Journal of Helminthology (2016) 90, 753–759 doi:10.

1017/S0022149X15001078
q Cambridge University Press 2016

Pseudosuccinea columella: experimental

co-infections of juvenile and pre-adult
snails with the digeneans Calicophoron
daubneyi and Fasciola hepatica
G. Dreyfuss*, P. Vignoles and D. Rondelaud
INSERM 1094, Faculties of Medicine and Pharmacy,
87025 Limoges, France
(Received 13 February 2015; Accepted 21 November 2015; First Published Online 23 February 2016)

Abstract

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 þ 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.

Introduction in several snail – parasite models. In the lymnaeid Galba

The co-infection of a host snail with miracidia of two
digeneans often causes the death of one parasite.
However, in several cases, the development or the death
of the first djgenean after its penetration into the snail
facilitates the growth of the second parasite and leads to
complete larval development of the first species, the
second or both (Lie et al., 1977a, b; Lie & Heyneman, 1982;
Southgate et al., 1989). The presence of natural co-
infections with two digeneans has already been observed
*Fax: 33.555.435863
E-mail: gilles.dreyfuss@unilim.fr
truncatula, several co-infections with Calicophoron micro-
bothrium and Fasciola hepatica have been reported by
Samnaliev et al. (1978). In the same way, Manga-Gonzalez
et al. (1994) have noted the presence of four co-infected G.
truncatula, i.e. three with F. hepatica and Plagiorchis elegans
and the other with Opisthoglyphe ranae and Notocotylus
reynai, among the 6291 snails they collected from Spain.
Natural co-infections of G. truncatula with Calicophoron
daubneyi and F. hepatica were also found in central France:
111 snails out of 24,764 collected between 1995 and 2002
(Rondelaud et al., 2004) and six snails out of 11,025
collected between 2012 and 2014 (Rondelaud et al., 2015).
In the laboratory, the use of two digeneans at miracidial

