First intermediate hosts of Paragonimus spp. in Vietnam and identification of intramolluscan stages of different Paragonimus species

First intermediate hosts of Paragonimus spp. in Vietnam and identification of intramolluscan... Background: Members of the genus Paragonimus require at least three hosts in their life-cycles. The obligatory first intermediate hosts are freshwater snails. In Vietnam, although seven Paragonimus species have been recorded, the natural first intermediate hosts of almost all species have not been confirmed. The aim of this study was, therefore, to investigate snail hosts of Paragonimus species in Vietnam, and to identify Paragonimus species at intramolluscan stages. Methods: Freshwater snails were collected from streams in Yen Bai and Quang Tri Provinces, where high prevalences of Paragonimus metacercariae in crab hosts have been reported. Snails were morphologically identified and then examined individually for Paragonimus cercariae using shedding and crushing methods. Chaetomicrocercous cercariae, the morphological class to which Paragonimus cercariae belong, were collected for morphological description and molecular species identification by analyses of ITS2 sequences. The infected snail species were identified based on analyses of nucleotide sequences of the cox1 gene. Results: Three snail species were found to be infected with Paragonimus cercariae at low infection rates, ranging between 0.07–1.0%. The molecular analyses identified them as Sulcospira quangtriensis and 2 species of subfamily Triculinae. In a phylogenetic tree, these two triculine snails were related to the genera Gammatricula and Tricula with low posterior probabilities. Thus we named them as Triculinae sp. 1 and Triculinae sp. 2. Cercariae from the three snail species, Sulcospira quangtriensis, Triculinae sp. 1 and Triculinae sp. 2, were molecularly identified as Paragonimus westermani, P. heterotremus and P. proliferus, respectively. The cercariae of the three species are morphologically similar to each other, but their daughter rediae can be distinguished by the length of the intestine and the number of cercariae per redia. The rediae of P. westermani have a long intestine and each contain 6–8 cercariae. In contrast, those of P. heterotremus and P. proliferus have a short intestine and each redia contain 10–12 and 5–6cercariae, respectively. Conclusions: Three snail species, Sulcospira quangtriensis, Triculinae sp. 1 and Triculinae sp. 2, serve as the first intermediate hosts of P. westermani, P. heterotremus and P. proliferus, respectively, in Vietnam. The length of the intestine of rediae and the number of cercariae per redia are valuable characteristics for distinguishing between larvae of these Paragonimus species. Keywords: First intermediate host, Paragonimus, Sulcospira quangtriensis,Triculinae * Correspondence: pndoanh@yahoo.com Institute of Ecology and Biological Resources, Viet Nam Academy of Science and Technology, Hanoi, Viet Nam Graduate University of Science and Technology, Viet Nam Academy of Science and Technology, Hanoi, Viet Nam Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Doanh et al. Parasites & Vectors (2018) 11:328 Page 2 of 8 Background different snail species. The host specificity and the mor- Lung flukes of the genus Paragonimus (Family Paragoni- phological features of different Paragonimus species in midae) are parasites of mammals, commonly cats and the snail hosts are discussed herein. canids, and occasionally humans. Members of this genus require at least three different hosts to complete their Methods life-cycles [1]. The first intermediate hosts are freshwater Collection and examination of snails snails in which the flukes develop through several stages Freshwater snails were collected in streams in An Lac (sporocyst, mother and daughter rediae, and cercariae). Commune (Luc Yen District, Yen Bai Province) and The cercariae belong to the microcercous type (subtype Huong Son Commune (Huong Hoa District, Quang Tri chaetomicrocercous), possessing an anterior stylet, a Province) (Fig. 1) where high infection rates of crab knob-like tail, spines on the body and tail, but lacking hosts with Paragonimus metacercariae were found [8]. unicellular glands [2]. Mature cercariae escape from the Snails were morphologically identified following Dang & snail hosts and penetrate suitable crab/crayfish hosts to Ho [13], and were examined for Paragonimus cercariae develop into an infective stage, metacercariae. Definitive using shedding and crushing methods [14]. Snail sam- hosts become infected by eating these crabs/crayfish ples were placed individually in 20 ml wide mouth plas- containing live metacercariae [1]. tic containers filled with 15 ml of water and left for 24 h. Regarding host specificity, Paragonimus flukes typic- The containers were checked under a stereomicroscope ally infect a wider spectrum of definitive and second at night and the next morning. If any chaetomicrocer- intermediate hosts, whereas specificity for the first inter- cous cercariae were found, they were used for morpho- mediate host is usually very strict and vary geographic- logical study. Afterwards, all snails were dissected under ally [1, 3, 4]. Thus, identification of snail hosts in each a stereomicroscope to identify cercariae that did not endemic region is essential for understanding of routes emerge, and other intramolluscan stages, such as rediae. of transmission. Live cercariae and rediae were observed under an Axio In Vietnam, Paragonimus metacercariae of seven spe- Lab A1 microscope (Carl Zeiss, Oberkochen, Germany). cies have been reported in northern and central prov- inces: P. heterotremus, P. westermani, P. bangkokensis, P. harinasutai, P. proliferus, P. vietnamensis and P. skrja- bini [5]. Of these, P. heterotremus and P. westermani are important causative agents for human paragonimiasis in Asian countries. The former species is commonest in northern provinces, and the latter in central provinces [5]. So far, there has been only one report of microcer- cous cercariae from “Oncomelania” snails in Vietnam, in 2002 [6], at a time when only metacercariae of P. hetero- tremus was found [6, 7]. The cercaria was, therefore, identified as P. heterotremus without molecular evidence [6]. However, subsequent surveys have revealed the pres- ence of six additional Paragonimus species, and metacer- cariae of two to four species has been found in the same area [5, 8]. Moreover, the taxonomy of the “Oncomela- nia” snails in Vietnam has been revised and remains un- certain [9–11]. They were re-identified as a new genus Pseudotricula [9]. Since the name Pseudotricula had been assigned previously [10], Dang and Ho [11] renamed their genus as Vietricula. Liu et