Trematodes obtained from the thiarid freshwater snail Melanoides tuberculata ( Müller , 1774 ) as vector of human infections in Thailand

Larval stages of trematodes obtained from the freshwater snail Melanoides tuberculata (Cerithioidea, Thiaridae) as intermediate host were studied by using cercarial emergence and crushing snails. Between December 2004 and September 2009 snails from one hundred twenty locations in Thailand were collected every two months for one year at each sampling site. Counts per unit of time method was used in this study, and the samples of snails were collected every 10 minutes per sampling by five collectors. The cercarial stages were examined using shedding and crushing methods. The infection rate was found to be 18.79%, i.e. 6,019 animals infected in a total of 32,026. Nine different types in eighteen species of cercariae were categorized, viz. are (1) Parapleurophocercous cercariae: Haplorchis pumilio, Haplorchis taichui, and Stictodora tridactyla; (2) Pleurophocercous cercariae: Centrocestus formosanus; (3) Xiphidiocercariae: Acanthatrium hitaense, Loxogenoides bicolor, and Haematoloechus similis; (4) Megalurous cercariae: Cloacitrema philippinum and Philophthalmus sp.; (5) Furcocercous cercariae: Cardicola alseae, Alaria mustelae, Transversotrema laruei, Apatemon gracilis, and Mesostephanus appendiculatus; (6) Echinostome cercariae: Echinochasmus pelecani; (7) Amphistome cercariae: Gastrothylax crumenifer; (8) Renicolid cercariae: Cercaria caribbea LXVIII; and, (9) Cotylomicrocercous cercariae: Podocotyle (Podocotyle) lepomis.


Introduction
The neglected tropical diseases (NTDs) represent the most common parasitic infections affecting the world's poorest people (Hotez et al. 2007). In addition to their detrimental effects on health, NTDs have a chronic debilitating effect by undermining the physical and cognitive development of individuals residing in areas infested with NTDs, especially for children and women of child-bearing age. An especially deleterious effect has been shown on their educational performance and future economic productivity (Hotez et al. 2007(Hotez et al. , 2009. It is important to note in this context that trematodes infecting humans, especially liver fluke and intestinal flukes, are highly prevalent in Southeast Asian countries (Wongratanacheewin et al. 2001;Chai et al. 2005). These infections have a major public health impact. It has been reported that the highest degree of infections with trematodes were discovered in the gastrointestinal tract of humans living in the north region of Thailand (Pungpak et al. 1998) and the most metacercarial species were found in cyprinoid fish in the north and northeastern regions (Srisawangwong et al. 1997b, Sukontason et al. 1999. The liver fluke Opisthorchis viverrini can cause chalangiocarcinoma, a kind of cancer in gall bladder (Sripa et al. 2010), while the intestinal fluke Haplorchis taichui is a possible agent of irritable bowel syndrome-like symptoms (Watthanakulpanich et al. 2010). However, Thai people have considerably underestimated these trematodes by continually eating Thai traditional food prepared from raw freshwater fish (Chuboon et al. 2005). So the prevalence of trematodes in Thailand has been a continual problem until now.

Life cycle of trematodes
Trematodes need three hosts to complete their life cycles. After their eggs hatch in the water source such as canals and streams, the miracidium, their first larval stage, will swim and find the first intermediate host, namely freshwater snails and terrestrial snails close to water. In particular, snail species of the genus Bithynia are known as intermediate hosts of the liver fluke Opisthorchis viverrini (Tesana 2002). Some edible mollusks, such as the viviparid Filopaludina spp. and the bivalve Corbicula spp., are known as the first and second intermediate hosts of echinostome intestinal fluke (Temcharoen 1992a, b, Krailas et al. 2008). Thus, we can call these trematode infections mollusk-transmitted diseases.
In the snails, they will develop from miracidium to sporocyst to redia and finally to cercaria. Cercaria will leave the snails, head to the second intermediate host, e.g. freshwater fish, and develop to be metacercaria, the infective stage. At least 18 species of cyprinoid fish act as the second intermediate hosts. If the vertebrates, like animals and humans, eat the infected raw fish, they will receive the metacercaria, which will not be digested by digestive enzymes. Instead, the trematodes will become adult and lay eggs, which are then emitted to the water again with the hosts'stool.

Survey of freshwater snails as the first intermediate hosts in Thailand
Several studies have been conducted on the fauna of mollusks with focus on their trematode infections (Wegeberg et al. 1999, Abdul-Salam et al. 2004). Not only in Asia but also Africa and Australia, these trematodes have been widely studied (Diaz et al. 2008, Derraik 2008. In Thailand, medically-important freshwater snails have been investigated since 1980 (Upatham et al. 1980, 1981, Nithiuthai et al. 2002, Krailas et al. 2003, Sri-aroon et al. 2005, Dechruksa et al. 2007, Krailas et al. 2008. It was found that because of their life cycle and host specificity, the distribution of trematodes depends on the presence of the first and second intermediate hosts, as well as the eating habit of local people ).

