INTRODUCTION
Fascioliasis is a worldwide parasitic disease caused by Fasciola gigantica and Fasciola hepatica. The latter species is common in temperate zones especially in Europe, the Americas, and Australia, whereas the former is the most prevalent species in tropical regions of Africa and Asia [1]. In Vietnam, human and animal fascioliasis has drawn a special interest. Indeed, the prevalence of human fascioliasis was found to be on the rise in the country. About 10,000 cases were detected in central Vietnam in 2011 alone [2]. Based on molecular data, F. gigantica and hybrid forms were identified.
The Lymnaeidae are one of the most widespread groups of freshwater snails and many of them act as intermediate hosts of important digenean trematodes that infect humans or livestock [3]. The correct taxonomic classification of these snails solely based on morphological characteristics was not always possible and the nomenclature of Lymnaeidae is extremely confused [4,5]. Additionally, environmental conditions are responsible for morphological variation which further complicates identification [5,6]. There are few studies dealing with morphology and phylogenetics of lymnaeids in Asia. Using morphological criteria, Thanh [7] recognized 2 common lymnaeid species, Lymnaea viridis and Lymnaea swinhoei, in northern Vietnam in 1980. Currently, the former is generally referred to as Austropeplea viridis and the second as Radix auricularia [8], and these are the names we shall use throughout this paper. An additional species, Lymnaea sp., was identified in Vietnam by Doanh [9] in 2012. Two species, A. viridis (as L. viridis) and R. auricularia (as L. swinhoei), were reported by some authors to act as intermediate hosts of Fasciola spp. in Vietnam [10,11]. Others indicated that only 1 of these species was an intermediate host [9,12]. In Taiwan, China, Japan, and Thailand, R. rubiginosa, R. auricularia, and A. viridis have been reported as hosts for Fasciola spp. [13-15]. Previous to this study, no analysis of molecular sequence data from lymnaeids had been done in Vietnam. Therefore, the aim of this study is to use both molecular and morphological criteria in order to identify the potential vectors of Fasciola spp. in the country.
MATERIALS AND METHODS
Snail sampling
Lymnaeid snails were collected in a variety of water bodies in 3 localities of Vietnam representing major climatic types: Thuong Tin district, Ha Noi capital (N: 20°84'75"; E: 105°90'87" - northern part); An Nhon district, Binh Dinh Province (N: 13°94'47"; E: 109°07'96" - central part); and Tieu Can district, Tra Vinh Province (N: 9°80'77"; E: 106°18'55" - southern part) (Fig. 1). Individual snails were morphologically sorted out, then examined for the presence of Fasciola spp. larval stages by stereomicroscopy. Representatives of each morphological group were individually fixed in 100% ethanol for subsequent molecular identification.
Morphological examinations
Snails were killed by immersion in hot water (70℃) for 15 sec and then transferred to water at room temperature. Stereomicroscopic examination was performed using an Olympus SZ51, SZX-ILL-B2 200 (Hai Ninh company, Ha Noi, Vietnam). The following criteria/measurements were recorded for each snail: form, apex whorls, body whorls, transparency, and the ratio of apex/body [16]. Pictures were taken with a digital camera Nikon D3x (Photogallerry Liège-Sauvenière, Belgium), and manipulated for presentation using Adobe Illustrator CS3 (TDT company, Ho Chi Minh, Vietnam). Morphological identifications were based on Burch [17].
Detection of Fasciola spp. larval stages by microscopy
All samples were checked under the stereomicroscope for cercariae. For this purpose, whole snails were crushed between 2 glass plates. Larvae were identified according to the keys of Schell [18].
