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International audience; In intertidal mudflats, during low tide exposure, microphytobenthos (MPB) migrate vertically through the surface sediment and form, with the heterotrophic bacteria, a transient biofilm. Inside this biofilm, multiple interactions exist between MPB and bacteria. These micro-organisms secrete a wide range of extracellular polymeric substances (EPS), which are major components of the biofilm matrix. In this study, we used a tidal mesocosm experiment in order to decipher the interactions of the MPB-EPS-bacteria complex within the biofilm. We tried to determine if the EPS could control bacterial activities and/or production and/or richness according to the age of the biofilm and to the immersion/emersion period. The dynamics of biomasses of MPB and prokaryotes, the bacterial production, the hydrolysis of predominating organic constituents in the dissolved organic carbon (DOC) pool (i.e., carbohydrates and polypeptides), and the bacterial structure were studied in relation to the different EPS fractions (carbohydrates and proteins: colloidal and bound) dynamics during 8 days. Our experiment had emphasized the influence of the environmental conditions (light, immersion/emersion) on the interactions within the biofilm and also on the effects on biofilm ageing. Bacterial production was always inhibited by the bound EPS-carbohydrate, especially during low tide. Our results suggest that the concentration and composition of EPS had a major role in the bacterial/MPB interactions: these interactions can be either positive or negative in order to regulate the productive phases of MPB and bacteria.

International audience; Numerous field-based investigations have highlighted that the production of extracellular polymeric substances (EPS) is physico-chemically and ecologically important for intertidal mudflats. EPS are largely secreted by marine benthic diatoms and their quantity and quality are environmental-dependant. This paper focused on the dynamic pathways, concentration rates and monosaccharides composition of colloidal, bound and residual carbohydrates extracted by using a cationic exchange resin from a diatom-dominated intertidal mudflat (Marennes-Oléron, France) during two different sampling periods: winter (February 2008) and summer (July 2008). A wide range of biotic and abiotic parameters were also studied to better understand the effect of environmental parameters, e.g., chlorophyll a, salinity, porewater amount, emersion time, luminosity, C:N ratio and tidal coefficient. Multiple colorimetric assays coupled to gas chromatographic analyseswere carried out to performthe biochemical characterizations. Firstly, the quantity of carbohydrates produced during winter (5.28 μg*μg chl a−1) was more important than during summer (2.04 μg*μg chl a−1). Yet, more proteins were found during summer for the colloidal and bound fractions (0.73 and 1.04 μg*μg chl a−1). Further investigations showed that the dynamic pathways were equivalent between winter and summer: bound carbohydrates (BC) quantities increased during the sediment emersion periods on the contrary to colloidal carbohydrates (CC) which tended to drop throughout the emersion time. The quality in monosaccharides was fraction-dependant, whatever the season. CC were always glucose-rich confirming their role of carbon source. BC were mainly composed of rhamnose whose the ratio increased during the emersion period, thus conferring adhesive properties to the extracellular matrix bounding diatoms cells. Residual carbohydrates (RC) were composed of various monosaccharides and a major increase of glucose content was found at the end of emersion, corresponding to intracellular C-storage in prevention to immersion times. Summer-RC were composed of fucose, a monosaccharide specific to these fractions and which was non-present during the winter campaign. Environmental parameters, such as salinity, pore water amount, and tidal coefficient could have a significant impact on the concentration rates and pathways of carbohydrates.

Gonadotropin-releasing hormone (GnRH), a key neuropeptide regulating reproduction in vertebrates has now been characterized in a number of non-vertebrate species. Despite the demonstration of its ancestral origin, the structure and the function of this family of peptides remain poorly known in species as distant as lophotrochozoans. In this study, two GnRH-related peptides (Cg-GnRH-a and CgGnRH-G) were characterized by mass spectrometry from extracts of the visceral ganglia of the Pacific oyster Crassostrea gigas. These peptides showed a high degree of sequence identity with GnRHs of other mollusks and annelids and to a lesser extent with those of vertebrates or with AKH and corazonins of insects. Both the mature peptides and the transcript encoding the precursor protein were exclusively expressed in the visceral ganglia. Significant differences in transcriptional activity of Cg-GnRH encoding gene were recorded in the ganglia along the reproductive cycle and according to trophic conditions with a higher level in fed animals compared to starved animals. This suggests the involvement of Cg-GnRHs as synchronizers of nutritional status with energy requirements during reproduction in oyster. Evidence for a role of Cg-GnRHs as neuroregulators and as neuroendocrine factors in bivalve is discussed. (C) 2012 Elsevier Inc. All rights reserved.