Freshwater Snails of the Thiaridae in Thailand
Many of the trematode infected freshwater snails are from the Cerithoidea, a large, essentially marine, group of caenogastropods with approximately 200 genera and with mainly pan-tropical distributions, which have been used also as model for evolutionary systematic studies (Glaubrecht 1996, 1999, 2000, 2011, Glaubrecht et al. 2009). To focus on the family Thiaridae, this group rep-resents, as became evident in recent studies (Glaubrecht 1996, 1999, 2011, Lydeard et al. 2002, Strong et al. 2008, 2011, one of the two (or three) independent invasions into and colonizations of freshwater habitats. Mainly distributed in Southeast Asia, they are to be considered, together with the Pachychilidae as most significant intermediate hosts for infections in humans. For instance, the thiarids Tarebia granifera and Thiara toucheana, as well as the pachychilid Brotia asperata, Brotia costula episcopalis and Brotia c. peninsularis were found to be the first intermediate hosts for lung flukes (Tang 1940, Tubangui et al. 1950, Davis 1971, Brandt 1974. In addition, the thiarids Tarebia granifera and Melanoides tuberculata are the first intermediate hosts for intestinal fluke and blood flukes (Malek andCheng 1974, Pointier andJourdane 2000). In Thailand, T. granifera and M. tuberculata have been reported as the first intermediate host for lung and intestinal flukes (Upatham et al. 1995, Dechruksa et al. 2007).

Melanoides tuberculata (Müller, 1774)
This taxon is common to freshwaters within its native distributional range that covers much of tropical Africa, Asia and the Oceania. It is now also present in much of the tropical and subtropical New World as a consequence of introductions that started during the last century (Madsen and Frandsen 1989). They were described as alien species around the world. Moreover, the species exhibits considerable polymorphism in shell ornamentation across its geographical range; however, at the same time among sites discrete lineages or 'morphs' of M. tuberculata can be separated by shell characters, such as coloration and ornamentation, apparently due to the predominantly parthenogenetic reproduction resulting from negligible intrapopulation variability in these traits (Samadi et al. 1999). For example, on Martinique Islands, each morph of M. tuberculata is different in terms of juveniles, growth rate and even parasite infection rate.
M. tuberculata is considered to be of medical significance, as most of the above cited parasites can affect humans. Although there can be considerable seasonal variation in the intensity of parasitism in these snails, the incidence of M. tuberculata with trematode parasites has been recorded to be as high as 92% (Derraik 2008). A checklist from 136 scientific published studies revealed that M. tuberculata could be host for flukes, identified as belonging to 17 families, 25 genera, and 37 species (Pinto and De Melo 2011). These trematodes are both animals and human parasites. Nevertheless, in Thailand there are only very few reports about Melanoides infection in certain specific areas of the country to date. However, investigations around Thailand have never been conducted. So, in the present study we have surveyed now the trematode infections of M. tuberculata all over Thailand. ery 10 minutes at each sampling site. The snail samples were then categorized into species according to their shell morphology. They were later examined for trematode infections.

Examination for parasitic infections
Parasitic infections were investigated by using snail shedding and crushing methods. Emerged cercariae were collected in dechlorinated water and observed for their swimming behavior (Krailas et al. 2003). Sporocysts and rediae were examined under a dissecting microscope.

Study of cercarial morphology
The cercariae were studied unstained or vitally stained with 0.5% neutral red and Semichon's acetic carmine & fast green. Descriptions of the morphology and anatomy of cercariae were based on the study of living cercariae that had escaped from the snails. Sample measurements (average size) in micrometers were taken from 20 specimens fixed in 10% formalin. Measurements in micrometers with averages in parentheses were taken from 20 specimens. Details of the cercariae were drawn using a camera lucida, and all their species were identified (Schell 1962, 1970, Nasir 1974, Yamaguti 1975Ito 1980). For scanning electron microscopy, cercariae were fixed in 2.5% glutaraldehyde phosphate buffer (0.1 mol/l, ph 7.4) at 4 °C for at least 2 hours and post-fixed in 1% osmium tetroxide in the same buffer for 2 hours at 4 °C. They were dehydrated through a graded series of ethanol, and then dried in a critical point dryer using liquid carbon dioxide as a transition medium. The specimens were coated with gold-palladium in an ion-sputtering apparatus (Polaron CPD 7501, UK), and then examined in a scanning electron microscope (Camscan mx 2000, UK or JEOL, JSM-5410 LV, Japan).

Melanoides tuberculata in Thailand
The thiarid Melanoides tuberculata was found in and sampled from study sites in five regions in Thailand, as shown in Table 1

Sampling sites
One hundred twenty locations in Thailand, which are used by humans as sources of water, were examined (snail collection sites). For the exact data, please refer to the first section of the Result section in this report. The positions of collection sites were recorded by GPS (Garmin PLUS III, Taiwan). The localities of the relevant samples were mapped on a dot-by-dot basis to a digitally reduced version of the drainage pattern map of Thailand, as developed in Dechruksa et al. (2013). This map was created using a Reliefkarte on the basis of the Global 30-Arc-Second Elevation Data (GTOPO30) from the U.S. Geological Survey and a river map from the Map/server Aquarius Geomar, and then compiled using Adobe Photoshop CS3 and Adobe Illustrator.

