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Field investigations in 14 wild watercress beds located in the French region of Limousin, a known endemic area for distomatosis, were performed for three years to determine the distance that cercariae of Fasciola hepatica can reach in water before their encystment on the host plant. Each bed was located on the course of an open drainage furrow, while snails (Galba truncatula) lived upstream around the emergence of a source. Five plant species were collected in early April and examined to find metacercariae. Most cysts were noted on Nasturtium officinale (188 on 48.7 kg of dripped plants), followed by Helosciadium nodiflorum (125 on 33.4 kg). On the other plant species, there were few larvae. Most cercariae encysted on the plants growing in the most upstream part of each bed, usually on the first 50 cm in length. When water in the beds was fast running, the distribution of metacercariae was more limited and their number was fewer than those in the beds fed by a slow flow of water. Cercariae were able to swim or were carried away by the current up to a mean of 5 m in slow-flow waters before encysting; this distance was only 4 m in faster waters. Plants growing on the most upstream section of a watercress bed located in a drainage furrow are the most used by cercariae for their encystment, when snails live around the emergence of a source. The speed of the water current affected the number and distribution of metacercariae in the bed.Fasciola hepatica : la dispersion des cercaires émises par le mollusque Galba truncatula.Des investigations de terrain dans 14 cressonnières naturelles localisées dans la région française du Limousin, zone connue d’endémie de distomatose, ont été effectuées pendant trois années pour déterminer la distance que les cercaires de Fasciola hepatica peuvent atteindre dans l’eau avant leur enkystement sur la plante hôte. Chaque cressonnière était située sur le cours d’une rigole de drainage superficiel, tandis que les mollusques (Galba truncatula) vivaient plus en amont autour de l’émergence d’une source. Cinq espèces végétales ont été recueillies au début d’avril et examinées pour y trouver des métacercaires. La plupart des kystes ont été observés sur Nasturtium officinale (188 sur 48,7 kg de plantes égouttées), suivis par Helosciadium nodiflorum (125 sur 33,4 kg). Sur les autres espèces végétales, il y avait peu de larves. La plupart des cercaires étaient enkystées sur les plantes qui poussaient dans la partie la plus en amont de chaque cressonnière (habituellement sur les premiers 50 cm de longueur). Lorsque l’eau courante dans les cressonnières était rapide, la distribution des métacercaires était plus limitée et leur nombre était inférieur à celui observé dans les stations alimentées par un débit d’eau lent. Les cercaires ont été capables de nager ou ont été entrainées par le courant jusqu’à 5 m en moyenne dans une eau à écoulement lent avant leur enkystement ; cette distance n’était que de 4 m dans les eaux plus rapides. Les plantes qui poussent sur la section la plus en amont d’une cressonnière située dans une rigole de drainage sont les plus utilisées par les cercaires pour leur enkystement lorsque les mollusques vivent autour de l’émergence d’une source. La vitesse du courant d’eau affecte le nombre et la distribution des métacercaires dans la cressonnière.

The lymnaeid Omphiscola glabra is known to be an intermediate host in the life cycle of Fasciola hepatica and knowing its distribution in the field is important to determine that of the parasite. Looking at indicator plants has been proposed by several authors as one of the most practical ways of carrying out a malacological survey in pastures of a farm. Field investigations on 361 French farms breeding cattle or sheep on acid soils were thus carried out for 36 years in March or April to list indicator plant species in seven types of habitats colonized by O. glabra , and determine the characteristics of their habitats (area, snail density) for each indicator plant. Six plant species: Agrostis stolonifera , Dactylis glomerata , Glyceria fluitans , Juncus acutiflorus , Juncus effusus and Lotus uliginosus , were examined in 3137 snail habitats. J. effusus was the best indicator species to detect snail populations in five habitat types, while J. acutiflorus was the best indicator for snail populations living in spring heads. The other four plant species characterized only a few populations. Snail habitats detected with J. acutiflorus , J. effusus or L. uliginosus were significantly larger in area, while the density of overwintering snails was significantly lower in habitats with G. fluitans . These initial results suggest that J. effusus is a good indicator for the detection of O. glabra in the acid soils of central France. However, additional studies are required to test its limitations in the field.

Transplantations of Omphiscola glabra from granite to marl, or vice versa, between farms known for cases of fasciolosis have shown differences in the maximum shell height of adult snails. To determine whether these differences were due to parasitism, soil geology, or a combination of both factors, four snail populations living on marl or granite were investigated over 12 months to specify the characteristics of egg-laying in spring, and to follow the shell growth of both snail generations. In both types of habitats, the number of egg-masses per adult snail and their size significantly decreased during the 7 weeks of snail egg-laying. During this period, each adult snail on marl laid a mean of 6.4 masses and 93.3 eggs. On granite, the mean values were 5.7 masses and 69.1 eggs, respectively. Two annual generations, the first in spring and the other in autumn, were noted in these habitats. The maximum shell height of adults was different: a mean of 19.8 and 20.5 mm on marl compared with 17.2 and 18.2 mm on granite. Soil geology thus plays a role in the egg-laying and shell growth of O. glabra .

Experimental co-infections of juvenile and pre-adult Pseudosuccinea columella with Calicophoron daubneyi and Fasciola hepatica (five miracidia of each digenean per snail) were carried out to determine the aptitude of this lymnaeid to ensure complete larval development of the former parasite, the latter or both. Snails infected with F. hepatica were found in the two groups of juveniles, i.e. 1 and 2 mm at exposure, and the four groups of pre-adults, i.e. 3–6 mm. The highest frequency of F. hepatica, i.e. 37.3%, was noted in the 4 mm group. Low frequencies were noted for C. daubneyi and co-infections of both digeneans in the 3, 4 and 5 mm groups. Two other groups of P. columella, measuring 3 and 4 mm at exposure, were also constituted to study the characteristics of these co-infections. Compared to controls infected only with F. hepatica, the frequency of this digenean infection and the mean number of metacercariae were significantly lower in co-infected snails, while the patent period was significantly shorter. In snails harbouring C. daubneyi only or both digeneans, lower values were noted for prevalence, the patent period and the number of metacercariae. Pre-adult P. columella (3–5 mm in shell height at exposure) were able to sustain larval development of C. daubneyi if they were co-infected with the sequence C. daubneyi +F. hepatica. Low values noted for the prevalence of C. daubneyi infection and the number of metacercariae would be in favour of a still incomplete adaptation between the snail population and the miracidial isolate.

Juvenile Pseudosuccinea columella, measuring 1 or 2 mm in height, were subjected to single-miracidium infections with Fasciola hepatica to determine the developmental pattern of redial generations and count free and live rediae according to their generation. Controls were constituted of juvenile Galba truncatula infected according to the same protocol. In the four groups, redial counts were performed in snails dissected every week from day 7 to day 49 post-exposure at 20 °C. Most infected snails showed a normal development of redial generations, whatever the lymnaeid species. In P. columella, the total number of live rediae on day 49 was 24.6 and 34.6 per infected snail in the 1 and 2-mm groups, respectively (instead of 11.5 and 18.8 rediae in the corresponding groups of G. truncatula). A single mother redia (R1a) producing only daughter rediae of the second generation was noted in each snail of three groups, while the 2-mm P. columella showed the presence of a single (43 snails/71) or two (28/71) R1a redia(e) within their bodies. The mean number of other mother rediae and that of daughter rediae of the second generation were low in the 1 and 2-mm groups of both lymnaeids. Besides, there was a delay in redial development. The development of two live R1a rediae in several P. columella from the 2-mm group needs to verify if this process would be specific to P. columella or would occur in other lymnaeid species known for their good susceptibility to the digenean and their larger size to allow harbouring rediae.