International audience; BACKGROUND: The Pacific oyster Crassostrea gigas (Mollusca, Lophotrochozoa) is an alternative and irregular protandrous hermaphrodite: most individuals mature first as males and then change sex several times. Little is known about genetic and phenotypic basis of sex differentiation in oysters, and little more about the molecular pathways regulating reproduction. We have recently developed and validated a microarray containing 31,918 oligomers (Dheilly et al., 2011) representing the oyster transcriptome. The application of this microarray to the study of mollusk gametogenesis should provide a better understanding of the key factors involved in sex differentiation and the regulation of oyster reproduction. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression was studied in gonads of oysters cultured over a yearly reproductive cycle. Principal component analysis and hierarchical clustering showed a significant divergence in gene expression patterns of males and females coinciding with the start of gonial mitosis. ANOVA analysis of the data revealed 2,482 genes differentially expressed during the course of males and/or females gametogenesis. The expression of 434 genes could be localized in either germ cells or somatic cells of the gonad by comparing the transcriptome of female gonads to the transcriptome of stripped oocytes and somatic tissues. Analysis of the annotated genes revealed conserved molecular mechanisms between mollusks and mammals: genes involved in chromatin condensation, DNA replication and repair, mitosis and meiosis regulation, transcription, translation and apoptosis were expressed in both male and female gonads. Most interestingly, early expressed male-specific genes included bindin and a dpy-30 homolog and female-specific genes included foxL2, nanos homolog 3, a pancreatic lipase related protein, cd63 and vitellogenin. Further functional analyses are now required in order to investigate their role in sex differentiation in oysters. CONCLUSIONS/SIGNIFICANCE: This study allowed us to identify potential markers of early sex differentiation in the oyster C. gigas, an alternative hermaphrodite mollusk. We also provided new highly valuable information on genes specifically expressed by mature spermatozoids and mature oocytes.

In Protostoma, the diuretic hormone 31 (DH31) signaling system was long considered as the orthologue of the chordate calcitonin (CT) signaling system. Using the Pacific oyster (Crassostrea gigas) transcriptomic database GigaTON (http://ngspipelines-sigenae.toulouse.inra.fr/), we characterized seven G-protein-coupled receptors (GPCRs) named Cragi-CTR1/7 and phylogenetically related to chordate CT receptors (CTRs) and to protostome DH31 receptors. Two CT Precursors (Cragi-CTP1 and Cragi-CTP2) containing two CT-type peptides and encoded by two distinct genes with a similar organization were also characterized. These oyster neuropeptides (Cragi-CT1/2) exhibit the two N-terminal paired cysteine residues and except CTP2 derived peptide (Cragi-CTP2dp) the C-terminal proline-amide motif typical of deuterostome CT-type peptides. All mature Cragi-CTs but Cragi-CTP2dp were detected in visceral ganglion (VG) extracts using mass spectrometry. Cell-based assays revealed that the formerly characterized oyster receptors Cg-CTR and Cragi-CTR2 were specifically activated by Cragi-CT1b and Cragi-CT2, respectively. This activation does not require the co-expression of receptor activity-modifying proteins (RAMPs). Thus, the oyster CT signaling appears functionally more closely related to the vertebrate CT/CTR signaling than to the (Calcitonin Gene Related Peptide) CGRP/CLR signaling. Gene expression profiles in different adult tissues and in oysters acclimated to brackish water suggest the potential implication of both Cg-CT-R/Cragi-CT1b and Cragi-CTR2/Cragi-CT2 in water and ionic regulations, though with apparently opposite effects. The present study represents the first comprehensive characterization of a functional CT-type signaling system in a protostome and provides evidence for its evolutionarily ancient origin and its early role in osmotic homeostasis.