Habitat at the study sites in Thailand
The study sites were usually found to be covered with big and medium trees that allow the sunlight to pass through to the stream. The average light intensity was >10,000 lux at noon. The current was swift in the rainy season, and water temperature was 21-28 °C. There were small to medium sized rocks all over the streams. The collected snails were found on the rocks, rough sand, and on aquatic plants. The physico-chemical quality of the environment and the water changed with the seasons and affected the study areas during the dry and flood season.

Parasitic infections
A total of 32,026 Melanoides tuberculata were collected and examined for trematode infections (Fig. 2). The cercarial infections were examined using shedding and crushing methods. The infection rate was 18.79% (6,019/32,026). Nine types and eighteen species of cercariae were categorized. They were (1) Table 2). Characteristics of cercariae were described from living cercariae, fixed cercariae and cercarial images from scanning microscope. Sizes of cercariae were measured for identification of cercarial species (Table 3). The behavior of cercariae was studied and reported for the physiological data of trematodes.

Haplorchis pumilio Looss, 1899 (Yamaguti, 1975)
Haplorchis pumilio (Fig. 3) were found from 371 M. tuberculata. The infection rate was 1.16% (371/32,026) ( Table 2). The body shape is oval, and its surface is covered with fine spines and sensory hairs. The pigment eyespots and pharynx are present. There are seven pairs of penetration glands, which are arranged in two longitudinal series with a ventral sucker and genital primordia. Their ducts are arranged in two bundles. Four of them were open through the dorsal wall, and four through the ventral wall of the oral sucker in two oblique symmetrical rows. The mouth aperture has transverse rows of spines. The ventral sucker and genital primordia are prevesicular. The excretory bladder has a rounded shape and is composed of fine pigments. No flame cells were found in the tail stem. The tail is long, attached to the dorsal end of the body, with lateral finfolds nearby and a dorso-ventral finfold for the greater distal portion. Cercariae were produced within rediae. Movement behavior: The cercaria moved by rolling up and springing the body back to move forward in a screwing motion for 2-4 seconds and then rested for 15-20 seconds on the surface of water. It survived up to 2-3 hours in the water after emergence. Cercaria stained with 0.5% neutral red c. Redia stained with 0.5 % neutral red d.-g. Images of Scanning Electronmicroscope Abbreviations: os -oral sucker, es -eye spot, p -pharynx, pg -penetration gland, vs -ventral sucker, eb -excretory bladder, lf -lateral finfold, ta -tail, df -dorsal finfold, re -redia, c -cercaria, b -body, sp -spine, sh -sensory hair (scale a, b = 100 µm, c = 10 µm). Nishigori, 1924(Yamaguti, 1975 Haplorchis taichui (Fig. 4) were found from 92 M. tuberculata, the infection rate was 0.29% (92/32,026) ( Table  2). Cercarial body is oval in shape, colored with orange yellow, and entirely covered with minute spines and sensory hairs. The oral sucker is situated ventrally in the head region. There are transverse rows of spines at the mouth aperture. The pigment eyespots and a pharynx are present. Seven pairs of penetration glands extend from the pharynx to the end of the body. There are two longitudinal rows with a ventral sucker and genital primordia; their ducts open on the anterior end of the body. The excretory bladder has a round shape and was composed of fine pigments. A long tail is attached to the dorsal end of the body, with lateral finfolds nearby and a dorso-ventral finfold for the greater distal portion. No flame cells are found in the tail stem. Cercariae were produced within the rediae. Movement behavior: Cercariae were escaped from the rediae. In the water, they floated on the surface or in the middle. The body part sank lower than the tail. The movement rolled around fast on the water, about 8 to 12 seconds, then rested for 5-10 seconds. It survived up to 2-3 hours in the water after emergence.

Stictodora tridactyla Martin & Kuntz, 1955 (Yamaguti, 1975)
Stictodora tridactyla (Fig. 5) were found from 2,182 M. tuberculata. The infection rate was 6.81% (2,182/32,026) ( Table 2). The body is oval in shape and yellowish brown in color. There are 3 rows of oral spines (4-6, 12-14, 22-24), and 7 pairs of penetration glands in 4 groups of 3:4:4:3. The penetration ducts are open near the oral sucker. The eight ducts are arranged in two bundles, four open through the dorsal wall, and four through the ventral wall. The ventral sucker is small. There is one pair of eye spots with coarse granules, with a small globular pharynx between the eye spots. The excretory bladder in the flattened V-shaped is situated at the end of the body. The tail is longer than the body with a bilaterial finfold and a dorso-ventral finfold. Both the dorsal and ventral finfolds arose at a short distance from the anterior and the posterior end of the lateral finfold. There is no flame cell, but 3-5 groups of pigment, an opening duct of the excretory bladder at the tip of the tail. Cercariae were produced within the rediae.

