Báo cáo khoa học: Molecular identification and expression study of differentially regulated genes in the Pacific oyster Crassostrea gigas in response to pesticide exposure doc

Molecular identification and expression study of
differentially regulated genes in the Pacific oyster
Crassostrea gigas in response to pesticide exposure
Arnaud Tanguy1, Isabelle Boutet1,2, Jean Laroche1 and Dario Moraga1
´
´
1 Laboratoire des Sciences de l’Environnement Marin (LEMAR), UMR-CNRS 6539, Institut Universitaire Europeen de la Mer, Universite de
´
Bretagne Occidentale, Plouzane, France
´
´
´
`
2 UMR CNRS-IFREMER 5171 ‘Genome, Populations, Interactions, Adaptation’, Station Mediterraneenne de l’Environnement Littoral, Sete,
France

Keywords
Crassostrea gigas; environment; gene
expression; pesticides; subtractive libraries
Correspondence
D. Moraga, UMR-CNRS 6539 Laboratoire
´
LEMAR, Institut Universitaire Europeen de
´
la Mer, Universite de Bretagne Occidentale,
´
Place Nicolas Copernic, F-29280 Plouzane,
France
Fax: +33 2 98 49 86 45
Tel: +33 2 98 49 86 42
E-mail :

(Received 3 August 2004, revised 5 November
2004, accepted 12 November 2004)

The effects of pesticide contamination on the metabolism of marine molluscs are poorly documented. We investigated the response of a marine
bivalve, the Pacific oyster, Crassostrea gigas, using a suppression subtractive hybridization method to identify up- and down-regulated genes after a
30-day exposure period to herbicides (a cocktail of atrazine, diuron and
isoproturon, and to the single herbicide glyphosate). A total of 137 unique
differentially expressed gene sequences was identified, as well as their associated physiological process. The expression of 18 of these genes was analyzed by RT-PCR under laboratory experimental conditions. The
metabolic functions they are associated with include xenobiotic detoxification, energy production, immune system response and transcription. This
study provides a preliminary basis for studying the response of marine
bivalves to long-term herbicide exposure in terms of regulated gene expression and characterizes new potential genetic markers of herbicide contamination.

doi:10.1111/j.1742-4658.2004.04479.x

For several decades, coastal ecosystems have been subjected to increased pesticide contamination, mainly
from agricultural practices. For example, herbicide
compounds such as atrazine, diuron, isoproturon, simazine, alachlor, metolachlor, and, more recently,
glyphosate, are widely applied to cereal crops and
reach coastal waters by runoff. Moreover, the exposure
of animals in these ecosystems to pesticides is never
limited to a single pollutant, but to an assortment of
chemicals from a variety of sources whose interactions
could result in additive, synergistic or antagonistic
effects with regard to toxic outcome. Pesticides, similar
to other xenobiotics, are metabolized by many
enzymes, including those of the cytochrome P450dependent monooxygenase system, flavin-containing
monooxygenase, prostaglandin synthetase, alcohol

dehydrogenase, esterases, and a variety of transferases
[1,2]. Pesticides are also known to alter the progression

of some human cancers by reducing immune defenses
against cancer [3]. They have also been reported to
affect reproductive and developmental processes in
wildlife, possibly by disrupting endocrine pathways
[4–6]. Some particular herbicides have been well studied in terms of toxicology effects in various organisms.
Atrazine [2-chlor-4-ethylamino-6-isopropylamino-1,3,5triazin] has a known toxicity to blood-forming organs
and the immune system, and can induce the production of cytokinines such as interferon c or tumor necrosis factor a [7,8]. In fish, atrazine affects different
tissues, particularly liver tissue which shows a substantial increase in the size of lipid inclusions followed
by lipoid degeneration, enlargment of the secondary

Abbreviations
AChE, acetylcholinesterase; SSH, suppression subtractive hybridization.

390

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A. Tanguy et al.

lysosomes, mitochondrial malformation and vacuolization, and a reduction in glycogen content [9]. Isoproturon[3-(4-ospropylphenyl)-1,1-dimethylurea] is a
nonhalogenated, lipophilic-substituted phenylurea herbicide that is used to protect cereal crops. It has a
strong affinity for interaction with membrane phospholipids [10]. It has been shown recently that high
concentrations of isoproturon could affect metall-othioneins by reducing their metal content in the aquatic
oligochaete Tubifex tubifex [11] and could affect
enzyme activities in amphibians [12]. Diuron [3-(3,4dichlorphenyl)-1,1-dimethyl-harnstoff] is a phenylurea
herbicide [13] used for pre- and post-emergence of
weeds in agriculture. The toxicity of this herbicide has
mainly been studied in water phytoplankton [14]. In
aquatic organisms, LC50 (48 h) values for diuron range

