Multi-parasite host susceptibility and multi-host

parasite infectivity: A new approach of the

Biomphalaria glabrata/Schistosoma mansoni
compatibility polymorphism.
A. Theron, A. Rognon, B. Gourbal, G. Mitta

To cite this version:

A. Theron, A. Rognon, B. Gourbal, G. Mitta. Multi-parasite host susceptibility and multi-
host parasite infectivity: A new approach of the Biomphalaria glabrata/Schistosoma mansoni
compatibility polymorphism.. Infection, Genetics and Evolution, Elsevier, 2014, 26, pp.80-88.
฀10.1016/j.meegid.2014.04.025฀. ฀halsde-00998747฀

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 1

Multi-parasite host susceptibility and multi-host parasite infectivity: a new

approach of the Biomphalaria glabrata / Schistosoma mansoni compatibility
polymorphism

A. THERON a,b, A. ROGNON a,b, B. GOURBAL a,b, G. MITTA a,b

a
CNRS, UMR 5244, Ecologie et Evolution des Interactions (2EI), Université de Perpignan,
France.
b
Université de Perpignan Via Domitia. 52, Ave Paul Alduy. 66860 Perpignan Cedex, France.

Corresponding author. Tel.: + 33 4 68 66 21 83; fax: + 33 4 68 50 22 81

E-mail address: [email protected] (A. Theron)

ABSTRACT
In this study, we analyze the degree of susceptibility/un-susceptibility of five strains of
Biomphalaria glabrata from different geographical origins successively challenged with a
panel of 4 Schistosoma mansoni strains. A total of 20 homopatric and heteropatric host-
parasite combinations were tested with exposure doses of 1, 10, 20, 30 and 50 miracidia per
individual host. By doing this, we characterized each B. glabrata strain by its “multi-parasite
susceptibility phenotype” that reflects better the efficiency of their defense mechanism against
not only one, but a diversity of schistosome stocks. In the same time, all the S. mansoni
strains used were characterized, by their “multi-host infectivity phenotype” that reflects the
level of infectivity they display when confronted to diverse snail populations. Based on these
results it is possible to select different homogenous stocks of snails with different spectrum of
susceptibility/un-susceptibility for several parasite strains. This will be a useful tool for future
functional studies conducted to understand the genetics and molecular basis of the
compatibility polymorphism in this host/parasite model.

Keywords:
Schistosoma mansoni
Biomphalaria glabrata
Compatibility polymorphism
Matching phenotypes
 2

1. Introduction

Schistosomiasis remains an important health problem that affects over 200 million
people worldwide (WHO, 2010). Schistosoma mansoni causes intestinal schistosomiasis and
as all trematode species, needs mollusks as first intermediate hosts to accomplish part of its
life cycle before infecting humans. For this species, only fresh water snails of the genus
Biomphalaria are potentially susceptible to an infection by the larval stages of the parasite.
However, within a same snail species, for example B. glabrata, the main intermediate host of
S. mansoni in South America, compatibility varies between populations, strains or individuals
(Basch, 1976; Theron et al, 1997). Snail/schistosome compatibility reflects both snail
susceptibility and schistosome infectivity and the observed polymorphism of compatibility
results from the complex phenotype-by-phenotype interactions occurring between each
individual host and each individual parasite (Basch, 1976; Theron and Coustau, 2005; Theron
et al., 2008). The immunologic mechanisms underlying compatibility have remained largely
unknown (see Bayne, 2009; Yoshino and Coustau, 2011). However recent studies have
succeed in identifying host immune recognition receptors and parasite antigens displaying a
high level of variability and which were described to be in interaction (Roger et al., 2008;
Moné et al., 2008, 2010). It was hypothesised that these molecules could be involved in the
expression of the B. glabrata / S. mansoni compatibility polymorphism (Mitta et al., 2012;
Hanington et al., 2012; Roger et al., 2008a,b,c; Moné et al., 2010; 2011).
Different B. glabrata laboratory strains (or isolates) show various degrees of
susceptibility to S. mansoni infection (Basch, 1976; Theron et al., 1997) and different strains
of S. mansoni display different levels of infectivity towards a particular strain of snail host.
Compatibility is strain specific (Theron and Coustau, 2005; Webster and Davies, 2001), one
snail strain that is highly susceptible to one particular strain of schistosome could be partially
or totally unsusceptible to another schistosome strain (Richards, 1984; Lie et al., 1979). On
the other hand, one parasite strain that is highly infective to a snail host strain could be less or
un-infective to another one. In spite of this overview about variation of compatibility, it
appears that very few studies have investigated the multi-parasite strain susceptibility of B.
glabrata stocks and conversely the multi-host strain infectivity of S. mansoni stocks.
The aim of the present study was to characterize and compare the degree of
susceptibility/un-susceptibility of different strains of B. glabrata when challenged with a
panel of S. mansoni strains. On the other hand, the infectious capacity of the different strains
of S. mansoni used was also evaluated when confronted to a panel of B. glabrata stocks. By
 3

