Stenomelania snails (Fisher 1885) have been reported from the coastal regions of the Indian Ocean and the Pacific Ocean, spanning India to Australia. Here, the species diversity and distribution of these snails in the south of Thailand are recorded. The snails were also examined for trematode infections in 13 locations in three Provinces, viz. Krabi, Trang and Satun, along the coast of the Andaman Sea. A total of 1,551 snails were in five morphs tentatively identified as Stenomelania aspirans, S. crenulata, S. punctata, S. torulosa and the closely-related Neoradina prasongi. With 10 infected snails, the trematode infection rate was 0.64%. The cercariae were categorised into three species from two morphologically-distinguishable types, viz. parapleurolophocercous cercariae (Haplorchis taichui and Procerovum cheni) and xiphidiocercariae (Loxogenoides bicolor), through the morphological characterisation of the larval stage. These trematodes were also analysed using the internal transcribed spacer subunit II region to confirm the species identity at generic and infrageneric levels.

Cerithioidea, Haplorchis taichui, Loxogenoides bicolor, parapleurolophocercous cercariae, Procerovum cheni, Stenomelania, Thiaridae, xiphidiocercariae

Trematode infections are major public health problems affecting humans in southeast Asia. At least 70 species of food- and water-borne trematodes, such as blood, intestinal, liver and lung flukes, are commonly found in various animals (Chai et al. 2005; Andrews et al. 2008; Johansen et al. 2010). Trematode infections depend not only on the habit of people, but also on the presence of first and second intermediate host species, resulting in the endemic spread of parasites, such as intestinal and liver flukes in Thailand. Two major agents of fish-borne infections are intestinal flukes belonging to Heterophyidae and liver flukes belonging to Opisthorchiidae. In a complex life cycle, trematode eggs are released by humans and animals. The first larval stage (miracidium) hatches from the egg in water and penetrates snails as the first intermediate host. A miracidium in embryonated eggs infects snails through passive uptake and subsequently hatches within hosts. The miracidium initially develops directly into sporocysts or rediae and then into cercariae that are released in water. In the second intermediate host, cercariae encyst and develop into infective metacercariae. They infect humans and animals via the consumption of raw fish or improperly cooked fish containing metacercariae (Dung et al. 2007; Skov et al. 2009; Tran et al. 2009; De et al. 2012).

In Thailand, medically important freshwater snails acting as the intermediate host of human and animal infections are reported from several taxa. For example, the opisthorchiid liver fluke Opisthorchis viverrini is found in freshwater Bithyniidae, i.e. Bithynia funiculata, B. siamensis goniomphalos and B. siamensis siamensis, in Thailand, Laos, Cambodia and Vietnam. Small intestinal flukes from Thiaridae serve as the first intermediate host. Some of them include Haplorchis pumilio (Looss, 1896; sensu Looss 1899), H. taichui (Nishigori 1924; sensu Witenberg 1930), Loxogenoides bicolor (Krull 1933; sensu Kaw 1945), Centrocestus formosanus (Nishigori 1924; sensu Price 1932) and Stictodora tridactyla (Martin & Kuntz, 1955), which are recorded from Tarebia granifera, Mieniplotia scabra, Melanoides tuberculata and M. jugicostis.

In the south of Thailand, Haplorchis taichui and H. pumilio are small intestinal flukes that are considered important causative agents of food-borne parasitic zoonoses. Two Cerithioidean snail families, namely, Thiaridae and Pachychilidae, were collected in a previous study. Parasitic infections were found in snail samples from 13 locations; six thiarid species, viz. Melanoides tuberculata (Müller, 1774), Melanoides jugicostis (Hanley & Theobald, 1876), Mieniplotia scabra (Müller, 1774), Sermyla riqueti (Grateloup, 1840), Neoradina prasongi (Brandt, 1974) and Tarebia granifera (Lamarck, 1822); and four pachychilid species, viz. Brotia sp. 1, Brotia sp. 2, Brotia wykoffi (Brandt, 1974) and Sulcospira housei (Lea, 1856). Three thiarid species, viz. M. tuberculata, M. jugicostis and N. prasongi, were infected with two intestinal flukes, viz. H. taichui and H. pumilio (Krailas et al. 2011, 2014).

Thiaridae is a group of cerithioidean gastropods, which are widely distributed and thriving in lotic (springs, creeks, rivers and streams) and lentic (lakes and ponds) habitats in tropic and subtropic regions (Glaubrecht 1996; Glaubrecht and Neiber 2019). This family includes snails belonging to Stenomelania (Fischer, 1885), whose members have elongated and pointed shells and are found near and in the brackish water environment of estuaries. Dey (2007) used shell morphology to identify four species, viz. Stenomelania torulosa, S. plicaria, S. punctata and S. aspirans. Haynes (2001) reported five species, viz. Melanoides (Stenomelania) arthurii (Brot, 1870), M. (Stenomelania) aspirans (Hinds, 1847), M. (Stenomelania) lutosa (Gould, 1847), M. (Stenomelania) plicaria (Born, 1778) and M. (Stenomelania) punctata (Lamarck, 1822), from the tropical Pacific Region. However, this freshwater snail taxon has insufficient data and a contentious taxonomy because its shell morphology is similar to that of other thiarid snails.

