Ecotoxicology (2011) 20:719–730
DOI 10.1007/s10646-011-0613-5
Impact of microcystin-producing cyanobacteria on reproductive
success of Lymnaea stagnalis (Gastropoda, Pulmonata)
and predicted consequences at the population level
Emilie Lance • Frederic Alonzo • Marion Tanguy
Claudia Gérard • Myriam Bormans
•
Accepted: 9 February 2011 / Published online: 22 February 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Our previous studies showed that microcystin
(MC)-accumulation in the gastropod Lymnaea stagnalis
and effects on life-history traits (survival, growth, and
fecundity) varied according to age, exposure pathway
(MC-producing cyanobacteria or dissolved MC), and
presence or not of additional non-toxic food. This study
investigated effects of exposure to MC-producing cyanobacteria or to dissolved MC of parent and of parent and egg
masses of L. stagnalis on hatching success, duration of
embryonic development and neonate survival. Secondly,
the potential impact of MC-producing cyanobacterial proliferations (blooms) on L. stagnalis population growth,
depending on bloom frequencies and recovery duration of
life traits after exposure, was evaluated using a modelling
approach. Experimental results showed that embryonic
development was shortened in case of parent exposure to
toxic cyanobacteria. Parent and eggs exposure to dissolved
MC extended embryonic development and reduced hatching percentage, suggesting a permeability of egg masses to
E. Lance (&) C. Gérard M. Bormans
UMR CNRS 6553 ECOBIO, Université de Rennes 1, 263
Avenue du Général Leclerc CS 74205, 35042 Rennes, France
e-mail: [email protected]
F. Alonzo
IRSN/DEI/SECRE/LME, Centre de Cadarache, Bat 159, BP3,
13115 Saint-Paul-Lez-Durance cedex, France
M. Tanguy
Department of Pathology and Microbiology, University of Prince
Edward Island, 550 University Avenue, Charlottetown, PE C1A
4P3, Canada
M. Tanguy
Laboratoire d’Ecotoxicologie, Université Le Havre, BP 540,
76058 Le Havre cedex, France
MC. Whatever exposure, neonate survival was reduced.
Neonates exposed to cyanobacteria accumulated MCs 24 h
after hatching, suggesting very early cyanobacteria ingestion. Modelling results showed that L. stagnalis population
growth was influenced by the recovery time of life-history
traits after exposure. When setting the latest at 6 weeks
according to previous experiments, a frequency of one to
four blooms per year strongly affected population dynamics and induced up to a 80-weeks delay compared to control in time required for populations to grow from 1 to 1000
individuals. Results are discussed in terms of impact of
intoxication pathways on parents, eggs and neonates, and
on population dynamics of L. stagnalis.
Keywords Cyanobacteria Microcystins Gastropods Fitness Population dynamics
Introduction
Biotic and abiotic stresses (e.g., parasitism, pesticides,
heavy metals) are known to frequently affect the fecundity
of gastropods and their progeny (e.g., hatching, survival),
as demonstrated for lymnaeid pulmonates (e.g., Singh and
Agarwal 1986; Gomot 1998; Russo and Lagadic 2004;
Leung et al. 2007; Coutellec et al. 2008; Pietrock et al.
2008). Microcystins (MCs), hepatotoxins produced by
cyanobacteria, are cyclic heptapeptides of which 80
structural variants have been identified differing by two
variable L-amino acids (e.g., MC-LR for leucine and arginine). MCs have been associated with acute and subacute
adverse effects in various aquatic and terrestrial organisms
(for review: Wiegand and Pflugmacher 2005). Previous
studies on the hermaphroditic oviparous Lymnaea stagnalis
showed a significant decreased fecundity during exposure
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to MC-producing cyanobacteria (Lance et al. 2007) and
dissolved MC-LR (Gérard et al. 2005). Due to accumulation of free (Zurawell et al. 1999; Gérard et al. 2005; Lance
et al. 2006) and covalently bound (Lance et al. 2010a, b)
MCs in L. stagnalis, one can expect a MC-transfer to the
progeny during vitellogenesis and oogenesis with potent
deleterious effects. Zhang et al. (2007) demonstrated a
MC-transfer from females to their embryos in the ovoviviparous gastropod Bellamya aeruginosa. Exposure of the
ovoviviparous prosobranch Melanoides tuberculata to
dissolved cylindrospermopsin (CYN), a cyanobacterial
hepatotoxin, induced an increase in the number of hatchlings released from parents, whereas a decrease was
observed after exposure to the CYN-producing cyanobacteria (Kinnear et al. 2007). Moreover, egg masses and
neonates originating from MC-intoxicated parents are
probably exposed to toxic cyanobacteria and to dissolved
toxin in the medium. Therefore, determining whether this
further MC-exposure may increase toxic effects remains
relevant. Zurawell (2001) did not demonstrate any impact
on survival of L. stagnalis embryos following egg exposure
to dissolved MC-LR (up to 10 lg L-1), but suggested that
higher MC concentrations could induce a toxin penetration
in egg masses. Chorion (i.e., outer membrane which surrounds the embryo) of fish eggs appears to be resistant to
MC penetration (Lecoz et al. 2008; Oberemm et al. 1999)
except for high exposure concentrations ([1 mg L-1)
resulting in deleterious effects (Wiegand et al. 1999).
We investigated adult L. stagnalis exposure to
MC-producing (10 lg L-1) cyanobacteria Planktothrix
agardhii or to dissolved MC-LR (33 lg L-1), and its
impact in terms of MC accumulation and negative effects
on fecundity, egg hatching (kinetics and rate), and neonate
survival. MC concentrations used were environmentally
relevant [e.g., respectively from 0.3 to 15 lg intracellular
MCs L-1 in French lakes (Briand et al. 2008; Lance et al.
