Live metacercariae of Fasciola hepatica are often used in biomedical research to study different aspects of immunology, physiology, and pathology in experimentally infected definitive hosts, or to determine the efficacy and safety of new molecules as anthelminthics in the treatment of fasciolosis in mammals. The cost of commercial production of these larvae needs to be taken into account to propose the lowest sale price possible to researchers and/or institutions. As the cost is closely dependent on the choice of a food source for breeding the intermediate hosts of F. hepatica (Rondelaud et al. 2002), it is useful to reduce it by the selection of the best method to feed snails. Several sources of high-quality food have already been proposed by different authors (Kendall 1949, 1965; Boray 1969; Lee et al. 1994; Rondelaud et al. 2004) for Galba truncatula, as this amphibious snail is the most used lymnaeid in European countries for experimental infections with F. hepatica. However, the use of these food sources often requires the presence of a technician, thus explaining the high cost of metacercariae (Rondelaud et al. 2002).

Romaine lettuce has been used for 25 years by our lab to feed G. truncatula (Abrous et al. 1998). As the cost of metacercariae obtained from these lettuce-reared snails was high (Rondelaud et al. 2002), the focus of our team is to reduce it by simplifying the breeding method for G. truncatula. The first step of this research is to obtain the best growth possible for these last snails because the mean shell height of experimentally infected G. truncatula was often significantly lower than that of unexposed controls (Abrous et al. 1998). As the growth of French G. truncatula living in communities with Omphiscola glabra was similar to that of G. truncatula forming monospecific populations (Rondelaud, unpublished data), two experiments were carried out under laboratory conditions to determine if the presence of several O. glabra in a breeding of infected G. truncatula had an indirect effect on the shell height of these last snails.

Table 1 gives the geographic origin of lymnaeids, the composition of the ten groups, the number of snails per group at the beginning of the experiments, and the presence or the absence of a snail infection with F. hepatica. The first experiment (A) was performed with snails originating from the same community, whereas the second (B) was made using snails from monospecific populations. The choice of four O. glabra for 46 G. truncatula was based on the results of a preliminary experiment, demonstrating that four O. glabra were sufficient to have the best growth for infected G. truncatula. All snails were collected from their habitats in November or in April, and measured 4 mm in height at the beginning of the experiments. Routine bimiracidial exposures were performed in four groups of G. truncatula, whereas the other groups of this last species and O. glabra were unexposed. All snail groups were raised during the first 30 days using the method by Abrous et al. (1998). Food was only constituted by leaves of cos lettuce, faded after their stay under wet conditions at 4–6°C. Some dead leaves of Glyceria fluitans (Poaceae) were also added in the boxes containing O. glabra, as this snail often fed on this grass. The breeding boxes were maintained at 20°C, under a natural photoperiod of 12 h. At day 30 post-exposure (PE), the surviving G. truncatula from infected groups were individually placed at 20°C in 35-mm-diameter Petri dishes, with 2–3 ml of spring water and a piece of faded-lettuce leaf per recipient. A routine daily surveillance was performed to change the water and to count metacercariae before their removal from dishes. The other snails were only counted at day 30. By contrast, all surviving snails were measured at day 45 under a stereomicroscope coupled with an image-processor (Aries, Châtillon, France) using the Esilab software.

Table 1 The characteristics of the ten groups of snails used for the two experiments
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The parameters studied were snail survival at day 30 of the experiment, the prevalence of F. hepatica infections (calculated using the ratio between the number of cercariae-shedding snails and that of surviving snails at day 30), the shell height at day 45, the date of the first cercarial shedding, the length of the patent period, and the number of metacercariae recorded for each group containing infected snails. A χ 2 test and one-way analysis of variance (Stat Itcf 1988) were used to determine levels of significance.

In experiment A, the survival rate of G. truncatula at day 30 of the experiment (Table 2) was significantly greater in unexposed controls than in exposed snails (Berneuil: χ 2=5.31, P<0.05; Saint Jouvent: χ 2=33.14, P<0.001). A similar finding was also noted in the B experiment (Saint Jouvent: χ 2=10.73, P<0.01 in the case of G. truncatula raised with O. glabra, and χ 2=26.08, P<0.001 for alone raised snails). Comparison of exposed G. truncatula demonstrated significantly greater survivals when snails are raised with O. glabra (experiment A: χ 2=16.83, P<0.001; experiment B: χ 2=7.99, P<0.01). By contrast, for each category of exposed snails separately considered (snails living with O. glabra, or alone raised), no significant differences between the rates recorded in both experiments were found. The prevalence of snail infections with F. hepatica was significantly greater when G. truncatula was bred together with O. glabra (experiment A: χ 2=15.67, P<0.001; experiment B: χ 2=31.60, P<0.001). Lastly, the shell height of cercariae-shedding snails at day 45 post-exposure did not significantly differ from that of unexposed controls, whereas the shell heights of snails living with O. glabra were significantly greater (experiment A: F=4.06, P<0.05; experiment B: F=4.57, P<0.05) than those raised alone.

Table 2 Several characteristics in the ten groups of snails in relation to the experiment and the lymnaeid species
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Table 3 gives the values of the other three parameters. The date of the first cercarial shedding and the length of the patent period did not show any significant variations, whatever mode of comparison. Even if the number of metacercariae recorded in the case of snails living with O. glabra was slightly higher, no significant difference was found.

Table 3 Mean values (SD) for three parameters characterizing snail infections with F. hepatica in four groups of G. truncatula
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When the two lymnaeids were bred together during the first 30 days of experiment, greater snail survivals, higher prevalences of F. hepatica infections and bigger shells for cercariae-shedding snails were noted for G. truncatula. Moreover, the origin of O. glabra (living in a snail community, or monospecific population) had no significant effect on the characteristics of snail infections in G. truncatula. These results indicated that the presence of several unexposed O. glabra in each box of G. truncatula had indirect effects on three characteristics of snail infections, and one may wonder about the causes of such changes for G. truncatula. In our opinion, the main reason would be of dietary nature. As O. glabra fed more willingly on lettuce than G. truncatula, the former species would facilitate the dietary pattern of the latter snail by damaging and eroding lettuce, so that G. truncatula might more easily eat the pieces of these leaves. An argument supporting this approach is the presence of numerous G. truncatula on lettuce in the boxes containing O. glabra as the first days of the experiment, whereas a few snails were only observed on lettuce when G. truncatula is raised without O. glabra.

Even if the variations in the mean numbers of metacercariae are insignificant, the greater survival of G. truncatula at day 30 and the higher number of cercariae-shedding snails in the two groups living with O. glabra allowed to obtain a higher total number of larvae than in groups of G. truncatula raised alone (a total of 8,409 and 8,671 metacercariae in the experiments A and B, respectively, instead of 3,271 and 3,043 larvae). The cercarial production of F. hepatica metacercariae was so highly increased in the case of mixed breeding of snails. However, these results can only be obtained with a 30-day cohabitation of two lymnaeids measuring 4 mm in height at the beginning of the experiment and the subsequent isolation of G. truncatula from the other species. Indeed, beyond day 30 at 20°C, there was a concurrence between the two lymnaeids in breeding boxes, as O. glabra, measuring 11–12 mm at day 30 PE, forced the other species to emerge from water and to take refuge on box walls, thus, entailing the death of most G. truncatula.