Morphological and molecular characterization of invasive Biomphalaria straminea in southern China

Please see Additional file 1 for translations of the abstract into the five official working languages of the United Nations.

Schistosomiasis is an endemic parasitic disease affecting almost 240 million people worldwide, and an additional 700 million people are at risk of infection. The infection is prevalent in seventy-eight developing, tropical and sub-tropical countries [1, 2]. Some species of the genus Biomphalaria act as obligatory intermediate hosts for Schistosoma mansoni Sambon, 1907, the parasite responsible for intestinal schistosomiasis in different parts of the world. In the absence of either the parasite or the snails, the life cycle cannot be maintained [3, 4]. Efforts are thus directed toward eradicating intermediate hosts using molluscicides and/or the parasite via treatment of infected populations using praziquantel (the drug of choice for schistosomiasis treatment) [5–8]. Biomphalaria species have a close association with freshwater habitats closely related to human activities [9, 10]. The natural histories of the various species of Biomphalaria snails have been studied extensively, although taxonomic difficulties and population-level variation are among the factors which have confounded this task [5]. A detailed knowledge of the geographic distribution of Biomphalaria snails is important for the control of intestinal schistosomiasis and its epidemiologic surveillance [11]. Biomphalaria snails are thought to have evolved in South America and are currently distributed throughout the continent as well as in central America, the Caribbean, Africa and the Middle East [12, 13]. In addition to South America and Africa, Biomphalaria spp. was also reported from Asia in Hong Kong, China 40 years ago [14], as a result of human-mediated accidental introduction, likely via aquatic plants imported from Brazil for commercial use [15]. The introduced snail was identified as B. straminea (Dunker, 1848), which is known by its invasiveness and colonization capabilities [16].

In China, in the years following its introduction, B. straminea dispersed through different watercourses in southern China. The snail was identified in numerous water bodies in Shenzhen City, Guangdong Province, China in 1981 [17], and in the next 2 years it showed a wider distribution in Shenzhen rivers, suggesting an origination from Hong Kong’s river system [18]. This distribution pattern confirms the ability of B. straminea to survive and form new colonies in southern China and its ability to spread to new water bodies [19]. The continuous rising in Earth’s surface temperature and climate change [20], as well as the continued rise in the mean annual temperature of China [21], may accelerate B. straminea spread due to its ability to adapt to different climate and ecological conditions [22]. In addition, the risks for B. straminea to be the source of S. mansoni transmission has been evaluated with the following concerns: (i) the involvement of China in numerous projects in Africa and the export of labor services to regions endemic with African schistosomiasis; (ii) S. mansoni infections have been reported in Chinese workers returning from Africa [23]; (iii) The presence of snails and infected humans in the same settings will increase the potential of S. mansoni transmission to be established in southern China in view of the role played by environmental changes such as climate change and increase in water manipulation and intensification of irrigation [23, 24].

A multidisciplinary approach for identification of species of medical importance combining morphological and molecular techniques is fundamental for our understanding of diseases epidemiology and surveillance. Proper identification of the intermediate host will help to map the potential schistosomiasis transmission foci in endemic areas as well as in areas without the disease but with abundant susceptible species [25]. For example, although Biomphalaria snails found in southern China were referred to as B. straminea, a new challenge was encountered. In 2015 a morphological and molecular study for seven Biomphalaria populations from Guangdong Province, China showed that, five of the populations are likely to be Biomphalaria kuhniana (Clessin, 1883) but not B. straminea [26]. The study suggested multiple colonization events and displacement of B. straminea by the closely related species, B. kuhniana. The latter species is also a neotropical snail described from Suriname, but reported in other South American countries [27]. Together, B. straminea, B. kuhniana, and B. intermedia (Paraense and Deslandes, 1962), are known as B. straminea complex [28]. The previous three species differ greatly in their susceptibility to infection with S. mansoni; for example, B. kuhniana is proven to be refractory to infection [27]. Thus, an urgent taxonomy of these species should be carefully examined through combined analysis of morphological and molecular variables, and their biomedical importance in the transmission of S. mansoni should be evaluated. Careful identification of Biomphalaria snails in southern China will lead to a better delineation based on biological profiles related to the potential transmission risk, and will help to develop an appropriate intervention for schistosomiasis surveillance or control in potential foci, particularly in areas of constrained resources.