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 754
exposure allowed successful co-infections to be obtained
with lymnaeid species which were age-resistant to
infection with either parasite. The snail Lymnaea glabra,
for example, is known to be susceptible to F. hepatica in its
first days of life only, i.e. , 2 mm in shell height (Boray,
1978), whereas it is completely resistant to C. daubneyi
infection, whatever snail age (Abrous, 1999). The co-
infection of pre-adult L. glabra, i.e. 3– 6 mm at miracidial
exposure, with these digeneans was successful and
resulted in complete larval development of F. hepatica,
C. daubneyi or both (Abrous et al., 1996, 1998, 1999).
Similar findings were also found with Lymnaea fuscus and
L. palustris when pre-adults were subjected to co-
infections with these two parasites (Dreyfuss et al., 2015).
In view of the above data, one may wonder if
lymnaeids susceptible to F. hepatica and resistant to
C. daubneyi can ensure larval development of either
digenean when they are co-infected with both parasites at
the pre-adult stage, i.e. 3 – 6 mm. To verify this possibility,
experimental infections were carried out using Pseudo-
succinea columella. This lymnaeid was age-resistant to
C. daubneyi infection and only young snails, measuring 1
or 2 mm at miracidial exposure, could ensure larval
development of the parasite up to cercarial shedding (Dar
et al., 2015b). In contrast, it is already known to be a
natural snail host of F. hepatica (Cruz-Reyes & Malek,
1987; Gutiérrez et al., 2002; Vázquez et al., 2014; Dar et al.,
2015a). Experimental infections of snails measuring
1 – 6 mm in shell height were carried out with a first
exposure to C. daubenyi and a second to F. hepatica,
according to the protocol used by Abrous et al. (1998)
for L. glabra.
Materials and methods
Snail collection
The population of P. columella originated from the
Lot River near Castelmoron, department of Lot, south-
western France. A total of 50 adult snails, measuring
10 – 15 mm in shell height, were collected in September –
October 2013 from two sites (448230 27.3100 N, 08320 2.4300 E
and 448230 31.1800 N, 08290 59.3000 E). In the laboratory, the
snails were placed in a 10-litre covered aquarium with five
snails per litre of permanently oxygenated spring water.
These aquaria were subjected to constant conditions:
temperature, 23 ^ 18C; light/dark period, 12 h/12 h. The
dissolved calcium concentration in spring water was
Table 1. Snail survival on day 30 post-exposure, prevalences of digenean infections and overall prevalence in six groups of
Pseudosuccinea columella co-infected with Calicophoron daubneyi (Cd) and Fasciola hepatica (Fh). For each group, 100 snails were
exposed on day 1.
Snail group (mm) Snail survival (%)
1 5.0
2 17.0
3 49.0
4 67.0
5 85.0
6 96.0
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G. Dreyfuss et al.
35 mg/l. Snails were fed on pesticide-free fresh lettuce
leaves ad libitum and the spring water in aquaria was
changed weekly. Egg masses laid by these adult snails
were collected and placed into small rearing aquaria.
Newly hatched snails fed on finely powdered lettuce and
those that attained 1 mm (24 h of life), 2 ^ 0.1 mm (5 days
of life), 3 ^ 0.1 mm, 4 ^ 0.1 mm, 5 ^ 0.1 mm or
6 ^ 0.1 mm in shell height, were used. A total of 1200
snails were subjected to experimental infections.
Parasite egg collection
Eggs of F. hepatica were collected from the gall bladders
of naturally infected cattle at the slaughterhouse of
Limoges, department of Haute Vienne, central France. To
obtain C. daubneyi eggs, adult worms were collected from
the rumen of infected cattle at the same slaughterhouse
and dipped in a physiological saline solution (0.9% NaCl,
0.45% glucose) before being placed at 378C for 3 h. Both
F. hepatica and C. daubneyi eggs were washed several times
with spring water and immediately incubated in the dark
at 208C for 20 days (Ollerenshaw, 1971).
Experimental protocol
The aim of the first experiment was to determine the
