Larval stage of trematodes obtained from brackish water snails in the central and east coast of the gulf of Thailand

Brackish water snail is one of the important intermediate hosts of trematode infections. This kind of snail along the Gulf of Thailand were examined for cercariae, the larval stage of trematodes. The aim of this study was to investigate cercarial infections in brackish water snails in the central and eastern side of the Gulf of Thailand. Snails were collected from 45 sampling sites (20 central areas, 25 eastern areas), between August 2013 and February 2014. A total of 8,532 snails were classified into 12 families, 23 genera and 32 species. Cercarial infections were investigated using shedding and crushing methods. The infection rate was 0.27% (23/8,532). The cercariae were categorized into 5 types and 8 species. The first type, Parapleurophocercous cercariae, consisted of Haplorchis taichui, Heterophyes cercaria I, and Metorchis intermedius. The second type, Xiphidiocercariae, consisted of Ascorhytis charadriformis. The third type, Furcocercous cercariae, consisted of Apharyngostrigea pipientis. The forth type, Cotylomicrocercous cercariae, consisted of Coitocaecum anaspidis. The fifth type, Echinostome cercariae, consisted of Hypoderaeum conoideam and Himasthla interrupta. Seven species of snails were found with trematode infections, comprising of Assiminea brevicula, Cerithidea cingulata, Cerithidea alata, Cerithidea djadjariensis, Cerithidea quadrata, Littorinopsis intermedia, and Sermyla riqueti.


INTRODUCTION
Thailand is situated between the Gulf of Siam and the Andaman Sea.Along the coasts line sand beaches, rock beaches, mangrove forests and river tributaries, providing abundant habitats where various aquatic fauna resided, including molluscs.It is well known that both gastropods and bivalves could be the first intermediate hosts of human and animal trematodes.The mollusc-transmitted diseases are very important for the veterinary and public health.Trematodes need two or three hosts to complete their life cycles.Trematode eggs hatch in the water.At the larval stage, they can swim to find the first intermediate hosts, such as freshwater snails, brackish water snails, and terrestrial snails.In the snails, miracidium develops into sporocyst to redia and finally to cercaria.Cercaria leave the snails and head for the second intermediate host, e.g.fish or amphibian.Then they develop into metacercaria.Animals and humans can be infected by eating metacercaria or by the penetration of cercaria.The study of brackish water snails as intermediate hosts of trematode was reported.Pirenella conica (Gastropoda: Potamididae), a small sea snail along the coasts of Sinai and Israel were infected with trematode of the family Heterophyidae, Echinostomatidae, Microphallidae, Notocotylidae, Haploporidae, Haplosplanchnidae, Cyathocotylidae and Strigeidae (Taraschewski and Paperna, 1981).Cerithidea cingulata of Kuwait Bay found with echinostome cercaria infection was reported as new cercaria species by Salam and Sreelatha (1999).Moreover, the snail, C. cingulata of Kuwait Bay were infected with 12 species of trematodes.They belong to the family Cyathocotylidae, Echinostomatidae, Haplosplanchnidae, Heterophidae, Microphallidae, Philophthalmidae, Plagiorchiidae and Schistosomatidae (Al-Kandari et al., 2000).Zeacumantus subcarinatus (Prosobranchia: Batillaridae), the southern creeper, was reported as a new echinostome cercaria (Marorelli et al., 2006).
In Thailand, mud whelks in the family Potamididae (Cerithidea cingulata, C. djadijarensis and C. charbonnieri) could be intermediate host of 3 groups of cercariae.They were separated by morphological characters.The first group was found with a cystogenous gland but with no eye spots and no collar spines.The second group was found with eye spots, 3-4 penetration glands, and a finfold tail.The third group was still undetermined (Sri-aroon et al., 2005).Those cercariae are not known species.And those surveys were limited to only some regions of Thailand (Sri-aroon et al., 2004(Sri-aroon et al., , 2005(Sri-aroon et al., , 2010)).Therefore, the aim of this study was to investigate and identify trematode infections in brackish water snails in Central and Eastern Thailand.The knowledge of trematode fauna can provide more understanding for veterinary and public health control.