this way we will be able to discriminate particular strains of snails that are more or less
efficient in their defense mechanisms against not only a single strain of parasite but against a
diversity of schistosome strains and then, to go beyond the classic approach that traditionally
characterize a strain either resistant or susceptible. This approach, concerning both the hosts
and the parasite may lead to the selection of snail phenotypes representative of different
spectrum of susceptibility/un-susceptibility and schistosome strains representative of different
capacities of infectivity. Such discreet phenotypes of multi-parasite susceptibility provide an
invaluable resource for future comparative genetics and functional studies to better
understand the genetics and molecular basis of the compatibility polymorphism in this
host/parasite model.

2. Materials and methods

2.1. Parasites and snails

Four strains of S. mansoni (Sm) from different geographical origins were used in this
study. Each was maintained in the laboratory since more than 10-20 years on their sympatric
strains of B. glabrata (Bg) as intermediate host and in mice (SWISS OF1) as definitive host.
These four homopatric host/parasite combinations are termed as follow: BgVEN / SmVEN
(origin Venezuela, Guaraca); BgGUA / SmGUA (origin Guadeloupe, Dans Fond); BgBRE /
SmBRE (origin Brazil, Recife) and BgBAR / SmLE (origin Brazil, Belo Horizonte). One
supplementary B. glabrata strain for which we do not dispose of its corresponding parasite
strain was also used: the BgBS-90 strain (origin Brazil, Salvador).

2.2. Compatibility trials

As snail infection rates vary with the parasite dose (Theron et al., 1997; 2008),
compatibility tests were conducted by dose-response curves obtained by challenging
individual snails (35-40 snails of 6-8 mm in diameter per treatments) with doses of 1, 10, 20,
30 and 50 freshly hatched miracidia (Mi) in 5 ml of water at 24-25° C for 8 h. Following
exposure to miracidia, snails were replaced in their original containers until their infection
status was assessed. For all experiments, infection of snails was evaluated by the presence and
count of mother sporocysts at 15 days post-exposure as previously described (Theron and
Gerard, 1994; Allienne et al., 2011). Briefly summarized, snails were relaxed in pond water
 4

containing excess of crystaline menthol for 12 hr. The snail body was removed and fixed in
modified Raillet-Henry solution. Using this technique, mother sporocysts were readily
observable as translucent white bodies within an opaque brown tissue background. The
number of developped mother sporocysts (containing young daughter sporocysts) present
within each snail was determined following exhaustive dissection of the snail including
deeper tissues. A previous histological study on BgGUA snails exposed to SmGUA strain and
fixed 48 hr later, showed that among the miracidia that have penetrated, 22% appeared well
developped without host reaction, 40% were encapsulated by the host hemocytes and 38%
degenerated without host reaction (Theron et al., 1997). One hour after exposure, 26% of the
miracidia had not penetrated the snail. The level of compatibility was quantified by the
proportion of infected snails and the intensity of infection by the number of developped
mother sporocysts (MSp) per snails. All the host-parasite combinations identified as
compatible by the presence of developing mother sporocysts at 15 days post-infection, were
able to produce cercariae. This has been verified during the laboratory cycling of the various
strains of S. mansoni on the different strains of snails and additionaly, during the experimental
selection of compatible phenotypes of snail hosts currently in progress.
For each of the 5 snail strains, compatibility was tested separately with their
homopatric schistosome strain and the 3 other heteropatric parasite strains respectively (for
the BS-90 snail strain, the 4 parasite strains used were heteropatric). In total, the following 20
host-parasite combinations were tested with exposure doses of 1, 10, 20, 30 and 50 miracidia
per host : (BgBAR / SmLE, BgBAR / SmVEN, BgBAR / SmBRE, BgBAR / SmGUA);
(BgVEN / SmVEN, BgVEN / SmLE, BgVEN / SmBRE, BgVEN / SmGUA); (BgBRE /
SmBRE, BgBRE / SmLE, BgBRE / SmVEN, BgBRE / SmGUA); (BgGUA / SmGUA,
BgGUA / SmLE, BgGUA / SmVEN, BgGUA / SmBRE); (BgBS-90 / SmGUA, BgBS-90 /
SmLE, BgBS-90 / SmVEN, BgBS-90 / SmBRE).