from 4.3 to 42 mgỈL)1 in fish and range from 1 to
2.5 mgỈL)1 in aquatic invertebrates. It was also shown
that its principal biodegradation product, 3,4-dichloroaniline, exhibits a higher toxicity and is also persistent
in soil, water and groundwater [15]. Significant inhibitions (9–12%) of brain acetylcholinesterase (AChE)
activity were also observed in response to diuron in
the juvenile goldfish (Carassius auratus) [16]. Glyphosate [N-(phosphonomethyl)glycine], also known as
‘Round-Up’, is a herbicide used to control grasses, herbaceous plants, including deep-rooted perennial weeds,
brush, some broadleaf trees and shrubs, and some
conifers. Glyphosate acts by preventing the plant from
producing an essential amino acid and by inhibiting the enzyme enolpyruvylshikimate-phosphate synthase which reduces the production of protein in the
plant, thereby inhibiting plant growth. Studies showed
that glyphosate caused the appearance of myelin-like
structures in Cyprinus carpio hepatocytes, swelling of
mitochondria and disappearance of the internal mitochondrial membrane in carp at both exposure concentrations [17]. However, little information about the
effect of glyphosate on marine invertebrate species is
available.
Despite the vast commercial use of these herbicides,
there is little published data about their effects on marine molluscs. In invertebrates, resistance to pesticides
by detoxification has previously been directly correlated with some enzyme biomarkers, such as mixed-function oxidase activity, glutathione S-transferase activity,
AChE or other esterase activity [18,19]. Aquatic
macro-invertebrates such as molluscs are widely used
in biological monitoring programs, but their use in
biomarker studies remains limited. To identify biomarkers of exposure, it is first necessary to identify
and describe the mechanisms of ecotoxicological damage and those involved in the response of the
FEBS Journal 272 (2005) 390–403 ª 2005 FEBS

Oyster response to pesticide exposure

organisms to the pollutant. In previous studies, we
demonstrated that atrazine and isoproturon could

select particular alleles at some enzymatic loci, such as
adenylate kinase, phosphoglucomutase and phosphoglucoisomerase [20].
In this paper, we report regulated genes involved in
the molecular response induced by herbicides in
C. gigas. As a first step, we identified the down and
up-regulated genes after one month of exposure to a
cocktail of globally employed herbicides using a suppression subtractive hybridization (SSH) method. We
then analyzed the regulation of specific herbicideregulated gene expression.

Results
Identification of herbicide-regulated genes
Two forward and reverse SSH libraries were made
from pooled digestive glands and gills of C. gigas after
30 days of exposure to a herbicide cocktail of atrazine,
diuron and isoproturon. Two other forward and
reverse SSH libraries were made from the same tissue
types from oysters exposed to glyphosate for 30 days.
The search for homology using the blastx program
revealed a total of 137 different sequences, including
56 sequences corresponding to known genes and 81
sequences corresponding to new expressed sequence
tags. The sequences obtained from the various SSH
libraries are listed in four tables: 30-days, up- and
down-regulated after exposure to an atrazine ⁄ diuron ⁄ isoproturon (ADI) cocktail (Tables 1 and 2), and
30-days, up-and down-regulated after exposure to
glyphosate (Tables 3 and 4). These genes regulated by
herbicide exposure can be assigned to six major cellular physiological functions. (a) Xenobiotic detoxification; (b) nucleic acid and protein regulation (including
transcription, cell cycle regulation and metabolism of
nucleic acid components); (c) respiration; (d) cell communication (including immune system and membrane
receptors); (e) cytoskeleton production and maintenance; and (f) energy metabolism. Several ribosomal

proteins were also found in both the forward and
reverse libraries from the two experiments. There were
more regulated genes from the libraries associated with
exposure to the ADI cocktail than in the libraries associated with exposure to glyphosate, particularly among
the down-regulated genes.
Expression of herbicide-regulated genes
The time-dependent expression of 13 genes that were
up-regulated by exposure to herbicide and five that
391


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A. Tanguy et al.

Table 1. Up-regulated genes identified in the SSH libraries of the
atrazine ⁄ diuron ⁄ isoproturon experiment (after 30 days of exposure)
with significant database matches.
BLASTX

Homolog (protein)

value

Xenobiotics detoxification
Glutamine synthetase
3e-33
Lysosomal associated protein
Unknown function
Clone 44 unnamed human protein product 6e-15

C380A1.2.2 Homo sapiens novel protein 0.048
ENSANGP00000014914
1e-6
ENSANGP00000024944
3e-10
Cellular cycle, protein regulation and transcription
RNA helicase
1e-108
Hypertension-related
1e-18
calcium-regulated gene (HSCR)
Elongation factor 2
3e-61
Respiratory chain
Cytochrome oxidase
9e-23
NADH dehydrogenase subunit 4
8e-94
Metabolism
ATP syntase beta subunit
1e-36
ATP syntase alpha subunit
8e-68
Cellular communication, membrane
receptors and immune system
b-1,3-Glucan binding protein
3e-6
Putative senescence-associated protein
5e-46
Cavortin

1e-5
Cytoskeleton
Tubulin alpha 3 chain
4e-17
Tubulin alpha 1 chain
5e-20
Tubulin beta
1e-50
Actin cytoplasmic A3
3e-84
Ribosomal proteins
Ribosomal protein L18a
6e-31
Ribosomal protein S18
5e-34
Ribosomal protein S7
2e-38
Ribosomal protein S17
3e-45
Ribosomal protein S6
4e-37
Unknown genes (27 sequences)a

GenBank
accession no

CB617403
CF369139
CF369128
CF369136

CF369137
CF369140
CF369125
CF369127
CF369143
AF177226
AF177226
CF369132
CF369142

CF369126
CF369134
CF369147
CF369131
CF369135
CF369141
CF369133
CF369129
CF369145
CF369146
CF369144
CF369138
CF369148 to
CF369174

a

Sequences with nonsignificant e-value (< 0.01) or with unknown
proteins.