The sex-determining system of Crassostrea gigas is still poorly known, especially regarding the potential influence of temperature. In order to address this question, mRNA expressions of actors of the molecular cascade (Cg-DMI, Cg-SoxE, Cg-beta-catenin, Cg-Foxl2/Cg-FoxI2os) and of Oyvlg, a germ cell marker, were investigated by real-time PCR in spat grown at different temperatures (18, 22, 25 and 28 degrees C). In parallel, gonadic differentiation, gametogenesis and sex ratios were assessed by histology at each of these temperatures. Whatever the temperature, Cg-DMI, Cg-SoxE, Cg-beta-catenin and Oyvlg expressions peaked at the same developmental stage, always after Cg-Foxl2/Cg-Foxl2os (around 40-44 dpf for spat grown at 18 degrees C). Temperatures increased the kinetics of first gonadic differentiation and gametogenesis. At 25 degrees C a significant switch occurred in sex ratio towards males and in the balance of expression between male and female genes, in favor of males. A slight gametogenesis disturbance was also observed. These results strengthen the hypotheses about the sex-determining time window and molecular cascade governing the development of C gigas, with notably the involvement of Cg-Foxl2/Cg-Foxl2os in the very early steps. They also suggest an influence of temperature on the oyster's sex determination which, associated to genetic control, would induce a mixed sex determination system (GSD +TSD). (C) 2013 Elsevier Inc. All rights reserved.

Knowledge of sperm maturation process is limited in the Pacific oyster Crassostrea gigas and major factors of fertilization success of this free spawning animal are unknown. We investigated proteins associated to spermatozoa by analyzing two cellular fractions obtained from a 40-80% Percoll gradient fractioning of germ cell of mature male gonads. Mature spermatozoa were enriched in the lower Percoll fraction while the upper fraction contained less mature or earlier germ cells. A 2-DE proteomic approach was used to identify differentially expressed proteins in both fractions. We screened out 31 differential proteins (P0.05) which included 14 up-regulated and 17 down-regulated proteins. Using MALDI-TOF/TOF MS and bioinformatics search against a C. gigas database, 13 and 8 proteins were identified for the up-regulated and down-regulated groups, respectively. In the spermatozoa enriched fraction, proteins regarding flagellum formation and control, energy production and Proteosome subunit beta were increased. In less mature germ cell fraction, proteins regarding developmental processes and chaperon molecules were mainly increased. Our results improve current knowledge of proteins associated with spermatozoa maturation related to zootechnical practices used in mollusk hatcheries.This is the revised version of the manuscript "Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas" by Kingtong et al. to the Journal of Proteomics. The corrections have been done by the team carefully. This work highlight the enrichment method of spermatozoa of Pacific oyster from stripped complex sample using Percoll gradient. The results reflexed developmental stages of germ cells in gonadal tubules of this species. We have used proteomic approach to identify differentially expressed proteins in mature spermatozoa fraction compared to less mature spermatozoa fraction which provided candidates of protein associated to mature spermatozoa in the Pacific oyster. This work improves the current knowledge in marine bivalve hatchery.

International audience; Insulin pathways were demonstrated from invertebrates to vertebrates to be involved in the regulation of numerous processes including storage metabolism and reproduction. In addition, insulin system may integrate variations of environmental conditions like dietary restrictions. In the Pacific oyster Crassostrea gigas, reproductive and storage compartments are closely intricated in the gonadal area and their respective development was found to be dependant of trophic conditions. For these reasons, C. gigas is an original and interesting model for investigating the role of insulin control in the balance between storage and reproduction and the integration of environmental parameters. On the basis of sequence conservation, we identified three potential elements of the oyster insulin pathway, Ras, Pten and p70S6K and we investigated their expression levels in various tissues. In the gonadal area, we used laser microdissection in order to precise the targeted contribution of insulin signaling to the restoration of storage tissue and to the control of vitellogenesis. Food deprivation during gametogenesis reinitiation stage led to reduced proliferations of gonia and also to modulate insulin signal by transcriptional activation of insulin pathway elements.

Despite their economic importance, only very little information is available regarding (neuro)endocrine mechanisms of reproduction in bivalve molluscs. To gain insights into the molecular control of gonadic development of these animals, G protein-coupled receptors (GPCR) expressed in the gonad of the pacific oyster Crassostrea gigas were investigated. One such receptor was cloned by RT-PCR using oligonucleotide primers derived from consensus sequences of various vertebrate (neuro)peptide receptors. This receptor named Cg-GnRH-related receptor (Cg GnRH-R) exhibits a high degree of amino acid sequence identity with both vertebrate GnRH receptors and insect AKH receptors. Quantitative RT-PCR shows a specific expression of Cg-GnRH-R in both male and female gonads during the reproductive cycle. This demonstrates for the first time the plausible involvement of a GnRH receptor orthologue in the control of reproduction in a protostomian invertebrate.