Stenomelania is distributed in the Oriental Region, from India to Western Pacific islands (Starmühlner 1976, 1979, 1984, 1993). Its reproductive mode, as taxonomically constituted currently, covers ovoviviparous species, which release numerous offspring as veliger larvae and euviviparous taxa, whose shelled juveniles hatch from a subhaemocoelic brood pouch. Normally, adult Stenomelania snails inhabit freshwater and brackish water environments of estuaries, where veliger larvae can be dispersed via marine currents. S. denisoniensis (Brot, 1877), which is found in Australia, releases shelled juveniles similar to those in other thiarid snails, such as Melanoides, Tarebia and Mieniplotia. Stenomelania species also exist in freshwater resources along the Andaman coast of southern Thailand, although there are only cursory remarks from there yet (Glaubrecht 1996, 2006; Bandel et al. 1997; Glaubrecht et al. 2009; Wiggering et al. 2019).

In this study, Stenomelania snails were investigated in 13 locations in three Provinces, viz. Krabi, Trang and Satun, near the Andaman Sea in the south of Thailand. They were also examined for trematode infections through the morphological characterisation and genetic identification of the snails and the parasitic larval stages of trematodes (cercariae). This study provided basic knowledge about the trematode fauna in Thailand and adjacent countries and the evolutionary potential of these parasites and their prevailing intermediate snail host.

Stenomelania is widespread in the Oriental Region, ranging from India to the south-western Pacific and Australia (Bandel et al. 1997; Glaubrecht et al. 2009). Wiggering et al. (2019) studied thiarid snails reported from Thailand and focused on N. prasongi (Brandt 1974) Stenomelania-like freshwater snail in comparison with Melanoides and Stenomelania species. In the present study, the thiarids resembling Stenomelania in south Thailand were examined to explore the occurrence of these snails and their infections with trematodes. Here, a parasitological approach, based on the morphological characteristics of the cercarial stages, was combined with a molecular approach and a preliminary phylogenetic analysis of the parasites obtained from the collected snails in Thailand was performed.

In this study, a total of 1,551 collected snails from 13 localities in the coastal of Andaman Sea were identified into five species: (1) S. cf. aspirans, (2) S. cf. crenulata, (3) N. aff. prasongi, (4) S. cf. punctata and (5) S. cf. torulosa. Interestingly, the distribution of the snail species exhibited a distinct pattern. In Satun Province, only S. cf. torulosa was found, whereas N. aff. prasongi was collected only in Trang Province. By contrast, all the taxa were observed in Krabi Province. Therefore, the presence of these species might be correlated with the circulation of sea currents. The flow of water along the Andaman coast is affected by the monsoon season, i.e. between January and May with a clockwise flow direction (northeast monsoon season) and between August and October with an anticlockwise direction (southwest monsoon season; Department of Marine and Coastal Resources, Thailand). Stenomelania produces veliger larvae and may represent a transitional stage in the invasion of freshwater habitats (Glaubrecht 1996, 2004; Bandel et al. 1997). Veligers move from one habitat to another via ocean currents.

Previous studies in Thailand found that thiarid snails, such as M. tuberculata, M. jugicostis, T. granifera, M. scabra and S. riqueti, are intermediate hosts of trematodes, which are categorised as types and species by using the characteristics of cercariae, viz. (i) paraplurolophocercous cercariae: H. taichui, H. pumilio and Stictodora tridactyla; (ii) pleurolophocercous cercariae: Centrocestus formosanus; (iii) virgulate xiphidiocercariae: Loxogenoides bicolor, Loxogenes liberum and Acanthatrium histaense; (iv) armatae xiphidiocercariae cercariae: Maritreminoides caridinae and M. obstipus; (v) furcocercous cercariae: Haematoloechus similes, Transversotrema laruei, Cardicola alseae, Alaria mustelae, Apatemon gracilis and Mesostephanus appendicalatus; (vi) megarulous cercariae: Cloacitrema philippinum and Philophthalmus gralli; (vii) echinostome-type cercariae: Echinochasmus pelecani; (viii) amphistome cercariae: Gastrothylax crumenifer; (ix) renicolid cercariae: Cercaria caribbea LXVIII; (x) cotylomicrocercous cercariae: Podocotyle (Podocotyle) lepomis and (xi) gymnocephalous-type cercariae (Dechruksa et al. 2007; Ukong et al. 2007; Krailas et al. 2011, 2014; Sritongtae et al. 2015; Veeravechsukij et al. 2018).