were down-regulated was analyzed by RT-PCR using
RNA from both the gills and digestive glands of oysters after 0, 7, 14, 21 and 30 days of pesticide exposure. First, an RNA pool of the eight oysters collected
at each exposure time was used to identify the tissue in
which the differential expression when compared to
the control was significant. RT-PCR was then carried
out on each oyster sample for the target tissues, to
estimate the variation in gene expression between samples. A summary of these results is presented in
Table 5 and Fig. 1. Among the 18 genes analyzed, only
392

Table 2. Down-regulated genes identified in the SSH libraries of
the atrazine ⁄ diuron ⁄ isoproturon experiment (after 30 days of exposure) with significant database matches.

value

GenBank
accession no

5e-70
8e-33
6e-18
3e-9
2e-9
3e-10

CF369178
CF369179
CF369181
CF369185
CF369187

CF369182

2e-4
3e-15
7e-10
4e-40

CF369175
CF369184
CF369183
CF369176

1e-4
9e-6
2e-52
2e-29

CF369177
CF369180
CF369186
CF369188
CF369189 to
CF369220

BLASTX

Homolog (protein)
Unknown function
EbiP2667 Anopheles gambiae
CG1524-PC Drosophila melanogaster

Unnamed protein Mus musculus
ENSANGP00000010310
AgCP1592 Anopheles gambiae
EbiP2667 Anopheles gambiae
Cellular communication, membrane
receptors and immune system
Tripartite motif protein TRIM2
Apoliphorin precursor protein
Transport protein Sec61 alpha subunit
Guanyl cyclase receptor
Ribosomal proteins
Ribosomal protein L38
Ribosomal protein L13
Ribosomal protein L7
Ribosomal protein S8
Unknown genes (32 sequences)a

a
Sequences with nonsignificant e-value (< 0.01) or with unknown
proteins.

two showed no clear regulation – fucolectin and ribosomal L18A. All of the other genes showed some regulation with exposure to herbicide. The regulation of
expression was strongly tissue-dependent for 10 genes,
and most of these are chiefly regulated in the digestive
gland (Table 5). Student’s t-test showed that despite
clear patterns of increased or decreased expression at
the different times of exposure, the differences in gene
expression are most significant when we compare samples exposed for 7 and 14 days with those exposed for
21–30 days. A summary of the significant value of statistical test is presented in Tables 6 and 7. Less significant differences can be explained by the variation seen
in expression among the samples. In the ADI (Fig. 1C)

and glyphosate (Fig. 1B) experiments, identified genes
from the SSH libraries were expressed differentially
compared to the control. In the control oysters, no significant variation in gene expression was observed
between samples from the different sampling periods,
though high inter-individual variations were detected
(Fig. 1A).

Discussion
Despite the intensity of pesticide use along the marine
coast, few studies have investigated the response to
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A. Tanguy et al.

Oyster response to pesticide exposure

Table 3. Up-regulated genes identified in the SSH libraries of the
glyphosate experiment (after 30 days of exposure) with significant
database matches.
BLASTX

Homolog (protein)

value

Xenobiotics detoxification
Glutamine synthetase
3e-33
Respiratory chain

NADH dehydrogenase subunit 4
8e-94
NADH dehydrogenase subunit 5
6e-16
Cytochrome oxidase
9e-23
Metabolism
ATP syntase beta subunit
1e-36
Cellular communication,membrane receptors
and immune system
b-1,3-Glucan binding protein
3e-6
Procathepsin L
1e-12
Meningioma expressed antigen 5
7e-19
Lipoprotein receptor related protein 5
2e-24
Fucolectin-1
6e-4
Cytoskeleton
Tubulin alpha 2 chain
4e-40
Tubulin beta
2e-96
Actin cytoplasmic A3
3e-84
Ribosomal proteins
Ribosomal protein L6

2e-33
Ribosomal protein S25
2e-14
Ribosomal protein S8
2e-29
Unknown genes (16 sequences)a

GenBank
accession no

CB617403
AF177226
AF177226
AF177226
CF369132

CF369126
CF369221
CF369226
CF369227
CF369228
CF369225
CF369141
CF369133
CF369223
CF369224
CF369222
CF369229 to
CF369244

a
Sequences with nonsignificant e-value (< 0.01) or with unknown
proteins.

Table 4. Down-regulated genes identified in the SSH libraries of
the glyphosate experiment (after 30 days of exposure) with significant database matches.
BLASTX

Homolog (protein)

value

Cellular cycle, protein regulation and transcription
ADP-ribosylation factor 2
1e-9
Ribosomal proteins
Ribosomal protein S3A
3e-25
Ribosomal protein L27
1e-22
Ribosomal protein L13
2e-52
Ribosomal protein 60S P2
2e-52
Ribosomal protein S3B
4e-45
Ribosomal protein 40S S2
2e-52
Unknown genes (nine sequences)a

GenBank
accession no

CF369249
CF369245
CF369246
CF369247
CF369252
CF369251
CF369248
CF369253 to
CF369261

a
Sequences with nonsignificant e-value (< 0.01) or with unknown
proteins.