al. [12] consid- ered that Vietricula snails are closely related to the genus Gammatricula. Thus, data on first intermediate hosts of Paragonimus in Vietnam are scant and unclear. The aim of this study was, therefore, to investigate snail hosts of Paragonimus spp. in Yen Bai and Quang Tri Provinces, where Paragonimus metacercariae are highly prevalent with the dominance of P. heterotremus and P. westermani, respectively [8]. In this survey, we found Fig. 1 Geographical locations of sampling sites cercariae of three Paragonimus species, each in a Doanh et al. Parasites & Vectors (2018) 11:328 Page 3 of 8 Photographs of the cercariae and rediae were taken GTR model) were unlinked across partitions, to allow using a Axiocam Erc 5s digital camera attached to a them to take different values. MrBayes was run for Axio Lab A1 microscope (Carl Zeiss, Oberkochen, 2,000,000 generations, after which the standard deviation Germany) and ZEN lite software (Carl Zeiss). Cercariae of split frequencies was below 0.01. Trees were sampled and rediae were drawn using Illustrator CS6. Measure- every 500 generations. ‘Burn-in’ was 25% of trees. A con- ments (in μm) of cercariae and rediae were taken. Live sensus tree was constructed, including all compatible cercariae were identified according to Schell [2]. After groups of taxa. morphological identification, a single cercaria and tissues from each infected snails were separately preserved in Results absolute ethanol for molecular identification of cercariae Identification of snails infected with chaetomicrocercous and snail hosts. cercariae The various snails collected belonged morphologically to Molecular analysis for identification of cercariae and snail Melanoides, Sulcospira and minute snails of the subfam- hosts ily Triculinae. Snails of the genus Melanoides were few Genomic DNA of chaetomicrocercous cercariae and in number and clearly identified as Melanoides tubercu- snails from 10 infected snails was extracted using a lata. In contrast, Sulcospira snails were abundant, occur- PureLink Genomic DNA Mini Kit (Invitrogen, Califor- ring at high densities, especially in larger rocky streams nia, USA) following the manufacturer’s protocol. The in- with high levels of water and fast flow. They usually at- ternal transcribed spacer 2 (ITS2) region of nuclear tached to the surfaces of stones. The two species of tri- ribosomal DNA from cercariae was amplified via poly- culine were commonly found attached to fallen leaves in merase chain reaction (PCR) with the primers 3S small tributary streams with slow water flow. 5'-CGG TGG ATC ACT CGG CTC GT-3' as a forward primer and A28 5'-CCT GGT TAG TTT CTT TTC Chaetomicrocercous cercariae were not found from CTC CGC-3' as a reverse primer [15]. A portion of the M. tuberculata in either studied site or from Sulcospira mitochondrial cytochrome c oxidase 1 (cox1) gene of the sp. in Yen Bai Province, but were found from five Sulcos- infected snail species was amplified via PCR with the pira sp. in Quang Tri, and from four and one triculine primers LCO1490 5'-GGT CAA CAA ATC ATA AAG snails in Yen Bai and Quang Tri Provinces, respectively. ATA TTG G-3' as a forward primer and HCO2198 The sequences obtained from five Sulcospira snails 5'-TAA ACT TCA GGG TGA CCA AAA AAT YA-3' as from Quang Tri Province were completely identical with a reverse primer [16]. PCR products were purified using each other. The sequences from four Triculine snails a QIAquick PCR Purification Kit (Qiagen, California, from Yen Bai Province were also identical with each USA) and sequencing reactions done using a Big-Dye other. The results of BLAST searches showed that the terminator cycle sequencing kit v3.1 (Applied Biosys- cox1 sequence of the Sulcospira snail from Quang Tri tems, California, USA). Both forward and reverse strands Province was highly similar (99%) to that of Sulcospira were sequenced directly by a Genetic Analyzer (Model quangtriensis (FJ377265), confirming the identification 3100, Applied Biosystems, California, USA), using the of this species as S. quangtriensis.The cox1 sequences of PCR primers as sequencing primers. Triculinae snails from Yen Bai and Quang Tri Provinces Sequences obtained from cercariae and snails were showed the highest similarity (but only 91% similarity) submitted to GenBank with accession numbers to Gammatricula snails (AF213342). The samples from LC3605000-LC360505. Basic Local Alignment Search Yen Bai and from Quang Tri Provinces differed by Tool (BLAST) searches in the server of the National 12.6% and were placed separately in the phylogenetic Center for Biotechnology Information (NCBI) were used tree (Fig. 2). The posterior probabilities of groups in to determine the most similar sequences from named which they occurred were rather low. The snail from species. The cox1 sequences of the two triculine snail Quang Tri was grouped with Gammatricula songi with a species showed low similarities to available sequences on value of only 53%, the snail from Yen Bai was grouped GenBank. These sequences were therefore aligned with with Tricula wumingensis with a posterior probability of related sequences, and were used to reconstruct a phylo- 56%. The genus Gammatricula is shown as monophy- genetic tree using MrBayes [17]. The substitution model letic, but with a posterior probability of only 27%. The was selected using MrModelTest2 [18]. The model se- genus Tricula is not monophyletic in this tree. This sug- lected was the general time reversal (GTR) model with gests that the snails from Yen Bai and Quang Tri Prov- gamma-distributed rate variation across sites. The data inces belong to different genera of the subfamily were partitioned by codon position (first, second and Triculinae, but their taxonomic status remains uncertain. third positions). Two parameters of the model (shape of We have temporarily labelled them as Triculinae sp. 1 the gamma distribution and substitution rates of the from Yen Bai Province, and Triculinae sp. 2 from Quang Doanh et al. Parasites & Vectors (2018) 11:328 Page 4 of 8 Fig. 2 Phylogenetic tree reconstructed from cox1 sequences of triculine snails using Bayesian inference analysis. Numbers at nodes represent posterior probabilities. Samples obtained in this study are printed in bold Tri Province. The snails infected with chaetomicrocercous ventral sucker. The latter was round and situated at cercariae and their habitats are shown in Figs. 3 and 4. close to the middle of the body. A total of 14 pene- tration grand cells were distributed in groups of 3 Prevalence and morphology of chaetomicrocercous and 4 cells antero-lateral to the ventral sucker. A cercariae from snails large I-shaped excretory bladder was located between Infection rates of the snails with chaetomicrocercous