2010c; Sabart et al. 2010), and concentration of dissolved
MCs varying from 0 to 140 lg L-1 over the world after
bloom collapse (Chorus and Bartram 1999; Zurawell et al.
1999; Hyenstrand et al. 2003)]. Adult exposure was followed or not by a further exposure of egg masses and
neonates. The discussion focuses on several hypotheses to
explain the negative effects on progeny according to the
intoxication pathway: (i) before oviposition, a MC-intoxication of oocytes and/or of fertilized eggs during egg
masses formation, (ii) after oviposition, a MC entrance into
egg masses, and (iii) after birth, a MC uptake by neonates
via cyanobacteria ingestion or dissolved MC exposure.
This study also aims to investigate potential impact of
chronic exposure to proliferations (blooms) of toxic cyanobacteria on population dynamics of L. stagnalis by
modelling adverse effects at the individual level (survival,
growth and reproduction) observed in the present study and
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E. Lance et al.
in a previous experiment (Lance et al. 2007). Models
simulated impact on L. stagnalis population growth over
3 years via two sets of simulations: (1) a low contaminated
medium (i.e., from one to three 3-week cyanobacterial
bloom(s) per 3-year) with a recovery time of life-traits after
exposure ranging from 1 to 70 weeks (i.e., snail lifespan),
and (2) a highly contaminated medium (i.e., from one to
four 3-week cyanobacterial bloom(s) per year) with a
6-week recovery of life-traits after exposure as extrapolated from Lance et al. (2007). The discussion focuses on
comparison with negative impact of proliferations of
MC-producing cyanobacteria on gastropod populations
observed in the field (Gérard et al. 2008, 2009; Lance et al.
2010c).
Materials and methods
Biological material
L. stagnalis adults were obtained from a laboratory population in the Experimental Unit of the Institut National de
Recherche en Agronomie (U3E INRA, Rennes). Prior to
the experiment, individuals (25 ± 3 mm shell length) were
isolated in glass containers filled with 35 mL of dechlorinated water, acclimated to the experimental conditions
(12/12 L/D, 20 ± 1°C) and fed on pesticide-free lettuce for
7 days. The filamentous cyanobacterium Planktothrix
agardhii (strain PMC 75-02) was cultured in 5-L flask and
suspensions were diluted to obtain a concentration of 10 lg
MC-LR equivalents (MC-LReq) per litre, measured by
HPLC as described in Lance et al. (2006). For dissolved
MC-exposure, MC-LR was obtained from Alexis Corporation (USA) and solubilized with 0.1% MeOH in
dechlorinated water for final MC-LR concentrations of
33 lg L-1.
Experimental set up
During 3 weeks, four groups of 40 snails individually
maintained in glass containers were submitted to varying
treatments: «control»(dechlorinated water and lettuce),
«cyano» (MC-producing cyanobacterial suspension),
«cyanolet» (MC-producing cyanobacterial suspension with
lettuce), «D33let» (MC-LR dissolved in dechlorinated water
with lettuce). These groups were divided in two sub-groups
according to further treatments on egg masses and neonates.
Egg masses were sampled every day, and maintained without
adult either in the adult contaminated medium (‘‘cyano/
cyano’’, ‘‘cyanolet/cyanolet’’, ‘‘D33let/D33let’’) or in
dechlorinated water (‘‘cyano/water’’, ‘‘cyanolet/water’’,
‘‘D33let/water’’). Media were renewed twice a week.
Impact of microcystin-producing cyanobacteria
MC accumulation in adult and neonate snails, in egg
masses and faeces
MC content was measured every week in two individuals
per treatment, in their egg masses and faeces, and in all
neonates from 2 egg masses at 24 h, 10 and 15 days after
hatching. MCs were extracted with 2 mL of 100% methanol. Each tissue was crushed in 1 mL of 100% MeOH and
then crushed again after 12 h at 4°C with 1 mL MeOH
added. Analysis by immuno-assay was performed as
described in Lance et al. (2006) with an ELISA Microcystin Plate Kit (Envirologix INC) with detection threshold of
0.05 lg L-1 and to the nearest 0.01 lg L-1 (Gilroy et al.
2000). All microcystins of the P. agardhii strain used were
detected and expressed in MC-LReq using MC-LR, given
by the supplier as standard. The accumulation in snail
bodies were calculated by taking into account extraction
recovery and possible matrix-induced signal enhancement
or suppression with the ELISA test, because of unspecific
binding to and/or denaturing of the antibodies. Control
snails, free of MCs, were freeze-dried and homogenized in
a mortar, spiked with MC-LR standard (5 lg g-1) (Lance
et al. 2006). The extraction was performed as described
previously and the recovery for the extraction was calculated. The matrix effect was checked by spiking control
snails with MC-LR standard (5 lg g-1) and the response
was compared to 100% methanol spiked with the same
amount. The average recovery was 74 ± 2.8% and matrix
effect was negligible (from 0.9 to 7.5% of differences
between matrix and methanol results).
Evaluation of the impact of MC exposure
on L. stagnalis reproductive success
Number of egg masses and eggs per mass: we did not
sample egg masses laid during the first week (week 0)
because ovum and spermatozoid maturation and fecundation probably occurred before the beginning of the intoxication. Thereafter, egg masses were sampled every day and
eggs were quantified. The index d0 represents the day of
laying, d1 the day of hatching of the first egg and df the last
day of hatching (= hatching of the maximal number of
eggs).
Hatching percentage at day n, hatch(dn), was calculated
each day from d1 to df as follows:
hatch(dn) = Number of individual hatched at dn/Number
of eggs at d0
The maximal hatching percentage corresponds to
hatch(df). The hatching duration corresponds to the number
of days between d1 and df. The duration of embryonic
development, Tegg, is the number of days between d0
and df.
