Tài liệu Báo cáo khoa học: Characterization of chitinase-like proteins (Cg-Clp1 and Cg-Clp2) involved in immune defence of the mollusc Crassostrea gigas docx

Characterization of chitinase-like proteins (Cg-Clp1 and
Cg-Clp2) involved in immune defence of the mollusc
Crassostrea gigas
Fabien Badariotti, Christophe Lelong, Marie-Pierre Dubos and Pascal Favrel
Universite
´
de Caen Basse-Normandie, IBFA, UMR M100 IFREMER, Physiologie et Ecophysiologie des Mollusques Marins, Laboratoire de
Biologie et Biotechnologies Marines, Caen, France
Glycoside hydrolase family 18 (GH18) is a phylogenet-
ically conserved group of proteins present in eukaryo-
tes, prokaryotes and viruses. The GH18 family is
characterized by a Glyco_18 domain adopting an
(a ⁄ b)
8
triose phosphate isomerase-barrel structure that
consists of a specific arrangement of eight parallel
b-strands, forming the barrel core, surrounded by eight
a-helices [1]. This family classification, based only on
similarities in amino acid sequences, groups together
chitinases and proteins devoid of catalytic activity due
to the substitution of a critical amino acid in the cata-
lytic centre. This latter singular class of proteins, called
chitinase-like proteins (CLPs), has been identified only
recently in plants [2], mammals [3], insects [4] and mol-
luscs [5]. CLPs have been implicated in many biologi-
cal processes, such as control of nodulation [2] and
growth ⁄ differentiation balance during development in
plants [6]. Insect CLPs such as imaginal disc growth
factors represent the first proliferating polypeptides
reported from invertebrates [7]. These mitogenic
growth factors cooperate with insulin to stimulate pro-

liferation, polarization and mobility of imaginal disc
cells in vitro. Imaginal disc growth factors may also
constitute morphogenetic factors controlling embryonic
and larval development, and could stimulate the cell
growth required for wound healing [8,9]. In mammals,
CLPs such as YM1 ⁄ 2 and YKL-40 (40 kDa mamma-
lian protein with the N-terminus YKL) [also known as
human cartilage glycoprotein-39 (HC-gp39) in humans]
are considered to be cytokines [10,11] involved in tis-
sue remodelling during pathological conditions [12,13].
Recently, the first lophotrochozoan CLP was identified
Keywords
chitinase-like protein; Crassostrea gigas;
immunity; lectin; mollusk
Correspondence
P. Favrel, Universite
´
de Caen Basse-
Normandie, IBFA, UMR M100 IFREMER,
Physiologie et Ecophysiologie des
Mollusques Marins, 14032 Caen cedex,
France
Fax: +33 231565346
Tel: +33 231565361
E-mail:
(Received 22 February 2007, revised
10 May 2007, accepted 23 May 2007)
doi:10.1111/j.1742-4658.2007.05898.x
Chitinase-like proteins have been identified in insects and mammals as non-
enzymatic members of the glycoside hydrolase family 18. Recently, the first

molluscan chitinase-like protein, named Crassostrea gigas (Cg)-Clp1, was
shown to control the proliferation and synthesis of extracellular matrix
components of mammalian chondrocytes. However, the precise physiologi-
cal roles of Cg-Clp1 in oysters remain unknown. Here, we report the clo-
ning and the characterization of a new chitinase-like protein (Cg-Clp2)
from the oyster Crassostrea gigas. Gene expression profiles monitored by
quantitative RT-PCR in adult tissues and through development support its
involvement in tissue growth and remodelling. Both Cg-Clp1- and Cg-
Clp2-encoding genes were transcriptionally stimulated in haemocytes in
response to bacterial lipopolysaccharide challenge, strongly suggesting that
these two close paralogous genes play a role in oyster immunity.
Abbreviations
Cg-Clp1 ⁄ 2, Crassostrea gigas chitinase-like protein 1 ⁄ 2; CLP, chitinase-like protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
GH, glycoside hydrolase; HC-gp39, human cartilage glycoprotein-39 (also called YKL-40); LPS, lipopolysaccharide; YKL-40, 40 kDa
mammalian protein with the N-terminus YKL.
3646 FEBS Journal 274 (2007) 3646–3654 ª 2007 The Authors Journal compilation ª 2007 FEBS
from the oyster Crassostrea gigas [5]. Interestingly, this
protein, named C. gigas chitinase-like protein 1 (Cg-
Clp1) was found to be involved in the control of
growth and remodelling processes in a manner similar
to its YKL-40 mammalian counterpart. These findings
argue for an early evolutionary origin and a high con-
servation of this class of proteins at both the structural
and functional levels. Given the multiplicity of CLPs
in humans and insects [14], we hypothesized that
homologues of the previously characterized Cg-Clp1
remain to be found in C. gigas.
In this article, we report the characterization of a
new CLP, named Cg-Clp2, from the oyster C. gigas.
The tissue distribution and temporal pattern of expres-