The influence of temperature and light on the growth, physiology and domoic acid (DA) production of the pennate toxic diatom Pseudo-nitzschia australis isolated from the English Channel was studied in semi-continuous culture. The effects of eight irradiances (35–400 μmol photons m2 s−1) and a temperature gradient (3.5–25.5 °C) were investigated. The highest growth rates (0.47–0.83 d−1) were observed between 18.6 and 13.5 °C, with an optimum irradiance for growth at 100 μmol photons m2 s−1. A decrease in the optimum growth temperature was observed with an increase in irradiance. Cell chlorophyll a content decreased at both low and high extreme temperatures and high irradiances. Stable and high values of Fv/Fm ratios below 21 °C highlight the great acclimation capacity of P. australis, which may explain its wide biogeographic distribution. The strain studied produced DA without nutrient limitation and during exponential growth although at lower levels than in other studies (0.15–2.0 pg DA cell−1). Results underline the importance of light–temperature interactions for growth and DA production. Toxin production increased with increasing temperature and light, and DA production increased exponentially with the growth rate. These results show that DA is not only produced under nutrient stress or by unhealthy cells in P. australis. This study underlines the need to take the ability of P. australis to produce DA during the exponential growth into consideration to understand the link between toxin production and bloom dynamics. These results will not only help explore the processes involved, but also help parameterize future models of growth and DA production especially for P. australis.

In dogfish, spermatogenesis progresses from a restricted germinative zone, which lines the dorsal testicular vessel. Single spermatogonia (As), including the spermatogonial stem cells (SSCs), produce successively paired (Ap), undifferentiated (Au4to Au512), and differentiated (Ad1to Ad8) spermatogonia and preleptotene (PL) spermatocytes through 13 mitoses. Dogfish spermatogonial subpopulations present classical morphological characteristics but cannot be distinguished on the basis of molecular markers. This characterization has been initiated in mammals despite the difficulty to separate each spermatogonial subpopulation. For instance, both glial cell-derived neurotrophic factor family receptor alpha 1 (GFRα1) and promyelocytic leukemia zinc finger protein (PLZF) are markers of undifferentiated spermatogonia, whereas receptor tyrosine kinase C-kit is a marker of differentiated spermatogonia. The aim of this study is to characterize spermatogonial markers and to differentiate several spermatogonial subpopulations. Dogfish cDNA sequences have been identified and validated by phylogenetic analyses forgfrα1,plzf,pou2, as well as for high-mobility group box proteins 2 and 3 (hmgb2and3) and for mini-chromosome maintenance protein 6 (mcm6). We have used the anatomical advantage of the polarized dogfish testis to analyze the expression of those markers by RT-PCR andin situhybridization.gfrα1,pou2, andplzfhave been detected in the testicular germinative zone, suggesting that spermatogonial markers are relatively well conserved among vertebrates but with a less restricted expression forplzf. Moreover,hmgb3andmcm6have been identified as new markers of differentiated spermatogonia. Finally, this first molecular characterization of spermatogonial subpopulations in a chondrichthyan model will be useful for further studies on the SSC niche evolution.

International audience; In vertebrates, epigenetic modifications influence gene transcription, and an appropriate DNA methylation is critical in development. Indeed, a precise temporal and spatial pattern of early gene expression is mandatory for a normal embryogenesis. However, such a regulation and its underlying mechanisms remain poorly understood in more distant organisms such as Lophotrochozoa. Thus, despite DNA in the oyster genome being methylated, the role of DNA methylation in development is unknown. To clarify this point, oyster genomic DNA was examined during early embryogenesis and found differentially methylated. Reverse transcriptase quantitative polymerase chain reaction indicated stage-specific levels of transcripts encoding DNA-methyltransferase (DNMT) and methyl-binding domain proteins. In addition, as highlighted by electronic microscopy and immunohistochemistry, the DNMT inhibitor 5-aza-cytidine induced alterations in the quantity and the localisation of methylated DNA and severe dose-dependent development alterations and was lethal after zygotic genome reinitiation. Furthermore, methyl-DNA-immunoprecipitation–quantitative polymerase chain reaction revealed that the transcription level of most of the homeobox gene orthologues examined, but not of the other early genes investigated, was inversely correlated with their specific DNA methylation. Altogether, our results demonstrate that DNA methylation influences gene expression in Crassostrea gigas and is critical for oyster development, possibly by specifically controlling the transcription level of homeobox orthologues. These findings provide evidence for the importance of epigenetic regulation of development in Lophotrochozoans and bring new insights into the early life of C. gigas, one of the most important aquaculture resources worldwide.

