POPULATION STRUCTURE OF BIOMPHALARIA GLABRATA ...

J. Moll. Stud. (1999), 65, 425–433

© The Malacological Society of London 1999

POPULATION STRUCTURE OF BIOMPHALARIA GLABRATA, INTERMEDIATE SNAIL HOST OF SCHISTOSOMA MANSONI IN GUADELOUPE ISLAND, USING RAPD MARKERS J. LANGAND 1 , A. THERON 1 , J.P. POINTIER 1 , B. DELAY 2 and J. JOURDANE 1 Centre de Biologie et d’Ecologie tropicale et méditerranéenne, UMR 5555, Université de Perpignan, 52 avenue de Villeneuve, 66860 Perpignan Cedex – France 1Centre d’Ecologie Fonctionnelle et Evolutive, CNRS, 1919 route de Mende, 34293 Montpellier Cedex 5 – France (Received 26 October 1998; accepted 30 December 1998)

ABSTRACT Nine populations of the snail Biomphalaria glabrata, sampled in 1992 and in 1994 in the Guadeloupean focus of schistosomiasis, were studied using random amplified polymorphic DNA (RAPD) markers. The detected polymorphism was low with 11 polymorphic markers. A canonical analysis of the genetic polymorphism showed a significant and large differentiation between the nine populations studied in a restricted area of 45 km2. A Mantel t-test indicated a significant correlation between the genetic differentiation and the geographical distance. A significant but smaller differentiation between a northern and southern group was also observed using canonical analysis. A comparison over time has been done.

INTRODUCTION The fragmentation of freshwater environments, their temporal instability and the reproduction patterns of snails are among the main factors likely to shape genetic structures of freshwater snail populations (Jarne & Delay, 1991). Snails are intermediate hosts of many species of trematodes. Several studies have shown that the susceptibility/resistance of snails and the infectivity of digenea are genetically determined (Richards, 1975a; Richards, Knight & Lewis, 1992). Theoretical studies have also shown that the structure of host populations could influence the compatibility, and hence the dynamics of parasite transmission (Thrall & Antonovics, 1995; Gandon, Capowiez, Dubois, Michalakis & Olivieri, 1996). Corresponding author: A. Théron. Laboratoire de Biologie Animale, UMR 5555 CNRS, Centre de Biologie et d’Ecologie Tropicale et Méditerranéenne, Université, 52 Av. de Villeneuve, 66860 Perpignan Cedex, France. Tel: 133 4 68 66 21 83 Fax: 133 4 68 66 22 81 E-mail: [email protected]

The snail Biomphalaria glabrata is the intermediate host of Schistosoma mansoni on the island of Guadeloupe, French West Indies. Adult worms develop in humans and in the rodent Rattus rattus. B. glabrata is an hermaphroditic pulmonate reproducing preferentially by cross-fertilisation (Paraense, 1955; Mulvey & Vrijenhoek, 1982; Vianey-Liaud, Dupuy, Lancastre & Nassi, 1987, 1991; Vianey-Liaud, Nassi, Lancastre & Dupuy, 1989; Vernon, Jones & Noble, 1995). The murine focus is located on the littoral part of the Grand Cul-de-Sac (Fig. 1). B. glabrata populations are present within a narrow band at the edge of a swampy forest (Pointier & Théron, 1979). The dynamics of these fragmented snail populations are strongly influenced by flooding and drying periods. These disturbances can be very different according to the locality and the year (Borel, 1990). Knowledge of the population structure of B. glabrata could improve understanding of the transmission dynamics of S. mansoni for which long term studies have reported large differences between localities (Théron, Pointier, Morand, Imbert-Establet & Borel, 1992). RAPD markers were used to quantify the genetic diversity between B. glabrata populations. The technique derived from PCR (Williams, Kubelik, Livak, Rafalski & Tingey, 1990; Welsch & McClelland, 1990) provides a particularly large number of polymorphic markers (Martin, Williams & Tansley, 1991; Bardakci & Skibinsky, 1994; van Oppen, Diekmann, Wiencke, Stam & Olsen, 1994; Catalàn, Shi, Armstrong, Draper & Stace, 1995; Link, Dixkens, Singh, Schwall & Melchinger, 1995). Several studies have shown that the RAPD technique can be applied successfully to gastropods (Langand, Barral, Delay & Jourdane,

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J. LANGAND, A. THERON, J.P. POINTIER, B. DELAY & J. JOURDANE

Figure 1. Map of the sampled sites for Biomphalaria glabrata at the edge of the swampy forest in Guadeloupe.