aptitude of juvenile and pre-adult P. columella to ensure
larval development of C. daubneyi and/or F. hepatica when
they were co-infected. Six groups of 100 snails each were
used in February 2014 (table 1). Each snail was first
subjected to five miracidia of C. daubneyi for 4 h at 238C in
3.5 ml spring water and secondly to five miracidia of
F. hepatica for another 4 h. The choice of ten miracidia for
this snail co-infection (five miracidia of each digenean)
was based on the results of a preliminary experiment
performed by Dar et al. (2015a) on P. columella. According
to these authors, the redial and cercarial production of
F. hepatica was greater when five miracidia were used to
infect each snail. Snails were then raised for 42 days in
individual 50-mm Petri dishes with 10 ml spring water
per recipient. A piece of pesticide-free fresh lettuce leaf
was placed in each dish. Petri dishes were then placed in
the same air-conditioned room at 23 ^ 18C as the parent
snails and were examined daily, so that spring water and
food could be changed if necessary. On day 42 post-
exposure (pe), surviving snails were dissected under a
stereomicroscope to detect the presence of larval forms of
Prevalence (%)
Cd only Fh only Cd þ Fh Overall prevalence (%): Cd þ Fh
0 20.0 0 0 þ 20.0
0 35.2 0 0 þ 35.2
8.1 32.6 4.0 12.2 þ 36.7
8.9 37.3 4.4 13.4 þ 41.7
2.3 14.1 1.1 3.5 þ 15.2
0 4.1 0 0 þ 4.1
 Digenean co-infections in Pseudosuccinea columnella
C. daubneyi, F. hepatica or both within their bodies. The
rediae of F. hepatica possessed well-developed pharynges,
collar rings and pairs of appendages in the third posterior
part of their bodies (Thomas, 1883). In contrast, the rediae
of C. daubneyi were shorter with small pharynges, and
their bodies had no collar or appendages (Sey, 1979).
The second experiment was carried out to determine
the characteristics of co-infections and follow the
dynamics of their cercarial shedding. As the best results
were noted in the 3 and 4 mm groups (see table 1), two
groups of 200 snails each were used in April 2014. Each
snail was exposed to the miracidial sequence used in the
first experiment. Two other groups of 100 snails each,
measuring 3 and 4 mm in shell height, respectively, were
exposed only to five F. hepatica miracidia per snail and
served as controls. No control group was constituted with
C. daubneyi because all experimental infections of 3 and
4 mm snails carried out with this digenean were negative
(Dar et al., 2015b). Snail exposure to miracidia and
maintenance were similar to those applied in the first
experiment. Spring water and food were changed, if
necessary, every day until snail death. When the first
cercarial shedding occurred, surviving snails were
subjected to a thermal shock every 3 days by placing
their Petri dishes at 10 – 138C for 3 h to stimulate cercarial
exit (Rondelaud et al., 2013; Vignoles et al., 2014). After
their emergence, cercariae were identified according to
the colour of their cysts (whitish or tawny for F. hepatica
and brown-blackish for C. daubneyi). They were counted
and removed from Petri dishes. At the death of each
infected snail, its shell was measured using callipers.
Data analysis
In the first experiment, the parameters were snail
survival on day 30 pe, the frequencies of C. daubneyi,
F. hepatica and both digeneans. Another parameter was
the overall prevalence of C. daubneyi infection, which took
into account the frequency of C. daubneyi only and that of
both digeneans. The same method was used to determine
the overall prevalence of F. hepatica infection. These
frequencies and prevalences were calculated in relation to
the number of snails surviving in each group on day 30
pe. A x2 test was used to compare the differences between
snail survival and overall prevalences.
In the second experiment, snail survival on day 30 pe,
the frequencies of C. daubneyi, F. hepatica and both
digeneans, the overall prevalence of each digenean
infection, the shell height of infected snails at their
