 B MH991970 This study SUT 0515067 B MH991981 This study SUT 0515077 B MH991985 This study SUT 0515079 C MH991978 This study SUT 0515087 B MH991983 This study SUT 0515090 B MH991976 This study SUT 0516106 A MH991982 This study 5 ee SUT 0516109 B MH991977 This study Loxogenoides bicolor Xiphidiocercariae SUT 05161188 MHOO1084 This study SUT 0516121A MH991974 This study SUT 0516125 A MH991980 This study SUT 0516128 B MH991972 This study SUT 0516129 B MH991979 This study SUT 0516130 B MH991971 This study SUT 0516139 B MH991973 This study SUT 0516145 B MH991975 This study . ee ; SUT 0516109 B MH991986 This study Loxogenes liberum Xiphidiocercariae SUT 0516143 B MH991987 This study 2%, é An By. : SUT 0516124 A MH991988 This study Maritreminoides obstipus Xiphidiocercariae SUT 05161388 MHO91989 This study ry . - KP 165437 Mei et al. (2015) Haplorchis pumilio Parapleurophocercous cercariae - KX815125 Le et al. (2017) SUT 0515090 B MH991968 This study Haplorchis taichui Parapleurophocercous cercariae | SUT 0516125A MH991969 This study - KX815126 Le et al. (2017) SUT 0515058 A MH991962 This study SUT 0515059 B MH991960 This study SUT 0515071 A MH991958 This study SUT 0515072 B MH991953 This study SUT 0515074 B MH991959 This study Stictodora tridactyla Parapleurophocercous cercariae | SUT 0515075 B MH991954 This study SUT OS15078:B MH991963 This study SUT 0515086 A MH991957 This study SUT 0516138 B MH991961 This study SUT 0516139 B MH991956 This study SUT 0516142 B MH991955 This study SUT 0516102 B MH991964 This study ; SUT 0516109 B MH991966 This study Centrocestus formosanus Pleurophocercous cercariae SUT 0516125 A MH991967 This study SUT 0516142 B MH991965 This study M. Karamian, S. Centrocestus sp. Pleurophocercous cercariae JQ390547 M. Sadjjadi and B. Farhangmenhr (unpubl.) Centrocestus sp. Pleurophocercous cercariae AY 245699 Dzikowski et al. (2004) Opisthorchis viverrini Pleurophocercous cercariae AY584735 Parvathi et al. 2008 Opisthorchis felineus Pleurophocercous cercariae EF688141 Katokhin et al. (2008) SUT 0515058 A MH991965 This study Philophthalmus gralli Megarulous cercariae : KF986200 Heneberg et al. (2014) Echinostome cercariae Echinostome cercariae SUT 0515086 A MH991991 This study Gymnocephalous cercariae Gymnocephalous cercariae SUT 0515059 B MH991990 This study Fasciola hepatica Gymnocephalous cercariae AM900370 Ali et al. (2008) M.D. Bargues and S. Fasciola gigantica Gymnocephalous cercariae re Miasiome (unpubl.) AM850108 Ali et al. (2008) zse.pensoft.net 430 The cercariae develop within sporocysts. The infection rate was 3.84% (579/15,076) (Table 3). Size range and average size (in micrometers, calculat- ed from 10 cercariae): Body 53-88 um (mean: 72 um) x 105-138 uum (mean: 117 um) Stylet 5—8 um (mean: 6 um) x 20-40 um (mean: 30 um) 23-40 um (mean: 33 wm) x 23-33 uum (mean: 29 um) 8—12 um (mean: 10 um) x 5-8 um (mean: 8 uum) 13—25 um (mean: 18 wm) x 8—20 um (mean: 16 um) Oral sucker Pharynx Ventral sucker Excretory bladder 18—55um (mean: 33 um) x 10-35 um (mean: 20 um) Tail 10-28 um (mean: 21 um) x 25-88 uum (mean: 44 um) Veeravechsukij, N. et al.: Trematodes in Thai Tarebia granifera 1.2 Loxogenes liberum Seno, 1907 (Fig. 4) 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 prephar- ynx, 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. The infection rate was 0.15% (23/15,076) (Table 3). Size range and average size (in micrometers, calculat- ed from 10 cercariae): Body 65-93 um (mean: 81 wm) Aves 1001 SUT 0516143 B, L. liberum " Mammalia 74 JF784192, L. spathulatum 1001_ kJ934792, L. linstowi A Human 47 An SUT 0516124 A, M. obstipus ? Unknow 52 a SUT 0516138 B, M. obstipus 8a SUT 0515059 B, gymnocephalous cercaria SUT 0515086 A, echinostome cercaria a| 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; [H2: second intermediate host. Cercarial types — a: virgulate xiphidiocercariae; b: armatae xiphidiocercariae; ¢: gymnocepha- lous cercariae; d: echinostome cercariae; e: megarulous cercariae; f: parapleurophocercous cercariae; g: pleurophocercous cercariae. zse.pensoft.net 454 Ventral sucker 35-48 um (mean: 41 wm) x 33-45 uum (mean: 41 um) Excretory bladder 28-45 um (mean: 39 um) x 25-43 uum (mean: 31 um) Tail 23-35 um (mean: 27 um) x 183-223 uum (mean: 199 um) 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 pleuropho- cercous cercariae had a length of approximately 380 bp. The ITS2 sequences of the remaining cercarial types, 1.e. megarulous cercariae, echinostome cercariae and gymno- cephalous cercariae, had a length of approximately 500 bp. The phylogenetic tree obtained from the neighbor-join- ing analysis (Fig. 18) was rooted with the nematode An- giostrongylus cantonensis (Chen, 1935) (GenBank acces- sion number: HQ540551.1). All trematode species from Thai populations of 7 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 highlight- ed in the following: — Specimens of S. tridactyla, C. formosanus, Centroces- tus