sion of the gene encoding Cg-Clp2 during oyster devel-
opment were established by real-time PCR and in situ
hybridization. In addition, the involvement of both
Cg-Clp2 and the previously identified Cg-Clp1 in oys-
ter immune defence was established.
Results
Isolation and sequence analysis of Cg-Clp2
full-length cDNA
RT-PCR with degenerate primers whose design was
based on the conserved amino acid sequences of the
catalytic domain of members of the GH18 family
resulted in the amplification of an expected 147 bp
sequence. Cloning and sequencing of this fragment
revealed an ORF showing amino acid sequence simi-
larity to members of the GH18 family. Subsequently,
specific primers deduced from this 147 bp sequence
were used to perform 5¢- and 3¢-RACE-PCR to obtain
the full-length cDNA. This experimental strategy has
been applied successfully in former studies, leading to
the identification of the two first C. gigas members of
the GH18 family, Cg-Clp1 (AJ971241) [5] and the chi-
tinase Cg-Chit (AJ971238) [15]. The complete 1697 bp
cDNA (AJ971235) revealed an ORF of 1425 bp, start-
ing with an ATG at position 117 and ending with a
TAA at position 1542. This ORF encodes a protein
named C. gigas chitinase-like 2, composed of 475
amino acids with a putative N-terminal 19 amino acid
signal peptide (Fig. 1). Cg-Clp2 contains one potential
recognition site for N-linked oligosaccharide [16] and
two potential recognition sites for O-linked oligosac-

charide [17] ( (Fig. 1).
Cg-Clp2 sequence identity with other proteins
Optimal alignment of Cg-Clp2 with Cg-Clp1 and other
GH18 family members revealed regions of significant
identity, especially in the Glyco_18 domain. The glu-
tamate residue known to be critical for chitinase activ-
ity [18] is substituted by a glutamine, suggesting that
this protein lacks chitinolytic activity, as was shown
previously for recombinant Cg-Clp1 [5] and other
CLPs [19]. Following the Glyco_18 domain, Cg-Clp2
displays an additional 90 amino acid C-terminal
sequence of unknown function (Fig. 1). Hence, the
overall structure of Cg-Clp2 is similar to that of
Cg-Clp1.
Expression of Cg-Clp2 transcripts during
development and in adult tissues
To gain insights into possible physiological functions
of Cg-Clp2, determination of its tissue distribution and
temporal pattern of expression during development
was performed by real time RT-PCR (Fig. 2A). Cg-
Clp2 transcripts were mainly expressed during larval
metamorphosis, in the mantle edge and the digestive
gland. During the reproductive cycle, expression was
high in gonads during the postspawning period but
not in stage I, when gonial multiplication starts [20].
To investigate which types of cell were responsible for
Cg-Clp2 expression in the mantle edge, in situ hybrid-
ization experiments were performed (Fig. 2B). Tran-
scripts were detected in both epithelial and conjunctive
cells of the mantle.