In this study, three trematodes species infecting snails at seven localities were reported: N. aff. prasongi in Trang, S. cf. punctata in Krabi and S. cf. torulosa in Trang and Satun Provinces. The three species from two trematode families were identified on the basis of the morphological characteristics of the emerged cercariae. The parthenitae at the larval stage (sporocysts or rediae) that produced the cercariae were observed. The two families were Heterophyidae (H. taichui and Procerovum cheni) and Lecithodendriidae (L. bicolor). The heterophyid trematode causes one of the fish-borne zoonoses which infect vertebrate animals, including humans and birds. Human infections are scattered and the major endemic areas are located in southeast Asia, including Thailand. Humans are infected by 13 genera, viz. Acanthotrema, Apophallus, Ascocotyle, Centrocestus, Cryptocotyle, Haplorchis, Heterophyopsis, Heterophyes, Metagonimus, Pygidiopsis, Procerovum, Stellantchasmus and Stictodora (Pearson 1964; Yamaguti 1971; Pearson and Ow-Yang 1982; Chai and Jung 2017).

In Thailand, H. taichui was first reported in 1971 from autopsy cases at Udonthani Provincial Hospital in the northeast region (Manning et al. 1971). Even though H. taichui is a small intestinal fluke, usually less than 5 mm in length, it can cause intestinal histopathology of hosts by mechanical and chemical irritations. It also induces chemical irritation by producing some substances that can act as antigens and toxins in the host’s body (Chai and Jung 2017). Moreover, this fluke can elicit inflammatory reactions, together with ulcers and superficial necrosis of the intestinal mucosa. Some reported cases in humans were from Chiang Mai in northern Thailand (Kliks and Tantachamrun 1974; Sukontason et al. 2005).

Since 1980, thiarid snails have been reported as medically important gastropods, especially H. taichui and their snail hosts M. tuberculata, M. jugicostis, M. scabra, T. granifera and S. riqueti. H. taichui is one of the most frequently-reported species in southeast Asia, including Thailand. The prevalence of H. taichui has been observed in every region in Thailand, where it is found more frequently in the southern part than other haplorchiinid species (Upatham et al. 1980, 1981; Kumchoo et al. 2005; Sri-aroon et al. 2005; Ukong et al. 2007; Dechruksa et al. 2007; Krailas et al. 2008, 2011, 2014, 2016; Wongsawad et al. 2009; Sritongtae et al. 2015; Veeravechsukij et al. 2018). In the present study, H. taichui infections were detected in S. cf. torulosa and N. aff. prasongi from four locations in Satun and one location in Trang Provinces. For the first time, H. taichui infections were observed in Stenomelania in Thailand.

Procerovum cheni, with P. varium as the type species, is a small fluke that belongs to the same subfamily Haplorchiinae (Looss 1899). Three species have been described: P. calderoni (Africa and Garcia 1935; Price 1940), P. varium (Onji and Nishio 1916) and P. cheni (Hsü 1950). P. calderoni was first reported in dogs, cats and two humans in the Philippines, whilst P. varium was described in the adult stage from experimental dogs infected with metacercariae from mullet fish in Japan (Price 1940; Onji and Nishio 1916). Procerovum differs from Haplorchis in terms of the structure of the ventro-genital complex that presents an expulsor and a gonotyle with numerous spines. As such, some species, previously included in Haplorchis, have been transferred to Procerovum, based on these differentiating characters. The occurrence of metacercariae in fishes and the development of adults from experimental hosts have been used to categorise trematodes under Procerovum (Hsü 1950a, b, 1951; Umadevi and Madhavi 2000). Here, morphological and molecular studies on cercariae were conducted to confirm the specific identity and prevalence of various infectious trematodes in the collected S. cf. punctata from Klong Nong Jik in Trang Province. One S. cf. punctata was infected with P. cheni, with a prevalence of 0.32% (1/310; Table 1) at this location. In previous reports, the first intermediate host of Procerovum was found to be either freshwater or brackish water thiarid snails, viz. M. tuberculata, Sermyla riquetti and Stenomelania denisoniensis (Velasquez 1973; Surin 1993; Umadevi and Madhavi 2000), which were similar to those found in the present study. Heterophyid flukes, including Haplorchis and Procerovum, cause erratic extra-intestinal parasitism, such as ocular parasitosis, in humans. The ocular infection of Procerovum was first reported in the Philippines. In South India, an ocular granuloma in a single patient was attributed to P. varium infection. Later, 42 children with ocular granulomatous inflammation were infected with this trematode and all of them were exposed to snail-infested water, for example, ponds and rivers. Molecular analysis was performed to identify the species causing granulomas and 13 of the 42 samples tested positive for P. varium (Arya et al. 2016). In our study, only one snail was infected were Procerovum. However, this trematode has not been reported in other thiarid snails in Thailand. This finding indicated that the resulting parasitic diseases are still largely neglected in tropical medicine, so further studies should be performed on the prevalence of various trematode-borne diseases in locations with snail occurrences in Thailand.