3. Results

3.1. Variations of susceptibility between B. glabrata strains (Fig. 1)

For all the host/parasite combinations tested, the dose-response curves generated
showed the same pattern: a rapid increase in infection rates between the doses 1 and 10-20
miracidia per snail then a plateau that leveled at different rates (Fig. 1).
 5

Each snail strain exhibits a particular scope of susceptibility when confronted to the 4
strains of schistosomes (Fig. 1). The BgBRE strain was 100% susceptible with 3 (SmLE,
SmVEN and SmBRE) of the 4 schistosome strains tested but very poorly susceptible to the
SmGUA (around 5%). At the opposite, BgBAR strain, 100% susceptible with its sympatric
schistosome strain (SmLE), was only 50% susceptible with SmVEN, 10% with SmBRE and
totally unsusceptible to SmGUA. BgVEN and BgGUA strains showed a similar pattern of
susceptibility (100% with SmLE and SmVEN, 80% with SmBRE and 40% with SmGUA).
The BgBS-90 stock was only partially susceptible to the SmLE strain of S. mansoni (53% of
infection with 20Mi) and totally unsusceptible to the 3 other strains of S. mansoni.
Excepted for the homopatric couple BgGUA/SmGUA that showed the lowest
compatibility level compared to the corresponding heteropatric combinations, the 3 other
homopatric couples (BgBRE/SmBRE, BgVEN/SmVEN and BgBAR/SmLE) were equally or
more compatible than their corresponding heteropatric couples.

3.2. Variations of infectivity between S. mansoni strains (Fig. 2)

By the side of the parasite, clearly the SmLE strain appeared as having the highest
capacity of infection whatever the snail strain used. Infection rates of 100% were obtained
with the dose of 10-20 Mi per snail with 4 snail strains BgBRE, BgGUA, BgVEN and BgBar.
SmLE was the only schistosome strain tested capable of infecting the BgBS-90 strain (53% of
infection with 20Mi). At the opposite, the SmGUA strain appeared as the less infective even
towards its sympatric host snails (40-50% of BgGUA infected at the plateau), equivalent
infection rates were obtained only with the BgBAR stock. SmGUA was very few infective for
BgBRE (around 5%) and un-infective towards the BgBAR and BgBS90 strains. SmVEN and
SmBRE strains showed high level of infectivity with the BgBRE (100%), BgGUA (80%) and
BgVEN (80%) snail stocks, low with the BgBAR strain (15%) and un-infective towards the
BgBS-90 strain.