Adult Galba truncatula ( ≥ 4 mm in shell height) were collected from 135 habitats for 3 years (2012–2014) to identify parasite species via the study of cercariae, and to determine the prevalence of each digenean infection in relation to the type of snail habitat (six types). A total of 323 infected snails and ten digenean species were noted in the bodies of 11,025 G. truncatula after their dissection. Snails with Calicophoron daubneyi and/or Fasciola hepatica were found in 20.7% and 12.5% of the habitats, respectively, and most of these infected snails were collected from rainwater-draining furrows and pools in meadows. The percentages were lower for snails with Echinostoma revolutum (9.6% of habitats) and Haplometra cylindracea (7.4%), and were less than 5% for those parasitized by any of the other five species of digenean. The highest prevalence of all digenean infections was noted in pools (9.4%), followed by furrows located in meadows (8.3%) and ponds (5.1%). The prevalence noted for each digenean infection varied with the type of habitat. In furrows located in meadows, the infection rate of C. daubneyi in snails (3.5%) was significantly higher than that of F. hepatica (2.2%). In pools, values greater than 1.5% were noted for C. daubneyi, H. cylindracea and Opistoglyphe ranae. In ponds, E. revolutum was the dominant species (prevalence, 2.5%). Parasite species richness in G. truncatula was greater in the Brenne Natural Regional Park than in the nearby region of Limousin (ten instead of eight). The distribution and prevalence of each parasite species were dependent on the type and location of each snail habitat.

Experimental infections of three EgyptianPseudosuccinea columellapopulations with sympatric miracidia ofFasciolasp., coming from cattle- or sheep-collected eggs, were carried out to determine the capacity of this lymnaeid to support larval development of the parasite. Using microsatellite markers, the isolates of Egyptian miracidia were identified asFasciola hepatica. Apart from being independent of snail origin, prevalences ranging from 60.4 to 75.5% in snails infected with five miracidia ofF. hepaticawere significantly higher than values of 30.4 to 42.2% in snails with bi-miracidial infections. The number of metacercariae ranged from 243 to 472 per cercarial-shedding snail and was independent of snail origin, parasite origin and miracidial dose used for infection. IfP. columellawas subjected to two successive bi-miracidial infections withF. hepatica, prevalence of infection was 63.3%, with a mean of 311 metacercariae per snail. These values were clearly greater than those already reported forRadix natalensisinfected with the same parasite and the same protocol. Successful experimental infection ofP. columellawithF. hepaticasuggests that this lymnaeid snail is an important intermediate host for the transmission of fascioliasis in Egypt.

Experimental infections of Lymnaea glabra (two populations) with Fasciola hepatica were carried out during seven successive snail generations, to determine if prevalence and intensity of snail infection increased over time through descendants of snails already infected with F. hepatica. Controls were descendants coming from uninfected parents and infected according to the same protocol. No larval forms were found in the bodies of control snails coming from uninfected parents. In contrast, prevalence and intensity of F. hepatica infection in snails originating from infected parents progressively increased from the F2 or F3 to the F6 generation of L. glabra. In another experiment carried out with the F7 generations of L. glabra and a single generation of Galba truncatula (as controls), the prevalence of F. hepatica infection and the total number of cercariae were lower in L. glabra (without significant differences between both populations). If the number of cercariae shed by infected snails was compared to overall cercarial production noted in snails containing cercariae but dying without emission, the percentage was greater in G. truncatula (69% instead of 52–54% in L. glabra). Even if most characteristics of F. hepatica infection were lower in L. glabra, prevalence and intensity of parasite infection increased with snail generation when tested snails came from infected parents. This mode of snail infection with F. hepatica suggests an explanation for cases of fasciolosis occurring in cattle-breeding farms where paramphistomosis is lacking and G. truncatula is absent.

Field investigations on the habitats colonized by Galba truncatula or Omphiscola glabra were carried out on 162 farms of the Limousin region, Central France, to determine whether there is currently a decline in the number and size of snail populations. Seven types of snail habitats were considered here. Compared to the numbers of snail populations recorded from 1976 to 1992, the values noted from 2013 to 2016 were significantly lower, with a decline rate of 34% for G. truncatula and 23% for O. glabra . Variations in this decline rate with the type of snail habitat were also noted. The greatest decreases in the numbers of snail populations were noted for spring heads located in meadows and for road ditches, while the lowest were noted for open drainage furrows present in meadows. The distribution of these habitats according to their area did not show any significant change over time. In contrast, overwintering snails were significantly less numerous in 2013–2016 in five types of habitats for G. truncatula and in three types only for O. glabra. Several causes underlie this population decline. Among them, the current development of mechanical cleaning in open drainage systems and road ditches, that of subsurface drainage in meadows, and regular gyro-crushing of vegetation around temporary spring heads were the most important.

A retrospective study on differentLymnaea glabrasamples collected from central France between 1993 and 2010 was carried out to determine the prevalence of natural co-infections withCalicophoron daubneyiandFasciola hepatica, and to specify the composition of redial burdens. Experimental infections ofL. glabraperformed during the same period of time were also analysed to study metacercarial production of each digenean in co-infected snails. Controls were naturally or experimentally co-infectedGalba truncatula. In natural co-infections, prevalence was 0.7% inL. glabra(186/25,128) and 0.4% inG. truncatula(137/31,345). Low redial burdens were found in these snails, withF. hepaticarediae significantly more numerous inL. glabrathan inG. truncatula(7.5 per snail instead of 5.2). In contrast, the total numbers ofC. daubneyirediae in both lymnaeids were close to each other (4.3 and 3.0 rediae, respectively). In experimentally co-infected groups, prevalence was greater inG. truncatulathan in the other lymnaeid (6.3% instead of 3.0%). Significantly shorter patent periods and lower metacercarial production for each digenean were noted inL. glabrathan inG. truncatula.However, in both lymnaeids, the two types of cercariae were released during the same shedding waves and several peaks during the patent period were synchronous. In spite of a greater shell height forL. glabra, metacercarial production of both digeneans in co-infected snails was lower than that inG. truncatula, thus indicating a still incomplete adaptation between these FrenchL. glabraand both parasites.

