• View in gallery

    Sampling localities of N. aperta. The stars indicate the sampling area in NK (TK) and UB (Ban Bung Khi Lek, KJ, and Kang Sapue).

  • View in gallery

    Maximum likelihood tree based on 18 published and 26 new partial cox1 gene sequences of N. aperta α-, β-, and γ-races. Accession numbers are shown for sequences obtained from a public DNA database. Sequences newly obtained for this study are indicated in bold. A published sequence of Tricula bollingi from Chiang Mai (AF339847) was used as an outgroup. CM = Chiang Mai; KC = Kracheh Province; KHM = Cambodia; KI = Khong Island; LAO = Laos; MR = Mun River; XBF = Xé Bang Fai.

  • 1.

    Attwood SW, Upatham ES, 2012. Observations on Neotricula aperta (Gastropoda: Pomatiopsidae) population densities in Thailand and central Laos: implications for the spread of Mekong schistosomiasis. Parasit Vectors 5: 126.

    • Search Google Scholar
    • Export Citation
  • 2.

    Urbani C, Sinoun M, Socheat D, Pholsena K, Strandgaard H, Odermatt P, Hatz C, 2002. Epidemiology and control of mekongi schistosomiasis. Acta Trop 82: 157168.

    • Search Google Scholar
    • Export Citation
  • 3.

    Davis GM, Kitikoon V, Temcharoen P, 1976. Monograph on “Lithoglyphopsisaperta, the snail host of Mekong River Schistosomiasis. Malacologia 15: 241287.

    • Search Google Scholar
    • Export Citation
  • 4.

    Tropmed Technical Group, 1986. Snails of medical importance in Southeast Asia. Southeast Asian J Trop Med Public Health 17: 282322.

  • 5.

    Attwood SW, Fatih FA, Campbell I, Upatham ES, 2008. The distribution of Mekong schistosomiasis, past and future: preliminary indications from an analysis of genetic variation in the intermediate host. Parasitol Int 57: 256270.

    • Search Google Scholar
    • Export Citation
  • 6.

    Attwood SW, 2010. Studies on the parasitology, phylogeography and the evolution of host-parasite interactions for the snail intermediate hosts of medically important trematode genera in Southeast Asia. Adv Parasitol 73: 405440.

    • Search Google Scholar
    • Export Citation
  • 7.

    Attwood SW, Ambu S, Meng X-H, Upatham ES, Xu F-S, Southgate VR, 2003. The Phylogenetics of Triculine snails (Rissooidea: Pomatiopsidae) from South-east Asia and Southern China: historical biogeography and the transmission of human Schistosomiasis. J Molluscan Stud 69: 263271.

    • Search Google Scholar
    • Export Citation
  • 8.

    Staub KC, Woodruff DS, Upatham ES, Viyanant V, 1990. Genetic variation in Neotricula aperta, the intermediate snail host of Schistosoma mekongi: allozyme differences reveal a group of sibling species. Am Malacol Bull 7: 93103.

    • Search Google Scholar
    • Export Citation
  • 9.

    Attwood SW, Kitikoon V, Southgate VR, 1998. Neotricula aperta (Gastropoda: Pomatiopsidae), the intermediate host of Schistosoma mekongi: allozyme variation and relationships between Khmer, Lao and Thai populations. J Zool (Lond) 246: 309324.

    • Search Google Scholar
    • Export Citation
  • 10.

    Attwood SW, 1999. Genetic variation in Neotricula aperta, the snail intermediate host of Schistosoma mekongi in the lower Mekong Basin. J Zool (Lond) 249: 153164.

    • Search Google Scholar
    • Export Citation
  • 11.

    Attwood SW, Johnston DA, 2001. Nucleotide sequence differences reveal genetic variation on Neotricula aperta (Gastropoda: Pomatiopsidae), the snail host of Schistosomiasis in the lower Mekong Basin. Biol J Linn Soc Lond 73: 2341.

    • Search Google Scholar
    • Export Citation
  • 12.

    Attwood SW, Upatham ES, Zhang Y-P, Yang ZQ, Southgate VR, 2004. A DNA-sequence based phylogeny for triculine snails (Gastropoda: Pomatiopsidae: Triculinae), intermediate hosts for Schistosoma (Trematode: Digenea): phylogeography and the origin of Neotricula. J Zool (Lond) 262: 4756.

    • Search Google Scholar
    • Export Citation
  • 13.

    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 27312739.