the ventral sucker and the end of the body (Fig. 5). cercariae were low, ranging between 0.07–1.0% (Table 1). These are typical characteristics of Paragonimus In Huong Son Commune, Quang Tri Province, the density cercariae. of the S. quangtriensis population is very high. Sulcospira In contrast, the daughter rediae collected from dif- quangtriensis collected in the rocky main stream were not ferent snail species differed from one another in the positive, but those from a small tributary were infected length of the intestine and the number of cercariae with chaetomicrocercous cercariae. per redia (Fig. 5). The rediae collected from S. quang- The chaetomicrocercous cercariae from different triensis possessed a long intestine (about 75% of the snail species morphologically resemble one another body length) and each redia contained 6–8 cercariae. (Fig. 5). In each case, the mature cercariae had a sty- The rediae from Triculinae sp. 1 and Triculinae sp. 2 let and a short tail, with the measurements presented each had a short intestine (about 20% of the redia in Table 2. The body was elongated-oval in shape. body length). Each redia from Triculinae sp. 1 con- The oral sucker at the anterior extremity was accom- tained 10–12 cercariae, double the number (5–6) seen panied by a long stylet and was larger than the in each redia from Triculinae sp. 2 (Fig. 5). Doanh et al. Parasites & Vectors (2018) 11:328 Page 5 of 8 Fig. 3 Freshwater snails infected with Paragonimus cercariae. a Triculinae sp. 1 from Yen Bai. b Triculinae sp. 2 from Quang Tri. c Sulcospira quangtriensis from Quang Tri Molecular identification of chaetomicrocercous proliferus (AB663672) and P. westermani (LC144899), cercariae respectively. The result of BLAST searches showed that ITS2 se- quences of cercariae from snails Triculinae sp. 1 (4 iden- Discussion tical sequences from 4 infected snails), Triculinae sp. 2 Previously, cercariae considered to be of P. heterotremus (1 infected snail) and S. quangtriensis (5 identical se- were reported from minute snails (3–4 mm) in northern quences from 5 infected snails) were completely (100%) provinces of Vietnam [6]. As outlined in the introduc- identical with those of P. heterotremus (AB82365), P. tion, the identity of these snails remains unclear. They Fig. 4 Habitats of snails in Quang Tri Province. a A rocky main stream with fast water flow. b Sulcospira quangtriensis on a stone of the main stream. c A tributary with slow water flow where Sulcospira and triculine snails live Doanh et al. Parasites & Vectors (2018) 11:328 Page 6 of 8 Table 1 Prevalence of Paragonimus cercariae in snails Location Snail species No. examined No. infected (%) Paragonimus species An Lạc (Yen Bai) Triculinae sp. 1 1200 4 (0.3) P. heterotremus Sulcospira sp. 1000 0 – M. tuberculata 65 0 – Huong Sơn (Quang Tri) Triculinae sp. 2 1520 1 (0.07) P. proliferus S. quangtriensis 500 5 (1.0) P. westermani S. quangtriensis 5000 0 – M. tuberculata 70 0 – Collected from the small tributary Collected from the main stream were first identified as Oncomelania snails [6], then were related to the genus Gammatricula. These might named as Pseudotricula [9] and re-named as Vietricula be new snail species. Further detailed studies on anat- [11]. Our results, based on molecular analyses of cox1 omy, biology, systematics and genetics are required to sequences, revealed that the minute Triculinae sp. 1 confirm the identities and taxonomic status of triculine snails infected with P. heterotremus from Yen Bai Prov- snails from Vietnam. The remaining snail species, in- ince was related to Tricula, and Triculinae sp. 2 snails fected with P. westermani, was clearly identified as Sul- infected with P. proliferus from Quang Tri Province cospira quangtriensis from Quang tri Province. Fig. 5 Cercariae (upper row) and rediae (lower row) of Paragonimus species. a P. heterotremus. b P. proliferus. c P. westermani. Scale-bars: 100 μm. Abbreviations: OS, oral sucker; VS, ventral sucker; PG, penetration gland; St, stylet; Ta, tail; EB, excretory bladder; I, intestine Doanh et al. Parasites & Vectors (2018) 11:328 Page 7 of 8 Species of the genus Paragonimus are regarded as The infection rates of snails with Paragonimus cer- exhibiting host specificity for their snail hosts at the cariae were low, between 0.07–1.0%, although those in superfamily level. Members of the P. westermani com- crab hosts in the same study area were very high, be- plex have only been found in cerithioidean snails but not tween 70–100% [8]. This situation is also found in other in the superfamily Rissooidea, within which are the first countries, such as Malaysia, Japan and China [21–23], intermediate hosts of other species (P. skrjabini, P. and helps to explain why natural first intermediate hosts ohirai, P. miyazaki, P. fukienesis, P. kellikotti, P. prolif- are known for only a few common species, such as P. erus, P. heterotremus, P. calienesis and P. mexicanus)[4]. westermani, P. heterotremus and P. ohirai [1]. In this There is also regional host specificity; P. westermani study, we identified the natural first intermediate hosts from the Philippines and Malaysia use thiarid snails of three species, P. westermani, P. heterotremus and P. while those from Japan, Korea, China and Taiwan ex- proliferus, in Vietnam. Among these, the first two spe- ploit pleurocercid snails as the first intermediate hosts cies are common and pathogenetic to humans while P. [2–4]. In Sri Lanka, P. westermani cercariae were re- proliferus is a rare species with a low infection rate in ported from snail Paludomus sp. of the family Palu- crab hosts in Huong Son Commune [8]. In China, the domidae, also belonging to the superfamily intermediate host of P. proliferus was identified as Tri- Cerithioidea [19]. In the present study in Vietnam, cula in experimental infection studies, but the taxonomy based on molecular and morphological analyses, cer- of these snails remains confused [1]. Our data clearly cariae of three Paragonimus species were found, each show that the natural first intermediate host of P. prolif- from a different snail species. Cercariae of P. wester- erus is a triculine snail, maybe Gammatricula or a mani were found from S. quangtriensis of the super- closely related taxon. family Cerithioidea, while P. heterotremus and P. Morphologically, cercariae of all Paragonimus species, proliferus were found from minute freshwater tricu- where known, resemble one another closely [1, 2]. Our line snails of the superfamily Rissooidea, reinforcing findings agree, with cercariae of the three Paragonimus the idea of host specificity at the superfamily level. species found in this study morphologically indistin- The snails Sulcospira quangtriensis and Triculinae