Asian Biomedicine Vol. 7 No. 1 February 2013; 97-104 DOI: 10.5372/1905-7415.0701.155 Original article

Occurrence and molecular identification of liver and minute intestinal flukes metacercariae in freshwater from -Mae Ai Agricultural Basin, ,

Chalobol Wongsawada, b, Pheravut Wongsawada, Somboon Anuntalabhochaia, Jong-Yil Chaic, Kom Sukontasond aDepartment of Biology, Faculty of Science, bApplied Technology in Biodiversity Research Unit, Institute of Science and Technology, Chiang Mai University, Chiang Mai 50202, Thailand, cDepartment of Parasitology and Tropical Medicine, Seoul National University College of Medicine, and Institute of Endemic Diseases, Seoul National University Medical Research Center, Seoul 110799, Korea, dDepartment of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50202, Thailand

Background: Fang-Mae Ai Agricultural Basin is located in Fang and Mae Ai districts, Chiang Mai province. There are many aquatic species distributed in this area, especially snails, crabs, and fish, which can serve as the first and second intermediate hosts of several trematodes. The roles of these intermediate hosts as related to parasitic infections in the area are not known. Objective: We determined the occurrence of liver flukes and minute intestinal fluke metacercariae in freshwater fish from Fang-Mae Ai Agricultural Basin. We also identified of metacercariae by using HAT-RAPD PCR method comparing DNA profiles of parasites. Materials and methods: Liver flukes and minute intestinal flukes were studied from the Fang-Mae Ai Agricultural Basin between October 2009 and September 2010. Fish specimens were seasonally collected and each fish was digested and filtered. The metacercariae were collected and counted under a stereo microscope and identified based on morphological characters. The genomic DNA of all parasites was extracted and purified from adult flukes and metacercariae. All extracted genomic DNA was detected by HAT-RAPD PCR using arbitrary primers to comparing DNA profiles between adult flukes and metacercariae. Results: Five species of metacercariae were found. There were one species of liver fluke, , and four species of minute intestinal flukes, viz. taichui, Haplorchoides sp., Centrocestus caninus, and Stellantchasmus falcatus. The prevalence of metacercarial infection was observed in the cool-dry season at 78.30%, followed by rainy and hot-dry seasons at 72.84% and 69.01%, respectively. The prevalence of trematodes were Haplorchoides sp. (37.43%), H. taichui (35.66%), C. caninus (3.80%), S. falcatus (1.40%), and O. viverrini (0.44%). Conclusion: Minute intestinal flukes accounted for high infection rates while, liver fluke, O. viverrini was of low infection rate. DNA profiles among metacercariae in Fang-Mae Ai Agricultural Basin correctly identified adult stages.

Keyword: Liver flukes, metacercaria, minute intestinal flukes, molecular identification, occurrence

Fang-Mae Ai Agricultural Basin is an important agriculture. Trematode life cycle is completed when agro-economics area in northern Thailand located in metacercariae (infective stage) infect fish, which are Fang and Mae Ai districts, Chiang Mai province. The eaten by a definitive host such as piscivorous birds topography consists of mountains over 1000 m. and fish-eating mammals (including humans) [1]. elevation and important watershed resources for There are many aquatic distributed in this area, especially snails, crabs, and . They serve Correspondence to: Dr. Chalobol Wongsawad, Department of as the first and second intermediate hosts of several Biology, Faculty of Science, Chiang Mai University, Chiang Mai trematode species. Chiang Mai province is known as 50202, Thailand. E-mail: [email protected] an endemic area of minute intestinal fluke infection. 98 C. Wongsawad, et al.