    • Search Google Scholar
    • Export Citation

 

 

 

 

A New Population and Habitat for Neotricula aperta in the Mekong River of Northeastern Thailand: A DNA Sequence-Based Phylogenetic Assessment Confirms Identifications and Interpopulation Relationships

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  • Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand; School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand

Neotricula aperta (Gastropoda: Pomatiopsidae), the snail intermediate host of Schistosoma mekongi, is found in Cambodia, Laos, and Thailand. We update information on the distribution of this species in the Mekong River and its tributary, the Mun River, in Thailand. DNA sequences of a portion of the mitochondrial cytochrome c oxidase subunit 1 were obtained from N. aperta collected from different locations and used to confirm species and strain identities. Specimens of the β-strain were found in the Mun River, whereas specimens of the γ-strain were found in the Mekong River. The γ-strain (with molecular confirmation of identity) is newly reported from Nong Khai Province, where it occurred in a habitat novel for this species: under paving slabs instead of under natural bed rocks, where agal aufwuchs is extensively located on the islet in the middle of the Mekong River. The new location is approximate 400 km upstream from the nearest previously known site for this species.

Introduction

Mekong schistosomiasis is an important waterborne parasitic disease caused by the blood fluke Schistosoma mekongi. Focal transmission of this disease is known from Khong Island, Champassac Province, Lao People's Democratic Republic (PDR) and Kratié, Stung-Treng, and Rattanakiri Provinces, Cambodia.1 S. mekongi uses the freshwater snail Neotricula aperta (family Pomatiopsidae, subfamily Triculinae) as an intermediate host to complete its lifecycle2,3; α-, β-, and γ-strains of N. aperta can be differentiated by shell size, shape, sculpture, mantle pigment patterns, ecology, time of development, and minor aspects of anatomy. Of these, the γ-strain is the most important vector for S. mekongi. In 1986, the Tropmed Technical Group reported that the α- and γ-strains were found in the Mekong River ranging from Savannakhet, Lao PDR to Kratié in Cambodia.4 The β-strain of N. aperta occurred exclusively in the Mun River in Phibun Mangsahan District, Ubon Ratchathani Province (UB), Thailand. In 2008, the reported range of the α- and γ-strains was extended northward to the Nam Hinboun in Khammouanne Province, Lao PDR and a total of 31 localities spanning nine river systems in Cambodia, Lao PDR and Thailand.5

Although transmission of Mekong schistosomiasis is not known from Thailand, populations of the potential intermediate host have been found. Routine activities of villagers living near the habitats of N. aperta potentially expose them to the risk of infection. To date, the known habitats of N. aperta in Thailand are limited to the Mun and Mekong Rivers in UB.3,4,6 N. aperta has not yet been reported from upstream portions of the Mekong within Thailand.

Conventional identification methods may not be adequate for N. aperta because of its small size, high prevalence of convergent evolution, and relatively high degree of intraspecific variation among anatomical characters.3 There are some studies of genetic variation among different strains of N. aperta based on allozyme differences,79 restriction fragment length polymorphism (RFLP),10 and nucleotide sequence differences.5,7,11,12 Attwood and others5 and Attwood and Johnston11 investigated genetic variation and the phylogenetic relationships of mitochondrial DNA sequences among the different strains of N. aperta from six populations11 and 31 sites5 of central and southern Lao PDR, eastern Cambodia, and northeast Thailand.

Here, we report molecular identification of N. aperta collected in northeastern Thailand in 2010–2012. The γ-strain was found at a new locality in the Mekong River, and the β-strain was found in the Mun River. DNA sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) of these snails were used to place the newly found populations in a phylogenetic context with previously studied populations. These genetic data will be important for epidemiological investigations of Mekong schistosomiasis.

Wherever it has been reported, N. aperta has been found on natural substrates, such as under natural bed rocks where agal aufwuchs are located on islets in the middle of the Mekong River.1 Here, we report a new non-natural substrate for N. aperta in the Mekong River in Thailand.

Materials and Methods

Sampling.

Samples were collected from four localities in two provinces in northeastern Thailand (Nong Khai [NK] and UB) (Figure 1 and Table 1). Live snails were identified on the basis of general characteristics, conchology, and habitat.3 Samples were preserved in absolute ethanol until DNA extraction.

Figure 1.
Figure 1.

Sampling localities of N. aperta. The stars indicate the sampling area in NK (TK) and UB (Ban Bung Khi Lek, KJ, and Kang Sapue).