The present study attempted to identify the species of Biomphalaria snails introduced to southern China using morphological and molecular identification methods. The early reports on Biomphalaria invasion to Hong Kong identified the snails as B. straminea [14, 32, 44]. Subsequent invasions and spread were reported from other water bodies in southern China such as Guanlan and Dasha rivers in Shenzhen City, Guangdong Province’s rivers [18]. This distribution scheme led to the hypothesis of multiple invasions and colonization including different roots of introduction. Moreover, recent molecular investigation of several Biomphalaria populations from Guangdong indicated that most of the species currently harboring water systems are B. kuhniana not B. straminea [26]. This conclusion merited further careful investigation because B. kuhniana has so far been shown to be refractory to S. mansoni infection [27, 45], whereas B. straminea is generally considered to be a susceptible species, albeit in some cases at a low level, that can nonetheless transmit schistosomiasis [46–50], and even sustain natural transmission in some settings [13]. It is worth noting, however, that the taxonomic confusion within Biomphalaria has led some to speculate that naturally-infected B. straminea found in Venezuela may have in fact been B. kuhniana [13]. The fact remains that based on current data, B. straminea is generally considered to be of higher biomedical concern.

The identification of Biomphalaria was performed through morphological study of five specimens from each location sites in Shenzhen, Guangdong Province. Shell diameters ranged from 7.59 ± 0.45 mm as in Shenzhen reservoir population to 9.70 ± 0.93 mm in Guanlan River population with significant differences among the populations. According to Paraense [28], the average shell of adult B. kuhniana is smaller than 7.5 mm while that of B. straminea is from 11 mm to 16.5 mm. Although almost all of the current measurements are higher than 7.5 mm (the average diameter of B. kuhniana), which may support the snails to be B. straminea, the shell diameter is of limited value in discrimination between B. straminea and B. kuhniana due to the difficulty in recognizing full-grown shells in these species of planorbids [28]. Also, studies by Dupouy et al. [51] on the African pulmonate snail Biomphalaria pfeifferi (Krauss, 1848) concluded that environmental components may influence shell morphology through phenotypic versatility and/or through impacts of local natural selection. The differences observed in the shell size of Biomphalaria collected in this study could be attributed to environmental factors as well as the snails’ ages. The data from internal anatomy are considered more informative than external features. For example, the number of prostate diverticula varies significantly between B. kuhniana and B. straminea, with 4 to 9 observed in B. kuhniana versus 9 to 18 in B. straminea [28]. In the present study, in all the snails collected, the number of prostate diverticula ranged from 10.8 ± 0.83 to 20.6 ± 2.3 and the diverticula were long and branched. Similarly, the anatomy of reproductive organs of Hong Kong snails of Biomphalaria revealed an average number of prostate diverticula ranged from 11.67 to 17.67 [52] and both are close to the numbers recorded for South American B. straminea (9–18 prostate diverticula) [28]. The corrugations of vaginal wall and of the spermiduct are very prominent, similar to B. straminea from Brazil. The shape and number of diverticula in Guangdong populations supports its close affinity to B. straminea rather than B. kuhniana (Fig. 4).

Molecular based taxonomy of snail intermediate hosts has been developed to overcome erroneous species identification resulting from morphometric measurements. Mitochondrial data are reliable and informative and its consideration in snails taxonomy stems from the fact that the COI mitochondrial gene proved to be an ideal barcode locus and default marker adopted by the Consortium for the Barcode of Life for all groups of organisms [53, 54] including Biomphalaria spp. [55–58]. In the current study, molecular evidence, retrieved from sequencing two mitochondrial loci (COI and 16S rRNA), were in harmony with anatomical evidence and suggested a close affinity between Guangdong Biomphalaria and B. straminea from Hong Kong of China and Brazil as showed in the cladograms of the data sets analyzed. The absence of existing COI data for B. kuhniana limits its use as an independent identifier although the analysis of this locus in combination with 16S rRNA gave a quite similar tree topology as obtained with COI alone. Moreover, COI analysis excluded the possibility of Guangdong populations of Biomphalaria to be B. intermedia. The haplotypes from the present study were clustered in one clade with B. straminea sequences from Hong Kong, Guangdong of China and Brazil. B. intermedia originated from Brazil (KF926179.1 [59]) was situated in a different clade basal to the previous one with a nodal support of 0.62 posterior probabilities (Fig. 5). The phylogenetic tree in Fig. 5 showed that the COI sequences of the samples collected were identical to those of B. straminea from Brazil and those previously deposited in GenBank for Chinese B. straminea (Fig. 5). It is worthy to mention that looking for similar sequences for COI sequences deposited by Atwood et al. [27] for B. kuhniana (KJ137284 − KJ137287) using the BLAST tool showed that those sequences are 98–100% identical to B. straminea isolates of China and Brazil indicating that the authors may misidentified the samples collected. For 16S rRNA, enough data were available in GenBank records in addition to the sequences obtained from samples of B. straminea complex obtained from Brazil and Colombia. Guangdong haplotypes were clustered in the same clade with Hong Kong isolates of B. straminea (KY697220.1, KY697223.1 and KY697236.1 [52]). Both Guangdong and Hong Kong isolates from China were clustered with Brazilian samples of B. straminea from Distrito Federal and Pernambuco and one GenBank isolate from Belem Para, Brazil (AY030213.1) with a major nodal support values. B. kuhniana sequences represented an in-group clade with a nodal support of 0.96 posterior probabilities. This tree construction indicates that the Guangdong populations are in close affinity with B. straminea present in Hong Kong and both share South American ancestors of B. straminea. This finding is in accordance with Zeng et al. [52] who identified two phenotypes of Biomphalaria snails (black- and red-colored shell Biomphalaria) in sites located at the border between Hong Kong and Shenzhen (few miles away from the samples collected in our study) and in the New Territories in Hong Kong, including places adjacent to the mainland China (30–40 miles away from our samples). The two phenotypes were genetically indistinguishable and were similar to B. straminea abundant in mainland China and South America and suggested that the B. straminea snails in Hong Kong are South American in origin.