exposure in marine organisms at the level of gene transcription. In this study, we characterized the response
of the Pacific oyster, C. gigas, to herbicide exposure
FEBS Journal 272 (2005) 390–403 ª 2005 FEBS

under experimental conditions. Using a suppression
subtractive hybridization method, we obtained 137
unique partial sequences of cDNA (56 corresponding
to known genes) encoding proteins being transcribed
in oysters after 30 days exposure to herbicides. Use of
this method in conjunction with differential display
PCR has previously identified 242 differentially
expressed genes in the zebra mussel, Dreissena polymorpha, treated with various contaminants such as
Aroclor 1254, 3-methylcholanthrene, chrysene and atrazine [21]. Our results are difficult to compare with

those obtained in D. polymorpha because of the higher
concentrations of pesticides (between 1 gỈL)1 and
2 mgỈL)1, according to the herbicide) and shorter time
of exposure (between 8 and 24 h) used by the authors.
In another study, 258 differentially expressed genes
were identified in C. gigas in response to exposure to
hydrocarbons for 7 and 21 days [22], and some of
these genes were also present in the herbicide SSH libraries (including glutamine synthetase and cathepsin).
In our experiments, we tested a cocktail of the three
more common herbicides detected in the French Atlantic estuaries and used concentrations that can be found
in seawater. The experiment using only glyphosate was
conducted to test the effect of this newly introduced
herbicide in French ecosystems. Glyphosate is known
to be not bioaccumulated, biomagnified or persisting
in a biologically available form in the environment. Its
mechanism of action is specific to plants and it is relatively nontoxic to animals [23]. But several studies have
demonstrated that glyphosate and more especially the
surfactants used to increase its efficacy showed that
glyphosate could be toxic for many organisms [24–26].
Isoproturon appears not to bioaccumulate in molluscs
[27], and the LC50 at 9 days in C. gigas larvae has
been estimated at 0.37 mgỈL)1. In acute tests, diuron
was had limited toxicity to fish and invertebrates [28].
Few results are available from chronic tests, especially
for aquatic invertebrates. The LC50 (at 48 or 96 h) for
diuron varied from 1 to 30 mgỈL)1 in fish and invertebrate species [29]. Fish species data are primarily
from acute exposures, and the lethal effects ranged
from 2.8 to 31 mgỈL)1 in 1–4 day exposures [28,30,31].
Glyphosate is considered relatively effective with little
to no hazard to animals [32]. However, at sublethal

concentrations, glyphosate affected the reproduction
and development of Pseudosuccinea columella snails
[32]. Glyphosate was also shown to be relatively nontoxic in certain animal species and presents virtually
no effects in some aquatic organisms (the 96 h LC50 in
rainbow trout, Oncorhynchus mykiss, and other fish
ranges from 86 to 168 mgỈL)1, and the 48 h LC50 was
780 mgỈL)1 in Daphnia) [33]. The LC50 for oyster
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A. Tanguy et al.

Table 5. Summary of the results of expression studies in the two tissues used in SSH experiments.
Gill

Digestive gland

Gene name

SSH library

ADI

Glyphosate

ADI

Glyphosate

Glutamine synthetase
ATP syntase beta
Coelomic factor
RNA helicase 2
Procathepsin L
Meningioma associated protein
Lipoprotein receptor
HSCR
Senescente associated prot
Lysosomal associated protein
Elongation factor 2
Apolipophorin
Tripartite motif protein TRIM2
Guanyl cyclase
ADP ribo
RiboL13

ADI and G (up)
ADI and G (up)
ADI and G (up)
ADI (up)
G (up)
G (up)
G (up)
ADI and G (up)
ADI (up)
ADI (up)
ADI (up)
ADI (down)

ADI (down)
ADI (down)
G (down)
ADI (down)

+
+
–
–
–
–
–
+
–
+
–
–
+
+
–
+

+
+
–
–
+
–
–
–

–
–
–
–
–
–
–
+

+
+
+
+
–
–
–
+
+
+
+
+
+
–
–
+

+
+
+
–

+
+
+
+
+
–
–
–
–
–
+
+

larvae has been estimated at more than 10 mgỈL)1 [34].
Glyphosate closely resembles naturally occurring substances and does not possess chemical groups that
would confer great reactivity or biological persistence,
and its chemical properties indicate that it is not bioaccumulate [35]. Although primarily aimed at reversibly inhibiting photosynthesis in plants [36], atrazine
has also been found to affect a variety of physiological
processes in aquatic animals. Atrazine accumulation has
been seen in a number of tissues [37,38]. In freshwater
invertebrates, atrazine affected hydromineral balance or
gill function in crabs [39,40], as well as hemocyanin
function [41]. In fish it affects hematology [42,43] and
metabolism [41,44,45]. More recently, low levels of atrazine have been shown to impair sexual development in
male frogs [6]. In the mollusc C. virginica, the 96 h
LC50 has been estimated at 1 mgỈL)1. The supposed
low level of toxicity of glyphosate to oysters, and the
weak concentration used in our experiment could
partly explain the difference observed in the number of

genes identified from the libraries (98 in the ADI SSH
libraries vs. 48 in the glyphosate SSH libraries).
In previous experiments studying the effect of atrazine on fish, the authors showed that the concentrations quantified in water were about 70% less than the
concentrations introduced in the tanks, probably due
to adsorption of atrazine to the surfaces of the tanks
and possibly due to removal by the fish themselves
[46]. Moreover, the authors suggested that some of the
atrazine added may be liable to biotic (e.g. bacteria)
and abiotic (e.g. light) degradation and therefore that
the physiological responses of the fish to atrazine may
be occurring at lower water concentrations than indicated. Until a better understanding of atrazine dynamics can be applied to our experimental set-up, we
prefer to discuss the physiological changes in C. gigas
in relation to the concentrations of pesticides introduced in our tanks. More, the fact that sea-water was
changed every day in our tanks and the corresponding
herbicide concentration was added at each water