721
The percentage of survival of neonate at n = 5, 10 and
15 days after df, Sneonate(df?n), was expressed as follows:
Sneonate(df?n) = Number of neonates surviving at df?n/
Number of neonates hatched at df
Modelling dynamics of L. stagnalis population
Parameters obtained from laboratory experiments
Parameter values used for simulation of L. stagnalis population growth were taken from laboratory exposures of
eggs, neonates, juveniles and adults to MC-producing (5
and 10 lg L-1) cyanobacteria with lettuce (this study;
Lance et al. 2007). We made the environmentally relevant
assumption that non toxic food sources for gastropods (e.g.,
phytoplankton, periphyton, detritus) (Dillon 2000) were
present in the medium during cyanobacterial proliferation.
According to this study, duration of embryonic development Tegg was set to 3 weeks in simulations for exposed
and control snails. Weekly egg survival rates (Segg) was
derived from hatch (df) as follows:
Segg ¼ ðhatchdf Þ1=Tegg :
Survival of neonates (from hatching to 2 weeks of age) per
week (Sneonate) was derived from Sneonate(df?15) as follows:
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Sneonate ¼ Sneonate ðdfþ15 Þ:
Juvenile and adult growth rates gjuv and gad (mm per week)
and fecundity rate f (eggs per week, taking account of the
proportion of reproducing adults) were taken from Lance
et al. (2007). Briefly, we investigated the effect of
consumption of MC-producing (5 lg MC-LReq L-1)
P. agardhii with lettuce on survival, growth and
fecundity of L. Stagnalis. Juveniles and adults (14 ± 1
and 25 ± 1 mm shell length respectively) were 5-weeks
exposed to P. agardhii, and further 3-weeks maintained in
safe medium. Growth and fecundity parameter values in
the third week of depuration significantly differed from
those measured during the two first weeks (Lance et al.
2007). As snails tended to recover during the third week in
safe medium, two periods of depuration (early depuration
and late depuration) were distinguished for simulations.
We assumed that shell length at birth Lb and at maturity
Lm were not affected by cyanobacterial exposure and were
set at respectively 1.25 and 25 mm (Zotin 2009; Koene
et al. 2007). Growth and reproduction rates used in
simulations (Lance et al. 2007) were lower than those
reported by Coutellec and Lagadic (2006) and Coutellec
et al. (2008). These parameter values applied continuously
during juvenile and adult stages lead to an age at maturity
of 34 weeks and to an average life-time fecundity of
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E. Lance et al.
approximately 500 eggs snail-1, which are close to those
observed in natural conditions (Berrie 1965; Brown 1979;
Dillon 2000; Zotin 2009). We assumed that there were
appropriate to simulate impact of toxic cyanobacteria on
population dynamics of L. stagnalis. Generation time of L.
stagnalis, i.e., the time it takes for freshly deposited eggs to
grow to reproducing adults, is then set to 34 weeks.
Because juvenile and adult survivals were not affected by
exposure to toxic cyanobacteria (Lance et al. 2007), we
used age-specific survival rates based on data measured
under constant culture conditions by Janse et al. (1988).
Survival data were fitted to a modified Weibull function as
follows:
R(t) ¼ exp[ ln2 ac tc where R(t) is the proportion of survivors at age (t). Agespecific survival rates S(t) were calculated as follows:
S(t) ¼ 1 ln2 cac tc1
where a is the age at 50% survival and c is the rate of
decrease of S(t) with age. Parameters a and c estimated by
Janse et al. (1988) were respectively 45.14 weeks and 2.90.
Simulating exposure of L. stagnalis to MC-producing
cyanobacterial blooms
The models simulate growing L. stagnalis populations
exposed to successive blooms of MC-producing cyanobacteria at different frequencies over a 3-year period. A first set
of simulations S1 considers a low contaminated medium
where blooms occur at yearly intervals: once, twice or thrice
over the 3 years. Each bloom of MC-producing cyanobacteria lasts for 3 weeks and is followed by the same succession
of exposure conditions, including 2 weeks of early depuration and a period of late depuration after which snails return
to the control condition. Parameters for each exposure
condition (bloom, early depuration, late depuration, unexposed) are presented above and in Table 1. In Lance et al.
(2007), snails had not fully recovered control values after the
3-week depuration period and the time required to do so is
not known. In order to test its impact on population
dynamics, duration of late depuration is simulated ranging
from 1 to 70 weeks [i.e., a snail lifespan (Janse et al. 1988)],
in increment of 1 week between 1 and 10 weeks and in
increment of 5 weeks from 10 to 70 weeks. A second set of
simulations S2 considers a highly contaminated medium
where one to four successive blooms occur each year at
6-week intervals, as observed in the Grand Lieu Lake, France
(Lance et al. 2010c). After the last bloom, late depuration is
set to 4 weeks before returning to control condition. This
extrapolated value corresponds to the time required to fully
recover if snails followed the trend observed between the two
first and the third week of depuration, as observed in Lance
et al. (2007).
Population models
Population models, performed using the Software MATLAB R2008a (MathWorks, France), describe a theoretical
population with no seasonal change in survival, growth and
reproduction rates. They assume a closed system with no
immigration or emigration, no density-dependence, no
competition, parasitism or predation pressure. Population
models are structured per age classes, where individuals
advance from one age class to the next at discrete, equidistant time intervals Dt = 1 week. The age and size distributions of the population are respectively described by
ni(t) and li(t), the number and size of individuals of age i at
time t. Models were constructed with 73 one-week stages
including egg, early life, juvenile and adult stages. Snails at
an age i [ 73 weeks are removed from the population, i.e.,
they die of old age (Janse et al. 1988).