1993; Vidigal, Neto, Carvalho & Simpson, 1994; Jones, Noble, Lockyer, Brown & Rollinson, 1997; Vernon, Jones & Noble, 1995; Stothard & Rollinson, 1996; Stothard, Mgeni, Alawi, Savioli & Rollinson, 1997; Hoffman, Webster, Ndamba & Woolhouse, 1998; Vidigal, Spatz, Nunes, Simpson, Carvalho & Neto, 1998). The

data analysis of the results achieved with this rather recent technique is still controversial (Perez, Albornoz & Dominguez, 1998). This is mostly due to the dominance of the RAPD markers (Clark & Lanigan, 1993; Tinker, Fortin & Mather, 1993; Williams, Hanafe, Rafalski & Tingey, 1993; Lynch & Milliga, 1994). With

POPULATION STRUCTURE OF B. GLABRATA

such dominant markers, heterozygous individuals cannot be distinguished from homozygous ones, and therefore, genotypic information is not available. This limits the use of classical indexes of population genetics, Fst, Fis, etc. based on allelic frequencies. Under these conditions we have considered RAPD profiles only, and use methods adapted to analyse such data. MATERIAL AND METHODS Snail material Nine localities (Fig. 1) were sampled in the swampy forest, at the end of the rainy season, with a two years interval (November 1992 and December 1994). These localities include: Pico (PIC), Dubelloy (DUB), Sauvia (SAU), Blain (BLA), Geffrier (GEF), Dans Fond (DFO), Gaoza (GAO), Belle Plaine (BLP), and Jacquot (JAC). The geographic distance between localities ranges between 0.2 km (Sauvia-Blain) and 11.5 km (Pico-Jacquot). Samples taken from each locality were considered as populations. A total of 306 snails were analysed, corresponding to 17 individuals per population per year.

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OP-B8, OP-B9, OP-B11, OP-B12, OP-B14, OP-B16, OP-B17, OP-B18), were tested on 18 individuals (2 per population) in order to detect polymorphism. The study was extended to all individuals to test then for primers detecting a clear polymorphism. We were not able to get again from the supplier one of the primers: OP-G15, and consequently we were not able to test all the individuals of the 1994 samples (the data obtained with this primer were then omitted in the comparison of the years 1992 and 1994). Data analysis The software GENEPOP V3 (Raymond & Rousset, 1996) was used to detect associations of RAPD marker pairs (option 2, sub-option 1) and to test the significance of the differentiation of population pairs (option 3, sub-option 4). Probabilities of differentiation of population pairs given by GENEPOP for each marker were combined according to the Fisher method to obtain a probability for all the markers. The diversity based on phenotypic frequencies was calculated for each marker with the Shannon index according to the formula: n

H 5 2S (pi 1n pi) i51

DNA extraction DNA was extracted from each individual snail as follows. Frozen snails were extracted from their shell. The headfoot was individually homogenised by crushing. The powder was mixed with 1 ml of extraction buffer (Tris 50 mM, EDTA 100 mM, pH 8) containing 1% of sodium dodecyl sulfate and 75 ml of Proteinase K at 10 mg/ml. The homogenate was incubated for 2–3 h at 55°C. DNA was isolated by phenol/ chlorophorm extraction, and precipitated with 3 volumes of cold absolute ethanol for 12 h at 220°C. DNA was recovered either with a Pasteur pipette or by centrifugation (30 min at 15,000 rpm). It was then washed twice in 70% ethanol, air dried and redissolved in 200 ml of buffer (Tris 10 mM, EDTA 1 mM, pH 8). RAPD markers technique PCR reactions were prepared in a volume of 25 ml containing 1 U of Taq-DNA polymerase (Promega), 2.5 ml of Taq buffer (Promega), 2.5 mM of MgCl2, 100 mM of each dNTP, 1 mM of primer and 15 to 60 ng of total DNA. Amplifications were carried out for 40 cycles and began by an initial denaturing stage at 92°C of 3 min 50 s. Each cycle included the following steps: 1 min at 92°C, 2 min at 35°C, 2 min at 72°C. The extension time was increased to 5 min during the final cycle. The products of amplification were analysed by electrophoresis in agarose gels (1%) stained with ethidium bromide. They were visualised by using ultraviolet light. First, 32 primers manufactured by the OPERON Society: the whole G kit (OP-G1 to OP-G20) and 12 primers from B kit (OP-B1, OP-B2, OP-B4, OP-B7,