Fig. 1. Analysis of differential expression of up- and down-regulated genes in C. gigas exposed to pesticides. Expression of the gene studied
is presented as the calculated ratios ODCgGSII ⁄ OD28S after RT-PCR. For each gene and each sampling time, the bar represents the average value of gene expression (ratios ODCgGSII ⁄ OD28S) for the eight samples and the error bars correspond to the standard deviation for
the eight samples at the sampling time considered. (A) Expression of the 17 studied genes in the control samples. 1, senescence associated
protein; 2, Trim2; 3, lysosomal associated protein; 4, elongation factor 2; 5, hypertension-related calcium-regulated gene (HSCR); 6, apoliphorin precursor protein; 7, glutamine synthetase; 8, ATP syntase; 9, coelomic factor; 10, RNA helicase; 11, guanyl cyclase receptor; 12, ribosomal protein L13; 13, lipoprotein receptor related protein; 14, meningioma expressed antigen 5; 15, ADP-ribosylation factor 2; 16,
procathepsin L. (B) Expression of the nine studied genes in the glyphosate experiment. 1, lipoprotein receptor related protein; 2, HSCR; 3,
meningioma expressed antigen 5; 4, glutamine synthetase; 5, ATP synthase; 6, coelomic factor; 7, ADP-ribosylation factor-2; 8, procathepsin
L; 9, ribosomal protein L13. (C) Expression of the 12 studied genes in the ADI experiment. 1, senescence associated protein; 2, Trim2; 3,
lysosomal associated protein; 4, elongation factor-2; 5, HSCR; 6, apoliphorin precursor protein; 7, glutamine synthetase; 8, ATP syntase; 9,
coelomic factor; 10, RNA helicase; 11, guanyl cyclase receptor; 12, ribosomal protein L13.

394

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Oyster response to pesticide exposure

A
14
12
10
8
6
4
2
0

1

12

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16

B

10

T0
T7

8

T14
T21

6

T30

4

2

0

1

14

2

3

4

5

6

7

8

9

C

12
10
8
6
4
2
0

1

2

3

4

FEBS Journal 272 (2005) 390–403 ª 2005 FEBS

5

6

7

8

9

10

11

12
395


Oyster response to pesticide exposure

change, allowed to maintain the oysters in a more or
less homogenous concentration of contaminants.

Most of the genes we identified function in the respiratory chain, cell communication, the immune system, or the regulation of protein or the cytoskeleton.
Only a few are specific to xenobiotic detoxification.
One, the glutamine synthetase gene was previously
shown to be associated with the response of various
organisms to pesticide exposure. The response seen in
our libraries was that more genes are up-regulated by
herbicides than are down-regulated. This pattern was
stronger in the ADI libraries compared to the glyphosate libraries. Glyphosate seemed not to have a strong
effect on C. gigas metabolism. However, some of the
genes involved in respiration and energy production
were highly expressed in response to herbicide exposure no matter which pesticide was used. Similar
results were observed in previous reports studying the
effect of other stress such as hydrocarbons [22] or
parasite infection [47], showing that any source of
stress generates an increase in energy production. A
comparison of the two experiments shows that the
same cellular functions were affected by herbicides
with only a few genes in common to both the ADI
and glyphosate response SSH libraries. These were glutamine synthase, ATP synthetase, b-1,3-glucan binding
protein and some housekeeping genes such as tubulin
and actin.
Among the 18 genes studied, 16 produced patterns
of differential time- or tissue-dependent expression.
Most of the significant differences observed in gene
expression appeared after 21–30 days, suggesting that
at the concentrations used herbicides only affect oyster
metabolism after long periods of exposure. Among all
the genes regulated was b-1,3-glucan binding protein,
also named coelomic factor, that has been widely studied in shrimp [48,49] and other crustaceans [50]. Lipopolysaccharide and b-1,3-glucan binding protein
combine to form LGBP, a pattern recognition protein,

that plays an important role in the innate immune
response of crustaceans and insects. This gene was previously identified in up-regulated SSH libraries from
C. gigas exposed to hydrocarbons [22] and parasites
[47]. The binding of lipopolysaccharide and b-1,3-glucan binding protein to form LGBP has been shown to
activate the prophenoloxidase cascade. The apolipophorin precursor protein we identified is also involved
in the activation of phenoloxidase in invertebrates [51].
The activation of the phenoloxidase cascade appears
to be a general response of C. gigas to stress exposure
whatever its nature, abiotic or biotic.
We also studied the expression of an RNA helicase
that is known to be a multifunctional protein involved
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A. Tanguy et al.