Citation: The American Society of Tropical Medicine and Hygiene 92, 2; 10.4269/ajtmh.14-0467

Table 1

Sampling details and accession numbers of N. aperta in the Mekong River and the Mun River, Thailand

SpeciesStrain*LocalitiesNumberAccession numbersSampling date
N. apertaγBan Bung Khi Lek, Mekong River, KR-UB6KM099100–KM099102 and KM099109–KM0991114/23/2010
N. apertaγMekong River, KJ-UB3 (KJL); 3 (KJH)KM099096–KM099099, KM099112, and KM0991134/24/2010
N. apertaβKang Sapue, Mun River, UB2KM099114 and KM0991154/30/2010
N. apertaβKang Sapue, Mun River, UB3KM099116–KM09911811/30/2012
N. apertaβKang Sapue, Mun River, UB3KM099119–KM09912111/27/2010
N. apertaγMekong River, TK-NK6KM099103–KM09910812/1/2012
N. apertaαMekong River, KR-UB3AF188210–AF1882124/22/1997
N. apertaβMun River, UB4AF188213–AF1882164/24/1997
N. apertaγKracheh Province, Mekong River, Cambodia3AF188217–AF1882195/2/1996
N. apertaγKhong Island, Laos2AF188220 and AF1882214/20/1997
N. apertaγMekong River, UB5AF188222–AF1882264/22/1997
N. apertaγXé Bang Fai River, Khammounne Province, Laos1AF1882273/27/1998

Strain identified by morphology.

Samples collected from this study.

Information from the GenBank database.11

DNA amplification and sequencing.

The whole-snail specimens (26 in total) were individually crushed, and DNA was extracted using the Nucleospin Tissue Kit (Macherey-Nagel GmbH & Co, Duren, Germany). The DNA was eluted in 100 μL distilled water, 5 μL of which was used for the polymerase chain reaction (PCR). A PCR specific for the partial cox1 gene sequence was conducted with primer set forward primer: 5′ ATG ATC TGG GTT AGT AGG TAC TG 3′and reverse primer: 5′ AGC AGG ATC AAA AAA AGC TGT A 3′, resulting in an amplicon size of 598 base pairs (bp). The primers were based on published sequences of N. aperta (AF188224).11

Amplification was done using a GeneAmp PCR System 9700 Thermal Cycler (Applied Biosystems, Singapore). The DNA template was initially denatured at 94°C for 5 minutes. The amplification procedure consisted of 35 cycles at 95°C for 30 seconds (denaturation), 53°C for 30 seconds (annealing), and 72°C for 30 seconds (extension), with a final extension at 72°C for 10 minutes. The PCR product was run on a 1% agarose gel to confirm amplification and estimate product length and quantity. DNA direct sequencing was performed using the Applied Biosystems 3730×I DNA Analyzer and ABI Big Dye, version 3.1 (Applied Biosystems, Foster City, CA) in both directions with the same PCR primers. The cox1 gene sequences of the N. aperta samples were analyzed using a standard nucleotide basic local alignment search tool (BLASTN) search through the National Center of Biotechnology Information. The sequences were also aligned and compared with the available sequence data for N. aperta in the GenBank Database using the multiple sequence alignment program ClustalW. A phylogenetic tree was constructed using the maximum likelihood method with MEGA, version 5.13 The Hasegawa–Kishino–Yano model, with non-uniformity of evolutionary rates among sites modeled by using a discrete γ-distribution (+G), was found to be the best model.13 Support for groupings within the tree was evaluated by bootstrapping with 1,000 resamplings. All sequences were submitted to the GenBank database (Figure 2 and Table 1).

Figure 2.
Figure 2.

Maximum likelihood tree based on 18 published and 26 new partial cox1 gene sequences of N. aperta α-, β-, and γ-races. Accession numbers are shown for sequences obtained from a public DNA database. Sequences newly obtained for this study are indicated in bold. A published sequence of Tricula bollingi from Chiang Mai (AF339847) was used as an outgroup. CM = Chiang Mai; KC = Kracheh Province; KHM = Cambodia; KI = Khong Island; LAO = Laos; MR = Mun River; XBF = Xé Bang Fai.