Cg-Clp1 and Cg-Clp2 mRNA levels are increased
in haemocytes after bacterial LPS challenges
As the two mammalian CLPs, YM1 and YKL-40,
were recently categorized as immune cytokines [10,11],
the possible involvement of Cg-Clp1 and Cg-Clp2 in
oyster immunity was investigated. Gene expression was
analysed by real-time RT-PCR in haemocytes at differ-
ent times after injection of bacterial lipopolysaccharide
(LPS) into the posterior adductor muscle and after an
in vitro LPS challenge.
A marked increase in Cg-Clp1 expression was
observed in vivo 9 h and 12 h after LPS injection
(Fig. 3A) relative to the respective controls. Cg-Clp1
was also transcriptionally stimulated in vitro, 6 h and
12 h after LPS addition, in comparison to unstimulat-
ed control haemocytes (Fig. 3B). However, this upreg-
ulation was substantially lower than that observed for
in vivo challenge. In contrast, Cg-Clp2 expression was
not affected by
in vivo LPS challenge (data not shown)
but, as compared to unstimulated haemocytes, was
stimulated in vitro 2 h after LPS addition (Fig. 3C).
Surprisingly, the Cg-Clp2 expression level was also
significantly enhanced in adherent nonstimulated
F. Badariotti et al. Oyster chitinase-like proteins
FEBS Journal 274 (2007) 3646–3654 ª 2007 The Authors Journal compilation ª 2007 FEBS 3647
Fig. 1. Multiple sequence alignment of Cg-Clp2 with members of the GH18 family. (A) The predicted amino acid sequence of Cg-Clp2 is aligned with the amino acid sequence of three
CLPs from the oyster C. gigas (Cg-Clp1), Drosophila melanogaster (IDGF4) and Homo sapiens (YKL-40), and with the sequence of the Drosophila chitinase Cht9. Conserved residues (iden-
tical to Cg-Clp2) are shaded in dark grey. Potential sites for N-glycosylation (NXT ⁄ S) and for O-glycosylation (S or T) are shaded in black. Amino acids of the predicted signal peptide are
shown in bold italic letters. Dashes indicate gaps in the amino acid sequence when compared with other sequences. The GH18 conserved sequence motif including the catalytic residues

is marked with a thick black line above the sequence alignment. Arrowheads indicate the positions of residues (D and E) required for catalytic activity in bacterial chitinases [18]. The black
dotted line delimits the Glyco_18 domain. The species abbreviations used are: Dm, Drosophila melanogaster; and Hs, Homo sapiens. GeneBank accession numbers: Cg-Clp1, AJ971241;
Dm IDGF4, NP511101; Hs YKL-40, NP001267; Dm Cht9, NP611543.
Oyster chitinase-like proteins F. Badariotti et al.
3648 FEBS Journal 274 (2007) 3646–3654 ª 2007 The Authors Journal compilation ª 2007 FEBS
haemocytes as compared to freshly harvested circula-
ting cells.
Discussion
In the present study, we identified a second oyster
CLP named Cg-Clp2. Comparative sequence analyses
with other GH18 family members show that Cg-Clp2
displays the same protein organization as the previ-
ously identified Cg-Clp1, with a Glyco_18 domain (in
a catalytically inactive form [5]) followed by an addi-
tional C-terminal sequence of about 90 amino acids of
unknown function. The high degree of identity of the
Cg-Clp1 and Cg-Clp2 Glyco_18 domains (84% iden-
tity) argues for a conservation of the tertiary structure
and associated biochemical properties (such as chitin
binding). Evidence for a high level of conservation of
the tertiary structure of CLPs during evolution is also
supported by the observation that both Cg-Clp1 and
its closest mammalian homologue YKL-40 present
A
B
Fig. 2. Expression of Cg-Clp2 mRNAs in adult tissues and during development measured by real-time quantitative RT-PCR. (A) Each value is
the mean + SE of three pools of four animals (tissues) or the mean of one pool of embryos or larva from one spawn. Expression levels are
related to 100 copies of GAPDH. (B) Localization of Cg-Clp2 mRNA expression in the mantle edge investigated by in situ hybridization.
Arrows indicate hybridization signals.
F. Badariotti et al. Oyster chitinase-like proteins