with pi the frequency of the phenotype i. If Hpop is the mean diversity per population, and if Ht is the diversity calculated for all populations, the part of the intrapopulational diversity is Hpop/Ht; the part of the interpopulational diversity is (Ht – Hpop)/Ht. Canonical analyses were carried out using individual data of presence/absence of RAPD markers. Permutation tests showed whether the groups actually exist or not, and which were the axes which discriminate between groups. The ratio between the sum of all canonical eigenvalues (given by the canonical analysis) and the sum of all unconstrained eigenvalues (given by the correspondence analysis) represented the part of the variance due to groups. Software CANOCO (Version 3.11, C. J. F. ter Braak, 1990) was used to perform canonical analyses and permutation tests. The software Praxis TM (Version 2.0, Praxème R & D, 1995) was used to carry out a global permutation test. A Mantel t-test (software R, program Mantel B 3.0, Alain Vaudor) was carried out to detect a correlation between geographic distances and Euclidean distances based on the frequencies of RAPD markers for each population pair.

RESULTS Intrapopulational results Five primers provided one or several polymorphic markers: two markers (G3.1 and G3.2) with OP-G3, two markers (G6.1 and G6.2) with OP-G6, two markers (G15.1 and G15.2) with OP-G15, one marker (G19.1) with OP-G15,

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two markers (B7.1 and B7.2) with OP-B7 and two markers (B8.1 and B8.2) with OP-B8. The repetition of the experiments for several individuals for each marker did not detect any problem of reproducibility. This polymorphism is low (32 primers tested, i.e. more than 400 bands), therefore problems of homology of actually different bands should be unlikely in our study. These markers detected polymorphism within each population (Table 1). Results achieved with the software GENEPOP V3 showed that none of these RAPD markers was significantly linked to another one. This means that each of these markers gives independent information. The genetic diversity obtained with Shannon index is of the same order of magnitude for each population, with a maximum of diversity for Blain and a minimum for Jacquot (Table 2).

marker. The software GENEPOP V3 showed that most of the population pairs were significantly differentiated (at the level of 5%), excepted between Sauvia-Blain, Blain-Dubelloy, Dubelloy-Dans Fond, Dubelloy-Gaoza, Dubelloy-Belle Plaine and Gaoza-Belle Plaine. A permutation test carried out with the software PRAXIS showed that the differentiation between the populations is highly significant (P , 0.0001). A canonical analysis was performed considering nine groups corresponding

Interpopulational results The polymorphic RAPD markers were broadly shared by all the populations. Differences between populations resulted mostly from quantitative differences of the frequencies of the bands. No population had an exclusive

Table 2. Shannon indexes of intrapopulational diversity in samples collected in 1992. The confidence interval is given at 1% with a t-test. Population

Shannon index

Pico Dubelloy Sauvia Blain Geffrier Dans Fond Gaoza Belle Plaine Jacquot mean diversity

0.501 0.499 0.442 0.571 0.355 0.516 0.382 0.457 0.303 0.447 6 0.085

Table 1. Frequencies of polymorphic RAPD markers in the B. glabrata populations sampled in 1992 and in 1994. Samples

RAPD markers

1992

G3.1

G3.2

G6.1

G6.2

G15.1

G15.2

G19.1

B7.1

B7.2

B8.1

B8.2

Pico Dubelloy Sauvia Blain Geffrier Dans Fond Gaoza Belle Plaine Jacquot

0.41 0.65 0.35 0.59 0.94 0.71 0.76 0.65 1

0.94 0.82 1 0.71 0.47 0.71 0.88 0.71 0.24

0.59 0.59 0.59 0.59 0 0.53 0.41 0.41 0

0.29 0.06 0.41 0.47 0 0.29 0 0 0

0.47 0.12 0.12 0.06 0.06 0.53 0.47 0.35 0.65

0.88 0.88 0.35 0.24 1 0.59 0.82 1 0.94

0.12 0.18 0.76 0.35 0.53 0.12 0.06 0.18 0

0.47 0.47 0.18 0.41 0.82 0.18 0.24 0.29 0.24

1 0.76 0.88 0.76 0.88 0.71 0.18 0.41 0.47

0.76 0.12 0.18 0.29 0.24 0 0 0 0

0.59 0.59 1 0.88 0.53 0.12 0.94 0.53 0.41

1994

G3.1

G3.2

G6.1

G6.2

G15.1a

G15.2a

G19.1

B7.1

B7.2

B8.1

B8.2

Pico Dubelloy Sauvia Blain Geffrier Dans Fond Gaoza Belle Plaine Jacquot

0.41 0.82 0.47 0.59 0.76 0.65 0.59 1 1

0.94 0.71 0.71 0.76 0.35 0.82 0.59 0.18 0.24

0.94 0.18 0.18 0.65 0.53 0.82 0.24 0.18 0.65

0.06 0.06 0.71 0.18 0.06 0.35 0 0 0

0.22 (9) 0.33 (9) 0 (7) 0.25 (8) 0.18 (11) 0.78 (9) 0.44 (9) 0.14 (7) 0 (11)