death, the lengths of the pre-patent and patent periods,
the total number of shed cercariae and the number of
shedding waves for each infected snail (Rondelaud et al.,
2009) were considered. The differences between snail
survival and overall prevalences were analysed using a
x2 test. Individual values recorded for the shell heights of
infected snails, the lengths of the pre-patent and patent
periods, the numbers of metacercariae and of shedding
waves were averaged and standard deviations were
established for each snail group and each type of infection
(C. daubneyi, F. hepatica or both). Normality of these values
was analysed using the Shapiro–Wilk test (Shapiro &
Wilk, 1965). According to results given by this test, one-
way analysis of variance or the Kruskal– Wallis test were
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755
used to establish levels of significance. All the statistical
analyses were performed using Statview 5.0 software
(SAS Institute Inc., Cary, North Carolina, USA).
The nomenclature proposed by Correa et al. (2011)
and that by Jones (2005) were used in the present study
to identify lymnaeids and paramphistome species,
respectively.
Results
Aptitude of co-infected P. columella to sustain larval
development of both digeneans
Table 1 gives the results of the first experiment. On day
30 pe, the survival of snails significantly increased
(x2 ¼ 268.47, P , 0.001) with increasing height of their
shells at miracidial exposure. Snails harbouring larval
forms of C. daubneyi were only found in the 3, 4 and 5 mm
groups and their frequency was low, from 2.3 to 8.9%. In
contrast, F. hepatica-infected snails were found in the six
groups and the frequency peaked at 37.3% in the 4 mm
group. Snails harbouring larval forms of both digeneans
were only noted in the 3, 4 and 5 mm groups, with low
frequencies: 1.1– 4.4%. If the three types of frequency are
pooled, the overall prevalence of C. daubneyi ranged from
3.5 to 13.4% and that of F. hepatica from 4.1 to 41.7%.
Significant differences between the overall prevalences
found in the six groups were noted for C. daubneyi
(x2 ¼ 21.98, P , 0.001) and F. hepatica (x2 ¼ 44.64, P , 0.001).
Characteristics of co-infections in the 3 and 4 mm groups
Compared to controls infected with F. hepatica only,
the survival of co-infected groups on day 30 pe was
significantly lower (3 mm: x2 ¼ 3.94, P , 0.05; 4 mm:
x2 ¼ 5.24, P , 0.05) (table 2). Moreover, the survival
of 4 mm snails was significantly greater than that of
3 mm groups (co-infections: x2 ¼ 8.86, P , 0.01; controls:
x2 ¼ 5.77, P , 0.05). In both co-infected groups, snails
harbouring larval forms of C. daubneyi, F. hepatica or both
digeneans were found. The highest frequencies were
noted for F. hepatica-infected snails: 25.0% and 35.0% in
the 3 and 4 mm groups, respectively, whereas the other
two categories of co-infected snails had only low values.
Non-significant differences in the overall prevalences of
both co-infected groups were noted for C. daubneyi and
F. hepatica. The same finding was also noted for controls.
In contrast, the prevalence of F. hepatica infection was
significantly higher in controls than in co-infected snails
(3 mm: x2 ¼ 4.47, P , 0.05; 4 mm: x2 ¼ 4.28, P , 0.05). The
mean shell height of co-infected and control snails at their
death ranged from 11.3 to 13.3 mm and no significant
difference was noted, whatever the mode of comparison.
In co-infected groups, quantitative variations in the
mean number of metacercariae were noted according to
the type of digenean infection (table 2). First, the highest
numbers were noted in snails harbouring F. hepatica only,
but this difference between the 3 and 4 mm groups was
not significant. In each category of shell heights
considered separately, the number of these larvae was
significantly lower (3 mm: H ¼ 7.85, P , 0.01; 4 mm:
H ¼ 13.00, P , 0.001) in co-infected snails with F. hepatica
only than in controls. Second, C. daubneyi metacercariae in
 756 G. Dreyfuss et al.