3.3. Variations of parasite intensities (Fig. 3, A, B)

Variations of parasite intensities (number of developed MSp per snails), are presented
in figure 3 only for the BgBRE host strain challenged by the 4 parasite strains and for the
SmBRE parasite strain infecting the 5 host strains at the different doses of miracidia. These
two examples were representative of the other combinations. For compatible combinations,
 6

the number of MSp increases with the miracidial doses at exposure. For the homopatric
couple SmBRE/BgBRE (Fig. 3 A) the mean number of MSp /snail for 10 Mi was of 3.58 ±
0.19 and raised to 16.18 ± 0.86 MSp for the dose of 50 Mi/snail. For the less compatible
combination, SmBRE/BgBAR (Fig. 3 B) the number of MSp remained stable around 2.5
whatever the miracidial dose was.
For the same parasite strain and at equivalent dose of Mi, variations exist as a function
of the host strain challenged and its level of susceptibility. Highest was the compatibility
level, highest was the mean parasite intensity. As an example (Fig. 3 B), the relationship
between mean parasite intensities and compatibility level for the SmBRE strain at the dose of
50 Mi/snail infecting the 4 host strains was as follow: 2.5 MSp and 16% of infection for
SmBRE/BgBAR, 8.0 MSp and 75% for SmBRE/BgVEN, 10.0 MSp and 90% for
SmBRE/BgGUA, 16.0 MSp and 100% for SmBRE/BgBRE.
We note that only a low proportion of miracidia presented to the snail host succeed in
developing to mother sporocysts and this proportion varies between parasite strains, host
strains and miracidial doses at exposure (Fig. 4). Including data from all the host strains and
all the miracidial doses, SmLE parasite strain showed the highest Mi/MSp transformation rate
with an average of 35%, SmGUA the lower (20%). Including data from all the parasite strains
and the miracidial doses, it is among the snails of the BgBRE strain that we observed the
highest rate of developing miracidia (35%) and among the snails of the BgBAR strain the
lowest (16%).

4. Discussion

Investigations on snail/schistosome compatibility between Biomphalaria species and

S. mansoni started several decades ago with the pioneer works of Files and Cram (1949),
Abdel-Malek (1950), then Richards (1970, 1973), and Basch (1976). Later, numerous studies
confirmed strong variations in host susceptibility or parasite infectivity, depending of the
geographic origin of the host and parasite strains used (Lie et al., 1979; Richard and Shade,
1987; Morand et al., 1996; Webster et al., 2001). Our study addressed another component of
this snail/schistosome compatibility polymorphism to the extent that the susceptibility /
unsusceptibility level of different strains of B. glabrata was determined when confronted to
the same panel of different S. mansoni strains. By doing this, we were able to characterize
each B. glabrata strain by what we defined as its “multi-parasite susceptibility phenotype”
and we may better compare the efficiency of their defense mechanism against not only one,
 7

but a diversity of schistosome stocks. In the same time, we characterized each of the S.
mansoni strain used by their “multi-host infectivity phenotype” that reflects the level of
infectivity they display when confronted to a diversity of snail stocks. Clearly the results
obtained, showed marked differences between both host and parasite strains in their capacity
to resist or infect the diversity of parasites or hosts proposed, respectively. This offers the
possibility to select experimentally new homogeneous susceptible/unsuceptible phenotypes of
snails for further studies.

4.1. A matching phenotype-phenotype interaction

As previously demonstrated for one of the host/parasite combination tested in this

study (i.e. SmGUA/BgGUA, see Theron et al., 2008), we note that all the dose–response
curves obtained for each of the 20 host/parasite couples analyzed showed the same shape.
They are characterized by a rapid increase of the infection rates between 1 and 10-20
miracidia per snail, then an asymptote that leveled off at different values as a function of the
strain used. For combinations with compatibility level lower than 100%, the parasite
intensities increased with increasing miracidial doses but not the proportion of infected hosts
(at 20, 30 and 50 miracidia/snail). This may indicate that there is no threshold dose for which
the parasite would be able to overwhelm the host defenses. Similar pattern of dose-response
curves with the plateau that leveled below 100% were obtained with higher miracidial doses
(80 and 100 Mi/snail, data not shown). Increasing the miracidial dose simply involves
sampling a larger fraction of the phenotypic diversity in the parasite strain. In other words, a
dose of 20 miracidia per snail contains, for all the S. mansoni strains, most of the possible
phenotypes that potentially match with the host phenotype challenged. These results agree
with the hypothesis that the success or failure of a challenge by one parasite mainly depends
on the matched/mismatched status of the host and parasite phenotypes (Bash, 1975; Theron
and Coustau, 2005; Theron et al., 2008; Mitta et al., 2012). Taking into account the little
water volume used (5 ml) for the exposition of individual snails to the miracidia, we assume
that all miracidia had contact with the host. Despite this high host/parasite contact, only a
small proportion of miracidia (between 20 and 40%) is not recognized by the innate defense
system of the host and develops into Msp, the other larvae are encapsulated or degenerated
(Theron et al., 1997). This means that within the same snail host, a specific defense response
occurs against some incompatible entered miracidia but without effect on neighboring
compatible larvae. The reason of the observed different fates of the nondeveloping MSp
 8

