# Characterization of South American Snails of the GenusBiomphalaria (Basommatophora: Planorbidae) andSchistosoma mansoni (Platyhelminthes: Trematoda) in Molluscs by PCR-RFLP

Authors: Roberta Lima Caldeira; Tatiana Maria Teodoro; Liana Konovaloff Jannotti-Passos; Pollanah M. Lira-Moreira; Christiane De Oliveira Goveia; Omar dos Santos Carvalho
Journal: BioMed Research International
 (2016)

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## Abstract

The identification of snails of the genusBiomphalaria can be done using morphological characteristics which depends on the size of the snails and skill and knowledge of researcher. These methods sometimes are not adequate for identification of species. The PCR-RFLP, using the ITS region of the rDNA, has been used to identify Brazilian species of the genusBiomphalaria. Nevertheless, there is a lack of information about snails from other Latin American countries. In addition, some snails may be infected bySchistosoma mansoni and when submitted to PCR-RFLP they show molecular profiles different from those previously standardized for the other mollusc species. In this work the molecular profiles of 15 species and the subspecies were established by PCR-RFLP of ITS-rDNA with the enzymeDdeI. Moreover, the molecular profiles of host species,B. glabrata,B. straminea,B. tenagophila, andB. prona, infected byS. mansoni were also established. The molluscs were dissected to permit morphological identification. These results contribute to a correct identification of snails of the genusBiomphalaria and detection of these snails infected byS. mansoni.


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## Body

## 1. Introduction

Despite therapeutic advances in the last decade, Schistosomiasis remains one of the most prevalent parasitic diseases worldwide and endemic in 76 countries and territories [1]. In Africa and Neotropical Region there are species of the genusBiomphalaria (Gastropoda: Planorbidae) which are intermediate hosts ofSchistosoma mansoni Sambon, 1907. In Latin America 24 species and one subspecies were registered (Table 1), four of them can be found naturally infected byS. mansoni, whereas six were found to be susceptible in the laboratory.Table 1
Molluscs of the genusBiomphalaria present in Latin America.
Species Geographical distribution aSusceptibility to Schistosoma mansoni Biomphalaria amazonica, Paraense 1966 Brazil, Bolivia, Colombia EI  Biomphalaria andecola (Orbigny, 1835) Bolivia, Peru, Chile NI  Biomphalaria cousini Paraense, 1966 Brazil, Ecuador EI  Biomphalaria edisoni (Estrada et al., 2006) Colombia NI  Biomphalaria equatoria (Cousin, 1887) Ecuador NI  Biomphalaria glabrata (Say, 1818) Antigua, Brazil, Curacao, Dominica, Guadeloupe, French Guiana, Haiti, Saint Kitts and Nevis, Martinique, Montserrat, Puerto Rico, Dominican Republic, Saint Lucia, Suriname, Venezuela S  Biomphalaria havanensis (Pfeiffer, 1839) Haiti, Mexico, Puerto Rico, Cuba, Venezuela EI  Biomphalaria helophila (Orbigny, 1835) Peru, Cuba, Costa Rica, Guatemala, Belize, Haiti, Mexico, Saint Thomas, El Salvador, Dominican Republic, Puerto Rico, Barbados, Nicaragua EI  Biomphalaria intermedia (Paraense & Deslandes, 1962) Brazil, Argentine NS  Biomphalaria kuhniana (Clessin, 1883) Suriname, Brazil, Venezuela, Panama, Colombia NS  Biomphalaria nicaraguana (Morelet, 1851) Nicaragua NI  Biomphalaria occidentalis Paraense, 1981 Brazil, Paraguay, Argentine NS  Biomphalaria oligoza Paraense, 1974 Bolivia, Brazil, Argentine EI  Biomphalaria orbignyi Paraense, 1975 Argentine, Uruguay EI  Biomphalaria obstructa (Morelet, 1849) Mexico, Puerto Rico, Guatemala, El Salvador, Belize, Cuba NS  Biomphalaria pallida (Adams, 1846) Jamaica, Cuba NI  Biomphalaria peregrina (Orbigny, 1835) Ecuador, Bolivia, Chile, Brazil, Paraguay, Peru, Uruguay, Argentine, Colombia EI  Biomphalaria prona (Martens, 1873) Venezuela S  Biomphalaria schrammi (Crosse, 1864) French Guiana, Guadeloupe, Brazil NS  Biomphalaria sericea (Dunker, 1848) Ecuador EI  Biomphalaria straminea (Dunker, 1848) Venezuela, Suriname, French Guiana, Guyana, Peru, Brazil, Paraguay, Argentine, Dominica, Grenada, Guadeloupe, Martinique, Dominican Republic, Trinidad, Uruguay, Costa Rica S  Biomphalaria subprona (Martens, 1899) Mexico, Guatemala NI  Biomphalaria tenagophila (Orbigny, 1835) Argentine, Paraguay, Uruguay, Brazil, Peru, Bolivia S  Biomphalaria tenagophila guaibensis Paraense, 1984 Brazil, Uruguay, Paraguay, Argentine NS  Biomphalaria trigyra (Philippi, 1869) Peru, Ecuador NS a: susceptible = S; not susceptible = NS; experimental infection = EI; not information = NI.The classical identification of snails of the genusBiomphalaria is based on morphological characteristics of the shell and the reproductive system [2]. However, this approach is complicated in cases of inadequate fixation or interspecific similarity. The Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP), directed to the internal transcribed spacer (ITS) region of the rDNA gene, has been used with success to resolve these cases. The molecular profile of Brazilian species of the genusBiomphalaria using this method has been established [3]. Thus, the specific profile of all these species together will be useful to facilitate interspecific identification in the genusBiomphalaria. Besides, the specific identification could be done by comparing the sequences between closely related species [4–6], as well as using the morphology associated with the species-specific PCR [7, 8].Furthermore, the snails which were collected in the field may be infected withS. mansoni during the prepatent period, and when they are submitted to the molecular identification, their DNA is simultaneously amplified with the DNA from the parasite. In this case the molecular profile differs from the profile established for the snail alone.The aim of the present work is to present the previously species-specific profiles established by PCR-RFLP of ITS-rDNA withDdeI and to establish the profiles forB. glabrata,B. tenagophila,B. straminea, andB. prona infected byS. mansoni.


