Changes in the Populations of Two Lymnaeidae and Their Infection by Fasciola hepatica and/or Calicophoron daubneyi over the Past 30 Years in Central France

Simple Summary Two parasitic diseases affecting humans and ruminants have a particular life cycle, because freshwater molluscs intervene in their transmission by ensuring the development of parasite larval forms. As the climate is changing, researchers have begun to investigate the effects of global warming on these snails and the larval forms of parasites that they harbour. Several authors have already conducted analyses of these diseases, but no field studies have been carried out so far. Therefore, in the present study, snail counts were conducted on 39 farms with acidic soils between 1976 and 1997, in 2013–2014, and in 2020–2021. The results showed that the number of snail populations decreased over time and that many populations have fewer and fewer individuals. This decline has also been faster in recent years. The infection of snails by one of the parasites has decreased over time. Conversely, snails are increasingly infected with the other parasite. These changes are due to the generalized use of a drug used to treat one of the diseases in ruminants and probably also due to the heatwave episodes that occurred for several years. As these larval forms are again infecting animals after their departure from the snails, practitioners must take these changes into account when treating ruminants for these diseases. Abstract Field investigations were carried out during three periods (from 1976 to 1997, in 2013–2014, and in 2020–2021) on 39 cattle-raising farms on acidic soils to track changes in the populations of two Lymnaeidae (Galba truncatula and Omphiscola glabra) and their infection with Fasciola hepatica and/or Calicophoron daubneyi. Compared to the survey between 1976 and 1997 on these farms, there was a significant decrease in the number of the two lymnaeid populations and the size of the G. truncatula populations in both 2013–2014 and 2020–2021. This decline was significantly faster in the last nine years than it was before 2013. The area of habitats colonized by G. truncatula showed no significant variation over the years, while that of habitats with O. glabra significantly decreased in the period covered by the three surveys. The prevalence of F. hepatica infection in snails significantly decreased over the years, while C. daubneyi infection increased over time in both lymnaeid species. These changes are due to the use of triclabendazole to treat fasciolosis in ruminants since the 1990s, and are probably a consequence of the successive heatwaves that have occurred since 2018 in the region.


Introduction
Among the parasitoses that freshwater snails can transmit, two helminthoses, i.e., distomatosis caused by Fasciola hepatica and paramphistomosis caused by Calicophoron daubneyi, have a wide distribution in temperate countries. Fasciolosis has been recognized for its endemicity in Western Europe from many years [1][2][3]. This infection has increased in significance over the last 20 years, and it is currently considered a public health problem for humans [4][5][6]. Paramphistomosis is traditionally considered to have a limited veterinary significance, at least when farm animals are kept in good nutritional and sanitary Animals 2022, 12, 3566 2 of 12 conditions [6]. In Western Europe, this parasitosis did not develop until the 1990s, although it has long existed in domestic animals [7][8][9][10]. In the United Kingdom and Ireland, the prevalence of paramphistomosis in cattle has increased dramatically in recent years and may exceed that of fasciolosis in some areas [11,12]. Similar observations in cattle herds were also reported for acidic soils in central France with a decrease in fasciolosis (from 25.2% to 12.6% between 1990 and 1999) and a corresponding increase in paramphistomosis (from 5.2% to 44.7%) [13].
These two parasitoses are known to infect the same definitive host (usually cattle), and sometimes they can be found within the same individual [7]. Moreover, they are transmitted by the same intermediate hosts. In central France, the most common host snail is Galba truncatula, but another lymnaeid, Omphiscola glabra, which lives in the same meadows as G. truncatula, can also be an intermediate host, generally when it is co-infected by miracidia of F. hepatica and those of C. daubneyi [14,15]. Samples of adult G. truncatula (>4 mm high) were taken in central France between 1989 and to determine the prevalence of snail infection by either of these two parasites. Of a total of 18,791 G. truncatula collected during this period, the average prevalence of F. hepatica infection was 5.1% (with annual variations ranging from 3.3% to 7.2%), while that of C. daubneyi was 3.3%, with an annual increase in this parameter since 1990, reaching 5.3% in 2000 [13].
Since the 2000s, a decline in the number of these two lymnaeid populations has been noted in central France. It was also observed in the number of overwintering snails in many populations [16,17]. In 2020-2021, this decline continued [18]. According to Vignoles et al. [18], this decrease in 2020-2021 could be partly due to the occurrence of successive heatwave episodes during the summer months of 2018 to 2020. These results raised two questions: what was the rate of the decrease in the number and size of lymnaeid populations over the past 30 years? and what were the consequences of this decline in snail infection by F. hepatica and/or C. daubneyi? To answer the first question, a retrospective study was carried out on 39 farms raising beef cattle on the acidic soils in central France; our team conducted three surveys between 1976 and 1997, in 2013-2014, and in 2020-2021. The second question was answered by studying the prevalence of these two parasitoses in snails collected on the same farms during the three survey periods.