(encapsulated vs degenerated) has until now received little attention. The absence of
hemocytic reaction around the degenerated MSp strongly suggests unidentified humoral
factors responsible for the nondevelopment of these larvae (Sullivan and Richards, 1981;
Vasquez and Sullivan, 2001). Such humoral reaction against MSp was always observed in
case of immune priming of B. glabrata (Sire el al., 1998; Portela et al., 2013). Indeed, MSp of
the challenge infection of B. glabrata degenerate progressively within the host tissues without
cellular encapsulation. Then, in case of "simultaneous" pluri-miracidial exposure (in our case
8 hours of exposition), not all miracidia penetrate the snail in the same time and we could
hypothesize that incompatible miracidia penetrating first and recognized as non-self should be
rapidly encapsulated while those penetrating later after this primary hemocyte-mediated
defense should be stopped in their development by the presence of toxic plasma molecules
and died undergoing gradual degeneration. Physiological unsuitability of the intermediate
host was also proposed as a possible factor for the nondevelopment of some MSp (Vasquez
and Sullivan, 2001). As emphasized by Basch (1976), compatibility is tested independently
for each entering miracidium and each exposed snail. The phenotype (susceptible vs
unsusceptible) of a host is expressed as a function of the parasite genotype it harbors and
reciprocally, the phenotype (infective vs uninfective) of a parasite is expressed as a function
of the genotype of the particular host that it enters.

4.2. Variations in host susceptibility and parasite infectivity

According to the pattern of the dose-response curves, the value of the infection rates at
the plateau for 20 Mi/snail was retained to define and compare the level of susceptibility/un-
susceptibility between the snail/schistosome combinations studied and a radar graph
presentation will facilitate such comparisons (Fig. 5 and 6). By the side of the host strains
(Fig. 5), a gradient of increasing multi-parasite susceptibility could be established. Snails of
the BS90 strain appeared as those having the best efficient internal defense system against the
four strains of S. mansoni tested. However we note that within this highly un-susceptible
strain, that was historically considered as "totally resistant" to both new and old world S.
mansoni strains (Ittiprasert and Knight, 2012) half of the B. glabrata snails was unable to
eliminate S. mansoni of the SmLE strain. The BgBAR strain, unsusceptible to SmGUA is
poorly susceptible to SmBRE and SmVEN, but totally susceptible to SmLE. The BgBRE
strain is 100% susceptible to three parasite strains but highly unsusceptible to SmGUA.
BgGUA and BgVEN strains show similar pattern and appear as the most susceptible to all
 9