Table 2. Snail survival on day 30 post-exposure, prevalence of each digenean infection, shell growth of
infected snails and number of shed cercariae (mean values ^ SD) in two groups of Pseudosuccinea columella
co-infected with Calicophoron daubneyi (Cd) and Fasciola hepatica (Fh), and in two groups of snails infected
with F. hepatica only (controls). Two hundred snails for each co-infected group and 100 snails for each control
group were exposed on day 1.

Co-infected groups Controls with F. hepatica only

Snail group (mm) 3 4 3 4

Snail survival (%) 54.0 68.5 66.0 81.0

Prevalence (%)
Cd 6.4 8.0
Fh 25.0 34.3 45.4 53.0
Cd þ Fh 4.6 4.3
Overall prevalence (%)
Cd 11.1 12.4
Fh 29.6 38.6 45.4 53.0
Shell growth (mm) of infected snails
Cd 11.3 ^ 2.1 13.2 ^ 2.4
Fh 12.6 ^ 1.9 12.8 ^ 2.0 12.8 ^ 2.4 13.3 ^ 2.1
Cd þ Fh 11.9 ^ 1.8 13.1 ^ 1.8
Number of shed cercariae
Cd 97.2 ^ 21.4 114.8 ^ 25.2
Fh 234.5 ^ 65.3 211.4 ^ 79.3 397.2 ^ 82.6 420.1 ^ 105.3
Cd þ Fh 49.6 ^ 8.4 (Cd) 62.4 ^ 19.8 (Cd)
þ92.7 ^ 17.8 (Fh) þ105.0 ^ 34.6 (Fh)

snails harbouring only this digenean were significantly
less numerous (3 mm: H ¼ 12.37, P , 0.001; 4 mm: H ¼ 4.74,
P , 0.05) than those found in co-infected individuals with
F. hepatica only, and their mean numbers in the 3 and 4 mm
groups were close to each other. Third, the lowest mean
numbers of metacercariae in co-infected groups were noted
for snails harbouring live larval forms of both digeneans.
Metacercariae of F. hepatica were significantly more
numerous (3 mm: H ¼ 14.48, P , 0.001; 4 mm: H ¼ 4.68,
P , 0.05) than those of C. daubneyi and no significant
difference between values recorded in both groups was
noted for either parasite. Significant differences between the
values found in snails with both digeneans and those
recorded in individuals with larval forms of a single parasite
were noted for C. daubneyi (3 mm: H ¼ 13.78, P , 0.001;
4 mm: H ¼ 17.34, P , 0.001) and F. hepatica (3 mm:
H ¼ 15.16, P , 0.001; 4 mm: H ¼ 6.31, P , 0.05).
In the four groups of snails, the length of the pre-patent
period ranged from 59.4 ^ 4.3 days to 62.7 ^ 7.4 days and
no significance between these lengths was noted (data not
shown). In contrast, the length of the patent period varied
with the type of digenean infection. In the 3 and 4 mm
snails harbouring larval forms of both parasites, the
lengths were 3.1 ^ 2.5 days and 4.4 ^ 1.7 days, respect-
ively. In snails harbouring C. daubneyi only, the respective
values were 10.7 ^ 5.9 days and 11.3 ^ 4.2 days. For each
category of digenean infection, no significant difference
between the 3 and 4 mm groups was noted. The longest
patent periods were found in co-infected snails harbour-
ing F. hepatica only (3 mm: 20.4 ^ 3.2 days; 4 mm:
18.2 ^ 4.1 days) and in controls (3 mm: 34.0 ^ 7.3 days;
4 mm: 37.2 ^ 8.5 days). In these last snails, the patent
period was significantly longer (H ¼ 6.31, P , 0.05)
in controls than in co-infected snails and did not
significantly differ from each other between both groups
of co-infected snails or between both control groups.
In co-infected groups, most snails harbouring
C. daubneyi only or both digeneans shed their cercariae
during a single wave and died after the last larva was
shed. In contrast, in individuals containing larval forms of
F. hepatica, several waves of cercarial shedding were
noted: 1 – 3 waves in the 3 mm group and 1 –5 in the 4 mm
group. The highest number of metacercariae was
noted for the second and third waves, respectively.
The number of shedding waves was greater in controls:
1 – 7 in the 3 mm group and 1 – 9 in the 4 mm group
(data not shown).
Discussion
Snail co-infection illustrates the parasitism of a mollusc
by two digenean species or a digenean and a proto-
strongylid. Depending on circumstances, the penetration
of both parasites can occur simultaneously. The second
parasite can also enter the snail several hours or several
days later. Lim & Heyneman (1972) and Combes (1982)
have listed consequences generated by these different
cases of miracidial infection in several snail – parasite
models. Among the different possibilities, snails resistant
to a parasite could become susceptible if they were first
infected with another parasite (Lie et al., 1977a, b). This
interference hypothesis was verified in several snail –
parasite models. Lie & Heyneman (1982) demonstrated
that infection of Biomphalaria glabrata with echinostome
miracidia can be advantageous for larval development of
Schistosoma mansoni in snail populations naturally
resistant to this latter parasite. Southgate et al. (1989)
also showed that it is possible to achieve successful
infection of Bulinus tropicus with Schistosoma bovis if
snails have previously been exposed to miracidia
of Calicophorum microbothrium. In the present study,