Citation: The American Society of Tropical Medicine and Hygiene 92, 2; 10.4269/ajtmh.14-0467

Results

The partial cox1 DNA sequences (553 bp excluding the primer sequences) of 26 specimens of N. aperta collected from four localities in the Mekong and Mun Rivers, Thailand have been deposited in GenBank under accession numbers KM099096–KM099121 (Figure 2 and Table 1). When aligned and compared with published data, 18 of our sequences proved to represent the γ-strain and 8 of our sequences proved to represent the β-strain (Figure 2 and Table 1). All β-strain samples came from the Mun River, and all γ-strain representatives came from the Mekong River. Similarities between sequences ranged from 97% to 99%. Variation within sampling sites was found in Ban Bung Khi Lek, Khammarat District (KR) and Khong Jeum District (KJ), UB. It is also worth noting that the populations were paraphyletic and coincided with the KR clades for the snails sampled in this study but not so with the GenBank KR data, which formed a separate clade. As a consequence, only snails of the β-strain from the Mun River population formed a monophyletic clade (Figure 2).

Morphological examination of N. aperta from KJ revealed two different patterns of mantle pigmentation. Consequently, the KJ snails were coded as marked pigmentation (KJH) and reduced pigmentation (KJL). Phylogenetic analysis showed that KJH fell into a single clade but that KJL separated into two clades.

A new population of N. aperta (γ-strain) is described here from Ban Tha Kathin (TK), Sri Chiang Mai District, NK Province well upstream of previously known populations in the Mekong River. Although the habitats of N. aperta in KR and KJ are under natural rocks located on islets in the middle of the Mekong River, specimens of N. aperta at the TK-NK site were found under the rocks, which were used together with concrete paving for prevention of soil erosion along the river bank.

Discussion

In northeast Thailand, three strains of N. aperta have been recognized (α, β, and γ) on the basis of shell size and body pigmentation.1 However, the small size of triculine snails leads to problems in assessing character states and technical difficulties in their identification. These problems together with convergent evolution in the group and a relatively high degree of intraspecific variation in anatomical characters suggest that nucleotide sequence data will be useful in resolving relationships in cases where anatomy is ambiguous.11 This is supported by our finding that N. aperta snails (γ-strain) from KJ-UB fell into two groups according to their mantle pigmentation; however, the molecular data failed to group the snails according to pigmentation.

DNA sequences of N. aperta γ-strain were examined for phylogenetic studies of triculine snails6 and evaluation of geographical patterns and to identify genetic subpopulations or clades.4 Mitochondrial genes were selected, because with their maternal pattern of inheritance and smaller effective population size, they were considered to represent potentially better records of phylogenetic events at the intraspecific level.

As in other studies on N. aperta phylogeny, the β-strain from the Mun River was clearly located in a different clade from the α- and γ-strains from the Mekong River. The genetic isolation of the β-strain appears to have a geographical basis.10 The Mun River populations seem more stable in these results than in the results of earlier studies,5 which might be because of the Pak-Mun Dam preventing dispersal of snails. The Pak-Mun Dam was opened in 1994 and may have affected our study more than earlier studies.11 However, the effect on β-strain diversity and long-term popular trends may need to be monitored.

Samples of the N. aperta γ-strain from KJ-UB and those from KR-UB fell into several different places on the phylogenetic tree, whereas those from TK-NK occupied a single region on the tree. Such a genetic subdivision of the N. aperta γ-strain was also recorded in the study by Attwood.10 Attwood10 suggested that the N. aperta γ-strain from the KR-UB site may have originated in smaller rivers draining the highlands of central Laos and that it then colonized the Mekong River and became a part of the γ-strain population in UB. This hypothesis suggests that N. aperta populations in the Mekong River are not stable and exist as a metapopulation. The lack of genetic diversity among snails from the TK-NK site might be explained by an origin in highland streams and lack of dispersal of snails from the lowland reaches of the Mekong River.10

This is the first report of the N. aperta γ-strain from the Mekong River in NK Province, Thailand. The identification was supported by molecular evidence from the mitochondrial cox1 gene. In addition, this is the first report of this snail from an anthropogenic habitat (i.e., paving slabs) rather than a natural substratum.

ACKNOWLEDGMENTS

We acknowledge the support of the Khon Kaen University Publication Clinic, Research and Technology Transfer Affairs, Khon Kaen University for assistance. The authors also thank Dr. David Blair for valuable suggestions.

  • 1.

    Attwood SW, Upatham ES, 2012. Observations on Neotricula aperta (Gastropoda: Pomatiopsidae) population densities in Thailand and central Laos: implications for the spread of Mekong schistosomiasis. Parasit Vectors 5: 126.

    • Search Google Scholar
    • Export Citation
  • 2.

    Urbani C, Sinoun M, Socheat D, Pholsena K, Strandgaard H, Odermatt P, Hatz C, 2002. Epidemiology and control of mekongi schistosomiasis. Acta Trop 82: 157168.

