Environmental DNA (eDNA) surveying has potential to become a powerful tool for sustainable parasite control. As trematode parasites require an intermediate snail host that is often aquatic or amphibious to fulfil their lifecycle, water-based eDNA analyses can be used to screen habitats for the presence of snail hosts and identify trematode infection risk areas. The aim of this study was to identify climatic and environmental factors associated with the detection of Galba truncatula eDNA. Fourteen potential G. truncatula habitats on two farms were surveyed over a 9-month period, with eDNA detected using a filter capture, extraction and PCR protocol with data analysed using a generalized estimation equation. The probability of detecting G. truncatula eDNA increased in habitats where snails were visually detected, as temperature increased, and as water pH decreased (P < 0.05). Rainfall was positively associated with eDNA detection in watercourse habitats on farm A, but negatively associated with eDNA detection in watercourse habitats on farm B (P < 0.001), which may be explained by differences in watercourse gradient. This study is the first to identify factors associated with trematode intermediate snail host eDNA detection. These factors should be considered in standardized protocols to evaluate the results of future eDNA surveys.

Fascioliasis is a zoonotic disease caused by liver flukes transmitted by freshwater lymnaeid snails. Donkey and horse reservoir roles have been highlighted in human endemic areas. Liver fluke infection in mules has received very limited research. Their role in disease transmission, epidemiological importance and Fasciola hepatica pathogenicity are studied for the first time. Prevalence was 39.5% in 81 mules from Aconcagua, and 24.4% in 127 from Uspallata, in high-altitude areas of Mendoza province, Argentina. A mean amount of 101,242 eggs/mule/day is estimated. Lymnaeids from Uspallata proved to belong to ribosomal DNA internal transcribed spacer (ITS) markers ITS-1 and ITS-2 combined haplotype 3C of Galba truncatula. These lymnaeids were experimentally susceptible to infection by egg miracidia from mules. Infectivity, number of cercariae/snail and shedding period fit the enhanced F. hepatica/G. truncatula transmission pattern at very high altitude. This indicates that the mule is able to maintain the F. hepatica cycle independently. Individual burdens of 20 and 97 flukes were found. Mule infection susceptibility is intermediate between donkey and horse, although closer to the latter. Anatomo-pathology and histopathology indicate that massive infection may cause mule death. Haematological value decreases of red blood cells, haemoglobin, leucocytes and lymphocytes indicate anaemia and strong immunosuppression. Strongly increased biochemical marker values indicate liver function alterations. The mule probably played a role in the past exchanges with Chile and Bolivia through Mendoza province. Evidence suggests that mules could contribute to the spread of both F. hepatica and G. truncatula to human fascioliasis-endemic areas in these countries.

Despite years of investigation into triclabendazole (TCBZ) resistance in Fasciola hepatica, the genetic mechanisms responsible remain unknown. Extensive analysis of multiple triclabendazole-susceptible and -resistant isolates using a combination of experimental in vivo and in vitro approaches has been carried out, yet few, if any, genes have been demonstrated experimentally to be associated with resistance phenotypes in the field. In this review we summarize the current understanding of TCBZ resistance from the approaches employed to date. We report the current genomic and genetic resources for F. hepatica that are available to facilitate novel functional genomics and genetic experiments for this parasite in the future. Finally, we describe our own non-biased approach to mapping the major genetic loci involved in conferring TCBZ resistance in F. hepatica.

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.

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.

Field investigations in two lymnaeid habitats located along river banks were carried out over three years to study snailcolonization in relation to water-level variations, to determine the number of annual generations, and to analyse the ability ofG. 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 variationsof water-level. However, only a few G. truncatula were observed following the daily retreat of water. Two annual generationsof G. truncatula, the first from the onset of July and the second in December, were noted in these habitats. When waterreceded each day, the mean distance covered by non-aestivating snails ranged from 25 to 44 cm. However, they escaped waterrise by seeking refuge in still emerged zones of their habitats. From July to October, snails following each day water recedingshowed a significantly quicker growth than that of G. truncatula which only moved in the same zone of their habitat. Populationsof G. truncatula living along these river banks would be completely adapted to their habitat. The escape of snails from waterrise between July and October might correspond to a physiological reaction of survival when a too rapid increase of water-leveloccurs.

Experimental introductions of Galba truncatula in new habitats were carried out over the 30 last years in the department ofHaute-Vienne (central France) to study the details of snail settlement and the outcome of these populations, and to determine ifthe susceptibility of these new colonies to experimental infection with Fasciola hepática did not change over time. A total of 51samples (100 adult snails each) were collected from three populations known for their high susceptibility to F hepatica infections (> 60 %). Each sample was introduced in March in an open drainage furrow (36 meadows) or a road ditch (15 sites) whichhad never been inhabited by snails. Introduced snails from 22 samples survived in 9 furrows and 13 road ditches. In these sites,the number of overwintering G. truncatula increased up to the-second or third year post-introduction and progressively decreasedin the following years to become stable from the sixth or seventh year (less than 40 snails per site). In 13 populations (outof 22), experimental infections of snails with F. hepatica did not result in significant variations in survival rates or in prevalencesof infections when compared to the respective rates recorded in the three original populations. Conversely, in the nine otherpopulations, experimental infections of snails resulted in a decrease in prevalence (< 40 %), whereas the survival rates of snailsat day 30 post-exposure did not change. This last result might be explained by a change in natural parasite pressure in the newhabitats, being probably lower than that existing in the three sites inhabited by the populations of origin.

Field and laboratory studies on 11 populations of Galba truncatula were performed to determine the survival rate of snails after aestivation, to specify the range of their shell heights, and to establish the percentage of G. truncatula which burrowed in soil when summer drying occurred. These investigations were performed using six lowland populations living in central France (alt. 206-282 m) and five highland populations, located in the Massif central (alt. 806-900 m). The survival rate of G. truncatula after flooding dried soil with water was significantly higher in the highland than in the lowland populations (69.5-80.7 % in road ditches, for example, instead of 29.3-33.0%). The mean shell heights of surviving snails and the durations of snail re-activation in water did not show any significant variation, whatever the origin of snail population. The percentages of snails buried in drying soil were higher in the highland population (13.0 % and 15.0 % of juvenile snails, for example, instead of 4.0 % and 7.7 % in lowland snails). In the lowland population only young snails buried, whereas 5.8-8.3 % of adults in the highland population were partially burrowing. The local climate of the sites studied in the Massif central, and the ability of snails to burrow into the mud when stagnant water disappeared in July, might explain the higher survival rate during aestivation.