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: pascal.favrel@unicaen.fr (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] (http://www.cbs.dtu.dk/services) (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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