    • Search Google Scholar
    • Export Citation
  • 3.

    Davis GM, Kitikoon V, Temcharoen P, 1976. Monograph on “Lithoglyphopsisaperta, the snail host of Mekong River Schistosomiasis. Malacologia 15: 241287.

    • Search Google Scholar
    • Export Citation
  • 4.

    Tropmed Technical Group, 1986. Snails of medical importance in Southeast Asia. Southeast Asian J Trop Med Public Health 17: 282322.

  • 5.

    Attwood SW, Fatih FA, Campbell I, Upatham ES, 2008. The distribution of Mekong schistosomiasis, past and future: preliminary indications from an analysis of genetic variation in the intermediate host. Parasitol Int 57: 256270.

    • Search Google Scholar
    • Export Citation
  • 6.

    Attwood SW, 2010. Studies on the parasitology, phylogeography and the evolution of host-parasite interactions for the snail intermediate hosts of medically important trematode genera in Southeast Asia. Adv Parasitol 73: 405440.

    • Search Google Scholar
    • Export Citation
  • 7.

    Attwood SW, Ambu S, Meng X-H, Upatham ES, Xu F-S, Southgate VR, 2003. The Phylogenetics of Triculine snails (Rissooidea: Pomatiopsidae) from South-east Asia and Southern China: historical biogeography and the transmission of human Schistosomiasis. J Molluscan Stud 69: 263271.

    • Search Google Scholar
    • Export Citation
  • 8.

    Staub KC, Woodruff DS, Upatham ES, Viyanant V, 1990. Genetic variation in Neotricula aperta, the intermediate snail host of Schistosoma mekongi: allozyme differences reveal a group of sibling species. Am Malacol Bull 7: 93103.

    • Search Google Scholar
    • Export Citation
  • 9.

    Attwood SW, Kitikoon V, Southgate VR, 1998. Neotricula aperta (Gastropoda: Pomatiopsidae), the intermediate host of Schistosoma mekongi: allozyme variation and relationships between Khmer, Lao and Thai populations. J Zool (Lond) 246: 309324.

    • Search Google Scholar
    • Export Citation
  • 10.

    Attwood SW, 1999. Genetic variation in Neotricula aperta, the snail intermediate host of Schistosoma mekongi in the lower Mekong Basin. J Zool (Lond) 249: 153164.

    • Search Google Scholar
    • Export Citation
  • 11.

    Attwood SW, Johnston DA, 2001. Nucleotide sequence differences reveal genetic variation on Neotricula aperta (Gastropoda: Pomatiopsidae), the snail host of Schistosomiasis in the lower Mekong Basin. Biol J Linn Soc Lond 73: 2341.

    • Search Google Scholar
    • Export Citation
  • 12.

    Attwood SW, Upatham ES, Zhang Y-P, Yang ZQ, Southgate VR, 2004. A DNA-sequence based phylogeny for triculine snails (Gastropoda: Pomatiopsidae: Triculinae), intermediate hosts for Schistosoma (Trematode: Digenea): phylogeography and the origin of Neotricula. J Zool (Lond) 262: 4756.

    • Search Google Scholar
    • Export Citation
  • 13.

    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 27312739.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Wanchai Maleewong, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail: wanch_ma@kku.ac.th

Financial support: This research was funded by Thailand Research Fund (TRF) Grant MRG5680085 (to Y.L.). O.S., W.K., P.M.I., P.J., L.S., and W.M. were supported by a TRF Senior Research Scholar Grant, TRF Grant RTA5580004, and Faculty of Medicine Grant, Khon Kaen University Grant TR57201.

Authors' addresses: Yanin Limpanont, Phiraphol Chusongsang, Yupa Chusongsang, and Jareemate Limsomboon, Southeast Asian Center for Medical Malacology, Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, E-mails: yanin.lim@mahidol.ac.th, phiraphol.chu@mahidol.ac.th, yupa.chu@mahidol.ac.th, and jareemate.lim@mahidol.ac.th. Oranuch Sanpool, Pewpan M. Intapan, Lakkhana Sadaow, and Wanchai Maleewong, Department of Parasitology, Faculty of Medicine and Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen, Thailand, E-mails: sanpoolor@yahoo.com, pewpan@kku.ac.th, sadaow1986@hotmail.com, and wanch_ma@kku.ac.th. Worasak Kaewkong, Department of Biochemistry, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand, E-mail: worasak_bc@hotmail.com. Penchom Janwan, Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand, E-mail: pair_wu@yahoo.com.

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