sp., H. taichui, O. viverrini, O. felineus (Rivolta, 1884) and H. pumilio, which all have cyprinoid fish as a second intermediate host, were grouped together with relatively high support. — The sequences of the echinostome cercaria and the gymnocephalous cercaria obtained from 7. granifera were grouped together with relatively high support. — This latter clade in turn formed a well-supported clade together with P. gralli and Fasciola hepatica Linnaeus, 1758 and Fasciola gigantica Cobbold, 1856 (for which we obtained data from previously published sequences). — A group of species with arthropods as second inter- mediate hosts, 1.e. L. bicolor, L. liberum, Lecithoden- drium spathulatum (Ozaki, 1929), Lecithodendrium linstowi Dollfus, 1931 and M. obstipus, formed a mod- erately supported group in the phylogenetic analysis. The relationships of species within this clade, howev- er, could not be resolved robustly. Discussion Thiarid gastropods, that transmit parasites of native birds, fishes or mammals, have frequently been reported as first intermediate hosts of trematodes affecting the respiratory, zse.pensoft.net Veeravechsukij, N. et al.: Trematodes in Thai Jarebia granifera intestinal and hepatic systems not only in some domestic animals but also in humans. As outlined in the Introduc- tion, 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. taichui, Loxogenoides bicolor, Centrocestus formosanus, Acanthatrium hitaense, Haematoloechus similes, Cloac- itrema philippinum, Transversotrema laruei, Stictodora tridactyla, Apatemon gracilis, Mesostephanus appendi- calatus, Cardicola alseae and Alaria mustelae (Dechruk- 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 vari- ous systematic, biogeographical and evolutionary studies but also brings them into special focus from a parasito- logical perspective. The present study aimed at bringing together the classical parasitological approach of the morphological characterization of the cercariae stages of trematodes ob- tained from their snail host, with a molecular parasitolo- gy 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 7: granifera was also able to colo- nize moderately saline habitats (brackish water). Without doubt, therefore, this thiarid is well established as an in- termediate host for several species of trematodes. We here focussed on the larval trematode infections found in this snail collected in various regions in Thai- land during two periods of field work. When we started the research in the first period (2004-2009), 7: granif- era was found in 18 sampling sites. In the second pe- riod (2014—2016), we not only went back to the same sampling sites but also added samples from new loca- tions. 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, 1.e. in populations at 51 sampling sites, infected snails were found, indicating the wide preva- lence of these trematodes in Thailand. As we mentioned above, only three species of trema- todes, viz. L. bicolor, S. tridactyla, C. formosanus, were found to commonly occur in 7Zarebia 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 collect- ing period five to ten years earlier, and recording infected snails in 18 of these sampling sites, we also found that Zoosyst. Evol. 94 (2) 2018, 425-460 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 (..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 7arebia granifera: Parapleurophocercous cercariae and pleurophocercous cercariae were reported to be commonly found also in oth- er freshwater snails in Thailand, such as e.g. Melanoides tuberculata (Krailas et al. 2014). In this study, three spe- cies of parapleurophocercous cercariae and one species of pleurophocercous cercariae were found in 7. granifera. Various reports have indicated the presence of para- pleurophocercous cercariae and some species of pleuro- phocercous cercariae of the intestinal trematodes Het- erophyidae, such as H. taichui, H. pumilio, S. tridactyla and C. formosanus (e.g. Chontananarth and Wongsawad 2013, Waikagul and Thaekham 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 hu- mans (Nithikathkul and Wongsawad 2008, Krailas et al. 2011, 2014, 2016). In this study, we found human trematodes, viz. H. tai- chui, H. pumilio, S. tridactyla and C. formosanus. Espe- cially 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. tai- chui is important for public health, as was shown in sev- eral 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 pa- tients 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 be- ing 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 epide- miology of zoonosis in general we recommend the study of snail intermediate hosts of human and animal trema- tode infections. It would be interesting to study whether there are geographically related higher or lower incidenc- 455 es of human infections, perhaps also correlated to infect- ed fishes in these areas. In contrast, known as parasites to animals