Some monogenetic trematodes, viz. Dactylogyrus sp., Agricultural Basin, and identification of metacercariae Gyrodactylus sp., and Paradiplozoon sp.; plus using the HAT-RAPD PCR method for comparing three species with metacercariae, viz. Centrocestus DNA profiles of these parasites [10]. caninus, Haplochis taichui, and Haplorchoides sp. and one species of nematode: Rhabdochona sp. were Materials and methods recovered from the Mae Ngat Somboonchon reservoir Fish specimens were seasonally collected with gill [2]. Recently, Centrocestus caninus, Haplorchis nets from eight sampling sites (Figure 1, site 1-4 Mae taichui, and Stellantchasmus falcatus were found Jai Stream, ; site 6 Mae Kok stream; site in cyprinoid fish from Mae Sa Stream, Mae Rim 5, 7, and 8 Mae Fang stream, Mae-Ai district) in the district, Chiang Mai province [3]. The prevalence of Fang-Mae Ai Agricultural Basin between October 2009 H. taichui and Haplorchoides sp. metacercariae and December 2010. This experiment followed the infecting cyprinoid fish at Mae Ngat and Mae Kuang guidelines of experiment by Chiang Mai Udomtara was 62.16% and 48.65%, respectively [4]. University. Fish specimens were identified by species Parasitic transmission has never been studied in using the handbook of Fishes of the Cambodian the Fang-Mae Ai Agricultural Basin. Molecular Mekong [11]. Each fish was digested with 1% of approaches, such as PCR have been developed for acid pepsin solution (99 ml of 0.85% sodium chloride detection of different parasite species. Specific DNA solution, 1 g of pepsin, and 1 ml of concentrated probes have been investigated for the detection of O. hydrochloric acid) and incubated at 37οC for one hour viverrini [5, 6] and Paragonimus heterotremus [7] and thirty minutes. The digested material was filtered in Thailand. A mitochondrial based multiplex PCR for through graded sieves to remove large particles and the detection and discrimination of Clonorchis rinsed twice with normal saline solution (0.85% NaCl). sinensis and O. viverrini has been documented [8]. The metacercariae were collected and counted under Specific primers derived from HAT-RAPD marker a stereo microscope and identified based on have been developed for detection and identification morphological characters. The prevalence and mean of H. taichui [9]. Our study was performed to density of metacercarial infections were also determine the occurrence of trematode infection, calculated. The seasonal variation on the prevalence especially liver fluke and minute intestinal flukes of metacercarial was analyzed by using ANOVA. metacercariae in fish from Fang-Mae Ai

Figure 1. Map of the eight sampling sites in the Fang-Mae Ai Agricultural Basin Vol. 7 No. 1 Molecular identification of intestinal metacercariae 99 February 2013

Genomic DNA of all parasites was extracted the presence of metacercariae. Eleven species of and purified from adult flukes and metacercariae, cyprinoid fish, viz. Barbonymus gonionotus, using the GF-1 Tissue DNA extraction kit (Vivantis, Esomus metallicus, Hampala macrolepidota, Malaysia), according to the manufacturer’s Henicorhynchus siamensis, Labiobarbus instructions. All extracted genomic DNA was detected siamensis, Mystacoleucus marginatus, Osteochilus by HAT-RAPD PCR using arbitrary primers to hasselti, brevis, Raiamas guttatus, Systomus compare DNA profiles between adult flukes and orphoides, and Systomus stoliczkanus and one metacercariae. The six arbitrary primers used were; species of Anabantid fish, Anabas testudineus, OPP 11, OPN 09, OPA 01, OPA 02, OPN 01, and were infected with metacercariae. Five species OPN 02. The reaction of HAT-RAPD PCR was of metacercariae were found; one species of liver carried out in a final volume of 20 L, with common fluke, Opisthorchis viverrini, and four species of PCR composition. The reactions were performed in minute intestinal flukes, viz. Haplorchis taichui, a MyCyclerTM Thermocycler (Bio-RAD) and PCR Haplorchoides sp., Centrocestus caninus and conditions performed as follows; 1 cycle of 94οC for Stellantchasmus falcatus. The highest total 2 minutes, 35 cycle of 94οC for 30 seconds, 48οC for prevalence of metacercarial infection was observed 45 seconds, 72οC for 1 minutes and 1 cycle of final in the cool-dry season with 78.30%, followed by the extension at 72οC for 7 minutes. HAT-RAPD PCR rainy and hot-dry seasons with 72.84%, and 69.01%, products were separated on 1.4% TBE agarose gel respectively. electrophoresis, stained with ethidium bromide, and The highest prevalence of metacercarial infection photographed with a Kodak Digital Camera Gel Logic based on each trematode species found were: 100. The amplified HAT-RAPD markers were Haplorchoides sp. (37.43%), H. taichui (35.66%), scored as present (1) or absent (0) for each sample. C. caninus (3.80%), and S. falcatus (1.40%). Ambiguous bands that could not be easily distinguished Contrastingly, O. viverrini was for the first time were not scored [12]. Phylogenetic relationships recorded from this area in the hot-dry season with a among each species were analyzed using the UPGMA prevalence of 0.44% (Figure 2). For statistical method in the Clustal W2. analysis, there were no significant differences (p >0.05) among the prevalence of metacercarial Results infections found in each season. Two thousand two hundred eighty two fish belonging to 27 species were investigated for