FEBS Journal 274 (2007) 3646–3654 ª 2007 The Authors Journal compilation ª 2007 FEBS 3649
similar biological activities on mammalian chondro-
cytes [5]. As YKL-40 is only composed of the sole
Glyco_18 domain, the C-terminal tail of C. gigas CLPs
may not noticeably contribute to the structure and the
function of these proteins. Interestingly, Cg-Clp1 and
Cg-Clp2 C-terminal regions share relatively low levels
of sequence identity (46%), probably as the result of a
lower pressure of selection during evolution. Neverthe-
less, these discrepancies may also account for slightly
distinct biochemical properties.
Analysis of mRNA distribution during development
and in adult tissues shows that Cg-Clp2 is expressed
A
B
C
Fig. 3. Real time quantitative RT-PCR
analysis of Cg-Clp1 and Cg-Clp2 mRNA
expression in haemocytes following bacter-
ial LPS challenges. In vivo experiment: time-
dependent effect of LPS (100 lg) injection
on Cg-Clp1 expression (A). Results are
means + SE of at least three oysters.
In vitro experiment: time-dependent effect
of LPS addition (final concentration
13 lgÆmL
)1
) to cell culture medium on
Cg-Clp1 (B) and Cg-Clp2 (C) expression.
Results are means + SE of three wells.

Statistical analysis of the results was per-
formed with Student’s t-test (*P < 0.05;
**P < 0.02).
Oyster chitinase-like proteins F. Badariotti et al.
3650 FEBS Journal 274 (2007) 3646–3654 ª 2007 The Authors Journal compilation ª 2007 FEBS
during metamorphosis, in the mantle edge and post-
spawning gonads. Metamorphosis represents the ulti-
mate stage of oyster development, and is characterized
by the degeneration of larval tissues, such as the velum
and the foot, and the remodelling of larval tissues to
produce adult tissues (i.e. the development of the gills
and the production of an adult shell), which is accom-
panied by significant growth of the soft body parts
[21]. The mantle edge governs shell formation and
body growth by the secretion of shell organic matrix
and by cell proliferation. As Cg-Clp2 appears to be
expressed in both epithelial and conjunctive cell types
of the mantle edge, this protein could orchestrate the
synthesis of extracellular components and ⁄ or the pro-
liferation of mantle cells, as was proposed for Cg-Clp1
[5]. The postspawning gonad is characterized by the
resorption of gonadic tubules and the rebuilding of
storage tissues [22]. The expression of Cg-Clp2 during
this particular period is somewhat reminiscent of the
finding that certain mammalian CLPs such as CLP-1
and MGP40 are specifically expressed during mam-
mary gland involution [23,24]. Considering Cg-Clp2
patterns of expression, this protein could be involved
in tissue growth and remodelling, as was formerly
postulated for Cg-Clp1 [5].

Messenger RNAs encoding Cg-Clp1 and Cg-Clp2
were upregulated in haemocytes after stimulation
with bacterial LPS. This supports a role for Cg-Clp1
and Cg-Clp2 in defence against Gram-negative bac-
teria in response to LPS. Nevertheless, it was
recently reported that commercial preparations of
LPS are often contaminated with peptidoglycan,
which actually constitutes the true immunostimula-
tory component in Drosophila [25]. Thus, we cannot
rule out the possibility that a similar situation occurs
in C. gigas.
The fact that Cg-Clp1 (and most likely Cg-Clp2) is
known to bind tightly and specifically to chitin [5]
strongly supports a role of this lectin in the immune
response to chitinous pathogens, such as fungi and
nematodes, as was postulated for its mammalian
homologue HC-gp39 [11]. Because bacteria do not
contain chitin, enhanced expression of Cg-Clp1 and
Cg-Clp2 in response to either LPS or peptidoglycan
stimulation might be considered as a general nonspe-
cific response of the organism to foreign invaders. On
the other hand, both LPS and peptidoglycan harbour
GlcNAc, the constituent of chitin, in their molecular
structure. A possibility is that Cg-Clp1 and Cg-Clp2
bind to bacteria via these cell wall components; if this
is so, the resulting overexpression of these lectins
should be considered as a specific immune response to
bacteria.
The fact that Cg-Clp1 stimulates the proliferation
and regulates the synthesis of extracellular matrix com-