International audience; Background: The Ice krill, Euphausia crystallorophias is one of the species at the base of the Southern Ocean food chain. Given their significant contribution to the biomass of the Southern Ocean, it is vitally important to gain a better understanding of their physiology and, in particular, anticipate their responses to climate change effects in the warming seas around Antarctica. Methodology/Principal Findings: Illumina sequencing was used to produce a transcriptome of the ice krill. Analysis of the assembled contigs via two different methods, produced 36 new pre-pro-peptides, coding for 61 neuropeptides or peptide hormones belonging to the following families: Allatostatins (A, B et C), Bursicon (alpha and beta), Crustacean Hyperglycemic Hormones (CHH and MIH/VIHs), Crustacean Cardioactive Peptide (CCAP), Corazonin, Diuretic Hormones (DH), the Eclosion Hormone (EH), Neuroparsin, Neuropeptide F (NPF), small Neuropeptide F (sNPF), Pigment Dispersing Hormone (PDH), Red Pigment Concentrating Hormone (RPCH) and finally Tachykinin. LC/MS/MS proteomics was also carried out on eyestalk extracts, which are the major site of neuropeptide synthesis in decapod crustaceans. Results confirmed the presence of six neuropeptides and six precursor-related peptides previously identified in the transcriptome analyses. Conclusions: This study represents the first comprehensive analysis of neuropeptide hormones in a Eucarida non-decapod Malacostraca, several of which are described for the first time in a non-decapod crustacean. Additionally, there is a potential expansion of PDH and Neuropeptide F family members, which may reflect certain life history traits such as circadian rhythms associated with diurnal migrations and also the confirmation via mass spectrometry of several novel pre-propeptides, of unknown function. Knowledge of these essential hormones provides a vital framework for understanding the physiological response of this key Southern Ocean species to climate change and provides a valuable resource for studies into the molecular phylogeny of these organisms and the evolution of neuropeptide hormones.

International audience; Two novel members of the oxytocin/vasopressin superfamily have been identified in the cephalopod Sepia officinalis. Oxytocin/vasopressin gene sequences were cloned by Race PCR. The two precursors we identified exhibit the classical organization of OT/VP superfamily precursors: a signal peptide followed by a nonapeptide and a neurophysin domain. The neurophysin domain is entirely conserved for the cuttlefish precursors, but the nonapeptides and the signal peptides differ. The first nonapeptide, called sepiatocin, is highly homologous to Octopus vulgaris octopressin. The second nonapeptide, called pro-sepiatocin, shows sequence homologies with a Crustacean oxytocin/vasopressin-like peptide identified in Daphnia culex and with a novel form of oxytocin described in New World monkeys. The expression of pro-sepiatocin is restricted to the supraesophageal and subesophageal masses of the brain whereas sepiatocin is expressed in the entire central nervous system. Sepiatocin, as described for octopressin, modulates the contractile activity of several muscles such as penis, oviduct and vena cava muscles; this suggests its involvement in reproduction and blood circulation. Pro-sepiatocin is released in the hemolymph; it is a neurohormone able to target numerous peripheral organs.

International audience; ex determination is poorly known in mollusks, lophotrochozoa and most hermaphrodites. In the oyster Crassostrea gigas, an irregular successive hermaphrodite, sex determination may occur at the end of a gametogenetic cycle to prepare the next cycle. To investigate further into these aspects we have focused on orthologs of SoxE and β-catenin, key players of the male and female gonadic differentiation respectively in mammals. Based on phylogenetic analyses, Cg-SoxE, the oyster's SoxE ortholog, is closely related to vertebrate SoxE genes while Cg-β-catenin, the oyster's β-catenin ortholog, is classified amongst lophotrochozoa. The mRNA expression measured by qPCR in the gonadic area during a gametogenetic cycle is maximal for Cg-SoxE when sex is indiscernible and for Cg-β-catenin in mature females. Both expressions are localized from early germ cells to spermatocytes and pre-vitellogenic oocytes, and potentially in somatic cells. Cg-β-catenin is also expressed in vitellogenic oocytes. These actors may be involved in early oyster gonadic differentiation, which includes sex determination. Our results enhance the understanding of sex determination in C. gigas and in mollusks and they provide additional knowledge in compared genomics of reproduction and in molecular phylogeny.