parasite strains. None of the snail strains used was totally (100%) susceptible to the four S.
mansoni strains respectively (Fig. 5).
Marked variations are also observed as related to the infectivity spectrum displayed by
the parasite strains, ranging from the SmGUA poorly infective or un-infective towards the 5
host strains, to SmLE that is able to infect 100% of the four host strains and half of the
BgBS90 snails (Fig. 6). Except for the BgGUA/SmGUA combination (37% of infection), all
the other homopatric host/parasite couples showed 100% of compatibility. Among the
homopatric combinations it is interesting to note that for the snail stock BgBAR that yields
the highest multi-parasite un-susceptibility level, its corresponding parasite strain (SmLE)
displays on the contrary the highest multi-hosts infectivity level. Such negative correlation
between host susceptibility and parasite infectivity was however not so apparent for the other
snail/schistosome homopatric couples.
We must emphazized that all the S.mansoni and B. glabrata strains used in this study
were maintained in the laboratory during several years and their infectivity and susceptibility
phenotypes are not representative of the corresponding natural populations (Theron et al.,
2008; Bech et al., 2010). However data collected during many years of routine maintenance
of S. mansoni strains in our laboratory do not show significant change in their compatibility
phenotypes after their establishment (personal observation). More precisely, previous
molecular analyses using 15 microsatellite markers carried out on the SmGUA/BgGUA
combination, have revealed that a loss of genetic diversity occured gradually from the field S.
mansoni isolate to the last laboratory generation analysed after three years of cycling (Bech et
al., 2010). What was interesting is the fact that, while the compatibility level decreases
sharply between the field isolate and the first laboratory generation, later the degree of
compatibility does not evolve and remains at the same level during all the successive
laboratory generations despite repeated bottlenecks at each maintenance cycle. Then, a
substantial level of compatibility polymorphism can be maintained independently of the
decrease of neutral genetic diversity (Bech et al., 2010). Such phenomenon could be linked to
recently described mechanisms able to generate high diversification levels in polymorphic
antigens of S. mansoni (Mitta et al., 2012) through somatic recombination events and
regulatory mechanisms occuring during their expression (see below) .

4.3. Possible underlying molecular mechanisms

 10

The co-evolution dynamics between hosts and parasites involves huge reciprocal
selective pressures on both protagonists. This co-evolutionary dynamics leads to an arms race
expected to shape molecular determinants playing a key role at the core of the interaction.
Relatively few reports have evaluated the impact of these reciprocal antagonistic pressures on
the molecules involved in host defense mechanisms and parasite infectivity. Recent molecular
approaches of the mechanisms underlying this compatibility polymorphism have identified a
group of polymorphic antigens (the SmPoMucs ) expressed by the miracidia of S. mansoni
that interact with polymorphic and/or diversified immune recognition molecules (the FREPs)
of B. glabrata (Roger et al., 2008a, b, c; Moné et al., 2011; Hanington et al., 2012; Mitta et
al., 2012). Co-evolutionary dynamics could also play on effector pathways as we
demonstrated a reciprocal adaptation between host reactive oxygen species (ROS) and
parasite ROS scavenger traits (Moné et al., 2011). Indeed, comparison between two
homopatric couples of B. glabrata/S. mansoni with different compatibility level showed that
higher ROS-production capacity by the host was associated with a higher ROS-scavenging
ability by the parasite (highly compatible couple) and in contrast, lower ROS production by
the host with lower ROS-scavenging by the parasite (less compatible combination).
Then compatibility polymorphism in B. glabrata/S. mansoni interaction could be the
result of multigenic and “multi-mechanism” processes in which the relative weight of
recognition mechanisms of the host and antigenic diversity of the parasite could be in balance
with the effector ability (ROS production) of the host and anti-effector mechanism (ROS
scavengers) of the parasite. This hypothesis could be tested in the different strains used in the
present study by analysing the polymorphism occurring on FREPS and SmPoMuc to evaluate
the investment of each strain in these recognition/antigenic variation processes. We could also
envisage more global transcriptomic or proteomic approaches to take into account the
mechanism variability we can have during co-evolutionary processes between these different
populations. This would be facilitated by the use of discreet host phenotypes selected for their
spectrum of susceptibility/un-susceptibility towards several strains of S. mansoni.

4.5. Selection of multi-parasite (un)-susceptible host phenotypes

A snail strain is not homogenous but composed of multiple host phenotypes in respect
of their susceptibility/un-susceptibility towards the diversity of parasite phenotypes, then, the
distribution of these phenotype frequencies within a host strain may be modified by selection.
Most of the studies on variation of snail susceptibility were interested in experimental
 11