Experimental infections of two South American lymnaeids (Lymnaea neotropica and L. viatrix var. ventricosa) with Paramphistomum daubneyi were carried out to determine if these snail species could sustain larval development of this digenean and, if so, to specify their potential for cercarial production. A French population of Galba truncatula infected and raised according to the same protocol served as controls. In both experiments, prevalence of P. daubneyi infections in snails did not significantly differ from each other. In snail groups evaluated for cercarial shedding (first experiment), a significantly lower number of shed cercariae was noted for L. neotropica, while those from G. truncatula and L. v. ventricosa did not differ significantly from each other. Dissection of infected snails at day 65 post-exposure at 20°C (second experiment) found significantly lower burdens of P. daubneyi rediae and cercariae in the bodies of L. neotropica than in those of G. truncatula and L. v. ventricosa. Compared to total cercarial production observed in dissected snails, the percentage of cercariae which exited from snails was 75.6% for G. truncatula, 21.6% for L. neotropica, and 91.4% for L. v. ventricosa. This last species seems to be a good candidate for metacercarial production of P. daubneyi., Des infestations expérimentales de deux limnées sud-américaines (Lymnaea neotropica et L. viatrix var. ventricosa) avec Paramphistomum daubneyi ont été réalisées pour déterminer si ces espèces peuvent assurer le développement larvaire de ce Digène et, dans l’affirmative, pour préciser leur potentiel dans la production cercarienne. Les témoins sont constitués par une population française de Galba truncatula infestée et élevée selon le même protocole. Dans les deux expériences, les prévalences de l’infestation par P. daubneyi chez les mollusques sont proches les unes des autres. Chez les groupes suivis pour les émissions cercariennes (première expérience), le nombre de cercaires émises est significativement plus faible chez L. neotropica, tandis que les chiffres notés chez G. truncatula et L. v. ventricosa ne présentent pas de différence significative. Si les mollusques infestés sont disséqués au 65ème jour post-exposition à 20 °C (seconde expérience), les charges rédienne et cercarienne trouvées dans le corps des L. neotropica sont significativement plus faibles que celles notées chez G. truncatula et L. v. ventricosa. Par rapport à la production cercarienne totale chez les mollusques disséqués, le pourcentage de cercaires qui sont émises est de 75,6 % chez G. truncatula, 21,6 % chez L. neotropica et de 91,4 % chez L. v. ventricosa. Cette dernière espèce semble être un bon candidat pour la production métacercarienne de P. daubneyi., Centro de Diagnóstico e Investigaciones Veterinarias

Experimental infections of six riverbank populations of Galba truncatula with Fasciola hepatica were carried out to determine if the poor susceptibility of these populations to this digenean might be due to the scarcity or the absence of natural encounters between these snails and the parasite. The first three populations originated from banks frequented by cattle in the past (riverbank group) whereas the three others were living on islet banks without any known contact with local ruminants (islet group). After their exposure, all snails were placed in their natural habitats from the end of October up to their collection at the beginning of April. Compared to the riverbank group, snails, which died without cercarial shedding clearly predominated in the islet group, while the other infected snails were few in number. Most of these last snails released their cercariae during a single shedding wave. In islet snails dissected after their death, the redial and cercarial burdens were significantly lower than those noted in riverbank G. truncatula. Snails living on these islet banks are thus able to sustain larval development of F. hepatica. The modifications noted in the characteristics of snail infection suggest the existence of an incomplete adaptation between these G. truncatula and the parasite, probably due to the absence of natural contact between host and parasite.

Cross-transplantations of Omphiscola glabra between two French departments (Haute-Vienne and Indre) were carried out to study snail settlement in these new sites during six years and to determine if this snail species was well adapted to its local parasites (Fasciola hepatica and Paramphistomum daubneyi). Local snails placed in new sites for each department were used as controls. In Indre furrows, the number of adults per population and the area of each habitat peaked on the third year post-introduction. Similar findings were also noted in the Haute-Vienne furrows occupied by local snails but peaks were only observed on the fourth year. In contrast, in Haute-Vienne sites populated by introduced snails, there was a progressive decrease in snail abundance and habitat area from the second year. The local O. glabra transplanted in Haute-Vienne kept their same degree de susceptibility to F. hepatica and P. daubneyi, thus demonstrating a good adaptation of these snails to their parasites. In other groups of transplanted snails, the first natural infections were only noted from the second or third year post-introduction and their prevalence progressively increased over time. Local adaptation of these latter snails to parasites would be more progressive in time and would stretch over several snail generations.

Four geographical strains of Galba truncatula living on riverbanks (the first on a sedimentary soil and the other three on an acid soil) were subjected to bimiracidial exposures with Fasciola hepatica to study their aptitude for cercarial shedding and to count metacercariae in snails dissected at day 42 post-exposure. All snails were reared in 14-cm Petri dishes at 24°C, with the same spring water (60–73 mg/l of Ca2+) and the same diet (grass and lettuce leaves). Metacercariae of F. hepatica were noted in the four populations after a cercarial shedding or after snail dissection. However, in spite of the breeding method used, the characteristics of snail infections varied with the origin of each geographical strain. For example, the shell heights of infected snails at day 42 were close to those found for the corresponding adults in the field (6.8–8.0 mm for the population living on the sedimentary soil, but only 4.6–5.5 mm for another strain originating from the acid soil). This variability may be explained by assuming that the diet of these riverbank G. truncatula would be different from that of snails living in swampy meadows. However, another hypothesis based on the influence of snail habitat on the characteristics of snail life cannot be excluded.

Three-year investigations were carried out in three populations of Galba truncatula to study their dynamics throughout the year when there was a single annual generation for the snail. These communities were living in permanently watered habitats, on acid soil and in lowland zones. In the three cases, egg-laying occurred from the end of June to the beginning of October and was closely associated with the summer fall in level and speed of running water. The highest numbers of egg-masses were noted at the end of July and the beginning of August. Newborns which hatched from these egg-deposits showed a strong decrease of their number from July to October and this diminution continues up to next June (at 3.4 snails per m2 of habitat). Juvenile snails measuring ≤ 2 mm in height were observed up to the end of next March. Compared to single annual generations of G. truncatula studied by other authors in zones of higher elevation, the longer period of egg-laying must be related to the local climatic and hydrologic conditions which are more favourable in lowlands than in elevation. The strong decrease in the number of snails throughout the year and the presence of a few ≤ 2-mm high juveniles during winter months might be explained by the characteristics of running water in these permanent habitats. Indeed, the high level and the great speed of running water during most months of the year would have an effect upon the number and the growth of these snails by causing unfavourable conditions for their life.

Bimiracidial infections of Lymnaea palustris and Lymnaea stagnalis (shell height at exposure, 4 mm) with Fasciola hepatica were carried out during six successive snail generations to determine if prevalence and intensity of snail infection increased over time through descendants issuing from eggs laid by parents already exposed to this digenean. Controls were constituted by a French population of Galba truncatula (a single generation) infected according to the same protocol. In a first experiment performed with the F1 to F5 generations of L. palustris, the prevalence and intensity of F. hepatica infection in snails progressively increased. Immature rediae and a few cercariae-containing rediae of the digenean were observed in L. stagnalis from the F3 generation, but no free cercaria was noted in the bodies of this lymnaeid from the F4 to F6 generations. In another experiment carried out with the F6 generation of L. palustris, the prevalence of F. hepatica infection and the number of shed cercariae were significantly lower in L. palustris than in G. truncatula. This mode of snail infection suggests an explanation for cases of human fasciolosis occurring in central France after the collection of wild watercress from beds where L. palustris was the sole lymnaeid.