Figure 2. Prevalence of metacercarial infections found in each season 100 C. Wongsawad, et al.

Haplorchoides sp. metacercaria were also found A molecular study using HAT-RAPD PCR was with H. taichui in the same fish species and were performed to generate DNA profiles and analyze found at all sampling site in all three seasons. Seven phylogenetic relationships among metacercariae fish species: Systomus orphoides, Henicorhynchus collected. This was done by comparing DNA profiles siamensis, Mystacoleucus ectypus, Labiobarbus of metacercariae found in adult stages. The pattern siamensis, Osteochilus hasselti, Barbodes of DNA profiles obtained from the primer OPP-11, gonionotus, and were recognized as OPN-09, OPA-01, OPA-02, OPN-01, and OPN-02 hosts of Haplorchoides sp. H. taichui was also found showed total polymorphism (Table 2) while the at all sampling sites in all three seasons and infected highest and lowest fragment number generated were six fish species: S. orphoides, H. siamensis, M. observed from OPA-02 and OPN-02 with 13 and ectypus, O. hasselti, B. gonionotus, and Raiamus seven fragments respectively. DNA profiles of all guttatus. C. caninus and S. falcatus were rare at metacercarial found in this study were the same as each sampling site in all three seasons. C. caninus those from adult stages. For O. viverrini metacercaria and S. falcatus were found only Rasbora metallicus found in S. stoliczkanus, the DNA profile showed and Anabas testudineus, respectively. O. viverrini the same pattern as those of adult flukes from northeast metacercariae were also found for the first time in S. Thailand (Figures 3 and 4). UPGMA phylogram stoliczkanus at only one sampling site (site 3) during analysis, based on HAT-RAPD profiles showed that the hot-dry season. The prevalence and mean density all metacercariae found in this study were of the same of metacercariae found in each fish species are shown branch as those of their adult clusters (Figure 4). in Table 1.

Table 1. Prevalence and mean density of metacercariae found in each fish species

Fish species Species of metacercaria No. infected/ % Mean examined Prevalence density

Anabas testudineus Stellantchasmus falcatus 11/11 100.00 15.00 Barbonymus gonionotus Haplorchis taichui 73/208 35.10 24.00 Haplorchoides sp. 135/208 64.90 15.04 Esomus metallicus Centrocestus caninus 29/29 100.00 11.72 Hampala macrolepidota Haplorchis taichui 54/129 41.86 11.85 Haplorchoides sp. 75/129 58.14 18.00 Henicorhynchus siamensis Haplorchis taichui 103/239 43.10 21.00 Haplorchoides sp. 136/239 56.90 12.13 Labiobarbus siamensis Haplorchis taichui 86/212 40.57 17.91 Haplorchoides sp. 126/212 59.43 13.13 Mystacoleucus marginatus Haplorchis taichui 233/532 43.80 14.00 Haplorchoides sp. 299/532 56.20 9.00 Osteochilus hasselti Haplorchis taichui 96/184 52.17 8.00 Haplorchoides sp. 88/184 47.83 11.00 Puntius brevis Centrocestus caninus 13/13 100.00 18.00 Raiamas guttatus Haplorchoides sp. 8/8 100.00 6.00 Systomus orphoides Haplorchis taichui 74/82 90.24 10.08 Systomus stoliczkanus Haplorchoides sp. 18/18 100.00 12.00 Opisthorchis viverrini 3/18 16.67 1.67 Vol. 7 No. 1 Molecular identification of intestinal metacercariae 101 February 2013

Table 2. Characteristics of random oligonucleotide primers used in the HAT-RAPD analyses.

