Collection of snails and adult worms of Fascioloides magna

The first South American population (strain U) of snails has been raised at CEDIVE since 1996 and originated from Paysandú, Uruguay (36°00′ S, 57°30′ W). The other population (strain SP) has been bred in the laboratory since 2008 and came from San Pedro, Buenos Aires, Argentina (33°40′ S, 59°39′ W). Two French populations of G. truncatula were also used. The first originated from a road ditch at Chézeau Chrétien, commune of Chitray, department of Indre (46°40′27″ N, 1°21′21″ E) and was used for molecular biology. The other population of G. truncatula was collected from a road ditch (45°55′33″ N, 2°2′33″ E) in the commune of Saint Michel de Veisse, department of Creuse, and was used for experimental infections. The habitat of this last population was located on siliceous soil so that the upper shell height of adults (8–9 mm) ranged in the same scale of values as that of South American adults. Snails of height 4 mm were collected from each population.

Eggs of F. magna were collected from adult flukes recovered from the livers of naturally infected red deer (Cervus elaphus) hunted in the district of Krivoklátsko (Central Bohemia, Czech Republic). Prior to hatching, the eggs were washed several times with spring water and were incubated for 12 days at 24°C in the dark (Ollerenshaw, Reference Ollerenshaw1971).

Molecular identification of South American lymnaeids by polymerase chain reaction–restriction fragment length polymorphism and sequencing

Five snails of each population (SP, U and G. truncatula) were used for molecular taxonomical determination. The entire foot of each fresh specimen was sectioned with a scalpel blade. Foot samples were processed using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). Lysis using proteinase K was performed overnight. Once extracted, the internal transcribed spacer (ITS)-1 segment of their nuclear ribosomal DNA was amplified using the following primers: ETTS2 (5′-TAACAAGGTTTCCGTAGGTGAA-3′) and ITS-1r (5′-CGAGCGAGTGATCCACCGC-3′), for forward and reverse senses, respectively (Carvalho et al., Reference Carvalho, Cardoso, Lira, Rumi, Roche, Berne, Müller and Caldeira2004).

The polymerase chain reaction (PCR) mix was prepared to have a final volume of 25 μl, containing 12.5 μl of Qiagen Multiplex, 1 μl of each primer, 5 μl of genomic rDNA and 5.5 μl of DNAse and RNAse-free water. The PCR reaction was performed using a Perkin-Elmer Thermo Cycler (Applied Biosystems, Warrington, Cheshire, UK) as follows: initial denaturation (95°C, 3 min); 35 cycles, each comprising denaturation (95°C, 45 s), annealing (55°C, 1 min) and extension (72°C, 2 min); final extension (72°C, 5 min).

Amplified products were digested with the endonuclease HpyF3I (DdeI) (Fermentas GmbH, St. Leon-Rot, Germany) which restricts the sites 5′…C ↓ TNAG…3′. This enzyme was chosen for the present study because it was previously reported as the most accurate for differentiating lymnaeid species (Carvalho et al., Reference Carvalho, Cardoso, Lira, Rumi, Roche, Berne, Müller and Caldeira2004). Incubation was performed at 37°C for 3.5 h into a final volume of 15 μl, containing 5 μl of DNA, 8.5 μl of DNAse and RNAse-free water, 1 μl of Tango buffer (Fermentas GmbH) and 0.5 μl of enzyme. All products were separated on 2% agarose gels using a 100 bp ladder and stained with ethidium bromide. They were visualized using a UV transilluminator coupled with the software GELSMART 7.0 (Clara Vision, Verrières le Buisson, France). Band size was measured with the online available software Gel Analyzer 2010a (http://www.gelanalyzer.com).

For each South American population (SP or U), two amplicons were chosen randomly and purified using a rapid PCR purification kit (Marligen Biosciences, Ijamsville, Maryland, USA) according to the manufacturer's instructions. Sequencing reactions were performed using the ABI Big Dye kits (Applied Biosystems) and the same forward and reverse primers as those employed in the PCR amplification of ITS-1. Both strands were sequenced utilizing the ABI Prism 3130xl genetic analyser (Applied Biosystems). The forward and reverse sequences obtained were aligned and analysed using the software FinchTV 1.4.0 (Geospiza Inc., Seattle, Washington, USA) and Clustal X 2.0.11 (EBI, Hinxton, Cambridgeshire, UK). Sequences were submitted to GenBank under the following accession numbers: JF960165 and JF960166 for population SP, and JF960167 and JF960168 for population U. A consensus sequence was then obtained for each population of South American snails and was compared to GenBank reported sequences using the online tool BLASTN (http://blast.ncbi.nlm.nih.gov).

Experimental infections of snails

Two experiments were carried out. In experiment A, the propensity of each snail species for F. magna infection was studied. One hundred and fifty snails, each measuring 4 mm in shell height, were randomly chosen from each population. Each snail was then exposed to two miracidia for 4 h at 20°C in 3.5 ml of spring water and then raised at the same temperature according to the method of Rondelaud et al. (Reference Rondelaud, Fousi, Vignoles, Moncef and Dreyfuss2007). Snails were finally raised in groups of ten individuals in 14-cm Petri dishes (volume of spring water, 60 ml) for 30 days. Food consisted of dried leaves of lettuce and dead leaves of grass (Molinia caerulea), while several stems of live Fontinalis sp. ensured oxygenation of the water layer. The dissolved calcium in spring water was 35 mg/l. The Petri dishes were placed in an air-conditioned room under the following conditions: a constant temperature of 20°C ( ± 1°C), natural photoperiod of 10 h light. At day 30 post-exposure (pe), each surviving snail was isolated in a 35-mm Petri dish with pieces of dead grass, lettuce and spring moss, and placed at 20°C. A daily surveillance was made, (1) to change spring water and food if necessary, and (2) to count the metacercariae in the dishes. When the first cercarial shedding occurred, the surviving snails were subjected to a thermal shock every 3 days by placing their Petri dishes at 10–13°C for 3 h (outdoors) to stimulate cercarial exit (this temperature was chosen according to our experience with cercarial shedding of snails infected with digeneans). At the death of each snail, the shell was measured using callipers.

Experiment B was carried out to determine cercarial production of F. magna in snails dissected at day 65 pe at 20°C. Each experimental group consisted of 100 snails. Snail exposure to miracidia and maintenance were similar to those of experiment A. At day 65, each surviving snail was dissected under a stereomicroscope to count free rediae, intraredial cercariae and free cercariae.

The two parameters calculated were snail survival at day 30 pe and the prevalence of F. magna infection (calculated in relation to the number of snails surviving at day 30 pe). Prevalence took into account the number of cercariae-shedding snails (CS snails) and the number of individuals containing cercariae but without shedding (NCS snails) in experiment A, and all snails carrying larval forms of F. magna in experiment B. For each parameter, the difference between the values recorded for the three snail groups was analysed using a χ² test.

In experiment A, the other parameters calculated were the growth of CS snails during the experiment, the length of the prepatent period (time of cercarial differentiation inside rediae), the length of the patent period (time during which cercariae exit from the snail), the life period after the last exit of cercariae and the total number of metacercariae. In experiment B, the number of free rediae, the quantity of free cercariae and the quantity of intraredial cercariae were considered. Individual values recorded for these last eight parameters were averaged and their standard deviations were calculated considering snail groups. One-way analysis of variance was used to establish levels of statistical significance calculated using the Statview 5.0 software (Deltasoft Meylan, France).