In the present study, both Guangdong populations from China and Brazilian B. straminea formed a clade of closely related species with a minimal genetic divergence between them (Figs. 5, 6 and 7). Moreover, the sequence divergence estimates (0.3%) and strong support for the relationship between these populations (as demonstrated by Bayesian posterior probability values) suggest that these are the same species, and closely related populations, rather than a separate species. Independent analyses of COI and 16S rRNA sequences showed low genetic variability with four and three distinct haplotypes, respectively for both genes, out of 60 sequences representing six different populations of Biomphalaria snails collected from Shenzhen. The low nucleotide diversity observed within the Guangdong populations (COI, 0.004488; 16S, 0.005587; COI + 16S rRNA, 0.002794) is consistent with the literature; in a recent study by Attwood et al. [26], the authors found three distinct COI sequences, with a nucleotide diversity of 0.00279, and only one unique 16S rRNA sequence.

The observed levels of genetic diversity across the Guangdong populations of Biomphalaria, suggests a more likely recent connection between these populations in view of the fast divergence of mitochondrial genes in pulmonates [60–62] which is expected in this recently introduced snail species. According to Attwood et al. [26], the observed divergence is relatively high given the small area of sampling, and they suggest that the diversity is not due to mutations occurring in Guangdong populations from China but rather was exhibited in ancestor Brazilian populations, indicating that the current Guangdong populations could represent multiple, independent, introductions followed by subsequent dispersal and colonization. This multiple colonization hypothesis is also supported by a previous study of 19 electrophoretlcally detected enzyme loci in four Hong Kong populations of B. straminea that showed high levels of variability among the populations. This variability resulted from multiple introduction events between 1973 and 1981 [44]. Although the relatively high genetic diversity at the COI (0.004488) locus, in comparison with other pulmonates, assumes divergence and mutations in ancestors’ snails at the introduction source, it can also be attributed to the reproductive traits of B. straminea and their high selfing rates. The presence of the populations in the same cladogram with B. straminea from Hong Kong (Fig. 5) may support the hypothesis that part of the snails currently distributed in Guangdong could be present as a result of peripheral dispersal of descendant snails from Hong Kong populations via passive transport or container ship traffic, or naturally due to connections between the water systems of the two adjacent localities. Finally, the genetic similarities among the snail populations strongly support the idea that these are derived from the original colonist populations via dispersal [63].

Preliminary experimental infection of filed captured-B. straminea from Shenzhen and their first generation with a Puerto Rican strain of S. mansoni showed, at different ratios of miracidial exposure, no infection in the exposed snails [64]. Although this finding suggests that the invasive B. straminea snails are incompatible with S. mansoni and thus there is a low risk of S. mansoni transmission in mainland China, it may not be appropriate to consider this as a final conclusion because the compatibility was tested with only one strain of S. mansoni from South America and the experiments were undertaken with the first generation of laboratory bred B. straminea. Further studies are needed to test the compatibility of different isolates of both Chinese Biomphalaria and S. mansoni from a variety of locations especially African strains because most of the imported cases of human infection are from Africa and the number is anticipated to increase due to the expansion in Chinese projects in Africa [23].

The reassessment of the identity of Biomphalaria species present in southern China using morphological and molecular delimitation procedures suggests that B. straminea is the only Biomphalaria species abundant in southern China. However, to validate the current conclusion and in order to obtain a more reliable barcoding database for future studies, COI sequences from original reference snails (for B. straminea and B. kuhniana) from South America are required, and should be deposited in GenBank or other repositories. The current finding is important from a public health perspective due to the biomedical importance of B. straminea as an intermediate host for Schistosoma mansoni, and the potential risk of Schistosoma mansoni transmission to mainland China in view of the increase in labor services exchange with African countries where the parasite is endemic. More studies should be conducted to understand the biology of this snail and its competitive capacity with other local snail species and to describe its range of expansion. Moreover, the susceptibility of these local populations to S. mansoni should be tested. Better understanding of the population structure of this intermediate host snail is important to elucidate its potential role in schistosomiasis transmission, considering factors that may affect population structure such as environmental and demographical stability. Finally, it is widely encouraged to increase surveillance for all entry ports and checking for any exotic species, in order to reduce the likelihood of future introductions of potentially invasive species [64, 65].