## 2. Material and Methods

### 2.1. Samples

Of the 24 species registered for Latin America, the Medical Malacological Collection (Fiocruz-CMM) has fifteen species and a subspecies:B. glabrata,B. tenagophila,B. occidentalis,B. schrammi,B. oligoza,B. peregrina,B. intermedia,B. straminea,B. kuhniana,B. amazonica,B. cousini,B. prona,B. edisoni,B. havanensis,B. orbignyi, andB. tenagophila guaibensis. The molluscs were dissected to permit morphological identification. DNA of specimens of the Fiocruz-CMM collection was cryopreserved.Biomphalaria glabrata,B. tenagophila, andB. straminea molluscs and AL, SJ, and LE strains ofS. mansoni used in this study were maintained and raised in the “Lobato Paraense” Mollusc Rearing of René Rachou Research Center, CPqRR/Fiocruz, in Belo Horizonte, MG, Brazil. The LE strain was isolated, in 1968, from a patient residing in Belo Horizonte, MG (Brazil). The SJ strain was isolated, in 1975, from naturally infected snails from São José dos Campos, São Paulo (Brazil). The AL strain was isolated in 1980 fromB. glabrata that originated from Alagoas state (Brazil). To obtain specimens ofB. glabrata,B. tenagophila, andB. straminea sheddingS. mansoni cercariae, experimental infection with LE, SJ, and AL strains, respectively, was carried out [9]. However, there was no population ofB. prona in the “Lobato Paraense” Mollusc Rearing, so the DNA of the snails and the parasite (LE strain) was mixed and amplified together to obtain the profile of this infected species. DNA of adult worms ofS. mansoni was used for control of amplification.Cercaria macrogranulosa, Cercaria caratinguensis, andCercaria ocellifera were obtained from field snailsBiomphalaria.