in various nuclear processes such as transcription, ribosomal RNA biogenesis and RNA export. Several tissue-specific RNA helicases have been described in the
literature, such as the myocyte enhancer factor-2 protein that acts as an inhibitor of cell proliferation and
cardiomyocyte hypertrophy and may also be involved
in cell cycle progression [52]. In previous studies, we
also showed that this gene was up-regulated by exposure to hydrocarbons, and that its over-expression was
greatest in the first week after PAH exposure [22].
We saw strong inhibition of guanyl cyclase receptor,
particularly in the gills. The inhibition of guanyl
cyclase has been shown to prevent increased enzyme
activity associated with the nitric oxide-mediated damage recovery process [53]. Nitric oxide (NO) is a
potent, bioactive molecule produced in the presence of
NO synthase, which constitutively mediates numerous
physiological functions. Over-production of NO (and
NO-reaction products) can be induced, and typically
leads to cell cycle arrest and apoptosis. In vertebrate

blood cells, an increase of extracellular levels of NO
was detected after exposure to the herbicide paraquat
[54]. Other studies have likewise shown that pesticides
could generate increasing NO levels which led to unrepaired damage caused by free radicals [55]. Our experiments were not designed to quantify the amount of
NO reaction products; however, the strong down-regulation of guanyl cyclase receptor that was measured
suggests an activation of physiological process involved
in free radical scavenging, especially NO, that could
have been generated during the herbicide exposure in
oysters.
ATPase is a large family of genes coding for different enzymes that all participate in the formation of
ATP. The enzyme identified from our libraries could
not be specifically identified from the partial sequence.
Nevertheless, over-expression of this gene was
observed in both experiments and in both tissue types
analysed after 15 days of pesticide exposure. In
another study, increased Mg2+-ATPase and Na+ ⁄ K+ATPase activity was detected in the liver and erythrocytes of rats exposed to the insecticides malathion and
anilofos [56]. Similar results were obtained in snails
exposed to fungicide and herbicides [57]. Because
ATPase is constitutively expressed and probably regulated by multiple environmental and ⁄ or physiological
factors, a more complete study of the regulation of its
expression has to be done before its potential use as a
biomarker for pesticide-exposure monitoring can be
determined.
Other interesting genes that were identified from our
libraries are the lysosomal associated protein and procathepsin L, a protein protease. Both are involved in
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A. Tanguy et al.

Oyster response to pesticide exposure

Table 6. Summary of the statistical test (Student’s t-test) performed on gene expression in the glyphosate experiment. The
gene expressions are compared by pair of sampling time. *, Significant value at 0.05%.
T0
Lipoprotein receptor
T7
T14
*
T21
*
T30
*
HSCR
T7
T14
*
T21
*
T30
*
Meningioma antigen
T7
T14
*
T21
*
T30
*
Glutamine synthetase

T7
T14
T21
*
T30
*
ATP synthase
T7
T14
*
T21
*
T30
*
Coelomic factor
T7
T14
T21
T30
*
ADP-ribosylation factor-2
T7
T14
T21
T30
*
Procathepsin L
T7
T14
*

T21
*
T30
*
Ribosomal protein L13
T7
T14
T21
*
T30
*

T7

T14

T21

*
*

*
*

*

*
*

*

*
*

*
*

*
*
*

*
*

*

*

*

*
*

*
*

*
*

lysosomal functions. Earlier studies have shown that

pesticides can affect lysosome membrane stability [58].
In insecticide-resistant Musca domestica strains, the
mechanism by which proteases may confer advantages
to insecticide resistant insects could involve providing
an increased supply of precursor amino acids from
FEBS Journal 272 (2005) 390–403 ª 2005 FEBS

proteolytic degradation products to the intracellular
pool, prior to the de novo synthesis of detoxifying
enzymes [59]. We previously showed that the lysosomal
associated protein was up-regulated in response to parasite exposure [47] suggesting that membrane stability
seems to be a target for stress factors. The lysosomal
membrane destabilization is a common parameter used
to to investigate the impact of environmental pollution
in disturbed ecosystems and is considered as a general
biomarker of stress [60,61].
We also identified the glutamine synthetase (GS) gene
as being up-regulated with pesticide exposure. GS performs the fundamental functions of ammonia fixation
and glutamine biosynthesis, but also plays a role in the
central nervous system where it clears the excitatory
neurotransmitters of glutamic acid [62]. In Arabidopsis
thaliana, GS was shown to be induced under amino acid
starvation conditions as well as by exposure to the herbicide acifluorfen [63]. In the fish, Cyprinus carpio, an
increase in GS was observed after several days of exposure to cypermethrin. At the same time, there was a
decrease in the amount of free amino acids coincident
with changes that occurred in the transamination process, again, related to the formation of nitrogenous end
products [64]. For the other differentially transcribed
genes identified in our libraries, we found no information in the literature concerning their regulation by pesticides. Some of these genes, such as meningioma
expressed antigen, hypertension-related calcium-regulated gene, and tripartite motif protein TRIM2, have been
described as being regulated in cell proliferation [65].

Their presence in our SSH libraries could suggest an
effect of pesticides on these physiological processes.
Interestingly, no sequence corresponding to AChE
was identified in our libraries. The monitoring of
AChE activity, particularly its inhibition, is commonly
used as a biomarker of pesticide exposure in experiments on marine species such as the mussel, Mytilus
sp. [66,67], the shore crab, Carcinus maenas [68] and
other invertebrates [69]. This result can be explained in
several ways. First, AChE has different distributions
and physiological roles in different species [70,71],
which results in a highly variable degree of inhibition
associated with toxic effects [68]. Also, AChE activity
is modulated by seasonal and nutritional variables [66].
Finally, in bivalves, only a few studies have been published on the use of AChE activity as a biomarker of
pesticide exposure, reflecting both low endogenous
activity and a relative insensitivity to inhibition by
these pollutants compared with other species [72–75].
The results given here provide a preliminary basis
for further study of detoxification processes and other
physiological responses to herbicides in the marine
397


Oyster response to pesticide exposure

A. Tanguy et al.

Table 7. Summary of the statistical test (Student test) performed
on gene expression in the atrazine ⁄ diuron ⁄ isoproturon experiment.
The gene expressions are compared by pair of sampling time.