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 Digenean co-infections in Pseudosuccinea columnella
pre-adult P. columella, measuring 3 – 5 mm at exposure,
were able to sustain larval development of C. daubneyi,
F. hepatica or both, when they were co-exposed to
both parasite species. Similar findings had already been
reported for several lymnaeid species, such as
G. truncatula (Augot et al., 1996), L. glabra (Abrous et al.,
1996, 1998), L. fuscus and L. palustris (Dreyfuss et al., 2015),
when pre-adults of these species were co-infected
according to the same protocol. All these results
demonstrated that simultaneous penetration of both
parasites in pre-adults of several lymnaeid species
allowed larval development of the first parasite, the
second or both. However, this ability did not seem to be
general in the family Lymnaeidae because co-exposures
of Radix balthica ( ¼ R. ovata) to C. daubneyi and F. hepatica
were always negative (Dreyfuss et al., 2015). The
development of both parasite species in several co-
infected snails may only be explained by the infectivity
of C. daubneyi miracidia, which would have to be
rather strong to withstand the immune response of the
snail and continue their development within the snail
body in spite of the simultaneous development of
F. hepatica larval forms. It is also possible that excretory/
secretory products of F. hepatica larval stages interfere
with the amoebocytes of the snail, thus diminishing
their killing ability and allowing the development
of C. daubneyi.
Compared to controls with F. hepatica only, the overall
prevalence of this parasite was significantly lower in co-
infected snails harbouring the sole larval forms of this
digenean: 29.6% and 38.6% compared to 45.4% and 53.0%
in controls. Similar results have already been reported
for pre-adults of L. glabra, L. fuscus and L. palustris when
they were subjected to the same co-infections and
harboured F. hepatica (Dreyfuss et al., 2015). In contrast,
the prevalence of this digenean in pre-adult G. truncatula
subjected to co-infections with C. daubneyi and F. hepatica
was clearly higher: 61% for Augot et al. (1996) and 42.2%
for Dreyfuss et al. (2015). The better prevalence
of F. hepatica infection reported in pre-adult P. columella
(the present study) and G. truncatula (Augot et al., 1996;
Dreyfuss et al., 2015) might be due to the fact that
these lymnaeids were natural intermediate hosts
of this digenean and could be infected easily at the
pre-adult stage, whereas L. glabra, L. fuscus and L. palustris
were only susceptible to F. hepatica within their first
days of life and were resistant at the pre-adult stage
(Boray, 1978; Dreyfuss et al., 2000). Low values noted
for the overall prevalence of C. daubneyi infection in
the 3 and 4 mm groups of P. columella may also be
interpreted by the same explanation, i.e. the susceptibility
of juveniles and the resistance of pre-adults to this
parasite (Dar et al., 2015b).
The patent periods noted in co-infected snails
harbouring only F. hepatica were significantly shorter
than those noted in controls only infected with this
digenean. In the case of C. daubneyi, the patent
periods noted in the present study agreed with those
noted in juvenile P. columella exposed only to C. daubneyi
miracidia (a mean of 8.4 – 10.6 days; unpublished data)
but were shorter than those noted in the common snail
host of this digenean, G. truncatula (a mean of 12.1 – 16.5
days; Abrous, 1999). The results noted in the present
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757
study might be explained by the degree of adaptation
between the snail and its parasite. Snails which shed
their cercariae during several waves and, consequently,
had a long patent period, are considered to be well
adapted to their parasite (Dreyfuss, 1994). In contrast,
those with short patent periods would be incompletely
adapted to the digenean (Dreyfuss, 1994). If this last
assumption is valid, the adaptation between the
population of P. columella used in the present study and
the miracidial isolate of C. daubneyi would be incomplete
at the present time.
The numbers of metacercariae noted in F. hepatica-
infected controls (397.2 and 420.1 cysts per snail) were
within the range of values, i.e. 243.9 – 472.1 cercariae shed
per snail, that Dar et al. (2015a) reported for three
Egyptian populations of P. columella infected with
sympatric isolates of miracidia. In co-infected snails
harbouring F. hepatica only, the values were significantly
lower: 211.4– 234.5 cercariae shed per snail. This finding
might be due to the miracidial dose used to infect
each pre-adult, i.e. a total of ten miracidia with five for
C. daubneyi and five for F. hepatica, and competition that
developed between sporocysts and rediae of both
parasites for sharing nutrients present in the snail body
(Rondelaud et al., 2009). Even if several miracidia might
degenerate after their penetration into the snail body, the
development of the other larvae would be delayed over