selection of host stocks yielding either 0% or approaching 100% infection frequencies when
exposed to only one particular strain of schistosome (Mascara et al., 1999; Richards et al.,
1992; Webster and Woolhouse, 1998). As we know that a host strain un-susceptible to one
parasite strain could be totally susceptible to another one, such an approach gives limited
information about the real efficiency level of its defense system against the diversity of
parasite phenotypes. Based on the results we obtained by exposing different snail strains to
different strains of schistosomes, the new idea would be to select different stocks of snails
showing different spectrum of susceptibility/un-susceptibility vis-à-vis of several parasite
strains. The figure 7 shows how selection patterns could be operated on the different strains of
B. glabrata used in order to produce homogenous snail stocks of different multi-strain (un)-
susceptibility types. Within the BgBS-90 strain, by successive elimination of the snails
susceptible to SmLE, we can select a snail stock with a phenotype (A) characterized by its
high defense efficiency since capable to eliminate four different strains of schistosome.
Eliminating snails susceptible to SmBRE and SmVEN within the BgBAR strain, we can
select another host phenotype (B), un-susceptible to three parasite strains (SmVEN, SmGUA
and SmBRE). Within both, BgGUA and BgVEN strains, elimination of snails susceptible to
SmGUA and SmBRE allows creating two snail stocks of phenotype (C) both un-susceptible
to two parasite strains. In the same way using the BgBRE strain and by successive elimination
of the few snails susceptible to SmGUA, we can select another phenotype (D) un-susceptible
to only one strain of schistosome. Finally with snails of the BgGUA and BgVEN, but in this
case by increasing the frequence of snails susceptible to both SmGUA and SmVEN, we will
select a phenotype (E) with the less efficient defense system since totally susceptible to the
four different strains of schistosomes.

5. Conclusion

Our study through reciprocal cross-infections between five strains of B. glabrata and
four strains of S. mansoni (i.e. 20 different homopatric and heteropatric host-parasite
combinations) confirms the high degree of compatibility polymorphism that occurs in this
host/parasite system. Differences in dose-response curves that characterize each snail stock
(i.e. increase rate of infection at low miracidial doses then level of the asymptote) could result
from the variations of the number and frequence of compatible combinations within each
strain of B. glabrata when confronted to the different strains of schistosomes.
 12

By using the same panel of parasite strains against each snail stock, our results
demonstrate that some B. glabrata strain have defense mechanisms more efficient than other
strains through probably a better capacity to recognize a larger diversity of parasite
phenotypes present within each schistosome strains and perhaps also a higher effector ability
in ROS production. Thus depending on the genotypic diversity in the host and parasite
isolates, the snail-schistosome compatibility would be determined by a combination of
changes and a balance in recognition and effector mechanisms that determine the success or
failure of the infection.
The compatibility polymorphism exists not only at this inter-strain level but also
between individual hosts of a same strain. A snail population is not homogenous with regard
to the susceptibility to schistosome miracidia. Within a strain, each individual host possesses
its specific capacity of defense against all (unsusceptibility) or solely a part (susceptibility) of
the phenotypic diversity of the miracidia from a parasite strain. The same diversity exists also
for the parasite strains with some of then being more infective toward the panel of B. glabrata
stock than other used in this study. Each parasite strain discriminates between the different
individual host phenotypes of a snail strain. This variation of susceptibility between and
within snail strains gives the opportunity, through experimental directional selection, to
develop new and more homogenous snail stocks differing by their multi-parasite (un-)
susceptibility phenotype (i.e., the number of schistosome strains with which there are not
partially, but totally (un-)compatible). This will be a useful tool for future comparative
functional studies conducted on different effectors mechanisms or recognition processes
involving other immune receptors or antigens to better understand the genetics and molecular
basis of the compatibility polymorphism in this host/parasite model.
To conclude and as previously mentioned, we must keep in mind that levels of
compatibility characterizing laboratory strains are not representative of their corresponding
field populations (Theron et al., 2008; Bech et al., 2010). Then we should proceed with
caution to the possibility of extrapolating results of such studies in order to discuss
epidemiological implications as for example, geographical compatibility variations and
colonization risk of new areas or disease spreading that might occur under effects of climate
and global changes (Morley, 2011). However, such laboratory studies on a diversity of
parasite and host strains, provide detailed informations on molecular determinants playing a
key role in the host-parasite compatibility interaction. This may contribute to the search for
molecular markers which might inform on the potential efficiency of the defense system of
 13

snails from a wild population and on the infectivity capacity of a schistosome population
towards snail hosts .