*, Significant value at 0.05%.
T0
Senescence associated protein
T7
*
T14
*
T21
*
T30
*
Trim2
T7
T14
T21
*
T30
*
Lysosomal associated protein
T7
T14
*
T21
*
T30
*
Elongation factor-2
T7
*
T14

*
T21
*
T30
*
HSCR
T7
T14
*
T21
*
T30
*
Apoliphorin precursor
T7
T14
*
T21
*
T30
*
Glutamine synthetase
T7
*
T14
*
T21
*
T30
*

ATP syntase
T7
T14
*
T21
*
T30
*
Coelomic factor
T7
T14
T21
*
T30
*
RNA helicase
T7
T14
*
T21
*
T30
*
Guanyl cyclase receptor
T7
*
T14
*

398

T7

T14

*

T21

Table 7. (Continued).
T0
T21
*
T30
*
Ribosomal protein L13
T7
T14
T21
T30
*

T7

T14

T21

*

*
*

*
*

*

*

*

bivalve, C. gigas. This is the first published investigation into the mechanisms of these responses at the
molecular level in this species. We will focus our next
efforts on a more complete study of the regulation of
these genes with respect to pollutant concentration,
their use in surveys of wild populations of oysters, and
also in the search for functional polymorphism.

Experimental procedures
*
*

Oyster conditioning and treatment

*
*
*

*

*
*

*

*
*
*

*

*
*

*
*

*

*

*
*

Adult Crassostrea gigas were collected from La Pointe du
Chateau (Brittany, France). After an acclimatization perˆ
iod of 7 days in aerated 0.22 lm filtered seawater at constant temperature and salinity (15 °C and 34&,
respectively), oysters were challenged as follows. Two
groups of 40 oysters were exposed to two experimental
conditions. One group was exposed to a cocktail of herbicides (ADI) composed of atrazine (2 lgỈL)1), diuron

(1 lgỈL)1), and isoproturon (0.5 lgỈL)1) that represent the
three most used and toxic herbicides detected in all
French Bay waters. The other group is exposed to
2 lgỈL)1 of glyphosate, a herbicide that has been recently
introduced in marine ecosystems, and for which no information about its toxicity at low concentration is known in
marine molluscs. Another group of 40 oysters was maintained in aerated 0.22 lm filtered seawater without contaminant as a control. The experiment lasted for 4 weeks
and no mortality was observed. Herbicide concentrations
were chosen based on data reported by the Bay of Brest
monitoring program [76] and by the National Observation
Network of IFREMER-France. The concentrations used
in our experiment correspond to the highest concentrations observed in the various French Bay waters and correspond to about 1 ⁄ 100 of the LC50 values observed for
the herbicides tested [77–79].

*

*

*

Suppression subtractive hybridization
After 4 weeks, total RNA was extracted from the digestive
gland and gills of a pool of 10 control and 10 exposed

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A. Tanguy et al.

Oyster response to pesticide exposure

oysters using RNAble (Eurobio, les Ulis, France) according to the manufacturer’s instructions. Poly(A+) mRNA
was isolated from total RNA using the PolyATtractÒ
mRNA Isolation System (Promega, Madison, WI, USA)
according to the manufacturer’s instructions. Both forward
and reverse subtracted libraries were made on 2 lg of
mRNA extracted from the 10 oysters collected after
30 days of exposure and pooled for RNA extraction.
mRNA from the gill and the digestive gland (1 lg from
each) were used for the construction of SSH libraries.
First and second strand cDNA synthesis, RsaI endonuclease enzyme digestion, adapter ligation, hybridization and
PCR amplification were performed as described in the
PCR-select cDNA subtraction manual (Clontech, Palo
Alto, CA, USA). The differentially expressed PCR products were cloned into pGEM-T vector (Promega, Madison, WI, USA). Two hundred white colonies per library
were cultured in Luria–Bertani medium (with 100 mgỈL)1
ampicillin) from which the vector was extracted using an
alkaline lysis plasmid minipreparation, and screened by
size after digestion. A total of 250 clones from forward and
reverse libraries were sequenced using a Li-COR IR2
(Sciencetech, Lincoln, NE, USA) and Thermo Sequenase Primer Cycle Sequencing Kit (Amersham Bioscience, Uppsala,
Sweden) and a AB3100 sequencer (PerkinElmer, Boston,
MA, USA) and Big Dye Terminator V3.1 Kit (PerkinElmer). All sequences were subjected to a homology search
through the blastx program ( />BLAST/).