Farms Studied
The 39 farms are located in the northwest or west of the Haute Vienne department ( Figure 1). Their altitude ranges from 155 to 241 m. These farms raised cattle on permanent grasslands not treated with lime, and the farming technique has not changed over the past 30 years. The subsoil of these farms is granitic or gneissic ( Table 1). The 178 permanent meadows within the perimeter of these farms are hygro-mesophilous, with a mesophilous zone extending over most of the area in each meadow. These grasslands are subjected to alternating grazing by ruminants and mowing during the summer. A surface drainage network, either supplied by one or more temporary sources or not, is present in each pasture. This network is only cleaned up every three, four, or five years, depending on its state of degradation. Water, which flows through drainage systems or road ditches, is generally present from early October to late June or early July, with a maximum from January to late April. The pH of this water varies from 5.8 to 7, while the concentration of dissolved calcium ions is usually less than 20 mg/L [19]. These meadows are subject to a continental climate, strongly attenuated by the wet winds coming from the Atlantic Ocean. The mean annual precipitation ranges from 850 to 1100 mm per year, while the mean annual temperature ranges from 10.5 • C to 11.5 • C for most farms [20].

Animals
On the 39 farms, the average number of beef cattle varied from 34 to 57 depending on the year (Table 1). These ruminants grazed outside all year and were driven to different meadows depending on the growth of grass [21]. These farms are locally considered "fluke farms" because of the frequency of fasciolosis outbreaks that affected cattle herds prior to the 2000s. According to local veterinarians who worked with these breeders, until 1990, prophylaxis against fasciolosis was performed by the administration of closantel, albendazole, and/or nitroxynil [22]. From 1994, these drugs were abandoned in favour of triclabendazole [22], and farmers have gradually used this product to treat their cattle against fasciolosis (Table 1). This last drug was first used annually. As cases of bovine fasciolosis have become rarer on these farms since 2008, most breeders have subsequently treated their animals only in cases of clinical fasciolosis or with the detection of parasite Animals 2022, 12, 3566 5 of 12 eggs in cattle stools. As a result, the periodicity of this treatment has changed over time, with an interval of up to two years between two successive triclabendazole intakes for each animal. Paramphistomosis only developed on these farms in 1994 and was not treated until 2008. The discovery of a few cattle with diarrhoea led thirteen breeders (out of thirty-nine) to deworm their cattle with oxyclonazide once or twice in the past ten years.
The two Lymnaeidae lived in the same meadows and often on the same surface drainage network. The habitats of G. truncatula are generally located at the upstream end of each drainage swale or each man ditch, while the O. glabra populations generally live on the course of the same swales ( Figure 2) with a distance of 8 to 25 m between the habitats of both lymnaeids [23]. The two snails also occupied other sites in these grasslands, such as slope rushes around temporary or permanent sources [23]. Pictures of these two habitat types have already been published in two reviews that our team has written on these species of Lymnaeidae [14,15].
prior to the 2000s. According to local veterinarians who worked with these breeders, until 1990, prophylaxis against fasciolosis was performed by the administration of closantel, albendazole, and/or nitroxynil [22]. From 1994, these drugs were abandoned in favour of triclabendazole [22], and farmers have gradually used this product to treat their cattle against fasciolosis (Table 1). This last drug was first used annually. As cases of bovine fasciolosis have become rarer on these farms since 2008, most breeders have subsequently treated their animals only in cases of clinical fasciolosis or with the detection of parasite eggs in cattle stools. As a result, the periodicity of this treatment has changed over time, with an interval of up to two years between two successive triclabendazole intakes for each animal. Paramphistomosis only developed on these farms in 1994 and was not treated until 2008. The discovery of a few cattle with diarrhoea led thirteen breeders (out of thirty-nine) to deworm their cattle with oxyclonazide once or twice in the past ten years.
The two Lymnaeidae lived in the same meadows and often on the same surface drainage network. The habitats of G. truncatula are generally located at the upstream end of each drainage swale or each man ditch, while the O. glabra populations generally live on the course of the same swales ( Figure 2) with a distance of 8 to 25 m between the habitats of both lymnaeids [23]. The two snails also occupied other sites in these grasslands, such as slope rushes around temporary or permanent sources [23]. Pictures of these two habitat types have already been published in two reviews that our team has written on these species of Lymnaeidae [14,15]. On the different pastures, the habitats of Lymnaeidae were not separated by a fence from the rest of each meadow, so that cattle had access to snail habitats during their grazing. On the different pastures, the habitats of Lymnaeidae were not separated by a fence from the rest of each meadow, so that cattle had access to snail habitats during their grazing.