Acknowledgements

The authors are indebted to E. S. Loker (Center for Evolutionary and Theoretical
Immunology, University of New Mexico, Albuquerque, USA) for providing the BS-90 stock
of snails, to Diana Ballen (Instituto Venezolano de Investigaciones Científicas (IVIC),
Caracas, Venezuela) for the snails and schistosomes from Venezuela and G. Oliviera
(Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais, Fundacao Oswaldo Cruz,
Brazil) for the BgBAR and SmLE strains. This work was funded by the ANR (grant # 25402
BiomGenIm; ANR-07-BLAN-0214-03), the CNRS, and the UPVD. The funders had no role
in the study design, data collection, data analysis, decision to publish, or preparation of the
manuscript.
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Legends to Figures

Fig. 1 – Infection rates (% ± 1 standart error) of individual snails of the five Biomphalaria
glabrata strains (BgBRE, BgVEN, BgGUA, BgBAR and BgBS-90) exposed to increasing
doses of miracidia (Mi) from the four strains of Schistosoma mansoni (SmBRE, SmGUA,
SmVEN and SmLE).

Fig. 2 – Infectivity (% of infected snails ± 1 standart error) of four strains of Schistosoma

mansoni (SmBRE, SmGUA, SmVEN and SmLE) towards the five Biomphalaria glabrata
host strains (BgBRE, BgVEN, BgGUA, BgBAR and BgBS-90) exposed to increasing doses
of miracidia (Mi).

Fig. 3 – Evolution of parasite intensities (mean number of mother sporocysts (Msp) per
infected snails ± 1 standart error): A) after exposure of BgBRE snails to increasing doses of
miracidia (Mi) from the four strains of Schistosoma mansoni (SmBRE, SmGUA, SmVEN and
SmLE); B) after exposure of Biomphalaria glabrata snails from the five strains (BgBRE,
BgVEN, BgGUA, BgBAR and BgBS-90) with increasing doses of miracidia (Mi) from the
SmBRE strains of Schistosoma mansoni. Percentages in brackets correspond to infection rates
of the host-parasite combinaison for a dose of 50 Mi/snail.

Fig. 4 – Average transformation rates of miracidia into mother sporocysts for the different
doses of miracidia (Mi) at exposure time: A) for each schistosome strains, all host strains
included; B) for each host strains, all schistosome strains included. Dashed lines represent the
total average for each strain including all miracidial doses (except for 1 Mi).

Fig. 5 – Radar graph representation of the multi-parasite susceptibility phenotypes

characterizing each Biomphalaria glabrata strain (BgBS-90, BgBAR, BgBRE, BgGUA and
BgVEN) vis-à-vis of the four strains of Schistosoma mansoni (SmLE, SmVEN, SmBRE and
SmGUA). Infection rate (%) and parasite intensity (number of mother sporocysts (Msp) per
snail) values are those corresponding to an exposure dose of 20 miracidia/snail.
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Fig. 6 – Radar graph representation of the multi-host infectivity phenotypes characterizing

each strain of Schistosoma mansoni (SmBRE, SmGUA, SmVEN and SmLE) vis-à-vis of the
five Biomphalaria glabrata strains (BgBS-90, BgBAR, BgBRE, BgGUA and BgVEN).
Infection rate (%) and parasite intensity (number of mother sporocysts (Msp) per snail) values
are those corresponding to an exposure dose of 20 miracidia/snail.

Fig. 7 – Selection patterns on each Biomphalaria glabrata strain, to create new discreet host
phenotypes: (A) from the BgBs90 strain, un-susceptible to 4 Schistosoma mansoni strains
(SmBRE, SmGUA, SmVEN and SmLE); (B) from the BgBAR strain, un-susceptible to 3
parasite strains (SmBRE, SmGUA and SmVEN); (C) from the BgGUA and BgVEN strains,
un-susceptible to 2 parasite strains (SmBRE and SmGUA); (D) from the BgBRE strain, un-
susceptible to one parasite strain (SmGUA); (E) from the BgVEN and BgGUA strains,
susceptible to 4 parasite strains ( see text for more details).
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Fig. 8