ponents of mammalian chondrocytes [5] endorses the
possibility that Cg-Clp1 promotes cell (haemocyte)
proliferation and ⁄ or tissue repair, both processes
occurring during immune responses [26,27]. Such a
role was also suggested for insect imaginal disc growth
factors [8,9,28]. As was observed for its murine
homologue (YM-1), which behaves as a chemotactic
cytokine that recruits cells to sites of inflammation and
promotes eosinophilia around larvae of nematode
parasites [10], mediation of immune cell (haemocytes)
migration or aggregation might also represent a
potential function for Cg-Clp1. Because Cg-Clp1 and
Cg-Clp2 are two close paralogues sharing a very sim-
ilar structure, the several roles predicted for Cg-Clp1
in immunity may also be relevant for Cg-Clp2.
Interestingly, haemocyte adhesion to the culture plastic
dish induces on its own a strong increase in Cg-Clp2
transcript expression, whereas no effect was detected
for Cg-Clp1. Such a surprising result was previously
observed for the oyster chitinase Cg-Chit [15]. This
in vitro assay somehow mimics haemocyte conversion
from circulating cells to cells that interact with and
adhere to each other or to a foreign target surface, as
is observed for encapsulation [29]. These ‘activated
haemocytes’ may become immunologically competent
cells capable of producing acute phase immune effec-
tors, as was recently reported for Manduca sexta plas-
matocytes, which express only the specific lectin
‘lacunin’ upon adhering to a foreign surface [30]. This
would explain why stimulation of Cg

-Clp2 transcript
expression is effective under in vitro but not in vivo
conditions, when only circulating cells are harvested
for gene quantification. On the contrary, the partial
failure of the in vitro cell culture conditions to elicit
LPS stimulation of Cg-Clp1 gene expression may be
due to the absence of pertinent haemolymph
circulating factors in these experimental conditions.
Indeed, such extracellular molecules could be necessary
for bacterial recognition as the first step in a process
leading to an increase in Cg-Clp1 transcript quantity.
This hypothesis is in agreement with the observation
that Drosophila host defence against Gram-negative
bacteria may involve the secretion in the haemolymph
of a pattern recognition receptor [31,32]. Alternatively,
one could postulate that Cg-Clp1 is expressed mainly
in nonadhering haemocytes.
Our results with C. gigas Cg-Clp1 and Cg-Clp2
suggest strongly that these proteins fulfil an important
function as immunity regulators and ⁄ or effectors in
molluscs. The structural similarities shared by these
two protein isoforms suggest they have similar
F. Badariotti et al. Oyster chitinase-like proteins
FEBS Journal 274 (2007) 3646–3654 ª 2007 The Authors Journal compilation ª 2007 FEBS 3651
biochemical mechanisms. In contrast, their discrete
responses to bacterial challenge hint at distinctive
physiological functions in immunity.
Experimental procedures
Animals
Adult C. gigas oysters were purchased from a local oyster

farm (Saint Vaast La Hougue, France). The embryonic and
larval stages were produced in the IFREMER shellfish
laboratory of Argenton (France).
RNA purification, reverse transcription, cloning
and sequencing
Total RNA was isolated from the oyster mantle edge using
Tri-Reagent (Sigma-Aldrich, St Louis, MO, USA) accord-
ing to the manufacturer’s instructions. mRNAs were isola-
ted using oligodT coupled to magnetic beads as described
by the manufacturer (Dynal, Invitrogen, Carlsbad, CA,
USA). Reverse transcription was carried out using oli-
go(dT)
17
as primer, 1 lg of mantle edge mRNA, and 200 U
of Moloney murine leukaemia virus reverse transcriptase
(Promega, Madison, WI, USA). cDNAs were used as tem-
plates for PCR amplifications using two degenerated prim-
ers designed to anneal to conserved consensus regions of
GH18 family members (chitinases and CLPs) from different
bilaterian species. The sense primer corresponding to the
LK(I ⁄ M)L(F ⁄ L)(S ⁄ T ⁄ R ⁄ C ⁄ W)VGG amino acid seque-
nce was 5¢-CTN AAR ATN CTN YTN WSN GTN GGN
GG-3¢, whereas the antisense primer corresponding to the
FDGLDLA amino acid sequence was 5¢-GGC NAG RTC
NAG NCC RTC RAA-3¢ (Y ¼ CorT,R¼ AorG,S¼
CorG,W¼ AorT,N¼ A or C or G or T). PCR was
performed in a total volume of 50 lL with 10 ng of mantle
edge cDNA in 10 mm Tris ⁄ HCl (pH 9.0), containing
50 mm KCl, 0.1% Triton X-100, 0.2 m m each dNTP, 1 lm
each primer, 2.5 mm MgCl