Protocol for Snail Investigations
The 39 farms were selected for this study because an outbreak of animal fasciolosis was diagnosed by local veterinarians in their cattle herds between 1976 and 1997. Three surveys were conducted on each farm. The first investigations were carried out during the above period on the overall area of each farm to identify populations of Lymnaeidae, measure the area of their habitats, and count adult snails belonging to the generation born during the previous autumn (overwintering snails) in each population. Pastures were then re-examined in 2013-2014 and 2020-2021, according to the same protocol. The first two surveys were conducted in late March or early April. In 2020-2021, temperature Animals 2022, 12, 3566 6 of 12 leniency enabled surveys to be conducted from the end of February to the end of March on the meadows of the 39 farms. This period was selected because the snail habitats were then waterlogged and only populated by adults of the overwintering generation. When a snail population was not found in 2013-2014 or 2020-2021 in a meadow, the breeder was questioned to find out the possible cause of this disappearance.
Adults higher than 4 mm for G. truncatula and 12 mm for O. glabra were counted by sight or using a colander (mesh size, 3 mm), depending on the height of the water layer. During the first 2 surveys, each count was performed by 2 people for 30 to 40 min in habitats located along a drainage network or a road ditch and by 1 person for 15 to 20 min in those located on pond edges and stream banks. In 2020 and 2021, each count was performed by 1 person for 15 to 20 min per habitat (when there was a population). The area of each habitat was then determined. Measuring areas occupied by G. truncatula or O. glabra is easy in the case of habitats located in drainage swales or along the banks of ponds and streams. When the shape of the habitat was irregular, the only solution was to draw a map and determine the area of this habitat according to its shape and dimensions.
On the 39 farms, snail counts and the determination of habitat areas were always conducted by a member of our team and students who already had at least two years of experience in collecting and identifying Lymnaeidae in the field. On each farm, the values recorded in the different sites occupied by each snail species were pooled without taking into account the type of habitat.

Protocol for Detecting Parasite Larval Forms in Snails
As herds of cattle graze in turn on the meadows of each farm, according to the growth of the grass, snail sampling was conducted in the different habitats of lymnaeids in order to obtain a sample of G. truncatula and another of O. glabra for each farm and for each survey period. Samples of 100 G. truncatula each (between 1976 and 1997, and in 2013-2014) or 50 snails each (2020-2021) were randomly collected within the perimeter of each farm, taking into account the number of snail habitats and the number of overwintering snails in each population. The same protocol was used in the case of O. glabra. Collected snails were over 4 mm high for G. truncatula and over 12 mm high for O. glabra. After being transported to the laboratory under isothermal conditions, the snails were dissected under a stereomicroscope to detect the presence of larval forms of F. hepatica and/or C. daubneyi. Rediae and cercariae belonging to either helminth were recognized based on our experience in identifying the larval forms of parasites in these two lymnaeid species [24,25].

Meteorological Data
The average monthly temperatures were provided by the Bellac weather station because it is the closest to the farms studied. The series of values considered concerned the temperatures recorded by the station from 1981 to 2010, in 2013-2014, and in 2020-2021 [26].

Statistics
Six parameters were studied. The first was the average monthly temperature during the three periods of investigation. Three other parameters were the number of populations for each snail species, the area of their habitats, and the density of overwintering snails in each population. The last two parameters were the prevalence of snail infection with F. hepatica or C. daubneyi. These parameters were noted, taking into account the farm investigated, the snail species, and the investigation period. Individual values noted for the habitat areas of each snail species during each survey were therefore reduced to an average and framed by a standard deviation for each farm. A similar protocol was used for the densities of overwintering snails per m 2 of habitat.
As these six parameters were recorded on the thirty-nine farms during three successive surveys, the data were first analysed using the Shapiro-Wilk normality test [27]. As the distributions of these values were not normal, Friedman two-way analysis of variance was used to establish the levels of statistical significance. The decline in the number of populations was calculated using the ratio of the number of populations in 2020-2021 to that recorded between 1976 and 1997. A similar protocol was used to determine the decline in the habitat area and the number of overwintering snails in the different populations of Lymnaeidae. Figure 3 shows the average monthly temperature for each investigation period. Temperatures were not significantly different from one investigation period to another. However, there was a more pronounced difference between temperatures in August and September (p = 0.08) and another between temperatures in June, September, and October (p = 0.10). These increases in temperature were therefore close to significance in summer and early autumn. age and framed by a standard deviation for each farm. A similar protocol was used for the densities of overwintering snails per m² of habitat.