Experimental infections of Galba sp. (origin, Colombia) with allopatric isolates of Fasciola hepatica from France or Fascioloides magna from the Czech Republic were carried out during five successive snail generations to determine if this lymnaeid might sustain complete larval development of either parasite. In snails exposed to F. hepatica, 7 of 400 snails harboured several rediae and only two snails contained a small number of free cercariae on day 50 post-exposure. In contrast, the intensity of F. magna infection in Galba sp. progressively increased from the F1 to F5 generations. Spontaneous cercarial shedding of F. magna occurred in 7 of 100 Galba sp. belonging to the F5 generation and the number of shed cercariae did not differ significantly from that noted in control Galba truncatula of French origin. Galba sp. from Colombia can be added to the list of potential intermediate hosts for F. magna.

Individual infections of Egyptian and French Pseudosuccinea columella with five miracidia of Calicoph- oron daubneyi were carried out to determine whether this lymnaeid was capable of sustaining larval development of this parasite. On day 42 post-exposure (at 23 C), infected snails were only noted in groups of individuals measuring 1 or 2 mm in height at miracidial exposure. Snail survival in the 2-mm groups was significantly higher than that noted in the 1-mm snails, whatever the geographic origin of snail population. In contrast, prevalence of C. daubneyi infec- tion was significantly greater in the 1-mm groups (15-20% versus 3.4-4.0% in the 2-mm snails). Low values were noted for the mean shell growth of infected snails at their death (3.1-4.0 mm) and the mean number of cercariae (

Lymnaea glabra is known to be a natural intermediate host of two flukes, Calicophoron daubneyi and Fasciola hepatica, in central France. But it can also sustain larval development of other digeneans. Adult snails were thus collected from 206 habitats in 2014 and 2015 to identify parasite species and determine the prevalence of each digenean infection in relation to the five types of snail habitats. Seven digenean species were noted in 321 infected snails (out of 17,647 L. glabra). Snails with F. hepatica or C. daubneyi were found in 14.5% and 12.6% of habitats, respectively. Percentages were lower for snails with Opisthoglyphe ranae (5.8%), Haplometra cylindracea (5.3%) and were less than 5% for those infected with Echinostoma revolutum, Notocotylus sp. or Plagiorchis sp. Prevalence noted for each parasite species varied with the type of habitat. The number of species in L. glabra was lower than that found in G. truncatula from the same region (7 instead of 10). The distribution and prevalence of each digenean species were thus dependent on the type and location of each snail habitat.

International audience; A retrospective study of the experiments performed during the past 15 years on infections of Galba truncatula with Fasciola hepatica was carried out to determine what susceptible populations of snails might be used for the commercial production of metacercariae, and to examine this metacercarial production in relation to the characteristics of snail infections. Of the four groups of snail populations studied, the ablest snails to sustain a complete larval development of F. hepatica originated from populations living on siliceous soils at 600 m and more in altitude. In contrast, snail populations living along river banks on siliceous soils were inappropriate due to the poor characteristics of snail infections (high snail mortality, low prevalence of snail infections, and low number of cercariae produced). Except for these last populations, 86-87% of cercaria-shedding (CS) snails in the other populations shed less than 300 cercariae, even if a maximum of 1,772 cercariae were obtained from a single snail. The date of the first cercarial shedding at 20 degrees C began during week 7 or 8 PE for 80.1-83.5% of CS snails. Most metacercariae (82.0-85.9% of the total production) were recorded during the first 10 days of the patent period. In these conditions, the authors collected metacercarial production up to the beginning of week 10 PE (20 degrees C) and did not use snails that shed their cercariae during the following weeks due to too low numbers of parasites. This method allows to have a continuous production of metacercariae over time by using successive groups of infected snails, each being separated from the other by a fortnight's time.

Bimiracidial infections ofLymnaea truncatulawith three isolates ofFasciola gigantica, originating from China, Egypt and Madagascar, were carried out to determine the effect of geographic origin of the parasite on the larval productivity of redial generations. The prevalences of experimental infections in snails exposed to strains from Madagascar, China and Egypt were 20.8%, 60.0% and 80.0%, respectively. At day 49 post-exposure (p.e.), the total number of free rediae in snails infected with the Egyptian isolate was significantly higher than that recorded in the Madagascan group. On the other hand, at day 49 p.e., the majority of cercariae in the Chinese and Egyptian groups were produced by R2a rediae (70.6% and 66.6% of cercariae produced by all live rediae), while, in the Madagascan group, the cercariae were produced mainly by the first redial generation. Snails infected with the Egyptian isolate of miracidia developed more live rediae and, consequently, could produce a higher number of cercariae. As a result,L. truncatulasnails were highly adapted to infections with the Egyptian and Chinese isolates ofF. gigantica.

Experimental infections of 1-mm high snails using three populations of Lymnaea (L. glabra, L. ovata and L. truncatula) and a cattle strain of Fasciola hepatica miracidia were carried out under laboratory conditions to determine if the snail species had an effect on the number of free rediae, their growth, and cercarial productivity in relation to each redial category (R1a, R1b, R2a, or R2b/R3a). The total number of rediae ranged from 6.4 to 7.5 per snail. The mean body length of rediae varied from 1–1.2 mm (R1a) to 0.3–0.4 mm (R2b/R3a). The width of the intrapharyngeal lumen also varied from 26.0–38.8 μm to 3.0–4.2 μm, respectively. The redial category had a significant effect on both measurements, whereas snail species only had a significant influence on body length. The mean number of cercariae produced by all living rediae at day 49 post-exposure ranged from 63.0 in L. glabra to 87.2 in L. truncatula. In L. ovata and L. truncatula, 55.8% and 58.6% of cercariae, respectively, were produced by R2a rediae, whereas 53.9% of cercariae in L. glabra were formed by the R1b rediae. When young snails were infected with F. hepatica, the species of snail had an effect on the number of living rediae, their length and their cercarial productivity.

Experimental infections of Lymnaea truncatula with Fasciola hepatica were performed to study the consequences of the presence of predators (sciomyzid larvae or zonitid snails) on the characteristics of larval F. hepatica development in surviving snails. Controls consisted of infected snails that were not subjected to predators. Compared to controls, the survival rate at day 30 post-exposure, the duration of cercarial shedding, and the number of cercariae shed by surviving snails were significantly lower when predators were present in snail breeding boxes, whatever the type of predator used. In contrast, the prevalences of Fasciola infections in snails, and the length of time between exposure and the onset of cercarial shedding showed no significant variation. The progressive development of a stress reaction in surviving snails against predators during the first 30 days of experimental exposure to F. hepatica would influence snail survival during the cercarial shedding period and, consequently, the number of cercariae shed by the snails.

Bimiracidial infections of French Lymnaea truncatula with a Madagascan isolate of Fasciola gigantica were carried out under laboratory conditions to study the growth of rediae and their larval productivity in relation to the different redial generations. The total numbers of rediae and their mean lengths significantly increased with the duration of infection until day 49 post-exposure (p.e.). Significant differences in the lengths between the different redial generations were noted. At day 49 p.e. (at 20 degrees C), the cercariae were produced by the first redial generation, while the productivity of other redial groups was delayed. This last finding shows a slow larval development of this Madagascan isolate of F. gigantica in this French population of L. truncatula.