International audience; Gametogenesis of the Pacific oyster Crassostrea gigas follows an annual cycle. In this work, we used contrasting dietary conditions (fed and unfed) for a preconditioning treatment in order to impact glycogen storage supporting reproductive pathways in diploid and triploid animals before a broodstock conditioning treatment. In diploid oysters, food deprivation during preconditioning led to reduced gonia proliferation as demonstrated by lower PCNA (Proliferating Cell Nuclear Antigen) expression and subsequently, the reproductive effort was significantly reduced. For triploid animals exhibiting arrested gametogenesis, dietary conditions impacted the ratio of α (animals with unlocked gametogenesis) and β (animals with locked gametogenesis). Thermal stress applied to these conditioned animals confirmed the weakness of diploid animals with high stored reserves and intense reproductive effort, versus low reproductive effort. Moreover, only low mortalities were observed for triploid animals whatever the reproductive status. The involvement of the insulin pathway in the regulation of gametogenesis and storage rebuilding was assessed by measuring an increased expression of CIR (C. gigas insulin related receptor) in unfed oysters.

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International audience; BACKGROUND: The cephalopod mollusk Sepia officinalis can be considered as a relevant model for studying reproduction strategies associated to seasonal migrations. Using transcriptomic and peptidomic approaches, we aim to identify peptide sex pheromones that are thought to induce the aggregation of mature cuttlefish in their egg-laying areas. RESULTS: To facilitate the identification of sex pheromones, 576 5'-expressed sequence tags (ESTs) were sequenced from a single cDNA library generated from accessory sex glands of female cuttlefish. Our analysis yielded 223 unique sequences composed of 186 singletons and 37 contigs. Three major redundant ESTs called SPα, SPα' and SPβ were identified as good candidates for putative sex pheromone transcripts and are part of the 87 unique sequences classified as unknown. The alignment of translated SPα and SPα' revealed a high level of conservation, with 98.4% identity. Translation led to a 248-amino acid precursor containing six peptides with multiple putative disulfide bonds. The alignment of SPα-α' with SPβ revealed a partial structural conservation, with 37.3% identity. Translation of SPβ led to a 252-amino acid precursor containing five peptides. The occurrence of a signal peptide on SPα, SPα' and SPβ showed that the peptides were secreted. RT-PCR and mass spectrometry analyses revealed a co-localization of transcripts and expression products in the oviduct gland. Preliminary in vitro experiments performed on gills and penises revealed target organs involved in mating and ventilation. CONCLUSIONS: The analysis of the accessory sex gland transcriptome of Sepia officinalis led to the identification of peptidic sex pheromones. Although preliminary functional tests suggested the involvement of the α3 and β2 peptides in ventilation and mating stimulation, further functional investigations will make it possible to identify the complete set of biological activities expected from waterborne pheromones.

International audience; Two types of exposures were performed to assess the effects of zinc released from sacrificial anode degradation: a chronic exposure, in which oysters were exposed to 0.53 ± 0.04 mg Zn L−1 for 10 weeks, and an acute exposure, where oysters were exposed to 10.2 ± 1.2 mg Zn L−1 for 1 week. At the end of the acute exposure experiment, 81.8% mortality was recorded. In contrast, no mortality was detected after 10 weeks exposure. Moreover, all of the immune system biomarkers studied, except the number of circulating haemocytes, were stimulated by a moderate level of zinc and inhibited by a high level. Our exposure conditions did not induce SOD or MXR mRNA expression in gills and digestive gland. However, an increase of MT mRNA is observed in these tissues. The results indicate that oysters are sensitive to acute zinc toxicity but are only moderately affected by a mild zinc concentration.

International audience; The Pavlovophyceae (Haptophyta) contains four genera (Pavlova, Diacronema, Exanthemachrysis and Rebecca) and only thirteen characterised species, several of which are important in ecological and economic contexts. We have constructed molecular phylogenies inferred from sequencing of ribosomal gene markers with comprehensive coverage of the described diversity, using type strains when available, together with additional cultured strains. The morphology and ultrastructure of 12 of the described species was also re-examined and the pigment signatures of many culture strains were determined. The molecular analysis revealed that sequences of all described species differed, although those of Pavlova gyrans and P. pinguis were nearly identical, these potentially forming a single cryptic species complex. Four well-delineated genetic clades were identified, one of which included species of both Pavlova and Diacronema. Unique combinations of morphological/ultrastructural characters were identified for each of these clades. The ancestral pigment signature of the Pavlovophyceae consisted of a basic set of pigments plus MV chl cPAV, the latter being entirely absent in the Pavlova + Diacronema clade and supplemented by DV chl cPAV in part of the Exanthemachrysis clade. Based on this combination of characters, we propose a taxonomic revision of the class, with transfer of several Pavlova species to an emended Diacronema genus. The evolution of the class is discussed in the context of the phylogenetic reconstruction presented. (C) 2011 Elsevier GmbH. All rights reserved.