time because of their high number and only some of these
rediae would produce cercariae, whereas the others
would still be immature when the infected snail died. An
element supporting this approach came from the report
by Rondelaud & Barthe (1982) on G. truncatula. According
to these authors, a delay in larval development of F.
hepatica occurred when five miracidia were used to infect
each snail, and this delay increased if the miracidial dose
for snail exposure became higher. The above hypothesis
proposed for co-infected snails with F. hepatica only can
also be used to explain the results noted in individuals
harbouring C. daubneyi only. Compared to values found
in co-infected snails with F. hepatica, the number of
C. daubneyi metacercariae was significantly lower in snails
that harboured this digenean only, and this result agreed
with the report by Augot et al. (1996) on G. truncatula co-
infected with C. daubneyi and F. hepatica. This last finding
can be explained by the redial burden, which was lower
for C. daubneyi than for F. hepatica. According to Abrous
et al. (1997), a total of 13 – 14 rediae generally developed
in single-miracidium infections of G. truncatula with
C. daubneyi, while a mean of 41 rediae was noted in snails
infected with F. hepatica according to the same protocol
(Augot et al., 1998). In co-infected snails harbouring live
larvae of both digeneans, the low values noted for either
parasite can be explained by the short length of the patent
period, as snail death occurred quickly in most snails, just
after the end of the first shedding wave.
In conclusion, pre-adult P. columella were capable of
sustaining larval development of C. daubneyi if they were
co-infected with the sequence C. daubneyi þF. 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 used in the present study and the
miracidial isolate.
 758
Conflict of interest
None.
References
Abrous, M. (1999) Les mollusques hôtes et les formes
larvaires de Paramphistomum daubneyi Dinnik, 1962
(Trematoda) dans le centre de la France. Influence
d’une co-infestation avec Fasciola hepatica Linné, 1758.
Doctorate Thesis, University of Limoges, France.
Abrous, M., Rondelaud, D. & Dreyfuss, G. (1996)
Paramphistomum daubneyi and Fasciola hepatica: the
effect of dual infection on prevalence and cercarial
shedding in preadult Lymnaea glabra. Journal of
Parasitology 82, 1026– 1029.
Abrous, M., Rondelaud, D. & Dreyfuss, G. (1997)
Paramphistomum daubneyi: the development of redial
generations in the snail Lymnaea truncatula. Parasitology
Research 83, 64 – 69.
Abrous, M., Rondelaud, D., Dreyfuss, G. & Cabaret, J.
(1998) Unusual transmission of the liver fluke, Fasciola
hepatica, by Lymnaea glabra or Planorbis leucostoma in
France. Journal of Parasitology 84, 1257– 1259.
Abrous, M., Rondelaud, D., Dreyfuss, G. & Cabaret, J.
(1999) Infection of Lymnaea truncatula and Lymnaea
glabra by Fasciola hepatica and Paramphistomum daubneyi
in farms of central France. Veterinary Research 30,
113 – 118.
Augot, D., Abrous, M., Rondelaud, D. & Dreyfuss, G.
(1996) Paramphistomum daubneyi and Fasciola hepatica:
the redial burden and cercarial shedding in Lymnaea
truncatula submitted to successive unimiracidial cross-
exposures. Parasitology Research 82, 623– 627.
Augot, D., Rondelaud, D., Dreyfuss, G., Cabaret, J.,
Bayssade-Dufour, C. & Albaret, J.L. (1998) Charac-
terization of Fasciola hepatica redial generations
(Trematoda: Fasciolidae) by morphometry and chae-
totaxy under experimental conditions. Journal of
Helminthology 72, 193–198.
Boray, J.C. (1978) The potential impact of exotic Lymnaea
spp. on fascioliasis in Australasia. Veterinary Parasitol-
ogy 4, 127– 141.
Combes, C. (1982) Trematodes: antagonism between
species and sterilizing effects on snails in biological
control. Parasitology 84, 151– 175.
Correa, A.C., Escobar, J.S., Noya, O., Velásquez, L.E.,
González-Ramı́rez, C., Hurtrez-Boussès, S. & Poin-
tier, J.P. (2011) Morphological and molecular charac-
terization of Neotropic Lymnaeidae (Gastropoda:
Lymnaeoidea), vectors of fasciolosis. Infection, Genetics
and Evolution 11, 1978– 1988.
Cruz-Reyes, A. & Malek, E.A. (1987) Suitability of six
lymnaeid snails for infection with Fasciola hepatica.
Veterinary Parasitology 24, 203– 210.
Dar, Y., Vignoles, P., Rondelaud, D. & Dreyfuss, G.
(2015a) Role of the lymnaeid snail Pseudosuccinea
columella in the transmission of the liver fluke Fasciola
hepatica in Egypt. Journal of Helminthology 89, 699– 706.
Dar, Y., Rondelaud, D., Vignoles, P. & Dreyfuss, G.
(2015b) Pseudosuccinea columella: age-resistance to
Calicophoron daubneyi infection in two snail popu-
lations. Parasite 22, 6 (6 pp.).
Downloaded from http:/www.cambridge.org/core. Australian Catholic University, on 20 Dec 2016 at 22:44:38, subject to the Cambridge Core terms of use, available at
http:/www.cambridge.org/core/terms. http://dx.doi.org/10.1017/S0022149X15001078
G. Dreyfuss et al.
Dreyfuss, G. (1994) Contribution à l’étude des émissions
cercariennes et de la charge parasitaire post-mortem
chez trois espèces de limnées infestées par Fasciola