sp. guishable. However, they can be distinguished at their 2livein the same waterbody, butwereinfectedwith redial stages by the length of the intestine and the num- different Paragonimus species, P. westermani and P. ber of cercariae per redia. These may be useful charac- proliferus, respectively. Also, cercariae of P. heterotre- teristics for species identification of larval stage of mus were found from a different snail genus/species. Paragonimus spp. in snail hosts. These may suggest host specificity at the genus/spe- cies level. The unidentified Sulcospira sp. in Yen Bai Conclusions Province may act as an intermediate host of P. wes- The natural first intermediate hosts of three Paragoni- termani there, and Triculinae sp. 1 may be present mus species were confirmed based on the molecular and somewhere in Quang Tri Province to maintain the morphological identification of both cercariae and snail life-cycle of P. heterotremus, of which eggs have been hosts. Freshwater snails Sulcospira quangtriensis, Triculi- foundinwildcatsin this area[20]. nae sp. 1 and Triculinae sp. 2 serve as snail hosts for P. Table 2 Measurements (μm) of Paragonimus spp. larval stages collected from snails Characteristic P. heterotremus P. proliferus P. westermani Cercaria Body 240–300 × 80–110 280–316 × 100–160 256–306 × 104–120 Oral sucker 50–70 × 50–60 60–75 × 60–75 64–80 × 64–80 Ventral sucker 28–30 × 28–30 35–50 × 35–50 40–48 × 40–48 Tail length 20–25 25–30 20–24 Stylet length 40–50 40–50 40–48 Redia Body 900–980 × 200–260 1000–1100 × 220–300 860–960 × 200–256 Pharynx 70–86 × 70–86 80–100 × 80–100 64–80 × 64–80 Intestine length 180–240 200–230 620–640 Intestine/body length (%) 20 20 75 No. of cercariae per redia 10–12 5–66–8 Doanh et al. Parasites & Vectors (2018) 11:328 Page 8 of 8 westermani, P. heterotremus and P. proliferus, respect- 6. Doanh PN, Le NT, The DT. Distribution of Paragonimus heterotremus and its intermediate hosts in the northwest region of Vietnam. Vietnam J Biol. 2002; ively, suggesting host specificity at the genus/species 24:14–22. (in Vietnamese with English abstract) level. It is difficult to identify cercariae to species based 7. De NV, Chau LV, Son DT. Study on the epidemiology, pathology, diagnosis, on morphology, but the length of the intestine of the re- and treatment of paragonimiasis in northern mountainous provinces of Vietnam. Record of scientfic research works 1996–2000. Nat Inst Malariol dia and the number of cercariae per redia are valuable Parasitol Entomol. 2000:560–94. (in Vietnamese with English abstract). characteristics for differentiation between species. 8. Doanh PN, Tu AL, Bui TD, Loan TH, Nonaka N, Horii Y, et al. Molecular and morphological variation of Paragonimus westermani in Vietnam with records Abbreviations of new second intermediate crab hosts and a new locality in a northern BLAST: Basic Local Alignment Search Tool; cox1: Mitochondrial cytochrome c province. Parasitology. 2016;143:1639–46. oxidase 1; GTR: General time reversible model; ITS2: Internal transcribed 9. Dang NT, Ho TH. The classification of snails belonging to the subfamily spacer 2; PCR: Polymerase chain reaction Triculinae (Hydrobiidae-Prosobranchia) in Vietnam. Vietnam. J Biol. 2006; 28:8–18. Acknowledgements 10. Ponder WF. A new genus and species of aquatic cave living snail from Special thanks are sent to Vietnam National Foundation for Science and Tasmania (Mollusca: Gastropoda: Hydrobiidae). Pap Proc R Soc Tasmania. Technology Development (NAFOSTED) for supporting this work. 1992;126:23–8. 11. Dang NT, Ho TH. Contribution to the study on Triculinae snail group Funding (Pomatiopsidae - Mollusca) in Tay Nguyen highland, Vietnam. Vietnam J This study was funded by Vietnam National Foundation for Science and Biol. 2010;32:14–8. (in Vietnamese with English abstract) Technology Development (NAFOSTED) under grant number 106.NN.05- 12. Liu L, Huo GN, He HB, Zhou B, Attwood SW. A phylogeny for the 2016.17 to PND. Pomatiopsidae (Gastropoda: Rissooidea): a resource for taxonomic, parasitological and biodiversity studies. BMC Evol Biol. 2014;14:29–59. Availability of data and materials 13. Dang NT, Ho TH. Freshwater snails in Vietnam. Hanoi: Natural Sciences and Data supporting the conclusions of this article are included within the article. Technology Publishing House. 2017; (in Vietnamese). The datasets used and/or analysed during the current study are available 14. Cort WW. A study of the escape of cercariae from their snail hosts. J from the corresponding author upon reasonable request. 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Institute of Ecology and Biological Resources, Viet Nam Academy of Science 23. Lou HQ, Hu Y, Jin YJ, Yu XT, Wang L, He XY, et al. Investigation on the hosts and Technology, Hanoi, Viet Nam. Graduate University of Science and with natural infection and species identification in Jinhua prefecture of Technology, Viet Nam Academy of Science and Technology, Hanoi, Viet Zhejiang Province. Chin J Parasitol Parasit Dis. 2011;29:348–52. Nam. Laboratory of Veterinary Parasitic Diseases, Faculty of Agriculture, University of Miyazaki, Center for Animal Disease Control, University of Miyazaki, Miyazaki, Japan. College of Marine and Environmental Sciences, James Cook University, Douglas, Australia. Tropical Diseases Research Centre, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. Received: 15 February 2018 Accepted: 14 May 2018 References 1. Blair D, Xu ZB, Agatsuma T. Paragonimiasis and the genus Paragonimus. Adv Parasitol. 1999;42:113–222. 2. Schell SC. How to know the trematodes. Dubuque: W. C. Brown Company Publishers; 1970. 3. Davis GM, Chen CE, Kang ZB, Liu YY. Snail hosts of Paragonimus in Asia and the Americas. Biomed Environ Sci. 1994;7:369–82. 4. Wilke T, Davis GM, Gong X, Liu HX. Erhaia (Gastropoda: Rissooidea): phylogenetic relationships and the question of Paragonimus coevolution in Asia. Am J Trop Med Hyg. 2000;62:453–9. 5. Doanh PN, Horii Y, Nawa Y. Paragonimus and paragonimiasis in Vietnam: an update. Korean J Parasitol. 2013;51:621–7. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Parasites & Vectors Springer Journals

First intermediate hosts of Paragonimus spp. in Vietnam and identification of intramolluscan stages of different Paragonimus species

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Biomedicine; Parasitology; Entomology; Tropical Medicine; Infectious Diseases; Veterinary Medicine/Veterinary Science; Virology
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Abstract