Laboratory investigations on Galba truncatula experimentally infected with Calicophoron daubneyi were carried out to study the influence of natural light and sky nebulosity on cercarial emergence in snails subjected every week outdoors to a thermal shock (a mean of 12 °C for 3 h) during the patent period. The same study was also performed in G. truncatula naturally infected with Haplometra cylindracea according to the same protocol. Compared to infected controls always reared indoors at 20 °C, the number of cercariae emerging from snails subjected outdoors to a thermal shock was significantly greater, whatever parasite species. Natural light had an effect on snails releasing C. daubneyi cercariae after the thermal shock because their numbers were significantly higher between 601 and 1,200 lx and for the highest nebulosity values (7–8 octas). In contrast, the type of light used during thermal shock did not influence cercarial emergence of H. cylindracea because the numbers of cercariae per shedding snail noted under natural light or a 3,000-lx artificial light did not significantly differ from each other. Most snails releasing H. cylindracea cercariae were significantly more numerous for light levels higher than 3,000 lx. As for Fasciola hepatica cercariae, natural light had a significant influence on cercarial emergence of C. daubneyi, and this behavioural particularity for cercariae of both digeneans might be due to the fact that both species develop in the same ruminants and the same snail host.

Experimental infections of Lymaea truncatula, using two susceptible snail populations (Berneuil, or Migné, central France) and four isolates of Fasciola hepatica miracidia differing in their mammalian host of origin (cattle, nutrias, rabbits, or sheep), were performed under laboratory conditions to determine whether the host of origin had an effect on the daily production of cercariae. Snails were each subjected to bimiracidial exposures and were then reared under semi-natural conditions (a constant temperature of 20 degrees C and natural photoperiod). Significantly lower values were noted in the rabbit groups for survival rates at day 30 post-exposure, as well as for prevalences of infection, snail growth. duration of shedding period, and the total numbers of cercariae these snails shed. The total number of cercariae shed by both nutria groups was significantly higher than those recorded in the six other infected groups. In the cattle, rabbit, and sheep (Berneuil only) groups, the peaks in the daily distribution of cercariae occurred between day 2 and day 4 after the first shedding, and the number of cercaria-shedding snails decreased with increasing number of shedding waves. In contrast, in the three other groups, the peaks were only observed between days 20 and 45. Snails shedding their cercariae during nine or more waves were numerous in these last groups. No infradian-type rhythm in the daily distribution of cercarial numbers over the shedding period was noted for any snail group. The highest production of F. hepatica cercariae in both nutria groups would be a consequence of a higher success rate of miracidia when they infected an allopatric population of snails. The absence of an infradian-type rhythm in the distribution of daily cercarial numbers in the eight groups suggests that this rhythm, if it occurs, would only be influenced by temperature and thus be limited to periods with optimal conditions for cercarial shedding.

Experimental infections of two South American lymnaeid populations with Fascioloides magna were carried out to determine whether these snails may sustain larval development of this digenean and, if so, to quantify their potential for cercarial production. The reference group was a French population of Galba truncatula infected and raised according to the same protocol. According to the internal transcribed sequence (ITS)-1 segment of their genomic rDNA, these South American populations were identified as Lymnaea neotropica (origin, Argentina) and Lymnaea viatrix var. ventricosa (origin, Uruguay). In the snail groups followed for cercarial shedding, longer prepatent periods and lower numbers of shed cercariae were noted in South American lymnaeids. In other snails dissected at day 65 post-exposure, the redial and cercarial burdens of F. magna found in the bodies of L. neotropica and L. v. ventricosa were significantly lower than those noted in G. truncatula. Compared to the total cercarial production noted in the dissected snails, the percentage of cercariae that exited from snails was 51.3% for G. truncatula, 32.2% for L. neotropica and 46.8% for L. v. ventricosa. The two South American species of snails can thus be considered as potential intermediate hosts of F. magna.

Bimiracidial infections of Lymnaea ovata with Fasciola hepatica were performed under laboratory conditions to determine the susceptibility of snails from six French populations to trematode infection. In five populations of L. ovata the prevalence of infection in the 1-mm groups ranged between 2.7% and 43.7% at day 35 postexposure; it decreased in the 2-mm snails and was zero in larger groups. In the snails from Thenay (periodically polluted brook) the prevalence of F. hepatica infection decreased from the 1-mm group to the 8-mm group (from 23.9% to 1.0%) and was zero in the 10-mm L. ovata. The total number of cercariae shed per snail was 18.3 in the 1-mm group, increasing to 117 in the 8-mm group. The latter findings could be interpreted as a consequence of periodic pollution in the brook of Thenay; pollution might disrupt the defense system of L. ovata and facilitate the subsequent larval development of F. hepatica.

International audience; Allopatric infections of French Galba truncatula with an Argentinean isolate of Fasciola hepatica were carried out to determine the infectivity of foreign miracidia in three snail populations differing by their susceptibility to French miracidia (two highly and one poorly susceptible populations). Sympatric infections of G. truncatula with French miracidia were used as controls. Compared to sympatric infections of G. truncatula, snail survival at day 30 post-infection in allopatric groups was significantly lower in a highly susceptible population and significantly greater in the other two. Prevalence in snails infected with the allopatric isolate was significantly lower (16.4-34.5 % instead of 58.6-72.1 %), whereas their patent period was significantly longer (a mean of 69.9-85.9 days instead of 6.4-20.7 days). The mean number of metacercariae was also higher in allopatric groups (236.5-897.3 per cercariae-shedding snail instead of 70.7-222.1). Owing to longer patent periods, the Argentinean isolate of F. hepatica was less pathogenic for these snails. The lower prevalence of infection, the longer patent period and the higher number of metacercariae noted in allopatric groups might be the consequence of an adaptive mechanism used by this digenean introduced to the New World to infect new populations of unusual intermediate hosts.

A total of 850 pre-adult Galba truncatula (shell height, 4 mm), originating from four French snail populations differing in shell height at the adult stage (from 6.5 to 12 mm), were individually subjected at 20°C to single-miracidium infections with Fascioloides magna. At day 75 post-exposure, the surviving snails were dissected, and rediae and cercariae were counted. Snail groups differed in shell growth during the experiment: from 1.8 ± 0.4 mm in group A up to 4.0 ± 1.1 mm in group D. The prevalence of F. magna infection, the numbers of free rediae and cercariae significantly increased together with increasing growth of infected snails during the experiment. Group A produced 1–6 first-generation rediae per snail and the mean daughter redia production ranged from 7.5 second-generation rediae (when a single first generation per snail developed) to 2.3 (6 first-generation rediae per snail). In contrast, up to ten first-generation rediae were noted in group D, and each mother redia gave daughter rediae with averages ranging from 1.5 (ten first-generation rediae per snail) to 13 (a single first generation per snail). In conclusion, the development of F. magna in G. truncatula exhibited both inter- and intrapopulation variability, where the development of rediae and cercariae was positively correlated with snail growth.