Table 1 Exposure conditions of model simulations and corresponding mean (?SE) weekly parameter values according to treatments performed
to L. stagnalis juveniles and adults (Lance et al. 2007) and to L. stagnalis adults followed by eggs/neonates exposure (this study)
Exposure conditions
X
Growth (mm week-1)
Unexposed
0.73 ± 0.05a
b
Bloom
Early depuration
Late depuration
Fecundity
(eggs week-1)
fX
Survival rate (week-1)
0.23 ± 0.03a
20.71 ± 3.53a
0.94 ± 0.03e
0.93 ± 0.02e
0.05 ± 0.01
b
b
f
0.78 ± 0.04f
0.01 ± 0.00
c
g
0.91 ± 0.05g
0.07 ± 0.02
d
e
0.93 ± 0.02e
Adults
gad,X
Juveniles
gjuv,X
0.32 ± 0.07
c
0.07 ± 0.01
d
0.22 ± 0.02
10.38 ± 2.31
c
4.48 ± 0.85
d
7.63 ± 1.26
Eggs
Segg,X
Neonates
Sneonate,X
0.88 ± 0.03
0.92 ± 0.06
0.94 ± 0.03
Correspondence between parameter values and exposure treatments are indicated via superscripted letter
see ‘‘Simulating exposure of L. stagnalis to MC-producing cyanobacterial blooms’’ section for survival rate from 3 weeks to death
a–d
Study of Lance et al. (2007) with treatments: acontrol clean water and lettuce; bcyanolet MC-producing cyanobacteria with lettuce,
depuration clean water and lettuce; cduring 2 weeks after exposure and dduring third week after exposure
e–g
This study with treatments: econtrol clean water and lettuce for parents and eggs and neonates, fcyanolet/cyanolet exposure of parents to MCproducing cyanobacteria and eggs and neonates further exposed, gcyanolet/water exposure of parents and eggs/neonates in clean water
123
Impact of microcystin-producing cyanobacteria
At t0, simulations are started with a control population in
asymptotic state (i.e., stable population growth rate and age
structure) and a size of 1 snail, as follows:
X
ni ð t 0 Þ ¼ 1
i
where ni (t0) are obtained using the right eigenvector to the
dominant eigenvalue of a Leslie matrix (Caswell 2001)
filled with survival and reproduction rates of control snails.
Size structure at t0 is determined in agreement with growth
of control snails, e.g., li¼4 ðt0 Þ ¼ lb at hatching, and sexual
maturity reached 34 weeks after hatching with
li¼37 ðt0 Þ ¼ lm :
(
liþ1 ðt0 Þ ¼ li ðt0 Þ þ gjuv;control if 4 i\37
liþ1 ðt0 Þ ¼ li ðt0 Þ þ gad;control if i 37
where gjuv and gad are growth rates in mm week-1.
Exposure condition Xi of age class i changes over time
depending on the simulated scenario. Over one time step, the
number of individuals in each age class is calculated as
follows:
8
< niþ1 ðt þ 1Þ ¼ ni ðtÞ Si;Xi
X
ni ðtÞ fi;Xi
: n1 ðt þ 1Þ ¼
i
where Si;Xi and fi;Xi are the survival and fecundity rates of
age class i under exposure condition Xi , with:
8
>
< Si;Xi ¼ Segg;Xi if 1 i\4
Si;Xi ¼ Sneonate;Xi if 4 i\6
>
:
Si;Xi ¼ 1 ln2cac ic1 8Xi if i 6
Eggs produced by all age classes are summed to calculate
the number of individuals of age i = 1. Snails start
reproducing when their sizes reach lm :
(
fi;Xi ¼ 0 if li ðtÞ\lm
fi;Xi ¼ fXi
if li ðtÞ lm
After hatching, sizes of individuals increases over time
following a bilinear growth function:
8
>
< l 4 ð t þ 1Þ ¼ l b
liþ1 ðt þ 1Þ ¼ li ðtÞ þ gjuv;Xi if li ðtÞ\lm
>
:
liþ1 ðt þ 1Þ ¼ li ðtÞ þ gad;Xi if li ðtÞ lm
Models calculate weekly changes in total population
P
number NðtÞ ¼ i ni ðtÞ. Population growth rates k are
calculated per week:
kðtÞ ¼
Nðt þ 1Þ
NðtÞ
and their geometric mean value k3y estimated over the
3-year simulation. k indicates the population persistence:
723
k [ 1, k \ 1 and k = 1 respectively indicates a growing, a
declining population and no change in population size
through time. Models calculate the time s1000 for the
population size to be multiplied permanently by 1000.
Difference in s1000 between an exposed and the control
population is defined as the delay-in-population-growth
index (Wennergren and Stark 2000).
Uncertainty on simulated values
Uncertainties on N ðtÞ; kðtÞ; s1000 and k3y were obtained using
a bootstrap method. Data (gjuv,X, gad,X, fX, Segg,X, Sneonat,X) for
each exposure condition X (unexposed, bloom, early depuration, late depuration) were simulated by randomly sampling values among raw observations (Table 1). Simulated
dataset was used to calculate N ðtÞ; kðtÞ; s1000 and k3y as
described previously. Distributions, mean values and 95%confidence intervals were built based on 5,000 repetitions of
this procedure. Difference in k3y or s1000 was considered
significant between simulations A and B when 0 was not
included in the confidence interval for the 5000 differences
k3y ð AÞ k3y ðBÞ or s1000 ð AÞ s1000 ðBÞ.
Data analysis
Experimental data did not follow a normal distribution
(according to the Kolmogornov–Smirnov test) and were
thus analysed for differences between treatment groups
using the Kruskall–Wallis (KW) test and 2 by 2 treatment
groups using (1) the Mann–Whitney U-test for the numbers
of egg per adult and the hatching delay, (2) the v2 test for
percentage of laying adult, rates of hatching and of neonate
survival at 5, 10 and 15 days. Differences were considered
as significant at p \ 0.05. Statistical analyses of data were
performed using the Software Statistica 7.0 (Stat Soft,
France). Data are reported as mean ± standard error
(±SE).