Primers Sequence No. of No. of % Polymorphism fragments polymorphic

OPP-11 AACGCGTCGG 8 8 100 OPN-09 TGCCGGCTTG 10 10 100 OPA-01 CAGGCCCTTC 9 9 100 OPA-02 TGCCGAGCTG 13 13 100 OPN-01 CTCACGTTGG 8 8 100 OPN-02 ACCAGGGGCA 7 7 100

Figure 3. DNA profiles generated by using six arbitrary primers: OPP-11 (a) OPN-09 (b) OPA-01 (c) OPA-02 (d) OPN-01 (e) OPN-02 (f). Lane M, 100 bp ladder; Lane 1, O. viverrini (adult); Lane 2, H. taichui (adult); Lane 3, C. caninus (adult); Lane 4, S. falcatus (adult); Lane 5, Haplorchoides sp. (adult); Lane 6, F. gigantica (adult);Lane 7, P. epiclitum (adult); Lane 8, O. viverrini (metacercaria); Lane 9, H. taichui (metacercaria); Lane 10, C. caninus (metacercaria); Lane 11, S. falcatus (metacercaria); Lane 12, Haplorchoides sp. (metacercaria). 102 C. Wongsawad, et al.

Figure 4. Cladogram constructed with 55 polymorphic fragments generated with the HAT-RAPD technique using UPGMA method from Clustal W2

Discussion of this area. Fang and Mae-Ai districts are located in The occurrence of trematode infections in the the basin of the , which is a mainstream river Fang-Mae Ai Agricultural Basin was already in this area. The Kok river originates in a mountainous determined and the most common was the group area and is running through small communities and of minute intestinal flukes with H. taichui, farmland, while the is mainstream of Chom Haplorchoides sp., C. caninus, and S. falcatus. The Thong district, which longer and goes through liver fluke, O. viverrini was rarely found in this area. numerous communities and farmland. The other reason The highest prevalence (100%) was found in for lower infection may be the intermediate host three species of trematodes, viz. Haplorchoides sp., diversity of these parasites. The number of snail C. caninus, and S. falcatus. Haplorchoides sp. intermediate host in the Kok river quite lower than metacercaria were always mixed infections along the Ping river. with H. taichui populations and it is a dominant Metacercarial infection of the minute intestinal species in cyprinoid fish [13]. These two flukes were flukes: H. taichui, Haplorchoides sp., C. caninus distributed at all sampling site in all three seasons, and S. falcatus were similar to previous reports, which whereas C. caninus and S. falcatus were rarely claimed that these trematodes were distributed in the distributed but found in all seasons and in all sampling northern region [3-4, 13]. Importantly, we found O. sites. C. caninus was found only from Rasbora viverrini for the first time from S. stoliczkanus at metallicus, but previously reported in P. brevis [14]. only one sampling site. This finding provides evidence In our study, S. falcatus was found in A. testudineus, that O. viverrini infection extended to northern agreeing with study from , Chiang Mai Thailand. The epidemiology and ecological distribution province whereas other reports recovered this parasite of these trematodes depend on several snail species from the half-beaked fish, Dermogenus pusillus [15, and cyprinoid fish that are the first and second 16]. For the mean density of trematode infection, H. intermediate host respectively. They are commonly taichui was the highest in B. gonionotus with mean found in all rivers in this basin. Raining and flooding density of 24.0 individuals / fish while 21.0 individuals are the main factors that affect the dispersion of / fish in He. siamensis. Mean density of trematode intermediate hosts. This combination of first and infection in Fang and Mae Ai districts were lower second intermediate host interaction enhances the than in Chom Thong and Mae Taeng districts [13]. possibility of parasitic infections. Our findings are The cause of lower infection in Fang and Mae-Ai supported by reports of host-parasite relationships that district than Chom Thong district may be the location indicate that the number of parasite infection depends Vol. 7 No. 1 Molecular identification of intestinal metacercariae 103 February 2013

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