Survey of Stenomelania Fischer 1885 (Cerithioidea, Thiaridae): The potential of trematode infections in a newly-recorded snail genus at the coast of Andaman Sea, South Thailand

Stenomelania snails (Fischer 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.


Introduction
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, cer-cariae encyst and develop into infective metacer cariae. 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).
Stenomelania is distributed in the Oriental Region, from India to Western Pacific islands (Starmühlner 1976(Starmühlner , 1979(Starmühlner , 1984(Starmühlner , 1993. Its reproductive mode, as tax-onomically 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(Glaubrecht , 2006Bandel 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.

Sampling sites
Stenomelania snails were collected from streams and rivers near the coastline of the south of Thailand in Krabi, Trang and Satun Provinces. The geographic coordinates (WGS84 datum) of the sampling sites were determined with a global positioning system (Garmin PLUS III, Taiwan).

Collection and determination of snails
Snail specimens were collected between February 2018 and February 2019 via hand picking, scooping and counts per unit of time sampling (Olivier and Schneiderman 1956). The samples were handpicked and scooped by five researchers every 10 min at each sampling site. The snails were then transferred and studied in the laboratory of the Parasitology and Medical Malacology Research Unit, Silpakorn University, Nakhon Pathom, Thailand (PaMa-SU: codens SUT). They were identified on the basis of their shell morphology.

Trematode infection analysis
The collected snails were examined for trematode infections by using shedding and crushing methods. The morphological characteristics of the trematodes were described on the basis of living cercariae that emerged from the snails. The studied cercariae were both unstained and vitally stained with 0.5% neutral red. The details of the cercariae were drawn with a camera lucida and identified in accordance with the methods described by Komiya (1961), Schell (1970), Yamaguti (1971Yamaguti ( , 1975, Ito (1980), Krailas et al. (2011Krailas et al. ( , 2014 and Veeravechsukij et al. (2018). The average size of 10 specimens fixed in 10% formalin was measured in micrometres by using an ocular micrometre. Some cercariae belonging to the identified trematode species were preserved in 95% ethanol for further DNA analysis.

Molecular analysis of cercariae
For molecular identification, genomic DNA was extracted from the preserved cercariae by using a DNeasy blood and animal tissue kit (QIAGEN, Germany). The nuclear internal transcribed spacer 2 regions (ITS2) were amplified via a polymerase chain reaction (PCR) with the following primers ITS2-F (5ʹ-CTT GAACGC ACA TTG CGG CCA TGG G-3ʹ) and ITS2-R: (5ʹ-GCG GGT AAT CACGTC TGA GCC GAG G-3ʹ; Sato et al. 2009). Reactions were set up in 50 μl volumes containing 0.5 μl of dNTPs (5 mM each), 2.5 μl of MgCl 2 (1.5 mM), 5 μl of Buffer A (10X Buffer A, Invitrogen, Thermo Fisher Scientific, USA), 2.5 μl of each primer (10 μM), 0.5 μl of Taq DNA polymerase (1.5 U/μl, Invitrogen) and 34.5 μl of ddH 2 O. The DNA samples were subjected to the fol-lowing: initial denaturation at 94 °C for 4 min; 35 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 30 s and elongation at 72 °C for 2 min (Sato et al. 2009); and a final elongation step at 72 °C for 10 min. Then, the PCR products were loaded on to 1% agarose gels for electrophoresis.
The ITS2 PCR products were sent to Biobasic (Canada) for sequencing analysis. The ITS2 consensus sequences were aligned in MEGA 10 by using MUSCLE (Edgar 2004) under default settings. A phylogenetic tree representing the species groups was constructed with neighbour-joining analysis based on p-distances with 3,000 bootstrap replicates.

Results
Geographical origin of the collected snails The snails were found at 13 sam pling sites in three Provinces, viz. Trang, Krabi and Satun ( Fig. 1, Table 1). The collected snails were tentatively categorised into five morphospecies, based on the analysis of the relevant thiarid taxa and comparison with the documented shell morphology. The following morphospecies were identified: morph a, Stenomelania cf. aspirans; morph b, S. cf. crenulata; morph c, Neoradina aff. prasongi; morph d, S. cf. punctata; and morph e, S. cf. torulosa (Fig. 2, Table 2).

Cercarial diversity and infection rates
The infected snails were reported from seven of the above sampling sites. The information on sampling sites, including geographic co ordinates and the number of infected snails, is presented in Table 1. A total of 1,551 snails were collected and examined for trematode infections.