Movement behavior:
The cercaria floated on the surface or in the middle of the water.
The body sank lower than the tail. The body moves by turning over left and right. The rolling movement is fast on the water, about 5-7 seconds, and rests for about 25-27 seconds. The body sinks on the surface of the water and then moves upside down.

Centrocestus formosanus Nishigori, 1924 (Yamaguti, 1975)
This parasite (Fig. 6) was found in 74 M. tuberculata. The infection rate was 0.23% (74/32,026) ( Table 2). Cercarial body is oval in shape. A pair of eyespots lay at the level of the pharynx. The oral sucker has two rows of oral spines similar to hooks of the tapeworm (rostellar hooks, four in the anterior and five in the posterior) on the dorsal wall of the mouth aperture. Short esophagus, the parenchymal body is spinulate, and yellowish brown in color. Acetabulum is found between the intestinal bifurcation and the excretory vesical. The bladder is a flattened V-shape. Seven pairs of penetration glands lay anterolateral to the acetabulum in front of an inverted V-shape. Cystogenous cells are distributed in the posterior part. The genital primordial part is somewhat elongated and triangular, between the acetabulum and the excretory vesicle. The tail is slender, with a very indistinct dorsal and ventral finfolds, both of which are more conspicuous in the distal half, with a tiny spike on the tip. Cercariae were produced within the rediae.

Acanthatrium hitaense
Size range and average size (in micrometers, calculated from 20 cercariae): Body: 53-92 mm (av. 78 mm) × 80-110 mm (av. 100 mm) Movement behavior: The cercaria floated on the surface or in the middle of the water. They moved by rolling up and springing the body back to move forward in a screwing motion for 45-60 seconds, and then rested for 2-5 seconds at the water surface. Some cercariae were stuck on the surface of the container, and moved by the oral sucker and ventral sucker. It survived up to 2-4 hours in the water after emergence. a. Drawing of cercaria structure b. Cercaria stained with 0.5% neutral red c. Redia stained with 0.5 % neutral red d.-g. Images of Scanning Electronmicroscope Abbreviations: os -oral sucker, es -eye spot, p -pharynx, pgpenetration gland, vs -ventral sucker, eb -excretory bladder, ta -tail, df -dorsal finfold, ti -tip, re -redia, c -cercaria, b -body, sp -spine (scale a, b = 100 µm, c = 10 µm). a. Drawing of cercaria structure b. Cercaria stained with 0.5% neutral red c. Sporocyst stained with 0.5 % neutral red d.-g. Images of Scanning Electronmicroscope Abbreviations: s -stylet, os -oral sucker, vi -vigulate gland, p -pharynx, pg -penetration gland, vs -ventral sucker, ebexcretory bladder, ta -tail, ti -tip, spo : sporocyst, c -cercaria, b -body, sp -spine (scale a, b = 25 µm, c = 50 µm). Kaw, 1945(Yamaguti, 1975 Loxogenoides bicolor (Fig. 8) was found from 2,373 M. tuberculata. The infection rate was 7.41% (2,373/32,026) ( Table 2). The body of cercaria is spinose and oval in shape. Its entire body is dotted with granules. The ventral sucker is smaller than the oral sucker. A virgular organ is located in the region of the oral sucker. A stylet is present. Three pairs of penetration glands exist: two anterior pairs and a posterior pair. The penetration glands had granules and ducts. The ducts opened near the tip of the stylet. There is a C-shaped genital primordium and a U-shaped excretory bladder. The tail is spinose, with slightly longer spines at the tip. Cercariae were produced within the sporocyst. Movement behavior: The cercaria moved by folding its tail and rolling up the body and moved from left to right quickly. In resting position, they floated on the surface or in the middle of the water. The body sank lower than the tail. The cercaria moved about 60-75 seconds, and rested for about 2-5 seconds. It survived up to 2-3 hours in the water after emergence.