only, xiphid- iocercariae 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 virgu- late 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 para- sites from the Lecithodendriidae, viz. L. bicolor, L. libe- rum 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 report- ed 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 par- asite is commonly known as the oriental avian eyefluke and it had been reported in connection with human acci- dental infections (Waikagul et al. 2006, Derraik 2008). Nollen and Murray (1978) reported that P. gralli para- sitized the conjunctival sac of various galliform and an- seriform 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, Pin- to and de Melo 2010, Krailas et al. 2014). In this study, we found P. gralli now also in the thiarid Zarebia granif- era 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 trema- todes of the Sanguinicolidae; they develop to cercariae in brackish-water and freshwater snails, while the defin- itive hosts were found in fishes. In this study, we found cercariae of three species, viz. C. alseae, A. mustelae and 7. 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, Anucherng- chai et al. 2017). Echinostome cercariae are distributed throughout Southeast Asia (Chai 2009). Most species mainly para- sitize avian hosts, such as migratory birds, but sometimes also infect mammals including humans. The echinos- tome 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 7arebia gran- zse.pensoft.net 456 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 in- fection with cercariae that morphologically are obviously attributable to Fasciola cercariae. However, the molec- ular identification showed that these cercariae were ac- tually 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 dis- tinguishable by being elongated spinose with a reniform collar, armed with a single or double row of spines sur- rounding 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 molec- ular 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., Cerato- Phallus sp., Gabbiella sp., Gyraulus sp., Lymnaea sp., and in Melanoides sp. (Frandsen and Christensen 1984). However, thiarid snails were never reported with fasci- olid trematode infections in Thailand. Even though, the morphology of gymnocephalous cercariae was obvious- ly 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 fresh- water snails of Zarebia 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 echi- nostome cercariae and gymnocephalous cercariae; only the furcocercous cercariae were not available for molec- ular 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 re- construction (Fig. 18), in which two characteristics are zse.pensoft.net Veeravechsukij, N. et al.: Trematodes in Thai Jarebia granifera most noteworthy: First, while the relationships of species within molecular clades found could not be resolved ro- bustly, 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 pleu- rophocercous cercariae, respectively (marked f and g in Fig. 18), 1.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 spe- cies have a bithyniid instead a thiarid snail as first inter- mediate host. In a second group cluster trematode species with vir- gulate xiphidiocercariae and armatae xiphidiocercariae, respectively (marked a and b in Fig. 18), 1.e. L. bicolor, L. liberum, Lecithodendrium spathulatum, L. linstowi and M. obstipus), which all have arthropods (Insecta or Crus- tacea) 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 cer- caria obtained from 7: granifera grouped together with relatively high support. However, no clear picture as to a correlation with their second intermediate hosts and de- finitive 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 trema- tode species are known to have an eupulmonate instead of a thiarid snail host. Interestingly, in this latter monophy- letic clade, formed by P. gral/li 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 in- termediate 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 oc- currences 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 world- wide (Adema et al. 2012). There is an urgent need for col- laboration 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 Zoosyst. Evol. 94 (2) 2018, 425-460 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 col- lected 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 evo- lutionary systematic analysis with data on reproductive biology, geographical distribution, morphology and mo- lecular phylogenies of 7’ 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. Acknowledgements This research was supported by the Research and Devel- opment Institute, Silpakorn University, Thailand. We also thank the Department of Biology, Faculty of Science, Sil- pakorn University. We are grateful for financial support from the Thailand Research Fund through the Royal Gold- en Jubilee Ph. D. 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