Results
MC in L. stagnalis adults and their faeces, egg masses
and in neonates
Accumulation in adult snails was higher after exposure to
toxic P. agardhii than after MC-LR exposure (respectively
57.34 ± 0.20 and 31.81 ± 0.89 lg g-1 DW in ‘‘cyano’’
and ‘‘cyanolet’’ group vs 0.10 ± 0.03 lg g-1 DW in
‘‘D33let’’ group). MCs were also detected in faeces of
intoxicated snails, in a higher concentration for the
‘‘cyano’’ group (1.39 ± 0.07 lg MC g-1 DW) versus
‘‘cyanolet’’ (0.20 ± 0.01 lg MC g-1 DW) and ‘‘D33let’’
123
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E. Lance et al.
(0.16 ± 10-3 lg MC g-1 DW). No MC was detected in
egg masses, in neonates originated from intoxicated adults
and hatched in free MC water, and in neonates hatched in
dissolved MC-LR. However, neonates accumulated MCs
from the first day after their birth when they were continuously exposed to toxic cyanobacteria: respectively
0.18 ± 0.05 and 0.33 ± 0.08 lg g-1 DW after 24 h of life
in the ‘‘cyanolet/cyanolet’’ and ‘‘cyano/cyano’’ groups, and
up to 0.50 ± 0.15 lg MC g-1 DW after 15 days in the
‘‘cyano/cyano’’ group.
Control
The weekly percentage of laying adults significantly varied
among treatment groups (p \ 0.05). Control snails showed
a significantly higher percentage of individuals laying (on
average 32.52 ± 5.21%) than snails exposed to toxic
P. agardhii with lettuce (on average 25.04 ± 3.74%), to
dissolved MC with lettuce (on average 8.82 ± 3.01%)
or to P. agardhii alone (on average 4.86 ± 2.74%) (all
p \ 0.05). The average number of eggs per individual
and per week was significantly lower in ‘‘cyano’’
(3.60 ± 0.75), ‘‘D33let’’ (5.89 ± 1.48) and ‘‘cyanolet’’
(14.65 ± 2.74) groups compared to control (23.43 ± 2.98)
group (all p \ 0.05).
Exposed parents+offspring
Exposed parents
Egg hatching after exposure to dissolved or intracellular
MCs
to cyanobacteria
to cyanobacteria
to cyanobacteria+ lettuce
to cyanobacteria+ lettuce
to dissolved MC-LR + lettuce
to dissolved MC-LR + lettuce
Parental exposures
100
percentage of hatching
Fecundity of L. stagnalis exposed to dissolved
or intracellular MCs
The maximal hatching percentage, hatch(df), was similar
between all groups with a mean of 82.10 ± 1.21% (Fig. 1,
p [ 0.05). The time between oviposition and the end of
hatching [at hatch(df)] as well as the duration of hatching
process were similar for eggs from controls and from snails
exposed to toxic cyanobacteria with lettuce or to dissolved
MC (Fig. 1, Table 2, p [ 0.05). However, egg masses laid
by snails exposed to toxic cyanobacteria alone reached
hatch(df) significantly earlier than controls, with longer
duration of the hatching process that started earlier (Fig. 1,
Table 2, all p \ 0.05).
80
60
40
20
0
0
5
10
15
20
25
days after laying
Fig. 1 Cumulated hatching percentage (% ± SE) of L. stagnalis
according to treatments: controls, exposed parents or exposed parents
and offspring to MC-producing P. agardhii, MC-producing
P. agardhii with lettuce, or dissolved MC-LR with lettuce
Table 2 Duration of embryo development and hatching, and neonate
survival (% ± SE) according to treatments to L. stagnalis adults
(MC-LR with lettuce, MC-producing P. agardhii without or with
lettuce) followed by eggs/neonates exposure to safe medium
Treatments
Time (days)
between d0 and d1
Control
13
D33let/water
Cyanolet/water
Parental and egg masses exposures
The maximal hatching percentage was significantly
reduced by further exposure of egg masses to dissolved MC
(respectively D33let/water, cyano/water and cyanolet/water) or by
eggs/neonates exposure to adult treatments (respectively D33let/
D33let, cyano/cyano and cyanolet/cyanolet)
Time (days)
between d0 and df
Neonate survival
at 15 days
7
19
88.15 ± 1.15
12
7
18
87.67 ± 1.24
11
8
18
82.90 ± 1.07
6
11
16
70.59 ± 1.41
D33let/D33let
12
14
25
77.78 ± 2.94
Cyanolet/cyanolet
Cyano/cyano
12
11
9
11
20
21
60.42 ± 1.68
33.80 ± 9.82
Cyano/water
Time (days)
between d1 and df
The index d0 represents the day of laying, d1 the day of hatching of the first egg and df the last day of hatching (= hatching of the maximal
number of eggs)
123
Impact of microcystin-producing cyanobacteria
725
compared to parent exposure alone (i.e., 63.25 ± 7.18%
for ‘‘d33let/d33let’’ vs 83.35 ± 5.77% for ‘‘d33let/water’’)
(Fig. 1, all p \ 0.05). Further exposure of eggs to dissolved
MC significantly extended the hatching duration that started at the same time but ended significantly later (Fig. 1,
Table 2, all p \ 0.05). Exposures of eggs to toxic cyanobacteria after parent exposure tended to reduce the maximal hatching percentage compared to parent exposure but
not significantly (Fig. 1, p [ 0.05). Further egg exposure to
toxic cyanobacteria, with or without lettuce, did not modify
the hatching duration compared to parent exposures only
(Fig. 1, Table 2, all p [ 0.05). However, exposure of egg
masses to cyanobacteria alone delayed the hatching that
started and ended significantly later (Fig. 1, Table 2, all
p \ 0.05).