0.88 (9) 0.75 (8) 0.29 (7) 0.29 (7) 0.90 (10) 0.33 (9) 0.89 (9) 1 (7) 1 (8)

0.06 0.24 0.41 0.18 0.53 0.06 0.06 0 0.12

0.53 0.71 0.29 0.47 0.82 0.35 0 0.71 0.29

0.71 0.76 0.65 0.88 0.88 0.65 0.18 0.41 0.35

0.82 0.12 0 0.06 0.29 0 0 0 0

0.41 0.59 0.82 0.82 0.71 0.47 1 0.35 0.71

a

The number in brackets indicates the number of individuals for which we got a result for the markers using the primer OP-G15 in 1994 (in the other cases, this number is always 17).


to the nine populations sampled in 1992 (Fig. 2). Series of 100 permutations indicated that there was a significant differentiation between the nine populations (P , 0.005) and that significant differentiation was observed as far as the fourth axis (at 5%). The four first axes of the canonical analysis described 11.5%, 5.3%, 4.2% and 2.4% of the total inertia, and 44.6%, 20.1%, 16.6%, 9.2% of the canonical inertia respectively. Populations were responsible for 25.9% of the total variance. the first axis of the canonical analysis separated the northern populations (PIC, DUB, SAU, BLA, GEF) from the southern ones (DFO, GAO, BLP, JAC). The map of the sampled localities shows a gap in the swampy forest (Fig. 1), at the border of which B. glabrata could be found, between Geffrier and Dans Fond localities. This discontinuity of the snail habitat seems sufficient to reduce the gene flow between the northern and southern populations and could be responsible for the observed differentiation at the first axis of the canonical analysis. G6.2, G15.1 and B8.2 markers are the most important on the first axis. The second axis isolated the Dans Fond population and especially that of Geffrier. B7.1, G6.2 and G15.1 markers were the most important on the

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second axis. The third axis clearly separated the population of Pico from the others. This differentiation was due to the marker B8.1. This marker was present in 76% of the Pico population but only in 1% of the other populations. Results observed on the fourth axis remain unclear. Taking into account the results of the previous canonical analysis, another analysis was carried out to check the separation between northern and southern groups. Eight canonical analyses were carried out in order to test the hypothesis of a gene flow interruption between populations along the swampy forest. Analyses were conducted at different levels: (i) between the northernmost population and the others, (ii) between the first two northernmost populations and the others, etc. The maximum part of the variance, 8.5% was observed when the separation is placed between Geffrier and Dans Fond. The northern and southern groups, as previously detected by the canonical analysis with populations are then confirmed. However, the populations being differentiated, almost any grouping of populations should show significantly differentiated groups. We then carried out permutations of populations divided into

Figure 2. Projection of nine Biomphalaria glabrata populations on the first two axes of the canonical analysis. The canonical analysis was carried out separating the individuals in nine groups corresponding to the nine populations sampled in 1992. The centroids of the populations were represented on the first two axes. the ellipses represent the confidence intervals at 95% of the centroids of the nine populations. They have been drawn dividing the radius of the ellipses containing 95% of the individuals of a same population (obtained with IDPlot II 1. Od4) by the root of the number of individuals in the population considered.

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two groups. The 255 possible combinations from nine populations were tested. The percentage of mean variance explained by these combinations was 3.5%. Only three combinations out of 255 (1.2%) displayed a greater inter-group variance. This rate is lower than the threshold of 5%, strengthening the validity of the northern and southern groups. Consequently, the variance actually due to the differentiation in two groups could be estimated to 5%. The B8.1, G15.1, G19.1 and G6.2 markers discriminated most these two groups. The Shannon index was used to analyse the distribution of diversity within and between populations and the diversity between the northern (Pico, Dubelloy, Sauvia, Blain and Geffrier) and southern groups (Dans Fond, Gaoza, Belle Plaine and Jacquot). Results showed that 75.3% of diversity was distributed within populations, 24.7% between populations, and 8.5% between the northern and southern groups. A significant correlation (Mantel t-test) was

observed between geographic and Euclidean distances using frequencies of RAPD markers (P 5 0.002).