### 2.2. Molecular Techniques

#### 2.2.1. DNA Extraction and PCR-RFLP Assay

Total DNA fromB. glabrata,B. tenagophila, andB. straminea infected byS. mansoni,B. prona, adult worms ofS. mansoni andC. macrogranulosa,C. caratinguensis, andC. ocellifera were extracted using Wizard Genomic Purification Kit (Promega, Madison, USA) with some modifications. The DNA of all samples was used as template in the PCR-RFLP assay. The entire ITS was amplified using the primers ETTS2 (5′ TAACAAGGTTTCCGTAGGTGAA 3′) and ETTS1 (5′ TGCTTAAGTTCAGCGGGT 3′) anchored, respectively, in the conserved extremities of the 18S and 28S ribosomal genes [10]. The PCR amplification was undertaken in a volume of 10 μL consisting of 1–10 ng template DNA, 10 mM Tris-HCl, pH 8.5, 200 μM of each DNTP, 1.5 mM MgCl2, 0.5 U of Taq DNA polymerase, and 50 mM KCl, together with 1.0 pmol of each primer. The reactions were covered with a drop of mineral oil and subjected to the following thermal cycling program: initial denaturation step for 3 min at 95°C, and then 32 cycles with annealing at 54°C for 1 min, extension at 72°C for 2 min, denaturation at 95°C for 45 sec, and a final extension step at 72°C for 5 min. A negative control (no template DNA) was included in all experiments. Three microliters of the amplification products were visualized on silver stained 6% polyacrylamide gels to check the quality of amplification. The remaining 7 μL was mixed with water, andDdeI (10–12 units) enzyme was added, together with 1.0 μL of the respective enzyme buffer. The digestion was performed for 3.5 h at 37°C and at 80°C for 20 min for enzyme denaturation and the digestion products were evaluated on silver stained 6% polyacrylamide gels [3].


## 2.1. Samples

Of the 24 species registered for Latin America, the Medical Malacological Collection (Fiocruz-CMM) has fifteen species and a subspecies:B. glabrata,B. tenagophila,B. occidentalis,B. schrammi,B. oligoza,B. peregrina,B. intermedia,B. straminea,B. kuhniana,B. amazonica,B. cousini,B. prona,B. edisoni,B. havanensis,B. orbignyi, andB. tenagophila guaibensis. The molluscs were dissected to permit morphological identification. DNA of specimens of the Fiocruz-CMM collection was cryopreserved.Biomphalaria glabrata,B. tenagophila, andB. straminea molluscs and AL, SJ, and LE strains ofS. mansoni used in this study were maintained and raised in the “Lobato Paraense” Mollusc Rearing of René Rachou Research Center, CPqRR/Fiocruz, in Belo Horizonte, MG, Brazil. The LE strain was isolated, in 1968, from a patient residing in Belo Horizonte, MG (Brazil). The SJ strain was isolated, in 1975, from naturally infected snails from São José dos Campos, São Paulo (Brazil). The AL strain was isolated in 1980 fromB. glabrata that originated from Alagoas state (Brazil). To obtain specimens ofB. glabrata,B. tenagophila, andB. straminea sheddingS. mansoni cercariae, experimental infection with LE, SJ, and AL strains, respectively, was carried out [9]. However, there was no population ofB. prona in the “Lobato Paraense” Mollusc Rearing, so the DNA of the snails and the parasite (LE strain) was mixed and amplified together to obtain the profile of this infected species. DNA of adult worms ofS. mansoni was used for control of amplification.Cercaria macrogranulosa, Cercaria caratinguensis, andCercaria ocellifera were obtained from field snailsBiomphalaria.