Two French populations of Galba truncatula were subjected to experimental infections with Egyptian and French isolates of Fasciola sp. miracidia, originating from cattle and sheep, to compare characteristics of snail infections in allopatric and sympatric groups. All sampled Egyptian isolates were identified as Fasciola hepatica using microsatellite markers. Compared to snails infected with French miracidia, snail survival at day 30 post-exposure was significantly greater in the Egyptian groups, while prevalence of infection was significantly lower (in an Egyptian group infected with cattle-derived miracidia) or did not show any significant differences in the other three cases. The total number of metacercariae was significantly higher in the four Egyptian groups. However, snail population and the mammalian origin of F. hepatica had also a significant effect on this parameter. The dissection of snail cadavers showed a significantly higher number of free rediae in the Egyptian groups, even if snail population also had a significant effect on the redial burden. Both Egyptian isolates of F. hepatica could easily develop in French snails, causing a low mortality in snails and inducing a metacercarial production higher than that noted in sympatric infections. However, the mammalian origin of F. hepatica eggs and the quality of snail populations as intermediate hosts had to be taken into account for studying local adaptation in reason of their effects on this process.

Two hundred Galba truncatula, originating from a French population, were subjected to single-miracidium infections with an allopatric isolate (origin, Argentina) of Fasciola hepatica. The control group was constituted by 100 snails coming from the same population and exposed to sympatric miracidia of F. hepatica according to the same protocol. Snail samples were collected bimonthly from both groups between day 14 and day 112 p.e. (at 20 C) and snail dissections were performed to count free rediae, intraredial morulae and free cercariae. Third and fourth generation rediae were significantly more numerous in the allopatric group, while the number of first generation rediae was significantly lower. In the sympatric group, the decrease in the number of intraredial morulae in the first, second and third redial generations was significantly faster. Free cercariae within the snail body were significantly more numerous in the sympatric than in the allopatric groups, whatever the date of snail dissection. The changes in redial development and cercarial production noted in the Argentinean group might be due to the evolution of South American flukes in a divergent way after the introduction of foreign infected ruminants in this continent from the 15th century., Facultad de Ciencias Veterinarias

International audience; Experimental infections of Galba truncatula (two populations) with Fasciola hepatica or Paramphistomum daubneyi were carried out to study the effect of water temperature changes (3 h at a mean of 12 °C every week) on cercarial shedding during the patent period. The results were compared with those of control snails infected according to the same protocol and always maintained at 20 °C. Compared to controls, a significant increase in the number of cercariae-shedding snails, a significantly longer patent period and significantly greater cercarial production were noted in temperature-challenged snails, regardless of the type of digenean infection. In contrast, the number of incompletely formed metacercariae was significantly higher in temperature-challenged snails than in controls. Incompletely formed metacercariae of F. hepatica consisted of cysts whose colour remained whitish after shedding (25.4% for temperature-challenged snails) or whose dome was flattened after encystment (74.6%). Those of P. daubneyi were totally dark brown or blackish after formation. These incomplete metacercariae might originate from young differentiating cercariae within the snail body (F. hepatica) or from cercariae which died just after encystment (P. daubneyi). The use of regular temperature changes for snails infected with F. hepatica or P. daubneyi must be monitored carefully during collection of metacercariae to select completely formed cysts for infecting definitive hosts.

Laboratory investigations on three aquatic populations of Galba truncatula, originating from the Peruvian Altiplano and French Massif Central, were carried out during three successive snail generations to determine if these populations might be successfully used for the metacercarial production of Fasciola hepatica under experimental conditions. High numbers of surviving snails at day 30 post-exposure (>70%), high prevalences of F. hepatica infections (>60%), and prolonged productions of cercariae for a mean period of 35 to 47 days were observed in the three populations, whatever the snail generation. In the Peruvian population, metacercariae of F. hepatica significantly decreased in numbers from a mean of 251 in the parent snails to 124 per snail in the F2 generation, whereas no significant variation was observed in the two French populations. As these aquatic snails rarely emerged out of water, the use of these populations for the commercial production of F. hepatica metacercariae was of great interest, because the daily time spent watching the breeding boxes of snails was clearly shorter, thereby reducing the cost of producing metacercariae compared with using amphibious snails reared with romaine lettuce.

Several experiments on the breeding of trematode-infected Galba truncatula for obtaining and packaging Fasciola hepatica and Paramphistomum daubneyi metacercariae were carried out to determine the more convenient methods to use for commercial production of these infective stages. Compared to the breeding of infected snails in aquaterraria, the use of 14-cm Petri dishes allowed a greater prevalence of snail infection and a higher number of metacercariae. The production of these larvae was still 2.3–3.4 times greater if infected snails were dissected during the patent period. The aspiration of these metacercariae at the extremity of a Pasteur pipette significantly shortens the time necessary for their transfer from Petri dishes to Eppendorf tubes. Using 14-cm Petri dishes, snail dissection and metacercarial aspiration for their transfer strongly reduce the cost price for metacercarial production of the trematodes Fasciola hepatica and Paramphistomum daubneyi.

Allopatric and sympatric infections of Lymnaea neotropica and Lymnaea viatrix var. ventricosa with Argentinean and French isolates of Fasciola hepatica were carried out to determine the capacity of these snails to produce metacercariae and to verify if this capacity changed with snail generation. The same process was also made with a French population of Galba truncatula known to be highly susceptible to French isolates of the parasite. In each lymnaeid species separately considered, the survival rate at day 30 post-exposure and prevalence of F. hepatica infection in the group infected with Argentinean miracidia were significantly greater than those recorded in the corresponding French one. Compared to infected G. truncatula, both South American lymnaeids had longer patent periods and produced a higher number of metacercariae. The highest infections were noted with L. v. ventricosa. In the three snail species, metacercarial production was more important with the Argentinean isolate of miracidia than with the French one. If three successive generations of L. v. ventricosa are exposed to the same French isolate of miracidia, cercarial production significantly increased from parents to the F2 generation, while the other characteristics of infection only showed insignificant variations. L. neotropica and L. v. ventricosa are better intermediate hosts for French F. hepatica than local G. truncatula. The numerical increase of shed cercariae in the F1 and F2 generations of L. v. ventricosa demonstrates a rapid adaptation of this species to the French isolate of the parasite., Facultad de Ciencias Veterinarias, Centro de Diagnóstico e Investigaciones Veterinarias

Natural infections of Galba truncatula with Haplometra cylindracea were followed from 2001 to 2009 to determine if their characteristics were similar when snails came from water collections frequented by Bufo bufo or by frogs and newts for their egg-laying. Snail samples were collected from both types of sites to count shed cercariae for three days and also free cercariae when snails were dissected. In sites only frequented by B. bufo, cercarial shedding occurred earlier than in those colonized by frogs and newts (March instead of April-May). In contrast, the number of cercariae shed during three successive days was significantly higher in May. This variation in the dates of cercarial shedding might be due, either to a synchronism between cercaria-releasing snails and the presence of the definitive host (tadpoles) in water collections, or to an earlier infection of overwintering snails in autumn by H. cylindracea miracidia in the case of toad-frequented sites.