Stafford (1905) classified L. bicolor as a trematode belonging to Lecithodendriidae when he reviewed Loxogenes and compared L. bicolor with L. arcanum (Kaw 1945). Yamaguti (1971) subsequently transferred it from Heterophyidae to Lecithodendriidae. This parasite is found in the terminal portion of the bile duct of frogs. It is regarded as an accidental parasite of the herring gull, which probably ingests an infected frog (Christensen 1981). Although Loxogenoides was first described in North America, it was studied in its adult form from a definitive and accidental avian host. In Thailand, L. bicolor from its snail intermediate host has been widely reported. Here, thiarid snails, such as M. tuberculata, M. jugicostis, M. scabra, S. riquetti and N. prasongi, act as the first intermediate hosts. Snails belonging to cerithioidean Pachychilidae are also infected with L. bicolor and three species (viz. Brotia costula, B. dautzenbergiana and B. wykoffi) have been reported (Dechruksa et al. 2007, 2013; Ukong et al. 2007; Krailas et al. 2011, 2014; Pratumsrikajorn et al. 2017; Veeravechsukij et al. 2018). Moreover, L. bicolor has the highest infection rate in infected thiarid snails. It also doubles or even triples the infection in their snail hosts when other trematodes are present. For example, L. bicolor infections doubled when it was combined with Stictodora tridactyla in M. tuberculata and L. bicolor was detected with S. tridactyla and Cardicola alseae in triple infections. S. tridactyla is a small intestinal fluke of the paraplurolophocercous cercaria type and C. alseae is a blood-dwelling trematode of the furcocercous cercariae type. In the present study, two locations in Trang Province had L. bicolor infections: one with N. aff. prasongi at Klong La 1 and three with S. cf. torulosa at Khao Ting Cave (Table 1).

Molecular analysis was conducted to confirm the results of cercarial identification, based on morphology, as this study aimed to combine classical morphology with molecular genetics, resulting in the conformation of cercarial infections by two distinct trematode families. As a noteworthy result, the nucleotide sequences of Haplorchis and Procerovum were found to be closely related. For phylogenetic analysis, some GenBank data, based on different parasite stages, such as metacercarial or adult stage (Van et al. 2009; Thaenkham et al. 2010; Buathong et al. 2019), were used. However, a similar phylogenetic pattern was observed and the relationships within the molecular clades of H. taichui could not be resolved clearly. All the samples originated not only from the locations in Satun Province, but also collected from the same snail species, viz. S. cf. torulosa. In a previous molecular genetic study, Van et al. (2009) found that Procerovum and Haplorchis are monophyletic. Thaenkham et al. (2010) reported a phylogeny of six species from Haplorchiinae by using the ITS2 region and other molecular markers (18S rDNA and 28S rDNA). They revealed the same topology of the phylogenetic tree. In our study, P. cheni was difficult to be clearly separated from the very closely relationed P. varium through molecular genetics. Furthermore, the sequences of H. taichui and P. cheni, obtained from Stenomelania, did not group together, although they were both of parapleurolophocercous cercaria type.

Stenomelania is considered a widely-distributed thiarid snail inhabiting freshwater and brackish environments in the tropical region of southeast Asia. Here, it is established as an intermediate host of trematode parasites along the Andaman coast in south Thailand. Information on the susceptibility of Stenomelania snails to food-borne zoonotic infections provides knowledge on public health in this region. Thus, the biodiversity and biology of thiarid snails should be further understood by studying their geographical distribution, morphological characteristics, molecular phylogenies and evolutionary associations with parasitic trematodes. Further in-depth evolutionary systematic analyses that involve the combination of data on reproductive biology, geographical distribution, morphology and molecular phylogenies of Stenomelania will enhance our understanding of the details of the host-parasite relationships of these snails as the first intermediate host populations in Thailand. Such analyses will also determine the role of parasitic infections in humans and animals in southeast Asia.

We are grateful for financial support from the Faculty of Science, Silpakorn University, Thailand (grant no. SRIF-JRG-2562-10) and to the Department of Biology, Faculty of Science, Silpakorn University. We thank our students in Parasitology and Medical Malacology Research Unit of SUT for their dedicated field and laboratory work. We are indebted to reviewers and the editor for their instructive comments and suggestions to the manuscript.