DNA extraction, PCR amplification, and sequencing
DNA was individually extracted from each snail type using the Chelex®-based DNA extraction technique [19]. A single individual was extracted for each shell type. Only a small piece of foot tissue was used for the extraction of DNA. The material was mechanically disrupted with the help of a Kimbe® Kontes disposable pellet mixer (Sigma-Aldrich, Chinh Chuong company, Ha Noi, Vietnam) in 100 µl of Chelex® 5% (Bio-Rad, TABC company, Ha Noi, Vietnam) and incubated for 1 hr and then for 30 min at 56℃ and 95℃, respectively, in a Master Gradient thermal cycler (Eppendorf, BCE company, Ha Noi, Vietnam). Then, the mixture was centrifuged at 13,000 g for 7 min. The supernatant, containing the DNA, was carefully transferred to another tube. The rDNA ITS-2 was amplified using primers previously described [4]. Reactions were performed in a total volume of 10 µl containing 5 µl Mastermix 2X (Fermentas, TABC company, Ha Noi, Vietnam) 0.4 µl MgCl, 1 µl dNTP, 1 µl of each primer, 0.6 µl water, and 1 µl of genomic DNA. The thermal cycling profile was 3 min at 94℃ followed by 35 cycles of 30 sec at 94℃, 30 sec at 55℃, 30 sec at 72℃, and a final elongation step at 72℃ for 5 min [20]. Amplified fragments were sequenced by Macrogen Inc., using an ABI 3730XL (Seoul, Korea).
Sequence analyses
Sequences were carefully edited "by eyes" and then aligned using CLUSTAL-W version 1.8 [21] and MEGA v5.2.2 [22] (S. McAllister Ave, Phoenix, Arizona, USA). Subsequently, minor corrections were manually introduced for a better fit of nucleotide correspondences in regions of simple sequence repeats. Tree construction was carried out in MEGA 5.2.2. The analyses were carried out using the sequences of our lymnaeid species together with 13 previously published ITS-2 sequences of closely related lymnaeid species. A sequence from Lymnaea stagnalis was used as an out-group (Table 1) [8,15,23-26]. A phylogenetic tree was constructed using the maximum likelihood (ML) method implemented in MEGA 5.2.2. The substitution model used was the Kimura 2 parameter model+gamma (allowing for unequal rates among sites). Clade support was assessed with 1,000 bootstrap replicates.
RESULTS
Morphological identification of lymnaeid snails
Three morphological types of snails were distinguished based on the key of Burch [17].
Type 1 (Fig. 2A): Shell is elongated and cylindrical. Shell height/width: 11-20/5-8 mm. There are 5 whorls; the last is large and inflated. Spire is high and aperture height/width: 7-11/4-5 mm. Aperture is large but not extended and moderately expanded. The outer margin is S-shaped. Columella generally twisted, is making a fold. This morphological description led to the identification of Lymnaea sp.
Type 2 (Fig. 2B): Shell is large but smaller than type 1. Shell height/width: 12-18/8-10 mm. Shell is thin with sharp apex. There are 4 whorls, the last inflated with convex angle in the left. Shell surface is smooth, thin and brown with clear ribs. Aperture is large, ear-shaped, and extended. Aperture height/width: 9-10/6-8 mm. Columella is curved in the middle. The lower part of the outer lip is rounded. This morphological description led to the identification of R. swinhoei (=L. swinhoei) Adam, 1866.
Type 3 (Fig. 2C): Shell is much smaller than the 2 other species. Shell height/width: 10-13/4-6 mm. There are 4.5-5 well-rounded whorls, the first of which are small compared to the swollen last whorl. Shell surface is smooth, thin and brown with clear ribs. The aperture is regularly oval without angles. Aperture height/width: 5-6/3-4 mm. The outer lip is sharp, regularly curved. Columella is generally straight, without a fold. This morphological description led to the identification of A. viridis (=L. viridis) Quoy and Gaimard, 1832.
Ribosomal DNA sequence analysis and phylogenetic tree construction
The molecular analyses based on the ITS-2 sequences strongly supported 3 morphological types. Their length of ITS2 sequences (type 1, 450; type 2, 470, and type 3, 451 bp) which were deposited in Genbank with the accession no. KF042385 (for type 1), KF042386 (for type 2), and KF042387 (for type 3), were various. The results of Blastn showed that type 1 had a 99% identity with R. rubiginosa (EU556316), supporting its identification as this species. Similarly, ITS2 sequence of type 2 showed 99% homology with those of R. auricularia (HQ637457). Type 3 was closest to A. viridis (GU167912) with 94% identity. In the phylogenetic tree (Fig. 3), type 1, 2, and 3 from Vietnam were clustered with R. rubiginosa, R. auricularia, and A. viridis, respectively, confirming their identification.