Interannual results The mean intrapopulational diversity did not change significantly between 1992 and 1994, even if the diversity of some populations could vary. The genetic diversity of a population in 1992 had no significant influence on its diversity in 1994 (P 5 0.19; ANOVA). The evolution of the intrapopulational diversity between 1992 and 1994 was not linked to the geographic distance between populations (P 5 0.96; ANOVA), suggesting independent dynamics even for neighbouring populations. As shown by a partition of the variance of pooled data between 1992 and 1994, the temporal factor was responsible for 7.1% of the diversity. The most influential markers in this temporal diversity were G6.2, B8.1 and G19.1.

Figure 3. Projection on the first two axes of canonical analysis of the nine populations sampled in 1992 and 1994. The ellipses represent the confidence intervals at 95% of the centroids of the 18 samples.


They also made important contribution to the diversity between groups. A canonical analysis was carried out using 18 groups including nine populations sampled in 1992 and 1994. A series of 100 permutations showed a significant differentiation between the 18 populations (P , 0.01) and also between the first axis at 5%. The two first axes represented 32.8% and 25.8% of the canonical variance respectively. The first axis still separated northern populations (PIC, DUB, SAU, BLA, GEF) from southern ones (DFO, GAO, BLP, JAC) (Fig. 3). Centroids of the two samples from the same site were generally close indicating a rather stable system. A canonical analysis showed a significant differentiation between the northern and southern groups (P , 0.01). Considering these two groups, only one axis represented 6.1% of the total variance. The most discriminating markers between the northern and southern groups were B8.1, G19.1 and G3.1. A series of 100 permutations showed that the canonical analysis did not allow discrimination between the 1992 and 1994 samples (P . 0.15). However, a significant differentiation between the 1992 and 1994 samples was observed for Belle Plaine at 1%, Sauvia at 5% and Jacquot at 5%. DISCUSSION Using isoenzymatic markers, Malek & File (1971) showed that B. glabrata was very variable in comparison with other congeneric species. This was later confirmed by Vidigal, Neto, Carvalho & Simpson (1994) for Brazilian B. glabrata using RAPD markers. These authors reported that less than 10% of the markers were shared by all the specimens studied. This variability was mostly observed between populations. The percentage of bands shared by the individuals from a population varied from 91.8% to 96.4%. The insular populations are generally characterised by a low genetic diversity. The genetic polymorphism within B. glabrata populations from four islands from the West Indies, including Guadeloupe, is lower than that detected on the American continent using isoenzymatic markers (Mulvey, Newman & Woodruff, 1988). Using RAPD markers, our data on B. glabrata populations from Guadeloupe confirmed this result since the use of 32 primers (i.e. more than 400 markers) resulted in only six (JAC) to eleven (DUB) polymorphic markers within a population.

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Mulvey & Vrijenhoek (1982) using isoenzymatic markers, have already reported a genetic micro-structure of the B. glabrata populations from Puerto Rico sampled on approximately 1200 km2. Recently, Hoffman, Webster, Ndamba & Woolhouse (1998) using RAPD markers, revealed extensive genetic variation in adjacent populations of B. pfeifferi along a 6 km stretch of a Zimbabwean river. Our study with RAPD markers has revealed a clear differentiation between nine populations from a restricted area smaller than 45 km2. Additionally, a significant and smaller differentiation between a northern and southern group was also observed. A gap within the snail’s habitat probably explains this pattern. These structures were maintained between the 1992 and 1994 samplings. A Mantel t-test has indicated a significant correlation between genetic differentiation and the geographical distance, suggesting that the dispersal of snails decreases with distance. RAPD markers associated to canonical analyses have proved powerful tools to solve some questions about population structure. The differentiation of snail populations which act as intermediate hosts for parasites could have an effect on the host-parasite relationships. Several studies have shown a greater compatibility in snail-schistosome sympatric combinations than in allopatric combinations demonstrating a local adaptation of parasites (Vera, Jourdane, Sellin & Combes, 1990; Manning, Woolhouse & Ndamba, 1995). In such a case, structure of snail populations could be an unfavourable factor for parasite migration, leading to a structure of parasite populations (Mulvey & Vrijenhoek, 1982). In this study, we demonstrated the structure of B. glabrata populations in the insular focus of Guadeloupe. This allows us to test in the future its consequences on the local adaptation and the genetic structure of S. mansoni at a very small geographic scale.

ACKNOWLEDGEMENTS We must thank P. Legendre and J. D. Lebreton for advice on multivariate analyses and canonical analyses respectively. We are also indebted to F. Renaud, T. de Meus and Y. Michalakis for valuable comments. This work received financial support from the CNRS (Sciences de la Vie, Programme Biodiversité) and the Ministère Français de l’Environnement (Comité EGPN) and the Ministère de la Recherche (PRFMMIP).

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