International audience; Experimental infections of Egyptian Radix natalensis with French miracidia of Fasciola hepatica were carried out to determine if this snail might act as an intermediate host in the life cycle of this digenean in Egypt. Single exposures of R. natalensis to miracidia (2/snail) and two successive exposures (a total of 4 miracidia/ snail) were performed using lymnaeids measuring 1 to 6 mm in height. Live larval forms of F. hepatica were noted in single- and double-exposed snails. In double exposures, a significant increase of snail survival on day 28 post-exposure (at 24 degrees C) and an decrease in prevalence were noted when the height of snails at exposure was increasing. Cercariae of F. hepatica were shed by these snails (90.7/snail) during a mean patent period of 24.3 days. All snails have released these cercariae during 2-13 waves of shedding. According to these results, R. natalensis can be considered a potential intermediate host of F. hepatica in Egypt.

Experimental infections of EgyptianRadix natalensiswith a French isolate ofFasciola hepatica(each snail was subjected twice to a bimiracidial exposure) were carried out to determine how many sporocysts grew in these snails and to study the developmental patterns of redial generations. Single-sporocyst infections were found in 69.3% (34/49) of infected snails, with equivalent numbers of normal and abnormal patterns. Snails with two- and three-sporocyst infections were 24.4% and 6.1%, respectively. In single- and two-sporocyst infections at days 42 and 56 post-exposure, the total redial burden was significantly higher in snails with a normal redial development. In two- and three-sporocyst infections, the overall maturity of rediae was delayed at days 42 and 56. The high frequency of abnormal patterns inR. natalensis(53.1% of all infected snails showed degeneration of a first mother redia) might be due to incomplete adaptation between the snail population and the parasite. The delayed redial maturity in two- and three-sporocyst infections can mainly be explained by the volume of the snail body, which would be insufficient to allow the simultaneous differentiation of most rediae over time.

Experimental infections of pre-adult Galba truncatula (two populations) using one, two, three or five miracidia of Fasciola hepatica per snail were carried out to determine the total number of cercariae produced by snails when they were sacrificed at day 70 post-exposure (at 24°C). When the number of miracidia used for each snail increased at exposure, significant numerical augmentations of live rediae and of intraredial differentiating cercariae were noted. In contrast, only insignificant differences between mean numbers of free cercariae were found. The number of metacercariae produced by each snail (after shedding and/or after dissection) increased in infections with up to three miracidia per snail in each population and strongly decreased in infections with five miracidia per snail. Compared to the other types of infections, the delay of cercarial differentiation noted in the five-miracidia groups was probably due to the volume of the snail host, which would be too small to assure the complete development of five sporocysts.

Single and double infections of juvenile Omphiscola glabra (Gastropoda: Lymnaeidae) with Paramphistomum daubneyi and/or Fasciola hepatica were carried out to determine the redial burden and cercarial production in snails dissected at day 60 or at day 75 post-exposure (p.e.) in the laboratory at 20°C. The results were compared with those obtained with single-miracidium infections by Fascioloides magna. Compared to F. hepatica, low values were noted at day 75 p.e. for the prevalence of snail infections with P. daubneyi (4.6–8.3% instead of 23.6–25.9%), the total number of free rediae (10.7–17.9 per snail instead of 26.3–34.7), and that of free cercariae (112.8–136.9 per snail instead of 177.8–248.5). Despite a greater number of free rediae at day 75 p.e. (36.2–45.6 per snail), the prevalences of snail infections with F. magna and cercarial production were similar to those noted for F. hepatica. The results concerning F. hepatica and P. daubneyi might partly be explained by a progressive adaptation of O. glabra to sustain the larval development of these digeneans over the years, as this snail is a natural intermediate host of F. hepatica and P. daubneyi in central France since 1995. Compared with the high number of fully-grown rediae of F. magna in O. glabra, cercarial production seemed limited and this might be explained by the presence of high numbers of rediae which reduced the avaibility of nutrients for cercarial differentiation within the snail.

International audience; Experimental infections of Galba truncatula with Fasciola hepatica, Fascioloides magna, or Paramphistomum daubneyi were carried out at 20 degrees C to determine if the use of 14-cm petri dishes for breeding lettuce-fed snails enhanced the characteristics of snail infections. Compared to infected snails raised in boxes up to day 30 post-exposure and later in individual 35-mm dishes, the survival of G. truncatula kept in 14-cm dishes and the shell height of cercariae-shedding snails during the first 45 days were higher, whatever the digenean species is. The consequence of such enhanced characteristics was a greater production of metacercariae in the case of F. hepatica (1.7 to 5.6 times higher) and P. daubneyi (2.3 times). In contrast, metacercariae of F. magna were few in number, whatever the method of snail breeding is, and this might be explained by a still incomplete adaptation between the parasite of Czech origin and the French population of G. truncatula. The use of these 14-cm dishes reduced the time necessary for snail maintenance and metacercaria collection, thus allowing a decrease in the cost price of these larvae for commercial production.

As large numbers of metacercariae of Fasciola hepatica are necessary for research, experimental infections of Galba truncatula and Pseudosuccinea columella with this digenean were carried out to determine the better intermediate host for metacercarial production and, consequently, the most profitable snail for decreasing the cost price of these larvae. Pre-adult snails (4 mm in shell height) originating from two populations per lymnaeid species were individually exposed to two or five miracidia, raised at 23 °C and followed for cercarial shedding up to their death. Compared to values noted in G. truncatula, the survival of P. columella on day 30 post-exposure was significantly greater, while the prevalence of F. hepatica infection was significantly lower. In the four P. columella groups, metacercarial production was significantly greater than that noted in the four groups of G. truncatula (347-453 per cercariae-shedding snail versus 163-275, respectively). Apart from one population of G. truncatula, the use of five miracidia per snail at exposure significantly increased the prevalence of F. hepatica in P. columella and the other population of G. truncatula, whereas it did not have any clear effect on the mean number of metacercariae. The use of P. columella for experimental infections with F. hepatica resulted in significantly higher metacercarial production than that noted with G. truncatula, in spite of a lower prevalence for the former lymnaeid. This finding allows for a significant decrease in the cost price of these larvae for commercial production.

International audience; Single-miracidium infections of Fascioloides magna in two populations of Galba truncatula were carried out under laboratory conditions to count free rediae and cercariae in snail cadavers just after death. Cercaria-shedding snails were in low numbers, and their shell height at day 60 p.e. was significantly greater than that of numerous infected snails that died without cercarial shedding. In snails that died between days 44 and 60 p.e. (at 20 degrees C), the numbers of second-generation rediae significantly increased with increasing shell heights of infected snails. First-generation rediae showed insignificant, quantitative variations, while scarce rediae of the third generation were only found in the highest snails. Cercariae were only produced by the second redial generation. In both groups of snails, free cercariae appeared from 6 mm of shell height, and their numbers increased in the upper classes up to 32.9 per snail. Metacercariae were only found from 9 mm of shell height and were in low numbers. The global cercarial production ranged from 163.5 to 210.0 in the highest classes of snail size from both groups and was limited, whereas the mean burdens of free rediae fluctuated from 39.5 to 43.9. The death of numerous infected snails without cercarial shedding might be explained by the presence of a very high number of second-generation rediae simultaneously growing within the body of these snails.