Results
As these six parameters were recorded on the thirty-nine farms during three successive surveys, the data were first analysed using the Shapiro-Wilk normality test [27]. As the distributions of these values were not normal, Friedman two-way analysis of variance was used to establish the levels of statistical significance.
The decline in the number of populations was calculated using the ratio of the number of populations in 2020-2021 to that recorded between 1976 and 1997. A similar protocol was used to determine the decline in the habitat area and the number of overwintering snails in the different populations of Lymnaeidae. Figure 3 shows the average monthly temperature for each investigation period. Temperatures were not significantly different from one investigation period to another. However, there was a more pronounced difference between temperatures in August and September (p = 0.08) and another between temperatures in June, September, and October (p = 0.10). These increases in temperature were therefore close to significance in summer and early autumn.   Table 2 shows the characteristics of snail populations during the three surveys. Compared to the values recorded before 1998 on the 39 farms, the total number of snail populations in 2020-2021 significantly decreased, by 54.2% for G. truncatula (Chi 2 = 78.0; p < 0.001) and by 34.5% for O. glabra (Chi 2 = 78.0; p < 0.001). This decline was faster during the last nine years than in the period between the first two surveys: a decrease of 34.6% for G. truncatula and 22.1% for O. glabra, compared to 30% and 15.9%, respectively. The average area of habitats occupied by G. truncatula did not show any significant variation between the three periods of investigation (Chi 2 = 0.80; p = 0.67). In contrast, the average area of the O. glabra habitat significantly decreased, by 33.4%, between the three surveys (Chi 2 = 20.2; p < 0.001). The number of overwintering snails per population showed a significant decrease of 79% for G. truncatula (Chi 2 = 23.0; p < 0.001) and 75.3% for O. glabra (Chi 2 = 14.2; p < 0.001) between the three surveys. The decrease in the size of each G. truncatula population was faster between 2013-2014 and 2020-2021 (69.2%) than in the period between the first two surveys (32.0%). In contrast, the rates of decline during the above two periods were close for O. glabra: 61.2% and 63.5%, respectively.

Prevalence of Parasite Infection in Snails
On the 39 farms studied (Table 3), the prevalence of infection with F. hepatica significantly decreased, by 95.9%, for G. truncatula (Chi 2 = 37.2; p < 0.001) between the first and third surveys. For O. glabra, the situation was quite different. Several immature rediae of F. hepatica were noted in 11 snails prior to 1998, while rediae and cercariae of Fasciola were noted during the other two surveys, with a significant decrease in prevalence between 2013-2014 and 2020-2021 (Chi 2 = 14.7; p = 0.006). Table 3. Number of adult snails infected with Fasciola hepatica or Calicophoron daubneyi and the prevalence of infection on 39 farms on acidic soils across 3 surveys. Snails collected were over 4 mm high for Galba truncatula and over 12 mm high for Omphiscola glabra. No snail infected with C. daubneyi (Table 3) was observed in samples collected between 1976 and 1997, regardless of the lymnaeid species. Infected snails were later noted with significant increases in prevalence for G. truncatula (Chi 2 = 55.5; p < 0.001) and O. glabra (Chi 2 = 31.8; p < 0.001) between 2013-2014 and 2020-2021.