Sampling sites and collection of snails
Snails were collected from 45 sampling sites (20 central areas, 25 east areas), between August 2013 and February 2014.The counts per unit of time method was used for snail sampling.Five collectors picked the snails by hand and scoop for 10 minutes each station.The sampling sites were mangrove forest, drainage, canal and tributary of river in Trat, Chanthaburi, Rayong, Chon Buri, Chachoengsao, Bangkok, Samut Pakran, Samut Sakhon and Samut Songkhram provinces of Thailand (Figure 1).The collected snails were transferred to the Parasitology and Medical Malacology Research Unit, Department of Biology, Faculty of Science, Silpakorn University, Thailand.Those snails were identified by shell morphology (Brandt, 1974;Upatham et al., 1983).

Examination for trematode infections
Trematode infections were investigated by using snail shedding and crushing methods.Cercariae were collected in dechlorinated water and examined under stereo-microscope and scored the infection rates.

Study of cercarial morphology
Cercarial morphology and anatomy were examined by stained cercariae.They were stained with 0.5% neutral red and identified under light microscope.The cercariae were fixed in 10% formalin, then measured and averaged from 10 specimens.The stained specimens were drawn using camera lucida.Scanning electron images of cercariae were taken by Camscan MX 2000 scanning electron microscope (UK).The cercariae were processed by fixed in 2.5% glutaraldehyde phosphate buffer at 4°C for at least 2 h and post-fixed in 1% osmium tetroxide at 4°C for 1 h, dehydrated through a graded series of acetone, then they were air dried.The specimens adhere to carbon stub, were coated with gold-palladium in an ion-sputtering apparatus (Polaron CPD 7501, UK), then examined under SEM.The species of trematodes were identified by their morphology and anatomy following Yamaguti (1975) and Ito (1980).

Morphology of cercariae
Characteristics of cercariae were described from fixed cercariae and living cercariae.Size of cercariae were measured for identification of cercarial species.

Metorchis intermedius Heinemann, 1937
The body was oval in shape and yellowish brown in color.
Four rows of spines were found around an oral sucker.There was a long prepharynx.Esophagus and ceca were not found.One pair of eye spots were apparently without lens.Fourteen penetration glands formed a compact median groups of 3:4:4:3 each.The ventral sucker was a prevesicular cell.The excretory vesicle was round, and lined with epithelia.Tail tubule median opened at a distance from the base of tail.The dorsal finfold began at the posterior end of basal swelling and passed over to the ventral finfold which reached the middle of the tail.The cercariae were produced within the redia (Figure 3).
The following figures are size ranges and average sizes

Heterophyes cercaria I Martin, 1959
The body was oval in shape and yellowish brown in color.The body surface was covered with tiny barbed and sensory hair.Pre-pharynx was found.Pharynx was small and round in shape.Eye spots were round and triangular in shape.Seven pairs of penetration glands were in two longitudinal rows.Their ducts were in 2 bundles.Acetabulum was not observed.Excretory vesicle was thick wall and V-shape.Tail was tubule with finfolds.The characteristics of finfolds were shown; the dorsal fin arose from the tail base, extended to the whole length of the tail, and continued with the ventral fin to the tail tip.The excretory duct opened at the tail tip.The cercariae were produced within the rediae (Figure 4).The following figures are size ranges and average sizes of 10 cercariae:

Apharyngostrigea pipientis (Faust, 1918) Olivier, 1940
The body was oval in shape.The tail was furcae, broad, and flat.There was a distinct pharynx.The esophagus reached half-way from the pharynx to acetabulum.A pair of unpigmented eye spots were anterolateral to acetabulum.There were 4 pairs of penetration glands.
The excretory bladder was small.The tail was a tubule branch opening on the dorsal edge of the furcae about halfway down to the furcal tip.There were furcal finfolds (Figure 6).The following are size rangs and average sizes of 10 cercariae:

Ascorhytis charadriformis (Young, 1949) Ching, 1965
The body was oval in shape and white in color.There was a long stylet.Four pairs of penetration glands lined near a ventral sucker.The ventral sucker was bigger than the oral sucker.The tail was long and round.The excretory duct opened at the tail-end.The cercariae were produced within the sporocyst (Figure 7).The following are size ranges and average sizes of 10 cercariae:

Coitocaecum anaspidis Hickman, 1934
The body was cylindrical in shape with a transparent body.The skin was covered with smaller spines.There was a stylet with double points at the oral sucker.The pre-pharynx was moderately long.The esophagus and intestine did not differ.There were 3 pairs of penetration glands.The excretory bladder was large with thick wall.
The tail was cup-shape.There was an adhesive organ at the tail end (Figure 8).The cercariae were produced within the sporocyst.The following are size ranges and average sizes of 10 cercariae:

Himasthla interrupta Loos-Frank, 1967
The body was cylindrical in shape and colorless.The skin was covered with spines and sensory hairs.There were collar spines around the oral sucker.There was a long esophagus.The intestines were bifurcated at the ventral sucker, and ending blindly near posterior.The excretory organs were distinct and branching from the anterior to posterior of the body.The excretory bladder was a small thick wall.Both the oral and ventral suckers were large, but the ventral sucker was bigger than the oral sucker.The tail was shorter than the body.Two excretory pores at the lateral sides were about one-fourth of the tail length from its base.The cercariae were produced within the daughter redia (Figure 9).The following are size ranges and average sizes of 10 cercariae: Body: 150-163 µm (av.151 µm) x 243-325 µm (av.270 µm)

Hypoderaeum conoideam (Bloch, 1982) Dietz, 1909
The body was round and colorless.There was no eye spot.There were two rows of collar spines.The first row had 15-20 spines, and the second row had 25-30 spines.There was a round pharynx.The esophagus was long; the bifurcate ceca terminated at the posterior end of the body.The ventral sucker was about two-third of the body length.The excretory vesicle was a simple sac.There was a Y-shaped excretory tube at the tail; the tube opened at the tail lateral, around one-fourth of the tail length from its base.The cercariae were produced within the rediae (Figure 10).The following are size ranges and average sizes of 10 cercariae:

DISCUSSION
The collected snails were separated into brackish and fresh water snails.They were collected from the river estuary and mangrove forests.Some fresh water snails found in this study are, for example, Melanoides tuberculata, Tarebia granifera and Filopaludina martensi.Normally, the fresh water snails were reported as the intermediate host of several trematodes, but we did not find trematode infections in this study.However, the present study confirmed the medical and veterinary importance of brackish water snails recorded in previous studies (Taraschewski & Paperna, 1981;Marorelli et al., 2006;Sri-aroon et al., 2010).In this study, either more species of snail or more species of trematodes were reported.
In terms of the species diversity of snails, we compared the species diversity of snails from the Eastern Coast and the Central Coast.We found that the Eastern Coast was more species diverse.Of the total thirty-two species found, six species of snails dispersed in the East Coast, the Central Coast, and the South of the Gulf of Thailand.These were Assiminea brevicula, Littorinopsis intermedia, Dostia violacea, Cerithidea cingulata, Sermyla riqueti and Tarebia granifera.They were common species generally found in the Gulf of Thailand (Sritongtae et al., 2015).More species of the Potamididae and Ellobiidae snails were found than others.Particularly, the family Potamididae were the most variation of snails found infected with trematodes, as previously reported (Sriaroon et al., 2010;Sritongtae et al., 2015).C. cingulata, C. djadjariensis and C. quadrata were known to be a host of human, bird and amphibian digenea.But this study showed that C. alata could be the intermediate host of bird trematode as well.Moreover, Littorinopsis intermedia was not reported with trematode infection in the past.Only L. scabra was mentioned.But only in this study L. intermedia was found infected with Ascorhytis charadriformis (amphibian trematode), while L. scabra was not.Thiarid snails, Sermyla riqueti were infected with Apharyngostiger pipentis (fish trematode) and Haplorchis taichui (human minute intestinal fluke), causing a major public health problem in Thailand and the other countries in South East Asia (Chai et al., 2005;Krailas et al, 2011).S. riqueti was reported with the highest infection rate in a previous study of cercarial infections of brackish water snails on the east coast of southern Thailand (Sritongtae  , 2015).In the present study, C. quadrata had the highest infection rate with Hypoderaeum conoideam (human echinostome fluke).It should also be noted here that identification into groups might not be sufficient.This study showed that identification at the species level was very useful to determine the exact species of trematodes and their intermediate hosts.

Table 1 .
Brackish water snalis were collected between August 2013 and February 2014 from the Central and Eastern Coasts of the Gulf of Thailand.

Table 2 .
The infection rates of brackish water snails as intermediate host.