Background: Members of the genus Paragonimus require at least three hosts in their life-cycles. The obligatory first intermediate hosts are freshwater snails. In Vietnam, although seven Paragonimus species have been recorded, the natural first intermediate hosts of almost all species have not been confirmed. The aim of this study was, therefore, to investigate snail hosts of Paragonimus species in Vietnam, and to identify Paragonimus species at intramolluscan stages. Methods: Freshwater snails were collected from streams in Yen Bai and Quang Tri Provinces, where high prevalences of Paragonimus metacercariae in crab hosts have been reported. Snails were morphologically identified and then examined individually for Paragonimus cercariae using shedding and crushing methods. Chaetomicrocercous cercariae, the morphological class to which Paragonimus cercariae belong, were collected for morphological description and molecular species identification by analyses of ITS2 sequences. The infected snail species were identified based on analyses of nucleotide sequences of the cox1 gene. Results: Three snail species were found to be infected with Paragonimus cercariae at low infection rates, ranging between 0.07–1.0%. The molecular analyses identified them as Sulcospira quangtriensis and 2 species of subfamily Triculinae. In a phylogenetic tree, these two triculine snails were related to the genera Gammatricula and Tricula with low posterior probabilities. Thus we named them as Triculinae sp. 1 and Triculinae sp. 2. Cercariae from the three snail species, Sulcospira quangtriensis, Triculinae sp. 1 and Triculinae sp. 2, were molecularly identified as Paragonimus westermani, P. heterotremus and P. proliferus, respectively. The cercariae of the three species are morphologically similar to each other, but their daughter rediae can be distinguished by the length of the intestine and the number of cercariae per redia. The rediae of P. westermani have a long intestine and each contain 6–8 cercariae. In contrast, those of P. heterotremus and P. proliferus have a short intestine and each redia contain 10–12 and 5–6cercariae, respectively. Conclusions: Three snail species, Sulcospira quangtriensis, Triculinae sp. 1 and Triculinae sp. 2, serve as the first intermediate hosts of P. westermani, P. heterotremus and P. proliferus, respectively, in Vietnam. The length of the intestine of rediae and the number of cercariae per redia are valuable characteristics for distinguishing between larvae of these Paragonimus species. Keywords: First intermediate host, Paragonimus, Sulcospira quangtriensis,Triculinae * Correspondence: pndoanh@yahoo.com Institute of Ecology and Biological Resources, Viet Nam Academy of Science and Technology, Hanoi, Viet Nam Graduate University of Science and Technology, Viet Nam Academy of Science and Technology, Hanoi, Viet Nam Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Doanh et al. Parasites & Vectors (2018) 11:328 Page 2 of 8 Background different snail species. The host specificity and the mor- Lung flukes of the genus Paragonimus (Family Paragoni- phological features of different Paragonimus species in midae) are parasites of mammals, commonly cats and the snail hosts are discussed herein. canids, and occasionally humans. Members of this genus require at least three different hosts to complete their Methods life-cycles [1]. The first intermediate hosts are freshwater Collection and examination of snails snails in which the flukes develop through several stages Freshwater snails were collected in streams in An Lac (sporocyst, mother and daughter rediae, and cercariae). Commune (Luc Yen District, Yen Bai Province) and The cercariae belong to the microcercous type (subtype Huong Son Commune (Huong Hoa District, Quang Tri chaetomicrocercous), possessing an anterior stylet, a Province) (Fig. 1) where high infection rates of crab knob-like tail, spines on the body and tail, but lacking hosts with Paragonimus metacercariae were found [8]. unicellular glands [2]. Mature cercariae escape from the Snails were morphologically identified following Dang & snail hosts and penetrate suitable crab/crayfish hosts to Ho [13], and were examined for Paragonimus cercariae develop into an infective stage, metacercariae. Definitive using shedding and crushing methods [14]. Snail sam- hosts become infected by eating these crabs/crayfish ples were placed individually in 20 ml wide mouth plas- containing live metacercariae [1]. tic containers filled with 15 ml of water and left for 24 h. Regarding host specificity, Paragonimus flukes typic- The containers were checked under a stereomicroscope ally infect a wider spectrum of definitive and second at night and the next morning. If any chaetomicrocer- intermediate hosts, whereas specificity for the first inter- cous cercariae were found, they were used for morpho- mediate host is usually very strict and vary geographic- logical study. Afterwards, all snails were dissected under ally [1, 3, 4]. Thus, identification of snail hosts in each a stereomicroscope to identify cercariae that did not endemic region is essential for understanding of routes emerge, and other intramolluscan stages, such as rediae. of transmission. Live cercariae and rediae were observed under an Axio In Vietnam, Paragonimus metacercariae of seven spe- Lab A1 microscope (Carl Zeiss, Oberkochen, Germany). cies have been reported in northern and central prov- inces: P. heterotremus, P. westermani, P. bangkokensis, P. harinasutai, P. proliferus, P. vietnamensis and P. skrja- bini [5]. Of these, P. heterotremus and P. westermani are important causative agents for human paragonimiasis in Asian countries. The former species is commonest in northern provinces, and the latter in central provinces [5]. So far, there has been only one report of microcer- cous cercariae from “Oncomelania” snails in Vietnam, in 2002 [6], at a time when only metacercariae of P. hetero- tremus was found [6, 7]. The cercaria was, therefore, identified as P. heterotremus without molecular evidence [6]. However, subsequent surveys have revealed the pres- ence of six additional Paragonimus species, and metacer- cariae of two to four species has been found in the same area [5, 8]. Moreover, the taxonomy of the “Oncomela- nia” snails in Vietnam has been revised and remains un- certain [9–11]. They were re-identified as a new genus Pseudotricula [9]. Since the name Pseudotricula had been assigned previously [10], Dang and Ho [11] renamed their genus as Vietricula. Liu et al. [12] consid- ered that Vietricula snails are closely related to the genus Gammatricula. Thus, data on first intermediate hosts of Paragonimus in Vietnam are scant and unclear. The aim of this study was, therefore, to investigate snail hosts of Paragonimus spp. in Yen Bai and Quang Tri Provinces, where Paragonimus metacercariae are highly prevalent with the dominance of P. heterotremus and P. westermani, respectively [8]. In this survey, we found Fig. 1 