Snail distribution
A. viridis is a common species found in various water bodies (rice fields, ditches, small canals, and submerged vegetable fields) and was present in the 3 areas investigated. R. auricularia was found in large canals in the northern part of Vietnam only, while R. rubiginosa was found only in small canals in the southern part (Table 2).
Prevalence of Fasciola spp. infection in field-collected snails
Table 3 shows that only 1 species (A. viridis) was infected with F. gigantica cercariae in the field with the infection rates of 0.11% (9/8,044) in Ha Noi and 1.57% (115/7,321) in Binh Dinh.
DISCUSSION
According to the key of Burch [17] reported in 1980, we identified 2 genera of lymnaeids in Vietnam: Austropeplea Cotton 1942 (columella generally straight, without a fold or plait at the apertural end) and Radix Montfort, 1810 (columella generally twisted, making a fold or plait at the apertural end). The genus Austropeplea was represented by a single species, A. viridis (=L. viridis). The 2 Radix species, R. swinhoei (=L. swinhoei) and R. rubiginosa (=Lymnaea sp.) had a quite similar morphology and consequently were difficult to identify according to morphological criteria only. This might be due to the phenotypic plasticity of the shell shape. Indeed, in numerous gastropod species, including the Lymnaeidae, the shell shape may vary according to environmental conditions [5,6]. In particular, shell morphology seems not to be critical for identifying species of the genus.
DNA-taxonomy is a reliable, comparable, and objective means for species identification in biological research and enabled to identify these species. Several gene regions have been used in taxonomic and phylogenetic studies of the Lymnaeidae, such as ITS-1, ITS-2, 16S, 18S, and CO1 [4,6,27]. Among them, the rDNA ITS-2 has been the most often used molecular maker in lymnaeid studies to date [27]. Our result has confirmed the correct name of 3 lymnaeid snail species in Vietnam based on molecular rDNA ITS-2 analysis, R. rubiginosa, R. auricularia, and A. viridis. Among them, R. rubiginosa is a new record for a freshwater snail fauna in Vietnam.
The 3 lymnaeids occur in somewhat different habitats. A. viridis is widely distributed [8, 35]. In our investigation, this species was common in all 3 regions (northern, central, and southern Vietnam). It can be found in rice fields, ditches, small canals, and submerged vegetable fields. By contrast, R. auricularia is found only in large stagnant canals. The species with the most restricted distribution was R. rubiginosa, which can be found at very low densities in small canals in southern Vietnam.
Based on microscopic examinations, R. auricularia (=R. swinhoei) and A. viridis (=L. viridis) were reported to be infected with Fasciola spp. in Vietnam [10,28]. Other authors reported that either R. auricularia or A. viridis could be infected with Fasciola spp. [9,12]. This was probably due to a lack of experience in mollusk identification; the name of the species was just given without pictures, description, or measurement, and the prevalence of infection. This point was also mentioned in a recent study [9]. Our results have shown that only A. viridis is infected with Fasciola spp. and at a low prevalence. The low prevalence observed might be a consequence of low sensitivity of microscopical examination in detecting intra-molluscan stages. Methods based on PCR might reveal higher incidences of infection [19], but this approach has yet to be used in the country.
In this study, we have confirmed that R. swinhoei (based on morphology) was in fact R. auricularia (based on PCR/sequencing). Although R. auricularia was reported to act as the intermediate host for F. gigantica in Africa, Asia, and USA, its role as the intermediate host for F. gigantica in Vietnam remains unclear because we did not find any infected individuals. The susceptibility of other lymnaeid snails to Fasciola spp. in Vietnam requires experimental studies.
The combined use of morphological and molecular data both under laboratory and field conditions, particularly on morphologically similar species of lymnaeid snails which act as potential intermediate hosts for Fasciola spp., should allow clarification of some key elements on the epidemiology of human and animal fascioliasis in Vietnam.