International audience; Experimental infections of Omphiscola glabra (preadult snails), originating from central France, to a Czech isolate of Fascioloides magna miracidia were carried out to determine if the local populations of O. glabra may ensure the larval development of this parasite and to compare these results with those noted for a natural snail host, Galba truncatula. The presence of experimentally infected snails was noted in the six populations of snails studied. However, only a few snails shed their cercariae (O. glabra 5.3 to 17.1%, G. truncatula 15.1% in the first population, and no shedding in the other). The shell heights of cercariae-shedding (CS) snails were significantly greater than those of other infected snails, for O. glabra as well as for G. truncatula. The number of metacercariae noted in each snail group was low and showed insignificant variations. When experimental infections of O. glabra were performed in relation to the shell height of snails (from 1 to 14 mm) at miracidial exposure, the prevalence of infected snails significantly decreased with increasing shell heights at exposure. However, the presence of CS snails was only noted from the 5-6 to the 9-10 mm groups, and the mean number of metacercariae per group ranged from 27 to 44.2. Despite the high infectivity of the Czech isolate of F. magna miracidia, there was an incomplete adaptation with the French G. truncatula and O. glabra used in this study, as the metacercarial production was low, and cercarial shedding only occurred for snails which showed a strong increase of their shell height during F. magna infections.

Field investigations in two lymnaeid habitats located along river banks were carried out over three years to study snail colonization in relation to water-level variations, to determine the number of annual generations, and to analyse the ability of G. truncatula to emerge from water. As these river banks were located 6.7 km upstream from a dam, water recedes each day, thus permitting the study of snails on banks for several hours per day. From March to December, snails followed seasonal variations of water-level. However, only a few G. truncatula were observed following the daily retreat of water. Two annual generations of G. truncatula, the first from the onset of July and the second in December, were noted in these habitats. When water receded each day, the mean distance covered by non-aestivating snails ranged from 25 to 44 cm. However, they escaped water rise by seeking refuge in still emerged zones of their habitats. From July to October, snails following each day water receding showed a significantly quicker growth than that of G. truncatula which only moved in the same zone of their habitat. Populations of G. truncatula living along these river banks would be completely adapted to their habitat. The escape of snails from water rise between July and October might correspond to a physiological reaction of survival when a too rapid increase of water-level occurs.

Field investigations on the control of Galba truncatula by Zonitoides nitidus were carried out over the past 30 years in the different types of lymnaeid habitats located in central France. When a layer of mowed vegetation was used to cover G. truncatula habitats at the end of June, the introduction of adult Z. nitidus (20/m2) eliminated lymnaeid populations after 2 years of control in habitats located in swampy meadows, around the heads of intermittent springs, in areas trampled by cattle, and along river or pond banks. In the case of wild watercress beds, 3 years were necessary. The best results (elimination of G. truncatula after a single year of control) were obtained using an association of snails (Z. nitidus + Oxychilus draparnaudi), or a mixed control (a first application of 0.1 mg/l CuCl2, followed 3 months later by the introduction of Z. nitidus). Recolonization of treated habitats by G. truncatula coming from downstream populations was noted 3 years following the last application of biological control. Apart from the regular introduction of Z. nitidus in several watercress beds, the use of this snail to control G. truncatula has not become generalized in cattle- and sheep-breeding farms. The reasons for this situation are probably the complexity of applying this control in the field by nonspecialists and the difficulty of selecting the period of snail control at the end of June due to frequent local rainfall at this time.

As most natural watercress beds in central France are located upstream of the permanent habitats of two lymnaeid species, Galba truncatula and Omphiscola glabra, field investigations were made from 1999 to 2004 on 67 beds to determine why the contamination of watercress with Fasciola hepatica is irregular over time in these sites, while definitive hosts, especially lagomorphs, are regularly found infected around them. Snails are able to migrate upstream in winter and spring towards the beds, and a 4-year survey demonstrated the existence of annual variation in the colonization of these sites by snails. In the 45 beds irregularly contaminated with F. hepatica over time, 37.7–62.2%, according to the year, were not populated by lymnaeids, in spite of the presence of snail populations downstream. G. truncatula was found alone in 8.8–13.3% of sites and was the first colonizing snail in 24.3–33.3% when the two lymnaeid species successively settled in these waterholes. The colonizing ability of O. glabra was more limited, as it was observed alone in 2.2% of beds and was the first colonizing snail in only 2.2–20% of them. The distances covered by these snails significantly increased with increasing migration time. After migration, a few overwintering snails (3.8% of G. truncatula and 6.8% of O. glabra) are able to colonize the beds, but their numbers decreased when the distance of migration was greater. O. glabra migrated more quickly and reached more watercress beds than G. truncatula. However, this did not influence the natural infections of snails, which were more frequent in the F1 of G. truncatula. The variability observed in the colonization of beds by snails might explain the regular or irregular contamination of wild watercress by metacercariae of F. hepatica. One of the factors which may explain this variability is the occurrence of showers in spring, so that a few snails are able to reach watercress beds after their upstream migration.

A retrospective study on 1,211 snails naturally infected with Fasciola hepatica in central France was carried out to determine the numbers of full-grown sporocysts from which infections derived, to specify the developmental patterns of redial generations (normal or abnormal) and to count live and free rediae. In the department of Haute Vienne (siliceous subsoils), most snails showed single-sporocyst infections, with normal (46.3%) or abnormal (33.9%) development of redial generations. Two-sporocyst infections were scarcer (a total of 10.6%), while snail co-infections with F. hepatica and Paramphistomum daubneyi (8.0%) were found since 1996. In the department of Indre (calcareous subsoils), single-sporocyst infections showing normal development of rediae were the most numerous (58.0%), while the frequency of single-sporocyst infections with abnormal development was strongly decreased (4.3%). Two-sporocyst infections (with normal development of generations) and co-infections with F. hepatica and P. daubneyi slightly increased in frequency. The redial burdens found in snails collected from the department of Indre were significantly higher than those noted in snails originating from the department of Haute Vienne, whatever the type of snail infection; and these increases in numbers especially concerned the rediae of the second and subsequent generations. The results might be explained by the lower calcium ion content (7–23 mg/l) present in waters from the department of Haute Vienne which would induce a slower growth of infected snails (the shell height of adults scarcely reached 8 mm) and, consequently, would create barely favourable conditions for the development of the first rediae of the first generation within snails. However, the quality of the diet provided as food for snails and its influence on the development of redial generations cannot be excluded.

Snails from two populations highly susceptible to Fasciola hepatica and their F1 generations were subjected to individual bimiracidial exposures to determine if changes noted in infection parameters were due to an effect imposed on the snail by the parasite, or to some other effect such as the food used for the snails. Apart from the higher survival of unexposed parents at day 30 post exposure (p.e.) and their higher shell heights at day 45 p.e., the differences between the survival rates of exposed parents, prevalences of infections, and shell sizes were not significant. In the F1 snails born to previously infected parents, the prevalences of F. hepatica infection and cercarial production were significantly lower than those noted for the F1 born to unexposed parents. The survival of these snails and their shell growth did not show any significant variation. The F1 snails born to previously exposed snails would have developed a partial resistance against F. hepatica and this process would probably be maximal in the first 2 weeks of larval development inside the snail.