Geographical locations of sampling sites cercariae of three Paragonimus species, each in a Doanh et al. Parasites & Vectors (2018) 11:328 Page 3 of 8 Photographs of the cercariae and rediae were taken GTR model) were unlinked across partitions, to allow using a Axiocam Erc 5s digital camera attached to a them to take different values. MrBayes was run for Axio Lab A1 microscope (Carl Zeiss, Oberkochen, 2,000,000 generations, after which the standard deviation Germany) and ZEN lite software (Carl Zeiss). Cercariae of split frequencies was below 0.01. Trees were sampled and rediae were drawn using Illustrator CS6. Measure- every 500 generations. ‘Burn-in’ was 25% of trees. A con- ments (in μm) of cercariae and rediae were taken. Live sensus tree was constructed, including all compatible cercariae were identified according to Schell [2]. After groups of taxa. morphological identification, a single cercaria and tissues from each infected snails were separately preserved in Results absolute ethanol for molecular identification of cercariae Identification of snails infected with chaetomicrocercous and snail hosts. cercariae The various snails collected belonged morphologically to Molecular analysis for identification of cercariae and snail Melanoides, Sulcospira and minute snails of the subfam- hosts ily Triculinae. Snails of the genus Melanoides were few Genomic DNA of chaetomicrocercous cercariae and in number and clearly identified as Melanoides tubercu- snails from 10 infected snails was extracted using a lata. In contrast, Sulcospira snails were abundant, occur- PureLink Genomic DNA Mini Kit (Invitrogen, Califor- ring at high densities, especially in larger rocky streams nia, USA) following the manufacturer’s protocol. The in- with high levels of water and fast flow. They usually at- ternal transcribed spacer 2 (ITS2) region of nuclear tached to the surfaces of stones. The two species of tri- ribosomal DNA from cercariae was amplified via poly- culine were commonly found attached to fallen leaves in merase chain reaction (PCR) with the primers 3S small tributary streams with slow water flow. 5'-CGG TGG ATC ACT CGG CTC GT-3' as a forward primer and A28 5'-CCT GGT TAG TTT CTT TTC Chaetomicrocercous cercariae were not found from CTC CGC-3' as a reverse primer [15]. A portion of the M. tuberculata in either studied site or from Sulcospira mitochondrial cytochrome c oxidase 1 (cox1) gene of the sp. in Yen Bai Province, but were found from five Sulcos- infected snail species was amplified via PCR with the pira sp. in Quang Tri, and from four and one triculine primers LCO1490 5'-GGT CAA CAA ATC ATA AAG snails in Yen Bai and Quang Tri Provinces, respectively. ATA TTG G-3' as a forward primer and HCO2198 The sequences obtained from five Sulcospira snails 5'-TAA ACT TCA GGG TGA CCA AAA AAT YA-3' as from Quang Tri Province were completely identical with a reverse primer [16]. PCR products were purified using each other. The sequences from four Triculine snails a QIAquick PCR Purification Kit (Qiagen, California, from Yen Bai Province were also identical with each USA) and sequencing reactions done using a Big-Dye other. The results of BLAST searches showed that the terminator cycle sequencing kit v3.1 (Applied Biosys- cox1 sequence of the Sulcospira snail from Quang Tri tems, California, USA). Both forward and reverse strands Province was highly similar (99%) to that of Sulcospira were sequenced directly by a Genetic Analyzer (Model quangtriensis (FJ377265), confirming the identification 3100, Applied Biosystems, California, USA), using the of this species as S. quangtriensis.The cox1 sequences of PCR primers as sequencing primers. Triculinae snails from Yen Bai and Quang Tri Provinces Sequences obtained from cercariae and snails were showed the highest similarity (but only 91% similarity) submitted to GenBank with accession numbers to Gammatricula snails (AF213342). The samples from LC3605000-LC360505. Basic Local Alignment Search Yen Bai and from Quang Tri Provinces differed by Tool (BLAST) searches in the server of the National 12.6% and were placed separately in the phylogenetic Center for Biotechnology Information (NCBI) were used tree (Fig. 2). The posterior probabilities of groups in to determine the most similar sequences from named which they occurred were rather low. The snail from species. The cox1 sequences of the two triculine snail Quang Tri was grouped with Gammatricula songi with a species showed low similarities to available sequences on value of only 53%, the snail from Yen Bai was grouped GenBank. These sequences were therefore aligned with with Tricula wumingensis with a posterior probability of related sequences, and were used to reconstruct a phylo- 56%. The genus Gammatricula is shown as monophy- genetic tree using MrBayes [17]. The substitution model letic, but with a posterior probability of only 27%. The was selected using MrModelTest2 [18]. The model se- genus Tricula is not monophyletic in this tree. This sug- lected was the general time reversal (GTR) model with gests that the snails from Yen Bai and Quang Tri Prov- gamma-distributed rate variation across sites. The data inces belong to different genera of the subfamily were partitioned by codon position (first, second and Triculinae, but their taxonomic status remains uncertain. third positions). Two parameters of the model (shape of We have temporarily labelled them as Triculinae sp. 1 the gamma distribution and substitution rates of the from Yen Bai Province, and Triculinae sp. 2 from Quang Doanh et al. Parasites & Vectors (2018) 11:328 Page 4 of 8 Fig. 2 Phylogenetic tree reconstructed from cox1 sequences of triculine snails using Bayesian inference analysis. Numbers at nodes represent posterior probabilities. Samples obtained in this study are printed in bold Tri Province. The snails infected with chaetomicrocercous ventral sucker. The latter was round and situated at cercariae and their habitats are shown in Figs. 3 and 4. close to the middle of the body. A total of 14 pene- tration grand cells were distributed in groups of 3 Prevalence and morphology of chaetomicrocercous and 4 cells antero-lateral to the ventral sucker. A cercariae from snails large I-shaped excretory bladder was located between Infection rates of the snails with chaetomicrocercous the ventral sucker and the end of the body (Fig. 5). cercariae were low, ranging between 0.07–1.0% (Table 1). These are typical characteristics of Paragonimus In Huong Son Commune, Quang Tri Province, the density cercariae. of the S. quangtriensis population is very high. Sulcospira In contrast, the daughter rediae collected from dif- quangtriensis collected in the rocky main stream were not ferent snail species differed from one another in the