2
and 1 U of Taq DNA poly-
merase (Eurogentec, Liege, Belgium). The reaction was
cycled between 94 °C, 50 °C and 72 °C (45 s, 60 s and 90 s,
respectively), and this was followed by an extension step at
72 °C for 5 min. After 40 cycles, a resulting 147 bp frag-
ment was isolated. Full-length cDNA was generated by 5¢-
and 3¢-RACE using the Marathon cDNA amplification kit
(Clontech, Takara, Mountain View, CA, USA). Double-
stranded cDNA from oyster mantle edges was ligated to
adaptors, and 25 ng of this template was used to PCR
amplify 5¢- and 3¢-RACE fragments using adaptor-specific
primers and gene-specific primers deduced from the ini-
tial 147 bp fragment sequence. PCR products were sub-
cloned into pGEM-T easy vector using a TA cloning kit
(Promega), and sequenced using ABI cycle sequencing
chemistry.
Real-time quantitative PCR
Quantitative RT-PCR analysis was performed using the
iCycler apparatus (Bio-Rad, Hercules, CA, USA). Total
RNA was isolated from oocytes, embryos, larvae and adult
tissues using Tri-Reagent (Sigma-Aldrich) according to the
manufacturer’s instructions. After treatment for 20 min at
37 °C with 1 U of DNase I (Sigma-Aldrich) to prevent ge-
nomic DNA contamination, 1 lg of total RNA was
reversed transcribed using 1 l g of random hexanucleotidic
primers (Promega), 0.5 mm dNTPs and 200 U of Moloney
murine leukaemia virus Reverse Transcriptase (Promega) at
37 °C for 1 h in the appropriate buffer. The reaction was
stopped by incubation at 70 °C for 10 min. The iQ SYBR

Green supermix PCR kit (Biorad) was used for real-time
monitoring of amplification (5 ng of cDNA template, 40
cycles: 95 °C for 15 s, 60 °C for 15 s) with the following
primers: QsCgClp1 (5¢-CTTCCTCCGCTTCCATGA-3¢)
and QaCgClp1 (5¢-CCATGAAGTCCGCGAATC-3¢); and
QsCgClp2 (5¢-GCATAGCGATGTGGACGA-3¢) and
QaCgClp2 (5¢-GAGGACCGAGACCGTGAA-3¢). The
abbreviations ‘Qs’ and ‘Qa’ refer, respectively, to sense and
antisense primers. Accurate amplification of the target
amplicon was checked by obtaining a melting curve.
Using QsGAPDH (5¢-TTCTCTTGCCCCTCTTGC-3¢) and
QaGAPDH (5¢-CGCCCAATCCTTGTTGCTT-3¢), a paral-
lel amplification of oyster glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (CGI548886) reference tran-
scripts was carried out to normalize the expression data of
Cg-Clp1 and Cg-Clp2 transcripts. The relative level of
expression of each target gene was calculated for 100 copies
of GAPDH transcript by using the following formula:
N ¼ 100 · 2
(Ct GAPDH ) cycle threshold transcript of interest)
.
In situ hybridization
A 1283 bp fragment corresponding to the most 3¢-end of
Cg-Clp2 was subcloned in pGEMT easy. This recombinant
plasmid was used as a template for the synthesis of biotin-
labelled sense and antisense cRNA probes according to the
manufacturer’s instructions (NEN Life Sciences, PE, Wal-
tham, MA, USA). Dissected C. gigas mantle edges were
fixed, dehydrated in an increasing alcohol series and xylene,
and embedded in paraplast. Seven-micrometre sections were