The survival at 5, 10 and 15 days of neonates from adults
exposed to dissolved MC or to cyanobacteria with lettuce
were similar to that of neonates from control snails (Fig. 2,
all p [ 0.05). However, survival of neonates from snails
exposed to toxic cyanobacteria alone was lower compared
to controls (Fig. 2, Table 2, p \ 0.05). The continuous
presence of dissolved MC-LR or of toxic cyanobacteria in
the medium of egg masses and of neonates reduced neonate
survival at 5, 10 and 15 days (Fig. 2, Table 2, all
p \ 0.05).
Control
Exposed parents+ offspring
percentage of survival of neonates
to cyanobacteria
to cyanobacteria
to cyanobacteria + lettuce
to cyanobacteria + lettuce
to dissolved MC-LR +lettuce
to dissolved MC-LR +lettuce
The number of alive 15 days old progeny per L. stagnalis
adult and per week was significantly lower when adult
snails have been exposed to toxic cyanobacteria without
(2.14 ± 0.38) or with lettuce (9.50 ± 2.81) and to dissolved 33 lg MC-LR L-1 (4.30 ± 0.72) vs control snails
(17.01 ± 3.54) (all p \ 0.05). Further exposure of egg
masses and neonates to toxic cyanobacteria alone or dissolved MC after parental exposure significantly decreased
the number of alive 15 days old progeny per adult snail:
0.90 ± 0.21 for ‘‘cyano/cyano’’ and 2.89 ± 0.84 for
‘‘d33let/d33let’’ (all p \ 0.05).
Effect of MC-producing cyanobacteria on population
growth of L. stagnalis
Neonate survival after exposure to dissolved
or intracellular MCs
Exposed parents
Reproductive success after exposure to dissolved
or intracellular MCs
100
90
80
70
60
Impact of bloom frequency and recovery time
on population growth in a low contaminated medium
Low contaminated medium corresponds to a frequency
from one to three blooms over 3 years. In our simulations,
control populations grew at a rate of 1.13 week-1. Exposure to MC-producing cyanobacteria significantly reduces
population growth rate (k). Average k over the simulated
3-year period decreases with increasing bloom frequency
and depuration time (Fig. 3). At the lowest bloom frequency (a single bloom over the 3 years), difference from
the control becomes significant (p \ 0.05) when snails take
more than 3 weeks after the end of toxic exposure to
recover control life-history parameters (Fig. 3). At higher
bloom frequency, average k is significantly reduced independent of depuration time. Even at fast 1-week recovery,
the reduction of k3y values predicted in population exposed
to two or three blooms over the 3 years are statistically
significant (p \ 0.05). Increasing depuration time affects
population growth rate to maximum reduction values of
2.12, 3.89 and 4.95% respectively with one, two and three
blooms over the 3 years. Those maximum effect levels are
reached respectively for depuration times of 11, 22 and
20 weeks above which, greater depuration times do not
cause further significant decrease in k3y (Fig. 3).
50
Impact of bloom frequency on population growth
in a highly contaminated medium
40
30
5
10
15
days after hatching
Fig. 2 Neonate survival (% ± SE) of L. stagnalis at an age of 5, 10
and 15 days in control, exposed parents or exposed parents and
offspring to MC-producing P. agardhii, MC-producing P. agardhii
with lettuce, or dissolved MC-LR with lettuce
Highly contaminated medium corresponds to a frequency
from one to four blooms per year over 3 years. Succession
of several blooms every year has significant effect on
predicted population size, with difference from the control
ranging over two to four orders of magnitude at the end of
123
E. Lance et al.
Population growth rate (per week)
over three years
726
1.13
1.13
A 1.13
1.12
1.12
1.12
1.11
1.11
1.1
1.1
1.1
1.09
1.09
1.11
A
B
1.09
C
1.08
1.08
1.08
1.07
1.07
1.07
1.06
1.06
1.06
1.05
0
10
20
30
40
50
60
70
1.05
A
0
10
20
30 40
50
60
1.05
0
70
D
10
20
30 40
50
60
70
Depuration time (weeks)
Fig. 3 Mean population growth rate (week-1) over 3 years
k3y depending on exposure scenario: control (A), one (B), two
(C) and three (D) bloom(s) of MC-producing cyanobacteria per
3-year] and on duration of late depuration (from one to 70 weeks for
snails to recover control life history parameters after exposure to
cyanobacterial blooms). 95% confident intervals are indicated in
dotted lines
the 3-year period (Fig. 4a). During exposure to MC-producing cyanobacteria, weekly population growth rate
remains at a value of *1.0 week-1 (e.g., the population
does not grow) for time periods which depend on the
number of successive blooms before reaching control value
(Fig. 4b). A transitory peak of growth (greater than control)
is observed when exposed juvenile cohorts reach maturity
with delay and reproduce concomitantly with unexposed
cohorts (Fig. 4b). Average population growth rate over the
3 years is significantly reduced compared to the control
tending towards the value of 1.0 week-1 with increasing
number of successive blooms per year (Fig. 5). The time
s1000 for the population size to be multiplied by 1000 is
significantly increased compared to controls in all exposure
conditions (Table 3). The delay ranging from 20 to up to
80 weeks, which is greater than two generation times in
case of four blooms per year (Table 3).