## 2.2. Molecular Techniques

### 2.2.1. DNA Extraction and PCR-RFLP Assay

Total DNA fromB. glabrata,B. tenagophila, andB. straminea infected byS. mansoni,B. prona, adult worms ofS. mansoni andC. macrogranulosa,C. caratinguensis, andC. ocellifera were extracted using Wizard Genomic Purification Kit (Promega, Madison, USA) with some modifications. The DNA of all samples was used as template in the PCR-RFLP assay. The entire ITS was amplified using the primers ETTS2 (5′ TAACAAGGTTTCCGTAGGTGAA 3′) and ETTS1 (5′ TGCTTAAGTTCAGCGGGT 3′) anchored, respectively, in the conserved extremities of the 18S and 28S ribosomal genes [10]. The PCR amplification was undertaken in a volume of 10 μL consisting of 1–10 ng template DNA, 10 mM Tris-HCl, pH 8.5, 200 μM of each DNTP, 1.5 mM MgCl2, 0.5 U of Taq DNA polymerase, and 50 mM KCl, together with 1.0 pmol of each primer. The reactions were covered with a drop of mineral oil and subjected to the following thermal cycling program: initial denaturation step for 3 min at 95°C, and then 32 cycles with annealing at 54°C for 1 min, extension at 72°C for 2 min, denaturation at 95°C for 45 sec, and a final extension step at 72°C for 5 min. A negative control (no template DNA) was included in all experiments. Three microliters of the amplification products were visualized on silver stained 6% polyacrylamide gels to check the quality of amplification. The remaining 7 μL was mixed with water, andDdeI (10–12 units) enzyme was added, together with 1.0 μL of the respective enzyme buffer. The digestion was performed for 3.5 h at 37°C and at 80°C for 20 min for enzyme denaturation and the digestion products were evaluated on silver stained 6% polyacrylamide gels [3].


## 2.2.1. DNA Extraction and PCR-RFLP Assay

Total DNA fromB. glabrata,B. tenagophila, andB. straminea infected byS. mansoni,B. prona, adult worms ofS. mansoni andC. macrogranulosa,C. caratinguensis, andC. ocellifera were extracted using Wizard Genomic Purification Kit (Promega, Madison, USA) with some modifications. The DNA of all samples was used as template in the PCR-RFLP assay. The entire ITS was amplified using the primers ETTS2 (5′ TAACAAGGTTTCCGTAGGTGAA 3′) and ETTS1 (5′ TGCTTAAGTTCAGCGGGT 3′) anchored, respectively, in the conserved extremities of the 18S and 28S ribosomal genes [10]. The PCR amplification was undertaken in a volume of 10 μL consisting of 1–10 ng template DNA, 10 mM Tris-HCl, pH 8.5, 200 μM of each DNTP, 1.5 mM MgCl2, 0.5 U of Taq DNA polymerase, and 50 mM KCl, together with 1.0 pmol of each primer. The reactions were covered with a drop of mineral oil and subjected to the following thermal cycling program: initial denaturation step for 3 min at 95°C, and then 32 cycles with annealing at 54°C for 1 min, extension at 72°C for 2 min, denaturation at 95°C for 45 sec, and a final extension step at 72°C for 5 min. A negative control (no template DNA) was included in all experiments. Three microliters of the amplification products were visualized on silver stained 6% polyacrylamide gels to check the quality of amplification. The remaining 7 μL was mixed with water, andDdeI (10–12 units) enzyme was added, together with 1.0 μL of the respective enzyme buffer. The digestion was performed for 3.5 h at 37°C and at 80°C for 20 min for enzyme denaturation and the digestion products were evaluated on silver stained 6% polyacrylamide gels [3].