Haematoloechus similis Looss, 1899 (Yamaguti, 1975)
Haematoloechus similis (Fig. 9) was found in 468 M. tuberculata. The infection rate was 1.46% (468/32,026) ( Table 2). This parasite was classified into Xiphidiocercariae. The body is ovate, and the surface is covered with spines. Cytogenous cell is not observed. A stylet is 30-32 µm long, with no virgulate gland. Six pairs of penetration glands of irregular shape are present, extending from the middle of the body to near the posterior end of body, each with large nuclei and fine granules. Their ducts are bundled, one on each side, opening near the tip of the stylet. Prepharynx is short, and pharynx poorly differentiated. Esophagus, ceca and genitalia are not developed. The excretory vesicle is Y shape. The flame cell formula is 2[(3+3+3)+(3+3+3)]=36. The tail is not finflod. Cercariae were produced within the sporocyst. Movement behavior: The cercaria floated on the surface or in the middle of the water. The body sank lower than the tail. It moved by folding its tail back to the body and turning its body to roll quickly from left to right, darting forward for about 15-20 seconds, and resting for about 10-15 seconds. It survived up to 1-2 hours in the water after emergence. Cloacitrema philippinum (Fig. 10) was found in 12 M. tuberculata. The infection rate was 0.04% (12/32,026) (Table 2). The body is elongate and muscular. There is no eye spot, and no spine on the body surface. There are long prepharynx and pharynx. The long ceca runs almost to the end of the body. There are numerous sensory papillae on the surface of body. The oral sucker has 12 opening ducts. The cystogenous cells were found all over the body. The thin wall of the excretory vesicle extends when moving, and the ventral sucker is bigger than the oral sucker. The long tail is inserted to the posterior end of the body. The clear vacuoles distributed along the tail, containing granules. There is an adhesive organ at the tip of the tail, with no lateral finfolds. Cercariae were produced within the rediae.
The body is elongated and white in color, with numerous minute spines on the half body at the posterior end. It has a muscular body, no eye spot, long prepharynx and pharynx, Y-shaped esophagus, and a large ventral sucker. The intestine runs almost to the end of the body. There are plenty of cyst glands along their bodies. The tail is the same length as the body but flexible, with various sizes of vacuole and granules along the tail. The adhesive gland cells were found. Cercariae were produced within the rediae. Cardicola alseae (Fig. 12) were found from 47 M. tuberculata which is equivalent to an infection rate of 0.1% (33/32,026) of the total number of the collected snails (Table 2). The small hook-liked body had an anterior organ, and was covered with minute spines. The longer spines were found in some parts of the body, the dorsal and ventral of the posterior end. The dorso-median finfold was observed in the middle part of the body. Many large granules were observed, with a penetration gland located at the middle part of the body. The excretory bladder was small. The tail is furcocercous; its furcae are shorter than the beginning of the tail, and sharp like animal crawls. Minute spines and sensory hairs were observed. A caudal body, longitudinal muscle, no flame cell, and furcal finfold were observed, with the opening duct of excretory bladder at the fork tail tip. Cercariae were produced within the sporocyst.

Alaria mustelae Bosma, 1899 (Yamaguti, 1975)
Alaria mustelae (Fig. 13) were found from 47 M. tuberculata which is equivalent to an infection rate of 0.15% (47/32,026) of the total number of the collected snails (Table 2). Cercarial body has a long shape. Unpigmented eyespots lay on the midway between two suckers in lateral fields, prepharynx short, pharynx small and muscular, esophagus rather long, ceca extending a short distance posterior to acetabulum. The oral sucker is slightly larger than the postequatorial acetabulum. There are two pairs of penetration glands, filled with fine granules. The duct openings on each side of the mouth are in spineless circumoral area. The body is covered entirely with spines and two irregular spines around the aperture of the ventral sucker. Genital primordium is a small mass of cells anterior to the excretory vesicle. Cercaria developed within the sporocyst. The tail stem was without spines; the furcae was irregularly spinose (long hair like), with no caudal bodies. The excretory pore was found at the fork tail tip. Movement behavior: The cercariae moved by rolling up and springing back the body to swiftly move forward  in a quick semi-circular motion. It then rested by floating with its head on top for a long time about 20-30 seconds then moved quickly about 7-15 seconds and rested by floating again. It survived up to 2-3 hours in the water after emergence. Velasguez, 1958(Yamaguti, 1975 Transversotrema laruei (Fig. 14) were found from 89 M. tuberculata, which is equivalent to an infection rate of 0.28% (89/32,026) of the total number of the collected snails ( Table 2). The body is in a bowl-liked shape and light brownish in color, with numerous spines like fish scales on the body surface. Many granules are observed. The genital pore of the seminal vesicle is at the anterior end of the body. There are very big round eye spots, and the ventral sucker is globular. There is a mouth on the ventral sucker. The esophagus is narrow and long, attached to the intestine. There are 1 pair of testes, with an ovary on the left side of the testes. A small excretory bladder is at the posterior end. The tail is longer than the body length with a fork tip, and one pair of appendage at the base of the tail. An adhesive pad was observed at the end of the tail. The furcal tails are spatulate. There are 4 flame cells, and an opening duct of the excretory bladder is on the furcal tail. Cercariae were produced within the rediae. Movement behavior: The cercaria floated on the surface or in the middle of the water. The body sank lower than the tail. It moved very fast by turning from left to right, and moving forward in a screwing motion for about 5-8 seconds, and resting about 2-3 minutes by floating with its head and tail folded together. The furcae floated upwards, moving by wavering on the container surface. Szidat, 1928(Yamaguti, 1975 Apatemon gracilis (Fig. 15) were found from 55 M. tuberculata, which is equivalent to an infection rate of 0.17% (55/32,026) of the total number of the collected snails ( Table 2). The cercarial body is oval in shape, and the prepharynx is practically absent. The anterior region has a cuticle spine. It has unpigmented eyespots. The tail stem contains about 16 caudal bodies. There are 4 pairs of penetration glands which lie between the acetabulum and genital primordia. The excretory bladder is very small. Cercariae were produced within the sporocyst. Movement behavior: The cercariae floated on the surface or in the water. The body sank lower than the spread- a. Drawing of cercaria structure b. Cercaria stained with 0.5% neutral red c. Redia stained with 0.5 % neutral red d.-f. Images of Scanning Electronmicroscope Abbreviations: gp -genital pore, es -eye spot, m -mouth, ov -ovary, vs -ventral sucker, te -testes, eb -excretory bladder, i -intestine, ap -appendages, ta -tail, fu -furca, p -pharynx, re -redia, c -cercaria (scale a, b = 100 µm, c = 20 µm).