Morphology of the infecting cercariae
The cercariae were categorised on the basis of their morphological and organ characters in accordance with previously-reported morphological descriptions (Komiya 1961;Schell 1970;Yamaguti 1971Yamaguti , 1975Ito 1980;Krailas et al. 2011Krailas et al. , 2014Veeravechsukij et al. 2018).   (1997) Haynes (2001) Morph e Stenomelania cf. torulosa Shell sculptured with strong spiral ridges, 8-12 whorls, the shell is always eroded, aperture ovate. Ramakrishna and Dey (2007) They were described as two distinct morphological cercarial types known and found to date and attributable to at least two distinct trematode families.

Type 1. Virgulate xiphidiocercariae cercariae
Lecithodendriidae Lühe, 1901(sensu Odhner 1910 1.1 Loxogenoides bicolor (Krull, 1933;Kaw 1945;Fig. 3) The body of this species was oval and covered with small spines. Brown granules were found underneath the skin of its body. Its oral sucker was globular and clearly observed with one stylet. The virgulate gland was presented in the anterior part of the body. The pharynx was round and small; however, the oesophagus was not found. Three pairs of penetration glands were located at two-thirds of the body and they had two anterior pairs with fine granules and a posterior pair with coarse granules. The ventral sucker was smaller than the oral sucker. The excretory bladder was U shaped and thick walled. The tail was flexible in length, but it was shorter than the body. Spines were observed on the body and excretory ducts opened at the end of the tail. The cercariae developed within sporocysts.
Size range and average size (in micrometres, calculated from 10 cercariae): Type 2. Parapleurolophocercous cercariae Heterophyidae (Leiper 1909;sensu Odhner 1914) 2.1 Haplorchis taichui (Nishigori, 1924;Chen 1936; Fig. 4) The body of this species was oval and brownish. Its mouth aperture was found at the oral sucker and covered with two rows of spines. The first row had six spines and the second row had five spines. Sensory hairs were observed on the ven-  The cercaria was oval. Its oral sucker was located at the anterior of the body and its mouth aperture was covered with three transverse rows of spines. The first row had four spines, the second row had five spines and the third row had six spines (4:5:6). A pair of pigmented eyespots was conspicuous from the anterior end and Table 3. Some characters of the infected trematodes found in this study and the reference sources (measurement in µm, n/a = no data). Eyespot 9 (7-10) × 11 (9-13) 9 (5-15) × 9 (5-15) 9 (8-11) × 6 (4-7) n/a Not found Not found the pharynx was presented. Seven pairs of penetration glands extended from the pharynx to the posterior end of the body. Numerous cystogenous glands in the cell were arranged in the middle third of the body and extended to the lateral fields of the body. The excretory system was mesostomate, the excretory bladder was saccular and thick walled and the tail was longer than the body. The lateral finfold was found at one-third of the tail trunk and the dorso-ventral finfold was located at the distal portion. The cercariae developed within rediae. Only one S. cf. punctata from Klong Nong Jik was infected. The infection rate was 0.06% (1/1,551; Tables 1, 3). Size range and average size (in micrometres, calculated from 10 cercariae):

Molecular analysis
The cercariae were studied using the ITS2 sequences ( Fig. 6 and Table 4). Three trematode species were categorised on the basis of their morphological and organ characters from 10 collected snails. The heterophyid trematodes consist of H. taichui and P. cheni. The ITS2 gene sequences of H. taichui and P. cheni were approximately 310-330 and 255 bp in length, respectively. The phylogenetic tree obtained from neighbour-joining analysis was rooted with the nematode Angiostrongylus cantonensis (GenBank accession number: AB700693). Unfortunately, L. bicolor, the virgulate xiphidiocercariae cercariae of Lecithodendriidae, could not be amplified. However, this trematode species was distinguished through morphological identification.
In the phylogenetic tree, the two H. taichui SUT172001E and SUT172002E clustered together, whereas H. taichui SUT172003E was more distant. These H. taichui samples, which grouped together with a relatively-high support, were collected from the same snail intermediate host, viz. S. cf. torulosa, but from different locations in Satun Province. The second heterophyid cercaria species, P. cheni (SUT172004D), grouped together with Procerovum sp. (GQ176376), P. cheni (HM004164, HM004165 and HM004164) and P. varium (HM004167, HM004168 and HM004169; Van et al. 2009;Thaenkham et al. 2010), with a high support. P. cheni and P. varium grouped together and could not be resolved unequivocally. Therefore, P. cheni (Hsü 1951) was confirmed morphologically on the basis of previously-published data.

Discussion
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, 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 trem-atode 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(Upatham et al. , 1981Kumchoo et al. 2005;Sri-aroon et al. 2005;Ukong et al. 2007;  2007; Krailas et al. 2008Krailas et al. , 2011Krailas et al. , 2014Krailas et al. , 2016Wongsawad 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(Hsü , b, 1951Umadevi 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(Dechruksa et al. , 2013Ukong et al. 2007;Krailas et al. 2011Krailas et al. , 2014Pratumsrikajorn 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.

Conclusion and outlook
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.