hepatica Linné ou par Fasciola gigantica Cobbold.
Doctorate Thesis, University of Limoges, France.
Dreyfuss, G., Abrous, M. & Rondelaud, D. (2000) The
susceptibility of Lymnaea fuscus to experimental
infection with Fasciola hepatica. Journal of Parasitology
86, 158– 160.
Dreyfuss, G., Vignoles, P. & Rondelaud, D. (2015)
Nouvelles données sur le rôle de plusieurs espèces
de limnées dans la transmission de la fasciolose.
Bulletin des Groupements Techniques Vétérinaires 77,
109– 116.
Gutiérrez, A., Yong, M., Perera, G., Sánchez, J. &
Théron, A. (2002) Fasciola hepatica (Trematoda:
Digenea): its effects on the life history traits of
Pseudosuccinea columella (Gasteropoda: Lymnaeidae),
an uncommon interaction. Parasitology Research 88,
535– 539.
Jones, A. (2005) Family Paramphistomidae Fischoeder,
1901. pp. 229– 246 in Jones, A., Bray, R.A. & Gibson,
D.I. (Eds) Keys to the Trematoda, vol. 2. London, CAB
International and the Natural History Museum.
Lie, K.J. & Heyneman, D. (1982) Acquired resistance to
echinostomes in four Biomphalaria glabrata strains.
International Journal for Parasitology 9, 533– 537.
Lie, K.J., Heyneman, D. & Richards, C.S. (1977a) Studies
on resistance in snails: interference by non irradiated
echinostome larvae with natural resistance to Schisto-
soma mansoni in Biomphalaria glabrata. Journal of
Invertebrate Pathology 29, 118 – 125.
Lie, K.J., Heyneman, D. & Richards, C.S. (1977b)
Schistosoma mansoni: temporary reduction of natural
resistance in Biomphalaria glabrata induced by irra-
diated miracidia of Echinostoma paraensei. Experimental
Parasitology 43, 54 – 62.
Lim, H.K. & Heyneman, D. (1972) Intramolluscan
inter-trematode antagonism: a review of factors
influencing the host – parasite system and its possible
role in biological control. Advances in Parasitology 10,
191– 268.
Manga-Gonzalez, Y., Gonzalez-Lanza, C. & Kanev, I.
(1994) Lymnaea truncatula, the intermediate host of
some Plagiorchiidae and Notocotylidae species
in Leon, NW Spain. Journal of Helminthology 68,
134– 141.
Ollerenshaw, C.B. (1971) Some observations on the
epidemiology of fascioliasis in relation to the timing of
molluscicide application in the control of the disease.
The Veterinary Record 88, 152– 164.
Rondelaud, D. & Barthe, D. (1982) Les générations
rédiennes de Fasciola hepatica L. chez Lymnaea
truncatula Müller. A propos des effets de plusieurs
facteurs. Annales de Parasitologie Humaine et Comparée
57, 245– 262.
Rondelaud, D., Vignoles, P. & Dreyfuss, G. (2004)
Fasciola hepatica: the developmental patterns of redial
generations in naturally-infected Galba truncatula.
Parasitology Research 94, 183– 187.
Rondelaud, D., Vignoles, P. & Dreyfuss, G. (2009) La
Limnée tronquée, un mollusque d’intérêt médical et
vétérinaire. Limoges: Presses Universitaires du Limousin.
 Digenean co-infections in Pseudosuccinea columnella
Rondelaud, D., Titi, A., Vignoles, P., Mekroud, A. &
Dreyfuss, G. (2013) Consequence of temperature
changes on cercarial shedding from Galba truncatula
infected with Fasciola hepatica or Paramphistomum
daubneyi. Parasite 20 (7 pp.).
Rondelaud, D., Vignoles, P. & Dreyfuss, G. (2015) Larval
trematode infections in Galba truncatula (Gastropoda,
Lymnaeidae) from the Brenne Regional Natural
Park (central France). Journal of Helminthology. DOI:
10.1017/S0022149X15000073.
Samnaliev, P., Kanev, I. & Vassilev, I. (1978) Inter-
actions between larval and parthenitae stages of
some trematodes in one and the same inter-
mediate host. Proceedings of the Fourth International
Congress of Parasitology, Warszawa, 19 – 26 August, A,
pp. 45 – 46.
Sey, O. (1979) Life-cycle and geographical distribution of
Paramphistomum daubneyi Dinnik, 1962 (Trematoda:
Paramphistomata). Acta Veterinaria Academiae Scien-
tarum Hungaricae 27, 115 – 130.
Downloaded from http:/www.cambridge.org/core. Australian Catholic University, on 20 Dec 2016 at 22:44:38, subject to the Cambridge Core terms of use, available at
http:/www.cambridge.org/core/terms. http://dx.doi.org/10.1017/S0022149X15001078
759
Shapiro, S.S. & Wilk, M.B. (1965) An analysis of variance
test for normality (complete samples). Biometrika 52,
591– 611.
Southgate, V.R., Brown, D.S., Warlow, R., Knowles, R.J.
& Jones, A. (1989) The influence of Calicophoron
microbothrium on the susceptibility of Bulinus tropicus
to Schistosoma bovis. Parasitology Research 75, 381– 391.
Thomas, A.P. (1883) The natural history of the liver fluke
and the prevention of rot. Journal of the Royal
Agricultural Society of England 19, 276– 305.
Vázquez, A.A., Sánchez, J., Pointier, J.P., Théron, A. &
Hurtrez-Boussès, S. (2014) Fasciola hepatica in Cuba:
compatibility of different isolates with two intermedi-
ate snail hosts, Galba cubensis and Pseudosuccinea
columella. Journal of Helminthology 88, 434–440.
Vignoles, P., Titi, A., Rondelaud, D., Mekroud, A. &
Dreyfuss, G. (2014) Fasciola hepatica: effect of
natural light level on parasite cercarial emergence
from temperature-challenged Galba truncatula.
Parasite 21, 8 (8 pp.).