Apatemon gracilis
ing fork tail. It moved by rolling up and springing back the body to swiftly move forward in a semi-circular motion. It then rested by floating with its head on top for about 12-15 seconds, then moved quickly about 3-6 seconds and rested by floating again.
14. Mesostephanus appendiculatus (Cicrea, 1916) Lutz, 1935 (Yamaguti, 1975) Mesostephanus appendiculatus (Fig. 16) were found from 3 M. tuberculata, which is equivalent to an infection rate of 0.009% (3/32,026) of the total number of the collected snails ( Table 2). The cercarial body is oval in shape and spinose. There are many spines on the surface and the oral sucker. Coarse granules and cytogenous gland are scattered inside the body. The pharynx is small and round. The prepharynx and esophagous are almost as long as the pharynx.
A large intestine, composed of two cecae, is terminated near a small excretory bladder. A ventral sucker vestigial is found to be in small groups. The tail is forked and longer than the body and the tail surface is covered with many spines. The tail stem is longer than the furca. The tail tubule opens at the tip of each tail furca in which no flame cell is found. Cercariae were produced within the sporocyst. 8-10 mm (av. 9 mm) × 11-18 mm (av. 15 mm ) Figure 15. Image of Apatemon gracilis; a. Drawing of cercaria structure b. Cercaria stained with 0.5% neutral red c. Sporocyst stained with 0.5 % neutral red d.-g. Images of Scanning Electronmicroscope Abbreviations: os -oral sucker, p -pharynx, es -eye spot, vs -ventral sucker, in -intestine, pg -penetration gland, eb -excretory bladder, ta -tail, cb -caudal body, fu -furca, b -body, sp -spine, spo -sporocyst, c -cercaria (scale a, b = 100 µm, c = 20 µm). a. Drawing of cercaria structure b. Cercaria stained with 0.5% neutral red c. Sporocyst stained with 0.5 % neutral red d.-g. Images of Scanning Electronmicroscope Abbreviations: os -oral sucker, p -pharynx, in -intestine, vs -ventral sucker, eb -excretory bladder, ta -tail, fu -furca, fffurcal finfold b -body, sp -spine, ex -excretory pore, spo -sporocyst, c -cercaria (scale a, b = 100 µm, c = 20 µm). Movement behavior: The cercaria floated on the surface or in the water. The body sank lower than the spreading fork tail. It moved by rolling up and springing back the body to swiftly move forward in a semi-circular motion for about 2-4 seconds. It then rested by floating with its head on top of the tail and slowly rotated its body to the bottom while lifting up its spreading fork tail. It rested for about 20-30 seconds and survived up to 2-3 hours in the water.  (Yamaguti, 1975) Echinochasmus pelecani (Fig. 17) were found from 19 M. tuberculata which is equivalent to an infection rate of 0.06% (19/32,026) of the total number of the collected snails (Table 2). Cercarial body is elongate, white in color, no eye spot, oral sucker with 3 opening of duct (1 median, 2 submediant), cystogenous cell containing rhabdites. Collar spine is not appearance. Esophagus was between pharynx and ventral sucker, ceca reaching to bladder, two main excretory tubes meet together before entering bladder, Genital primordia are two mass behind ventral sucker, no penetration gland, flame cell pattern not determine. Tail is the same length as body, flexible, vacuole appearance along the tail. Cercariae were produced within rediae.  (Yamaguti, 1975) Gastrothylax crumenifer (Fig. 18) were found from 8 M. tuberculata which is equivalent to an infection rate of 0.02% (8/32,026) of the total number of the collected snails (Table 2). Cercariae were liberated from the rediae. The body shape is ovate and large. The eye spots have conical lens with yellow pigment through the body with a smooth surface. The ceca ended 0.14-0.17 mm away from the posterior end of the body, with symmetrical testes at the level of the ceca end. The oral sucker is equal to the ventral sucker. The tail inserted to the posterior end of the body. There are various sizes of vacuole through the tail. Movement behavior: The cercaria floated on the surface or in the water. It moved by wavering on the surface of the water for around 8-10 seconds, and then rolling up and springing back for about 5-10 seconds. It survived up to 3-4 hours in the water after emergence. The cercariae were photo-sensitive. They shrank rapidly in changing light conditions.