Discussion
Several factors may explain the changes in the number and size of lymnaeid populations on the 39 farms studied. Most of these changes are related to human activity on the grasslands. According to Dreyfuss et al. [16,17], the mechanical cleaning of the surface drainage system and/or the extension of underground drainage in many grasslands may be responsible for the disappearance of many snail populations and a reduction in their size since the 1970s. Another more recent factor is climate change, with the succession of heatwave episodes over recent years resulting in a decline in biodiversity, which is linked to the extinction of numerous species (see review by Johnson [28]). Several authors have already analysed the impact that climate change will have on the development of fasciolosis and host snails in several countries [29][30][31][32] because this parasitosis is highly dependent on temperature and precipitation [33]. According to projections by Cordellier et al. [34], the increase in evaporation, the decrease in oxygen concentrations due to increased water temperatures, and changes in precipitation patterns are likely to affect the survival and reproduction of Lymnaeidae, as well as other freshwater molluscs in northwestern Europe. These climatic disturbances will also lead to changes in the distribution of native freshwater species, with some migrating to colder locations, while others may have limited distribution. Non-native species may take over and expand their distribution areas [34].
Our observations regarding the declines in the number of both lymnaeid populations and in the size of the G. truncatula populations are consistent with data reported by our team on many farms on acidic and sedimentary soils in central France [16,17]. The new insight offered by this study concerns the rapidity of this decline over the last nine years. Similarly, habitats with O. glabra showed a significant reduction in their mean area over the same period, while sites occupied by G. truncatula showed no significant variation in their area. Several assumptions can be made to explain these results. In our opinion, the occurrence of heatwave episodes in 2018, 2019, and 2020 [35][36][37] are likely at the root of the changes observed in the populations of both species. These successive episodes of heat waves over several years may have led to the progressive extinction of some snail populations and the numerical decrease in the number of individuals in other populations due to the death of many snails belonging to the spring generation during habitat aestivation. Under these conditions, the survivors would have laid fewer eggs in the fall, and there consequently would have been fewer overwintering snails in successive years.
The reduction in the area of O. glabra habitats over the past nine years can also be explained by the above hypothesis, assuming a lower number of snails in their habitat and, consequently, a lower occupancy of the habitat area. On the other hand, the lack of significant variation in the area of G. truncatula habitats must be related to their particular location in grasslands on acidic soils. According to Moens [38] and Vareille-Morel et al. [23], many G. truncatula habitats are located at the upstream end of swales, with or without a temporary source in the case of surface drainage or around temporary springs on the hills surrounding these grasslands.
The changes in the infection rates of both Lymnaeidae are more complex to interpret. The first factor is triclabendazole, which local breeders have used progressively since the 1990s to control F. hepatica infection in ruminants. This drug is known to be highly effective against adult forms of the parasite and also in juveniles up to 1 week old [39,40]. The use of this product has resulted in a sharp decrease in cases of cattle fasciolosis over the years [41,42]. As farmers switched from broad-spectrum anthelmintics to triclabendazole to treat fasciolosis in their cattle, the number of ruminants infected by C. daubneyi showed a marked increase over the years [7,13] because the two parasites are often present in the same animals. This also affected the infection rates of G. truncatula and O. glabra. The prevalence of infection with F. hepatica gradually decreased in G. truncatula over time (from 4.7% to 3.3% between 1990 and 2000 [13]), whereas that with C. daubneyi gradually increased (from 0.8% to 5.3% between 1991 and 2000 in the same grasslands [13]). The second factor is more difficult to comment on, as the 19 G. truncatula and 12 O. glabra infected with F. hepatica were only collected from 5 and 2 farms, respectively, while the grasslands of the 39 farms were investigated in 2020-2021 (Table 3). The most valid hypothesis would posit that the low prevalence of these lymnaeids could be a direct consequence of the sharp decrease in cases of animal fasciolosis found in central French farms [42]. However, unfavourable conditions for the survival of lymnaeids and the embryogenesis of parasite eggs cannot be excluded, because the snail habitats on many farms remained dry for a long period during the autumn months in 2018, 2019, and 2020, meaning that individuals of the overwintering generation would have been unlikely to be infected.
Lower prevalence values in O. glabra than in G. truncatula have already been noted by Abrous et al. [43,44] in farms on acidic or sedimentary soils when the two lymnaeids were living in the same environment (a surface drainage swale on acidic soil, for example). These results are related to the susceptibility of each lymnaeid because the miracidia of both parasites are more attracted to G. truncatula than to the other species [45][46][47].

Conclusions
There has been a significant decrease in the number and size of lymnaeid populations in central France over the past 30 years, and this decline was significantly faster in the last 9 years than before 2013. The prevalence of F. hepatica infection in G. truncatula has also decreased over the years, while C. daubneyi infection has increased over time in both lymnaeid species. These changes are due to the use of triclabendazole to treat fasciolosis in ruminants since the 1990s and are likely also a consequence of the successive heatwaves that have occurred in the region since 2018.