[A Retrospective Study on Planorbarius metidjensis and Its Role as a Potential Intermediate Host in the Transmission of Schistosoma haematobium in the Souss-Massa Region (Morocco)]. / Étude rétrospective sur Planorbarius metidjensis et son rôle comme hôte intermédiaire potentiel dans la transmission de Schistosoma haematobium dans la région du Souss-Massa (Maroc).

A retrospective research study was carried out by our team on Planorbarius metidjensis to highlight its epidemiological role as a potential host snail of Schistosoma haematobium in the Souss-Massa region. Contrary to the habitats colonized by Bulinus truncatus, the main host snail, those of P. metidjensis are located at the altitude of above 300 m. The best results of the experimental infections with S. haematobium were obtained using juvenile planorbids of 2 to 3 mm in diameter and a dose of five miracidia per snail. No snail naturally infected with the parasite was found when dissecting 3,457 adult bulinids and 2,470 adult planorbids. Despite the location of its habitats in altitude, P. metidjensis is a potential intermediate host of S. haematobium and it is important to follow these populations, as those of B. truncatus, in the future to avoid possible reoccurrence of indigenous cases of the disease.

Une étude rétrospective des travaux de notre équipe sur Planorbarius metidjensis a été réalisée afin de souligner son rôle épidémiologique comme mollusque hôte potentiel de Schistosoma haematobium dans la région du Souss-Massa. À l'inverse des habitats colonisés par Bulinus truncatus, le principal mollusque hôte, ceux de P. metidjensis sont localisés en altitude (plus de 300 m). Les meilleurs résultats de l'infestation expérimentale avec S. haematobium ont été obtenus en utilisant des planorbes juvéniles de 2 à 3 mm en diamètre et une dose de cinq miracidiums par individu. Aucun individu infesté naturellement par le parasite n'a été trouvé lors de la dissection de 3 457 bulins adultes et de 2 470 planorbes adultes. Malgré la localisation de ses habitats en altitude, P. metidjensis est un hôte intermédiaire potentiel de S. haematobium, et il importe de surveiller dans l'avenir ces populations, comme celles du bulin, afin d'éviter la réapparition éventuelle de cas autochtones de la maladie.

Calicophoron daubneyi and Fasciola hepatica: characteristics of natural and experimental co-infections of these digeneans in the snail Lymnaea glabra.

A retrospective study on different Lymnaea glabra samples collected from central France between 1993 and 2010 was carried out to determine the prevalence of natural co-infections with Calicophoron daubneyi and Fasciola hepatica, and to specify the composition of redial burdens. Experimental infections of L. glabra performed 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-infected Galba truncatula. In natural co-infections, prevalence was 0.7% in L. glabra (186/25,128) and 0.4% in G. truncatula (137/31,345). Low redial burdens were found in these snails, with F. hepatica rediae significantly more numerous in L. glabra than in G. truncatula (7.5 per snail instead of 5.2). In contrast, the total numbers of C. daubneyi rediae in both lymnaeids were close to each other (4.3 and 3.0 rediae, respectively). In experimentally co-infected groups, prevalence was greater in G. truncatula than in the other lymnaeid (6.3% instead of 3.0%). Significantly shorter patent periods and lower metacercarial production for each digenean were noted in L. glabra than in G. 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 for L. glabra, metacercarial production of both digeneans in co-infected snails was lower than that in G. truncatula, thus indicating a still incomplete adaptation between these French L. glabra and both parasites.

Aptitude of Lymnaea palustris and L. stagnalis to Fasciola hepatica larval development through the infection of several successive generations of 4-mm-high snails.

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.

Pseudosuccinea columella: experimental co-infections of juvenile and pre-adult snails with the digeneans Calicophoron daubneyi and Fasciola hepatica.

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.

Larval trematode infections in Galba truncatula (Gastropoda, Lymnaeidae) from the Brenne Regional Natural Park, central France.

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.

Fasciola hepatica: the redial generations in juvenile Pseudosuccinea columella.

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.

Lymnaea glabra: progressive increase in susceptibility to Fasciola hepatica through successive generations of experimentally infected snails.

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.

Differences in the compatibility of infection between the liver flukes Fascioloides magna and Fasciola hepatica in a Colombian population of the snail Galba sp.

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.

Role of the lymnaeid snail Pseudosuccinea columella in the transmission of the liver fluke Fasciola hepatica in Egypt.

Experimental infections of three Egyptian Pseudosuccinea columella populations with sympatric miracidia of Fasciola sp., 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 as Fasciola hepatica. Apart from being independent of snail origin, prevalences ranging from 60.4 to 75.5% in snails infected with five miracidia of F. hepatica were 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. If P. columella was subjected to two successive bi-miracidial infections with F. hepatica, prevalence of infection was 63.3%, with a mean of 311 metacercariae per snail. These values were clearly greater than those already reported for Radix natalensis infected with the same parasite and the same protocol. Successful experimental infection of P. columella with F. hepatica suggests that this lymnaeid snail is an important intermediate host for the transmission of fascioliasis in Egypt.

Natural light influences cercarial emergence of Calicophoron daubneyi but not that of Haplometra cylindracea from temperature-challenged Galba 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.

Fasciola hepatica: development of redial generations in experimental infections of Pseudosuccinea columella.

Experimental infections of Egyptian Pseudosuccinea columella with one or two miracidia of Fasciola hepatica per snail were carried out to determine the developmental pattern (normal or abnormal) of redial generations and specify the number of free rediae developing in snails according to their generation. Controls were constituted by a French population of Galba truncatula infected according to the same protocol. Most infected P. columella showed a normal development of redial generations (96.2-98.1 vs 75.5-85.7% for G. truncatula). In each redial category, free rediae were more numerous in P. columella than in G. truncatula, and their number were also greater in the two-miracidia groups than in single-miracidium infections for each lymnaeid considered separately. This increase in redial production was mainly due to the number of first mother (R1a) rediae producing daughter rediae only: 2 per P. columella (vs one redia in G. truncatula) in single-miracidium groups and 3.1 (vs 1.9) in the two-miracidia groups. In P. columella, the mean total number of free rediae developing in single-miracidium and bimiracidial infections was 77.2 and 117.6, respectively (instead of 33.5 and 52.1 rediae in G. truncatula). The number of F. hepatica rediae present in P. columella was related to the number of fully grown sporocysts and the quantity of R1a rediae which developed into the snail body.

The production of mammalian trematode infective stages by the snail Galba truncatula.

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.

Growth rate of the intermediate snail host Galba truncatula influences redial development of the trematode Fascioloides magna.

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.

Variations in local adaptation of allopatric Fasciola hepatica to French Galba truncatula in relation to parasite origin.

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.

Comparative strategies and success of sympatric and allopatric Fasciola hepatica infecting Galba truncatula of different susceptibilities.

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.

Lymnaea neotropica and Lymnaea viatrix, potential intermediate hosts for Fascioloides magna.

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.

Paramphistomum daubneyi: characteristics of infection in three lymnaeid species.

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.

Intermediate snail hosts of French Fasciola hepatica: Lymnaea neotropica and Lymnaea viatrix are better hosts than local Galba truncatula.

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.

Local adaptation of the trematode Fasciola hepatica to the snail Galba truncatula.

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.

Haplometra cylindracea (Zeder, 1800) (Trematoda:Plagiorchiidae): variation in the dates of cercarial shedding for overwintering Galba truncatula.

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.