## 3. Results and Discussion

The PCR amplification resulted in a product of approximately 1200 pb forBiomphalaria, one of 800 pb forS. mansoni, and both fragments for infected molluscs (data not shown). The RFLP profiles obtained by digesting rDNA ITS withDdeI in Figure 1 allow the following: (1) to identify noninfectedB. glabrata,B. tenagophila, B. straminea, andB. prona, by observation of species-specific fragments (Lanes 2, 3, 4, and 5); (2) to establish the species-specific profile ofS. mansoni (Lanes 6 and 11); and (3) to detect by the presence of overlapping species-specific fragments the infection byS. mansoni inB. glabrata,B. tenagophila,B. straminea, andB. prona (Lanes 7, 8, 9, and 10).Figure 1
6% silver stained polyacrylamide gel showing restriction profiles obtained by digestion of the ITS region of DNA ribosomal withDdeI. Lane 1: molecular size markers Phi X 174/HaeIII; Lane 2:Biomphalaria glabrata; Lane 3:Biomphalaria tenagophila; Lane 4:Biomphalaria straminea; Lane 5:Biomphalaria prona; Lane 6: adult worm ofSchistosoma mansoni; Lane 7:B. glabrata infected byS. mansoni; Lane 8:B. tenagophila infected byS. mansoni; Lane 9:B. straminea infected byS. mansoni; Lane 10: DNA ofB. prona with DNA ofS. mansoni; Lane 11: adult worm ofS. mansoni. The numbers on the left of the gel represent the value in base pairs (bp).All 15 species and the subspecies ofBiomphalaria were dissected and their identification confirmed by analysis of specific diagnostic characters established for each species. In association with morphological identification, the profile of PCR-RFLP was established for these species and is shown in Figure 2.Figure 2
6% silver stained polyacrylamide gel showing restriction profiles obtained by digestion of the ITS region of DNA ribosomal withDdeI. Lane 1: molecular size markers Phi X 174/HaeIII; Lane 2:Biomphalaria glabrata; Lane 3:Biomphalaria tenagophila; Lane 4:Biomphalaria tenagophila guaibensis; Lane 5:Biomphalaria occidentalis; Lane 6:Biomphalaria schrammi; Lane 7:Biomphalaria oligoza; Lane 8:Biomphalaria peregrina; Lane 9:Biomphalaria intermedia; Lane 10:Biomphalaria straminea; Lane 11:Biomphalaria kuhniana; Lane 12:Biomphalaria amazonica; Lane 13:Biomphalaria cousini; Lane 14:Biomphalaria prona; Lane 15:Biomphalaria edisoni; Lane 16:Biomphalaria havanensis; Lane 17:Biomphalaria orbignyi. The numbers on the left of the gel represent the value in base pairs (bp).Studies that incorporate morphological and molecular techniques in taxonomic analysis can generate data that allow a better interpretation and understanding of the biological diversity of the organisms under study. In fact, both the molecular and morphological taxonomy, if properly applied, successfully achieve the same goal [11]. In previous studies, the diagnosis ofS. mansoni in molluscs has been performed using the LS-PCR [12], the conventional PCR assays for amplification of the Sm1–7 repeated sequence [13], and Loop-Mediated Isothermal Amplification [14] and otherwise the most frequent technique used to the identification ofBiomphalaria is the PCR-RFLP.This study has demonstrated the usefulness of the PCR-RFLP technique in the diagnosis of infection byS. mansoni in molluscs concurrently with identification of the four intermediate hosts,B. glabrata,B. tenagophila,B. straminea, andB. prona (Figure 1). In addition it was possible to identify a unique profile for the cercariae ofS. mansoni,C. macrogranulosa,C. caratinguensis, andC. ocellifera, obtained from snailsBiomphalaria collected in the field, after amplification of the ITS region of the rDNA digestion individually with the enzymesDdeI,AluI,HaeIII,RsaI, andHpaII (data not published).Thus, this molecular biology technique has great utility for generating new knowledge about the taxonomy of molluscs of the genusBiomphalaria. Further, from the genetic analysis of various species ofSchistosoma andBiomphalaria, it was observed that intraspecific genetic polymorphism of the parasite is limited while in the mollusc, it is very pronounced, showing the higher relevance of molluscan genetics over parasite genetics in determining the epidemiology of the disease [15]. For example, in adultB. glabrata, resistance toS. mansoni has been shown to be a dominant single-gene trait that is inherited by Mendelian genetics. In contrast, in juveniles, the genetics of resistance has been shown to involve 5 to 6 genes each with multiple alleles [16]. Additionally, Ittiprasert and Knight report reversing the resistance phenotype of resistant BS-90B. glabrata by applying stress in the form of a mild heat pulse before they were exposed toS. mansoni, rendering these snails susceptible [17].



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*Source: 1045391-2016-11-17.xml*