Cercaria caribbea LXVIII (Cable, 1963) (Yamaguti, 1975)
Cercaria caribbea LXVIII (Fig. 19) were found from 45 M. tuberculata which is equivalent to an infection rate of 0.14% (45/32,026) of the total number of the collected snails (Table 2). Cercariae developed in the sporocyst. Its body is flat with yellow pigment and numerous minute spines on the surface of the body, with no eye spot, a small sucker of 28-36 mm, a short prepharynx, a pharynx of 12-14 mm, plenty of small cephalic glands in the middle of the body, a short excretory vesicle split into two since the upper of the acetabulum. The tail is straight, and is longer than the length of the body, with no lateral finfold, and no flame cells. a. Drawing of cercaria structure b. Cercaria stained with 0.5% neutral red c. Redia stained with 0.5 % neutral red d.-g. Images of Scanning Electronmicroscope Abbreviations: os -oral sucker, p -pharynx, es -eye spot, vs -ventral sucker, ga -genital atrium, in -intestine, vs -ventral sucker, ta -tail, re -redia, c -cercaria (scale a, b = 100 µm, c = 10 µm).  (Yamaguti, 1975) Podocotyle lepomis (Fig. 20) were found from 3 M. tuberculata which is equivalent to an infection rate of 0.009% (3/32,026) of the total number of the collected snails (Table 2). Cercariae developed in the sporocyst. The body is cylindrical in shape, clear white in color, with no sensory hair, spine, 6 papillae on the head, and rough granules present on the body. The stylet is present in oral sucker. There were 2 rows of sensory papillae around the oral sucker, with a long prepharynx. Pharynx is round. The ceca extended to the posterior end, with 5 pairs of penetration glands. 2 of them were not stained with 0.5% neutral red while 3 of them were stained with 0.5% neutral red. Excretory vesicle had a thick wall and open dorsally at the tail, the flame cell formula is 2[(2+2)+(2+2)]. The tail is short, only half the length of the body, cup-shaped, with an adhesive gland present at the end of the tail for attaching. Movement behavior: The cercaria floated with the ventral upside. It moved by floating with its head and tail folded together, and then sprang up. Normally it creeps on the surface of the container using the oral and ventral sucker. The cercaria floated for about 15-20 seconds, and rested for about 10-15 seconds. It survived up to 2-3 hours in the water.