cut and mounted on aminosilane-coated slides. Sections
were rehydrated, and endogenous peroxidase activity was
blocked by incubating sections in 0.3% hydrogen peroxide
in methanol for 30 min at room temperature. Slides were
then washed and incubated in a blocking solution accord-
ing to the manufacturer’s instructions. Hybridization was
performed overnig ht at 55 °C. Biotin-labelled probes were
detected using a streptavidin–horseradish peroxidase conju-
gate. Peroxidase activity was revealed by 3,3¢diaminobenzi-
dine substrate (Sigma-Aldrich).
Oyster chitinase-like proteins F. Badariotti et al.
3652 FEBS Journal 274 (2007) 3646–3654 ª 2007 The Authors Journal compilation ª 2007 FEBS
Quantification of mRNA levels in haemocytes
after bacterial LPS challenge
In vivo challenge
Animals were injected with 100 lg (in 100 lL of NaCl ⁄ P
i
)
of Escherichia coli 026:B6 LPS (Sigma-Aldrich) into the
posterior adductor muscle, through a hole drilled in the
shell. NaCl ⁄ P
i
-injected oysters were used as controls. After
injection, animals were placed in sea water (12 °C). At four
time points after LPS injection (3 h, 6 h, 9 h and 12 h),
haemolymph samples from three animals were withdrawn
from the pericardic region using a 45-gauge needle and cen-
trifuged at 1000 g for 2 min (Eppendorf 5810R centrifuge,
fixed angle rotor F45-30-11) in order to separate cells from
the haemolymph fluid.

In vitro challenge
Primary haemocyte culture was performed as previously
described, with some modifications [33]. Haemolymph was
recovered from the pericardic region of 90 oysters using a
45-gauge needle, and then subsequently transferred to a
sterile tube and simultaneously diluted 1 : 3 in cooled sterile
anticoagulant modified Alsever’s solution (115 mm glucose;
27 mm sodium citrate; 11.5 mm EDTA; 382 m m NaCl).
Haemocytes were rapidly plated at 4 · 10
6
cells per 9.5 cm
2
well, to which three volumes of sterile artificial sea water
were added to allow cell attachment. Cultures were main-
tained at 15 °C in a humidified incubator (CO
2
-free). After
60 min of incubation, cells were washed with Hanks-199
medium modified by the addition of 250 mm NaCl, 10 mm
KCl, 25 mm MgSO
4
, 2.5 m m CaCl
2
, and 10 mm Hepes; the
final pH was 7.4, and the osmolarity was 1100 mOsmolÆ L
)1
.
Cells were then covered with fresh medium supplemented
with l-glutamine (2 mm), concanavalin A (2 mm), strepto-
mycin sulfate (76.1 IUÆmL

)1
) and penicillin G
(100 IUÆmL
)1
), and were incubated (CO
2
-free) at 15 °C.
Haemocyte monolayers were then treated for 30 min with
culture medium containing bacterial LPS (1 lgÆlL
)1
in
NaCl ⁄ P
i
, final concentration 13 lgÆ mL
)1
). Control (medium
without LPS) haemocyte monolayers were run in parallel.
After 30 min, culture media were exchanged for fresh
media. Haemocytes were lysed for total RNA extraction
with Tri-Reagent (Sigma-Aldrich) at different time points
of the experiment: haemocytes in suspension, immediately
after haemocyte adhesion, 30 min, 1 h, 2 h, 3 h, 6 h and
12 h after adhesion (control haemocytes), or 30 min, 1 h,
2 h, 3 h and 6 h after addition of LPS to the medium.
Statistical analysis
Results were expressed as means + SE and analysed using
Student’s t-test. The significance level was set as stated in
the legend to Fig. 3.
Acknowledgements
This study was financially supported by the ‘Conseil

Re
´
gional de Basse-Normandie’, the ‘Agence de l’eau
Seine-Normandie’ and FEDER Presage No. 4474
grant (program PROMESSE). The authors are
indebted to all staff of the Argenton IFREMER
experimental hatchery for the production of oyster
embryos and larvae. The authors thank Christophe
Fleury and Emeline Furon (University of Caen) for
technical assistance.
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