positive, but those from a small tributary were infected length of the intestine and the number of cercariae with chaetomicrocercous cercariae. per redia (Fig. 5). The rediae collected from S. quang- The chaetomicrocercous cercariae from different triensis possessed a long intestine (about 75% of the snail species morphologically resemble one another body length) and each redia contained 6–8 cercariae. (Fig. 5). In each case, the mature cercariae had a sty- The rediae from Triculinae sp. 1 and Triculinae sp. 2 let and a short tail, with the measurements presented each had a short intestine (about 20% of the redia in Table 2. The body was elongated-oval in shape. body length). Each redia from Triculinae sp. 1 con- The oral sucker at the anterior extremity was accom- tained 10–12 cercariae, double the number (5–6) seen panied by a long stylet and was larger than the in each redia from Triculinae sp. 2 (Fig. 5). Doanh et al. Parasites & Vectors (2018) 11:328 Page 5 of 8 Fig. 3 Freshwater snails infected with Paragonimus cercariae. a Triculinae sp. 1 from Yen Bai. b Triculinae sp. 2 from Quang Tri. c Sulcospira quangtriensis from Quang Tri Molecular identification of chaetomicrocercous proliferus (AB663672) and P. westermani (LC144899), cercariae respectively. The result of BLAST searches showed that ITS2 se- quences of cercariae from snails Triculinae sp. 1 (4 iden- Discussion tical sequences from 4 infected snails), Triculinae sp. 2 Previously, cercariae considered to be of P. heterotremus (1 infected snail) and S. quangtriensis (5 identical se- were reported from minute snails (3–4 mm) in northern quences from 5 infected snails) were completely (100%) provinces of Vietnam [6]. As outlined in the introduc- identical with those of P. heterotremus (AB82365), P. tion, the identity of these snails remains unclear. They Fig. 4 Habitats of snails in Quang Tri Province. a A rocky main stream with fast water flow. b Sulcospira quangtriensis on a stone of the main stream. c A tributary with slow water flow where Sulcospira and triculine snails live Doanh et al. Parasites & Vectors (2018) 11:328 Page 6 of 8 Table 1 Prevalence of Paragonimus cercariae in snails Location Snail species No. examined No. infected (%) Paragonimus species An Lạc (Yen Bai) Triculinae sp. 1 1200 4 (0.3) P. heterotremus Sulcospira sp. 1000 0 – M. tuberculata 65 0 – Huong Sơn (Quang Tri) Triculinae sp. 2 1520 1 (0.07) P. proliferus S. quangtriensis 500 5 (1.0) P. westermani S. quangtriensis 5000 0 – M. tuberculata 70 0 – Collected from the small tributary Collected from the main stream were first identified as Oncomelania snails [6], then were related to the genus Gammatricula. These might named as Pseudotricula [9] and re-named as Vietricula be new snail species. Further detailed studies on anat- [11]. Our results, based on molecular analyses of cox1 omy, biology, systematics and genetics are required to sequences, revealed that the minute Triculinae sp. 1 confirm the identities and taxonomic status of triculine snails infected with P. heterotremus from Yen Bai Prov- snails from Vietnam. The remaining snail species, in- ince was related to Tricula, and Triculinae sp. 2 snails fected with P. westermani, was clearly identified as Sul- infected with P. proliferus from Quang Tri Province cospira quangtriensis from Quang tri Province. Fig. 5 Cercariae (upper row) and rediae (lower row) of Paragonimus species. a P. heterotremus. b P. proliferus. c P. westermani. Scale-bars: 100 μm. Abbreviations: OS, oral sucker; VS, ventral sucker; PG, penetration gland; St, stylet; Ta, tail; EB, excretory bladder; I, intestine Doanh et al. Parasites & Vectors (2018) 11:328 Page 7 of 8 Species of the genus Paragonimus are regarded as The infection rates of snails with Paragonimus cer- exhibiting host specificity for their snail hosts at the cariae were low, between 0.07–1.0%, although those in superfamily level. Members of the P. westermani com- crab hosts in the same study area were very high, be- plex have only been found in cerithioidean snails but not tween 70–100% [8]. This situation is also found in other in the superfamily Rissooidea, within which are the first countries, such as Malaysia, Japan and China [21–23], intermediate hosts of other species (P. skrjabini, P. and helps to explain why natural first intermediate hosts ohirai, P. miyazaki, P. fukienesis, P. kellikotti, P. prolif- are known for only a few common species, such as P. erus, P. heterotremus, P. calienesis and P. mexicanus)[4]. westermani, P. heterotremus and P. ohirai [1]. In this There is also regional host specificity; P. westermani study, we identified the natural first intermediate hosts from the Philippines and Malaysia use thiarid snails of three species, P. westermani, P. heterotremus and P. while those from Japan, Korea, China and Taiwan ex- proliferus, in Vietnam. Among these, the first two spe- ploit pleurocercid snails as the first intermediate hosts cies are common and pathogenetic to humans while P. [2–4]. In Sri Lanka, P. westermani cercariae were re- proliferus is a rare species with a low infection rate in ported from snail Paludomus sp. of the family Palu- crab hosts in Huong Son Commune [8]. In China, the domidae, also belonging to the superfamily intermediate host of P. proliferus was identified as Tri- Cerithioidea [19]. In the present study in Vietnam, cula in experimental infection studies, but the taxonomy based on molecular and morphological analyses, cer- of these snails remains confused [1]. Our data clearly cariae of three Paragonimus species were found, each show that the natural first intermediate host of P. prolif- from a different snail species. Cercariae of P. wester- erus is a triculine snail, maybe Gammatricula or a mani were found from S. quangtriensis of the super- closely related taxon. family Cerithioidea, while P. heterotremus and P. Morphologically, cercariae of all Paragonimus species, proliferus were found from minute freshwater tricu- where known, resemble one another closely [1, 2]. Our line snails of the superfamily Rissooidea, reinforcing findings agree, with cercariae of the three Paragonimus the idea of host specificity at the superfamily level. species found in this study morphologically indistin- The snails Sulcospira quangtriensis and Triculinae sp. guishable. However, they can be distinguished at their 2livein the same waterbody, butwereinfectedwith redial stages by the length of the intestine and the num- different Paragonimus species, P. westermani and P. ber of cercariae per redia. These may be useful charac- proliferus, respectively. Also, cercariae of P. heterotre- teristics for species identification of larval stage of mus were found from a different snail genus/species. Paragonimus spp. in