Discussion
8
indirect energy trade-offs induced by higher
energy demand for coping with toxic stress. For
a similar ingestion of toxic cyanobacterial cells
A
a
B
C
D
E
Log N
4
2
0
0
20
40
60
80
100
120
140
160
Weeks
1.4
b
1.4
1.2
A
1
0
1.2
E
1
40
80
Weeks
123
(1)
6
Population growth
rate (per week)
Fig. 4 a Dynamics of L.
stagnalis population (log N,
N = number of individuals)
over three years and b weekly
population growth rate k
(week-1) depending on
exposure scenario [control (A),
one (B), two (C), three (D) and
four (E) bloom(s) of MCproducing cyanobacteria per
year] with 4 weeks of late
depuration. 95% confident
intervals are indicated in dotted
lines
Intoxication of freshwater gastropods by intracellular or
dissolved MCs and its impact at the individual (e.g., lifehistory traits) and sub-individual (e.g., MC accumulation,
histopathology) levels have been recently investigated in
the laboratory (Zurawell 2001; Gérard and Poullain 2005;
Gérard et al. 2005; Lance et al. 2006, 2007, 2008, 2010a,
b). This and previous experiments reported stronger impact
on fecundity in L. stagnalis exposed to MC-producing
cyanobacteria alone vs exposed to MC-producing cyanobacteria with lettuce or to dissolved MC. This could be
explained by:
120
160
0
40
80
Weeks
120
160
Impact of microcystin-producing cyanobacteria
Frequency among
5000 bootstrapped simulations
0.3
727
E
D
C
B
A
0.2
0.1
0
1.05
1.07
1.09
1.11
1.13
Population growth rate (per week) over three years
Fig. 5 Distribution frequency among 5000 repetitions of bootstrapping of mean population growth rate over 3 years k3y depending on exposure
scenario [control (A), one (B), two (C), three (D) and four (E) bloom(s) of MC-producing cyanobacteria per year] with 4 weeks of late depuration
Table 3 Mean (?SE), among 5000 repetitions of bootstrapping,
s1000 (weeks) (i.e., time for the population size to be multiplied by
1000), and delay in population growth index (i.e., difference between
s1000 of exposed snails and of controls relative to generation time) of
L. stagnalis, depending on exposure scenario [control; one, two, three
and four bloom(s) of MC-producing cyanobacteria per year with
4 weeks of late depuration]
s1000
Delay in population
growth relative to
generation time
Control
58.98 ± 1.52
x
One bloom per year
79.87 ± 1.52*
0.61
Two blooms per year
90.01 ± 1.35*
0.91
Three blooms per year
100.49 ± 1.53*
1.22
Four blooms per year
133.32 ± 8.59*
2.18
*significant differences (p \ 0.05) compared to controls
(2)
(Lance et al. 2006), the concomitant ingestion of lettuce
would allow both MC detoxification (e.g., Pflugmacher
et al. 1998; Wiegand et al. 1999; Setlikova and
Wiegand 2009) and life trait maintaining.
direct toxic effect of MCs on gonadic cells. The genital
gland is known to be the second major site of MC
accumulation in gastropods after the digestive gland
(Chen et al. 2005; Xie et al. 2007; Zhang et al. 2007).
Accumulation of bound MCs in the genital gland of
L. stagnalis exposed to intracellular or dissolved MCs
have been recently reported (Lance et al. 2010b). After
ingestion and disruption of cyanobacterial cells in the
gizzard of gastropods, a fraction of released MCs can
enter the digestive gland for being accumulated or
excreted in the faeces (Carriker 1946; Zurawell et al.
2006). Here, we demonstrate the presence of MCs in
tissues (up to 57.34 ± 0.20 lg g-1 DW) and faeces
(up to 1.39 ± 7.10-2 lg g-1 DW) of L. stagnalis after
ingestion of toxic cyanobacteria. Processes by which
MCs pass to the genital gland are unknown but most
likely involve haemolymph transport. Genital glands
of snails exposed to dissolved MCs were less
MC-contaminated than those of snails exposed to toxic
cyanobacteria (Lance et al. 2010b) and impact on
fecundity was twice lower but remained severe compared to the controls. Intoxication by dissolved MCs
can occur through oral water intake [8–12 ll g-1 h-1
for L. stagnalis (De Witt 1996)] but also through
penetration via epidermal cells, particularly where
tegument is thinner as in the highly vascularized
pulmonary cavity (Dillon 2000).
MC-contaminated spermatozoids and oocytes are likely
to be either irreversibly damaged or viable but dysfunctioning. In oviparous gastropod species eggs may be contaminated during vitellogenesis, and during construction of
egg masses by accessory sexual organs (i.e., albumen, nidamental and prostate glands, spermatheca). After internal
fertilization, eggs are enclosed in perivitelline fluid (containing proteins and calcium) from the albumen gland and
coated by a perivitelline membrane (Dillon 2000). Eggs are
further encapsulated in the oviduct with mucopolysaccharides and mucoproteins. As MCs are known to covalently
bind to proteins phosphatase via the dehydroalanine moiety
of MCs to the sulfhydril group of cysteine or methionine
(Dietrich and Hoeger 2005; Ernst et al. 2005), MC covalent
binding to mucoproteins of eggs is highly possible. Intoxication of embryos by MCs may therefore have three origins
(i.e., contaminated gametes, MC-transfer in developing
embryos and MC-entrance in released egg masses) probably
varying depending on exposure (i.e., MC dissolved in medium or ingestion of toxic cells by parent snails). However, the
origin of MC-intoxication of embryos and its impact remain
scarcely investigated. Only Zhang et al. (2007) demonstrated
a MC-transfer from mother to embryos in the brood pouch
of oviduct of the ovoviviparous prosobranch Bellamya
aeruginosa sampled in a lake submitted to toxic
123
728
cyanobacterial blooms. Our study reveals impact on development and hatching kinetics of embryos with decreased
neonate survival after parental exposure to toxic cyanobacteria, suggesting a MC-transfer from gastropod females to
embryos. This study did not reveal any impact of the parental
exposure on hatching percentage as demonstrated by Coutellec and Lagadic (2006) and Leung et al. (2007) in L.
stagnalis exposed to xenobiotics. However, egg hatching
started far earlier compared to controls and hatching duration
was greatly extended after parent exposure to P. agardhii. In
the case of the ovoviparous M. tuberculata exposed to CYN,
the number of hatchlings released from parent snails
increased, suggesting that stress due to this cyanotoxin
induced abortion (Kinnear et al. 2007). Further investigations are needed in order to assess how MCs can affect the
functioning of genital gland and accessory sexual organs as
well as gametogenesis, vitellogenesis and embryogenesis of
freshwater invertebrates.