Discussion
Melanoides tuberculata Müller, 1774 is a benthic freshwater thiarid native to Africa and Asia. Its original native range seemed uncertain but nonetheless wide, including parts of Africa, the Mediterranean, Asia and the Pacific Islands (Pace 1973, Clench 1969. Glaubrecht (1996) hypothesized that its origin lies rather in Asia than Africa, given its natural occurrences and the history of Thiaridae; see also Glaubrecht et al. (2009) and Glaubrecht (2011). It had also become established in several other countries. (Facon et al. 2004, Derraik 2008. Aquarium plants exchange by humans caused the brake down of natural dispersal barriers to these snails (Kolar and Lodge 2001). Moreover, Melanoides had also adapted well to new environments. For example, on Martinique Island M. tuberculata was surveyed in 1979 and 2003. While there were only two morphs found in 1979, in 2003 there were nine morphs. Population dynamics and distribution of M. tuberculata were studied in many areas, where they dominated the streams, ponds, and lakes (e.g. Supian and Ikhwanuddin 2002, Eldblom and Kristensen 2003, Facon et al. 2003. Melanoides tuberculata was found to be intermediate host for a number of trematode parasites (e.g. Pinto andDe Melo 2011, Ukong et al. 2007). As a consequence, the introduction of M. tuberculata leads to new parasitic cycles in humans in the invaded area. However, there are also reports that show the efficacy of M. tuberculata as a biocontrol agent against the schistosome (blood fluke) vector snails Biomphalaria glabrata (Pointier and Jourdane 2000).
In Thailand, Brandt (1974) reported that thiarid snails were found in lakes, ponds, marshes, canals, streams, rivers, and other sources of river such as waterfalls. In the present work, we did a smaller scale of investigation than Brandt's. More than thirty thousand of M. tubercu- a. Drawing of cercaria structure b. Cercaria stained with 0.5% neutral red c. Sporocyst stained with 0.5 % neutral red d.-f. Images of Scanning Electronmicroscope Abbreviations: s -stylet, os -oral sucker, p -pharynx, penetration gland, vs -ventral sucker, eb -excretory bladder, ta -tail, ad -adhesive organ, spo -sporocyst, c -cercaria (scale a, b = 100 µm, c = 10 µm). lata snails were collected from rice paddies, drainages, ponds, canals, water reservoirs, marshes, streams, waterfalls, and rivers in Thailand. The classification of M. tuberculata was performed as reported by Brandt (1974). It was quite clear to distinguish M. tuberculata from other thiarids, although among these snails there were quite different shell morphologies in terms of ribs, color, pigmentation and even size. Although, based on the shell, the destinction of discrete several morphs was possible (see e.g. Pointier 1989, 1993, Samadi et al. 1999), all of these morphs were still considered to be conspecific within M. tuberculata. It is hoped that molecular techniques will help us to eventually solve this question of intraspecific versus intraspecific variation.
In this study, the recovery of adult trematode stages are not completely recorded, but the morphological distinction of cercariae are quite clear from the unstained, stained and electronmicroscopic images, allowing to classify the eighteen species of cercariae from this thiarid snail species into nine types of cercarial morphology, as compiled in the Result section.
In the present study, we also found human trematodes, viz. Haplorchis taichui, Haplorchis pumilio, Centrocestus formosanus and Cercaria caribbea LXVIII. Especially the H. pumilio human minute intestinal fluke (371/6,019 = 6.16%) showed a high level of prevalence in Thailand. As they complete their complex life cycle not only in humans but also in other vertebrates, it is difficult to control their infection. H. taichui is another important minute intestinal fluke. Three cases of humans were reported with mucosal ulceration, mucosal and submucosal haemorrhages, fusion and shortening villi, chronic inflammation and fibrosis of submucosa; in addition, there was a report of the pathology in the small intestine of patients caused by H. taichui (Sukontason et al. 2005). In earlier reports, Haplorchis spp. were found to be of high prevalence of infection in the north of Thailand (Chontananarth and Wongsawad 2010). In the present report, we now found Haplorchis infections in every region of Thailand.
The minute intestinal flukes were reported not only in Asia but also in South America. The life cycle of H. pumilio was studied from redia to adult under natural and experimental conditions in the digestive gland of Melanoides tuberculata, collected from Agasanta, Venezuela (Diaz et al. 2008). It seems that M. tuberculata was one of the important intermediate host snails of humans and animal trematodes in the world.
For animal parasites, the cercariae with the highest prevalence were L. bicolor (2,373/6,019 = 39.43%), an amphibian trematode. However, the other animal parasites were also very important for public health. For example, the eye fluke Philophthalmus spp. are parasites of birds, using a snail intermediate host and birds as the definitive host. However, they also occurred in human and other animals. Human infection by these eye flukes occur via direct contact with the eye by cercariae in the water or by ingestion of cercariae in contaminated water (Alicata 1962, Waikagul et al. 2006, Derraik 2008. One of heterophyid trematodes, Stictodora tridactyla was reported that they occasionally infested brackish water and marine snails, while metacercariae encyst in fish, with the definitive hosts being birds and mammals including humans (Chai et al. 1988, Abdul-Salam et al. 2000. S. tridactyla also infected M. tuberculata with other cercariae as well. The infection rate of S. tridactyla is 6.81% (2,182/6,019). In our previous study, we found S. tridactyla in M. tuberculata and even more in M. jugicostis ).
The highest infection rate of parasite is 7.41% (2,373/6,019) with Loxogenoides bicolor being the most common parasite found in the present study. This parasite is one of the Xiphidiocercariae, being produced by trematodes from the superfamily Plagiorchioidea (Schell 1962, Malek andCheng 1974). They were found in other thiarid snails, such as Thiara (i.e. Plotia) scraba at Erawan Waterfall, Kanchanaburi, Thailand .
Interestingly, it can cause double infection or even triple infection in M. tuberculata together with other trematodes. We found a total of 326 double infections and 13 triple infections in M. tuberculata (Table 4). S. tridactyla and L. bicolor were found to be common in double infections, while S. tridactyla, L. bicolor and C. alseae were commonly found triple infections. C. alseae is a blood-dwelling trematode. This parasite is furcocercous cercariae. It is produced by trematodes from the family Sanguinicolidae. Found in freshwater fishes they were reported to have sporocysts that developed in the visceral mass of the snail Oxytrema silicula from Alsae River, Oregon, USA (Meade 1967). In Thailand, C. alseae was also found in the thiarid snail Tarebia granifera at Erawan Waterfall ). The others furcocercous cercariae Alaria mustelae, Transversotrema laruei, Apatemon gracilis, and Mesostephanus appendiculatus, can also be found in M. tuberculata. These parasites, such as Transversotrema laruei and Apatemon gracilis, were found with metacercariae in brackish and freshwater fishes. The adult stages of these flukes inhibit the small intestine of their bird hosts (Smith and Hickman 1983).
Although the counts per unit of time method (Olivier and Schneiderman 1956) used to measure the density of the snail population in the marked areas does not represent the total population, our observations were performed all year round. That way we were able to document seasonal variation in the intensity of parasitism in M. tuberculata. Thus, we consider our data of cercariae infection rates from this research to be reliable. Since most of the above cited intestinal flukes affect humans and animals alike (Derraik 2008), in conclusion we can state that the thiarid snail Melanoides tuberculata can doubtlessly be considered as of considerable medical significance in Thailand and elsewhere.