snail hosts. These may suggest host specificity at the genus/spe- cies level. The unidentified Sulcospira sp. in Yen Bai Conclusions Province may act as an intermediate host of P. wes- The natural first intermediate hosts of three Paragoni- termani there, and Triculinae sp. 1 may be present mus species were confirmed based on the molecular and somewhere in Quang Tri Province to maintain the morphological identification of both cercariae and snail life-cycle of P. heterotremus, of which eggs have been hosts. Freshwater snails Sulcospira quangtriensis, Triculi- foundinwildcatsin this area[20]. nae sp. 1 and Triculinae sp. 2 serve as snail hosts for P. Table 2 Measurements (μm) of Paragonimus spp. larval stages collected from snails Characteristic P. heterotremus P. proliferus P. westermani Cercaria Body 240–300 × 80–110 280–316 × 100–160 256–306 × 104–120 Oral sucker 50–70 × 50–60 60–75 × 60–75 64–80 × 64–80 Ventral sucker 28–30 × 28–30 35–50 × 35–50 40–48 × 40–48 Tail length 20–25 25–30 20–24 Stylet length 40–50 40–50 40–48 Redia Body 900–980 × 200–260 1000–1100 × 220–300 860–960 × 200–256 Pharynx 70–86 × 70–86 80–100 × 80–100 64–80 × 64–80 Intestine length 180–240 200–230 620–640 Intestine/body length (%) 20 20 75 No. of cercariae per redia 10–12 5–66–8 Doanh et al. Parasites & Vectors (2018) 11:328 Page 8 of 8 westermani, P. heterotremus and P. proliferus, respect- 6. Doanh PN, Le NT, The DT. Distribution of Paragonimus heterotremus and its intermediate hosts in the northwest region of Vietnam. Vietnam J Biol. 2002; ively, suggesting host specificity at the genus/species 24:14–22. (in Vietnamese with English abstract) level. It is difficult to identify cercariae to species based 7. De NV, Chau LV, Son DT. Study on the epidemiology, pathology, diagnosis, on morphology, but the length of the intestine of the re- and treatment of paragonimiasis in northern mountainous provinces of Vietnam. Record of scientfic research works 1996–2000. Nat Inst Malariol dia and the number of cercariae per redia are valuable Parasitol Entomol. 2000:560–94. (in Vietnamese with English abstract). characteristics for differentiation between species. 8. Doanh PN, Tu AL, Bui TD, Loan TH, Nonaka N, Horii Y, et al. Molecular and morphological variation of Paragonimus westermani in Vietnam with records Abbreviations of new second intermediate crab hosts and a new locality in a northern BLAST: Basic Local Alignment Search Tool; cox1: Mitochondrial cytochrome c province. Parasitology. 2016;143:1639–46. oxidase 1; GTR: General time reversible model; ITS2: Internal transcribed 9. Dang NT, Ho TH. The classification of snails belonging to the subfamily spacer 2; PCR: Polymerase chain reaction Triculinae (Hydrobiidae-Prosobranchia) in Vietnam. Vietnam. J Biol. 2006; 28:8–18. Acknowledgements 10. Ponder WF. A new genus and species of aquatic cave living snail from Special thanks are sent to Vietnam National Foundation for Science and Tasmania (Mollusca: Gastropoda: Hydrobiidae). Pap Proc R Soc Tasmania. Technology Development (NAFOSTED) for supporting this work. 1992;126:23–8. 11. Dang NT, Ho TH. Contribution to the study on Triculinae snail group Funding (Pomatiopsidae - Mollusca) in Tay Nguyen highland, Vietnam. Vietnam J This study was funded by Vietnam National Foundation for Science and Biol. 2010;32:14–8. (in Vietnamese with English abstract) Technology Development (NAFOSTED) under grant number 106.NN.05- 12. Liu L, Huo GN, He HB, Zhou B, Attwood SW. A phylogeny for the 2016.17 to PND. Pomatiopsidae (Gastropoda: Rissooidea): a resource for taxonomic, parasitological and biodiversity studies. BMC Evol Biol. 2014;14:29–59. Availability of data and materials 13. Dang NT, Ho TH. Freshwater snails in Vietnam. Hanoi: Natural Sciences and Data supporting the conclusions of this article are included within the article. Technology Publishing House. 2017; (in Vietnamese). The datasets used and/or analysed during the current study are available 14. Cort WW. A study of the escape of cercariae from their snail hosts. J from the corresponding author upon reasonable request. The sequences Parasitol. 1922;8:177–84. obtained from cercariae and snails were submitted to GenBank with 15. Bowles J, Blair D, McManus DP. A molecular phylogeny of the human accession numbers LC3605000-LC360505. schistosomes. Mol Phylogenet Evol. 1995;4:103–9. 16. Folmer O, Black M, Hoeh W, Lutz RA, Vrijenhoek RC. DNA primers for Authors’ contributions amplification of mitochondrial cytochrome c oxidase subunit I from diverse Conceived and designed the study: PND, HVH, LAT, YH, DB and YN. metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3:294–9. Collected data: PND, HVH, LAT and NVD. Analyzed the data: PND, HVH and 17. Huelsenbeck JP, Ronquist F. MrBayes: Bayesian inference of phylogenetic LAT. Wrote the paper: PND, LAT, YH, DB and YN. All authors read and trees. Bioinformatics. 2001;17:754–5. approved the final manuscript. 18. Nylander JAA. MrModeltest v2. Program distributed by the author. Sweden: Evolutionary Biology Centre, Uppsala University; 2004. Ethics approval and consent to participate 19. Iwagami M, Rajapakse RPVJ, Yatawara L, Kano S, Agatsuma T. The first Not applicable. intermediate host of Paragonimus westermani in Sri Lanka. Acta Trop. 2009; 109:27–9. Competing interests 20. Doanh PN, Hien HV, Tu LA, Nonaka N, Horii Y, Nawa Y. Molecular The authors declare that they have no competing interests. identification of the trematode Paragonimus in faecal samples from the wild cat Prionailurus bengalensis in the Da Krong Nature Reserve, Vietnam. J Helminthol. 2016;90:658–62. Publisher’sNote 21. Kim JS. An ecological study of Paragonimus in Malaysia. Korean J Parasitol. Springer Nature remains neutral with regard to jurisdictional claims in 1978;16:47–53. published maps and institutional affiliations. 22. Tomimura T, Sugiyama H, Yokota M. Parasitological survey of the first intermediate host of Paragonimus westermani in Iga area of Mie prefecture, Author details Japan. Nihon Juigaku Zasshi. 1989;51:315–26. 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How to know the trematodes. Dubuque: W. C. Brown Company Publishers; 1970. 3. Davis GM, Chen CE, Kang ZB, Liu YY. Snail hosts of Paragonimus in Asia and the Americas. Biomed Environ Sci. 1994;7:369–82. 4. Wilke T, Davis GM, Gong X, Liu HX. Erhaia (Gastropoda: Rissooidea): phylogenetic relationships and the question of Paragonimus coevolution in Asia. Am J Trop Med Hyg. 2000;62:453–9. 5. Doanh PN, Horii Y, Nawa Y. Paragonimus and paragonimiasis in Vietnam: an update. Korean J Parasitol. 2013;51:621–7.

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Parasites & VectorsSpringer Journals

Published: May 30, 2018

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