Toxicant transfer from the parent to eggs might increase
sensitivity of offsprings to MCs as well as continuous
exposure of egg masses and during early stages post
hatching. Egg masses exposure to dissolved MC-LR after
parental exposure decreased hatching percentage and
increased hatching duration (compared to those incubated
in non toxic water). These results suggest a MC-LR penetration in egg masses. The MC-uptake by fish eggs has
been demonstrated for Danio rerio exposed to 2.5 mg
14
C-labelled MC-LR L-1, followed by an activation of the
detoxification system (Wiegand et al. 1999). In gastropods,
no impact was observed by Zurawell (2001) on embryo
survival of L. stagnalis after egg exposure to low MC-LR
concentrations (up to 10 lg L-1). However, the author
suggested that at higher concentration MC-LR may penetrate in the egg mass via the tunica capsule which imbibes
water over time in order to soften the egg mass and to
allow snails hatching (Dillon 2000). MC-penetration in egg
masses may impair the common matrix surrounding the
egg cells or the perivitelline membranes enveloping each
egg within the mass. The exposure of egg masses to toxic
cyanobacteria induced a delayed hatching and was less
severe than dissolved MC-LR exposure. Toxic cyanobacteria cannot penetrate in egg masses but few MCs are
released in the medium due to the senescence of some cells
(Chorus and Bartram 1999).
Parent exposure to toxic cyanobacteria and dissolved
MC-LR decreased neonate survival, and additional effect
occurred when egg masses stayed in the toxic parental
medium. Exposure to toxic cyanobacteria induced a MC
accumulation in neonates from the first day after hatching,
suggesting that they can ingest cyanobacteria very early in
their life. This ingestion led to a twice lower percentage of
survival compared to neonates exposed to uncontaminated
123
E. Lance et al.
water. Previous experiment on 2-month old juveniles of
L. stagnalis showed no effect on survival after exposure to
dissolved MC-LR (Gérard et al. 2005) and to toxic cyanobacteria (even if growth was reduced) (Lance et al.
2007). The 15-day old snails have probably less physiological capacities to detoxify and to survive under cyanobacterial stress than 2-month old snails.
In the field, most freshwater gastropods may be MCintoxicated from their birth with detrimental effect on their
life traits (survival, growth, and fecundity) (Gérard and
Poullain 2005; Gérard et al. 2005; Lance et al. 2007, 2008),
since their breeding season generally occurs during the
bloom period, in late spring or early summer (Berrie 1965;
Calow 1978). Cascading effect on the gastropod populations were evaluated via the simulations of 3-years
dynamics of L. stagnalis populations submitted to 3-weeks
MC-producing (5-10 lg L-1) blooms at frequencies ranging from one bloom per 3-years to four blooms per year.
The results clearly demonstrated a reduced population
growth rate and a delayed growth of up to 80 weeks
(compared to controls) to reach a population of 1000
individuals (starting with 1 snail). Our simulations were
environmentally relevant: (1) bloom frequency was taken
from a field study (Lance et al. 2010c), (2) intracellular MC
concentration were in accordance with concentrations (i.e.,
from 0.3 to 15 lg L-1) measured in French lakes contaminated by toxic cyanobacteria (Briand et al. 2008;
Lance et al. 2010c; Sabart et al. 2010), (3) dissolved MCLR concentration corresponded to what may result in the
water column after the collapse of blooms and consequent
cell lysis, i.e., concentration varying from 0 to 140 lg L-1
(Chorus and Bartram 1999; Zurawell et al. 1999;
Hyenstrand et al. 2003), and (4) we simulate gastropod
exposure to cyanobacteria with a non toxic food source
(e.g., macrophytes, phytoplankton, periphyton, detritus),
which is likely in natural conditions (Dillon 2000). The
models were based on the assumption that populations
were under optimal trophic and spatial resources. However,
in natural conditions environmental factors (e.g., food
availability and density-dependence) may have additive or
synergistic effects and increase impact of toxic blooms on
gastropod populations. This may lead to the decline of some
gastropod species in case of recurrent blooms as observed by
Gérard et al. (2008) and Lance et al. (2010c). Not only may
the dynamics of gastropod populations be affected but also
the structure of gastropod communities. The model results
demonstrated that time required for snails to recover after
exposure influenced the level of impact on population
dynamics. Therefore, species with high MC-detoxification
abilities may be favored in medium exposed to recurrent
cyanobacterial blooms. Consequently, toxic cyanobacteria proliferations may indirectly influence competitive
Impact of microcystin-producing cyanobacteria
interactions in favouring the most resistant or tolerant gastropods to the detriment of the most sensitive ones.
Conclusion
This study confirms the differential effect of MCs on
gastropods according to age and intoxication pathways.
Intoxication of gastropods during cyanobacterial blooms
may occur at embryo stage via egg-mass contamination in
mother and in the medium, and at neonate stage mainly via
consumption of toxic cyanobacteria. We modelled the
L. stagnalis population dynamics using life-history traits
reported for egg, neonate, juvenile and adult stages in this
and our previous studies. The models revealed both the
influence of detoxification abilities on population growth of
gastropods, and the population decline in case of recurrent
exposure to cyanobacterial blooms. Therefore toxic cyanobacteria proliferations may constitute a determinant
factor in the regulation of gastropod populations, changing
the community structure and leading to their decline in
case of severe recurrent blooms, with probable consequences on the functioning of the whole ecosystem.
Acknowledgments We gratefully thank the Institut National de
Recherche en Agronomie (INRA, Rennes, France) for providing
individuals of Lymnaea stagnalis and the Museum National d’Histoire Naturelle (Paris, France) for providing P. agardhii strain.
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