Pesticide detoxification gene expression analysis
by semiquantitative RT-PCR
Total RNA was extracted from the gill and digestive gland of
eight control and eight exposed oysters at days 0, 7, 15, 21
and 30 days using a method based on extraction in guanidium isothiocyanate [80]. For each oyster, 50 lg of total
RNA were reverse transcribed using the oligo(dT) anchor
primer 5¢-GACCACGCGTATCGATGTCG>ACT16V-3¢

and M-MLV reverse transcriptase (Promega, Madison, WI,
USA). Amplification of 18 regulated genes was carried out
using cDNA from both the control and exposed samples in
2 mm MgCl2, with 5 pmol of each primer. The names and
primer sequences for the genes studied are listed in Table 8.
For an internal PCR control, 28S ribosomal DNA was
amplified under the same conditions with sense (5¢-AAGG
GCAGGAAAAGAAACTAAC-3¢) and antisense (5¢-GT
TTCCCTCTAAGTGGTTTCAC-3¢) primers. The resulting
PCR products were electrophoresed in a 0.5· TBE ⁄ 1.5% agarose gel, and visualized with UV light after staining with
ethidium bromide. To minimize differences in RT efficiency,
template for all the PCRs were done on 2 lg of the same
reverse transcribed product. The number of PCR cycles needed to show differential expression between the control and
exposed samples was the same (40 cycles) for each gene
except for the 28S, where 25 cycles were used to avoid band
intensity saturation for optical determination. Band intensities were quantified using the gene profiler software (version 4.03, Scanalytics, Inc., Lincoln, NE, USA).

Table 8. Sequences of the primers used in the expression study.
Gene name

SSH library

Sense primer

Antisense primer

Glutamine synthetase
ATP syntase beta
Coelomic factor
RNA helicase 2

Procathepsin L
Meningioma
associated protein
Lipoprotein receptor
Fucolectin
RiboL18A
HSCR
Senescente
associated prot
Lysosomalassociated
protein
Elongation factor2
Apolipophorin
Tripartite motif
protein TRIM2
Guanyl cyclase
ADP ribo
RiboL13

ADI and G (up)
ADI and G (up)
ADI and G (up)
ADI (up)
G (up)
G (up)

5¢-GTGCATCAAAGAATTTTGGATAC-3¢
5¢-AGAGAAGTGGCAGCTTTCGCTCAGTTTGG-3¢
5¢-CTCGGCAAAGAAACCGCTGGTTCCTCCCA-3¢
5¢-GAGACGTCCAGGAAATCTTCCGCAACACC-3¢

5¢-CAGAGTGTGCACTAGCATGCGGTCCCGT-3¢
5¢-AGGTGTCCTAGATACCGGCCATGTACCA-3¢

5¢-TGCAATAATTTTTGAAGCCCCGG-3¢
5¢-TTAGCATCTGTGGCCTCTGTGATTTGTCC-3¢
5¢-GCCCCTACCATAACATAGAGGACCCCTGG-3¢
5¢-CAAATCTACCTGCACGAGCCACTCTGTGC-3¢
5¢-CACAACCTGGTCGCCGACCGCGGGGACT-3¢
5¢-TGGACACCTTAGAGACGGTGGCCAGAC-3¢

G (up)
ADI and G (up)
ADI and G (up)
ADI and G (up)
ADI (up)

5¢-AGCCTTGATGAGCCAAGGGCAGTGACCT-3¢
5¢-CATGGCTTCGAATTGATCTTGGAGCTGT-3¢
5¢-ATACCGTGACCTGACATCCGCTGGTGCT-3¢
5¢-CCTTTTTAGCAGTGACTTTCCGTTGCAA-3¢
5¢-TTGCAACGACTGCAGTCATCAGTAGGGT-3¢

5¢-GGCCCGACGGGTGTCTCTCCAGACCCGT-3¢
5¢-TAACCTCCAAAAACTTGCACGTCGGCAA-3¢
5¢-GCACAGTTCCCCTACAGTCCCGCTTTAG-3¢
5¢-CACGTCTCAGCAGGGAGAATAATCCCGA-3¢
5¢-GAGCTCAGCGAGGACGGAAACCTCGCGT-3¢

ADI (up)

5¢-CCAATCAGGTAGGCCTTCATGGAGAGGA-3¢

5¢-CCCAGAGATCCTCCAAGAGACAGCCAGT-3¢

ADI (up)
ADI (down)
ADI (down)

5¢-ATCTGGAGAGCACATCATTGCTGGTGCA-3¢
5¢-ACATCGAGGAAGAGTTTTCTATCCTGGA-3¢
5¢-ACATCGCTGAGAATGTCAACGGGGATAT-3¢

5¢-CTTTCTGGCCTCTCCAACATCCATGCCA-3¢
5¢-ATGCCAAGGTAGTTTATGATGATCGAGA-3¢
5¢-TCTCCTACGATCATCTCACCGTCACCGA-3¢

ADI (down)
G (down)
ADI (down)

5¢-GGTTGTCAATTTATTGAATGATCTCTACA-3
5¢-CATTTTGACTCGTCCCGATAAACAGGCA-3¢
5¢-AACTCAGTTGATTGGCCTAGTTATGCCA-3¢

5¢-CCTCGGATCTTGAGACCCTCGACTGGA-3¢
5¢-GTTTTACCGGCAGCATCCAACCCCACCA-3¢
5¢-TTGGGCTTGTTGGCCTCCTCCTGTGCCT-3¢

FEBS Journal 272 (2005) 390–403 ª 2005 FEBS

399


Oyster response to pesticide exposure

Statistical analysis
To compare mRNA expression between treatment and
time of exposure, a statistical analysis using Student’s
t-test was performed on the expression data. For each
treatment and each gene, differences were tested by sampling date pairs (first date vs. second date, third date vs.
second date, etc.).

Acknowledgements
This research program was financially supported by
´
the Region Bretagne, the interregional program
MOREST (Summer Mortality of juvenile oyster Crassostrea gigas, Grant number 02-2-500022) and the
´ ´
Conseil General du Finistere. The authors are grateful
to Brenda J. Landau for English corrections.

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