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Biomineralization integrates complex physical and chemical processes bio-controlled by the living organisms through ionic concentration regulation and organic molecules production. It allows tuning the structural, optical and mechanical properties of hard tissues during ambient-condition crystallisation, motivating a deeper understanding of the underlying processes. By combining state-of-the-art optical and X-ray microscopy methods, we investigated early-mineralized calcareous units from two bivalve species, Pinctada margaritifera and Pinna nobilis, revealing chemical and crystallographic structural insights. In these calcite units, we observed ring-like structural features correlated with a lack of calcite and an increase of amorphous calcium carbonate and proteins contents. The rings also correspond to a larger crystalline disorder and a larger strain level. Based on these observations, we propose a temporal biomineralization cycle, initiated by the production of an amorphous precursor layer, which further crystallizes with a transition front progressing radially from the unit centre, while the organics are expelled towards the prism edge. Simultaneously, along the shell thickness, the growth occurs following a layer-by-layer mode. These findings open biomimetic perspectives for the design of refined crystalline materials. STATEMENT OF SIGNIFICANCE: Calcareous biominerals are amongst the most present forms of biominerals. They exhibit astonishing structural, optical and mechanical properties while being formed at ambient synthesis conditions from ubiquitous ions, motivating the deep understanding of biomineralization. Here, we unveil the first formation steps involved in the biomineralization cycle of prismatic units of two bivalve species by applying a new multi-modal non-destructive characterization approach, sensitive to chemical and crystalline properties. The observations of structural features in mineralized units of different ages allowed the derivation of a temporal sequence for prism biomineralization, involving an amorphous precursor, a radial crystallisation front and a layer-by-layer sequence. Beyond these chemical and physical findings, the herein introduced multi-modal approach is highly relevant to other biominerals and bio-inspired studies. ispartof: ACTA BIOMATERIALIA vol:142 pages:194-207 ispartof: location:England status: published

International audience; In living organisms, calcium carbonate biomineralization combines complex bio-controlled physical and chemical processes to produce crystalline hierarchical hard tissues (usually calcite or aragonite) typically from an amorphous precursor phase. Understanding the nature of the successive transient amorphous phases potentially involved in the amorphous-to-crystalline transition requires characterization tools, which are able to provide a spatial and spectroscopic analysis of the biomineral structure. In this work, we present a highly sensitive coherent Raman microscopy approach, which allows one to image molecular bond concentrations in post mortem shells and living animals, by exploiting the vibrational signature of the different carbonates compounds. To this end, we target the nu_1 calcium carbonate vibration mode and produce spatially and spectroscopically resolved images of the shell border of a mollusk shell, the Pinctada margaritifera pearl oyster. A novel approach is further presented to efficiently compare the amount of amorphous carbonate with respect to its crystalline counterpart. Finally, the whole microscopy method is used to image in vivo the shell border and demonstrate the feasibility and the reproducibility of the technique. These findings open chemical imaging perspectives for the study of biogenic and bio-inspired crystals.

The orbicular batfish (

Background Tenacibaculum maritimum is a fish pathogen known for causing serious damage to a broad range of wild and farmed marine fish populations worldwide. The recently sequenced genome of T. maritimum strain NCIMB 2154T provided unprecedented information on the possible molecular mechanisms involved in the virulence of this species. However, little is known about the dynamic of infection in vivo, and information is lacking on both the intrinsic host response (gene expression) and its associated microbiota. Here, we applied complementary omic approaches, including dual RNAseq and 16S rRNA gene metabarcoding sequencing using Nanopore and short-read Illumina technologies to unravel the host–pathogen interplay in an experimental infection system using the tropical fish Platax orbicularis as model. Results We showed that the infection of the host is characterised by an enhancement of functions associated with antibiotic and glucans catabolism functions but a reduction of sulfate assimilation process in T. maritimum. The fish host concurrently displays a large panel of immune effectors, notably involving innate response and triggering acute inflammatory response. In addition, our results suggest that fish activate an adaptive immune response visible through the stimulation of T-helper cells, Th17, with congruent reduction of Th2 and T-regulatory cells. Fish were, however, largely sensitive to infection, and less than 25% survived after 96 hpi. These surviving fish showed no evidence of stress (cortisol levels) or significant difference in microbiome diversity compared with controls at the same sampling time. The presence of T. maritimum in resistant fish skin and the total absence of any skin lesions suggest that these fish did not escape contact with the pathogen, but rather that some mechanisms prevented pathogens entry. In resistant individuals, we detected up-regulation of specific immune-related genes differentiating resistant individuals from controls at 96 hpi, which suggests a possible genomic basis of resistance, although no genetic variation in coding regions was found. Conclusion Here we focus in detail on the interplay between common fish pathogens and host immune response during experimental infection. We further highlight key actors of defence response, pathogenicity and possible genomic bases of fish resistance to T. maritimum.

International audience; Cultivated pearls frequently exhibit morphological irregularities making obvious that mineral deposition was irregularly distributed onto nucleus surface. Taking advantage of experimental cultivations with short durations (from 10 days to few months), these irregular deposits predating occurrence of the nacre were investigated in Polynesian pearls by biochemical characterizations and a series of physical methods. Diversity in the resulting data suggests that various in‐depth alterations of the biomineralization mechanism may have occurred during the grafting process, leading to diversity in the biochemical pathways to nacreous deposition. This allows a precise discussion of current views about pearl formation. The “reversed shell theory” is formally disproved through point to point comparison with development of the shell growing edge. Similarity of pearl formation with “regeneration” or “shell repair” is also discussed, emphasizing the differences between these concepts.

The orbicular batfish (Platax orbicularis), also called ‘Paraha peue’ in Tahitian, is the most important marine fish species reared in French Polynesia. Sudden and widespread outbreaks of severe ‘white-patch disease’ have occurred since 2011 in batfish farms one to three weeks after the transfer of juveniles from bio-secured hatcheries to lagoon cages. With cumulative mortality ranging from 20 to 90%, the sustainability of aquaculture of this species is severely threatened.In this study, we describe for the first time the isolation from diseased batfish of several strains belonging to the species Tenacibaculum maritimum, a major pathogen of many marine fish species. Histopathological analysis, an experimental bath challenge and a field monitoring study showed that T. maritimum is associated with white-patch disease. Moreover, molecular and serological analyses performed on representative strains revealed some degree of genetic diversity among the isolates, a finding of primary importance for epidemiological studies and for the development of management and control strategies such as vaccination.

Advancement and diversification of the aquaculture industry is reliant on the development of captive breeding and rearing protocols for novel fish species. Orbicular batfish (Platax orbicularis), a major emerging species in Polynesian aquaculture, live in brackish and marine waters around coral reefs, and are highly prized by Pacific Island communities for their high-quality meat. The present study describes the larval growth of P. orbicularis from hatching until 16 days after hatching (DAH) using meristics and thyroid hormone (T3) quantification. Our study highlighted that metamorphosis of P. orbicularis is critical to their production in aquaculture, as found for other species. Levels of the thyroid hormone T3 in P. orbicularis reached a peak at 12 DAH (i.e. hormonal metamorphosis). This peak occurred in concert with important morphological changes and increased mortality and growth between 9 and 12 DAH, clearly illustrating the sensitivity of this fish during metamorphosis. Overall, our study sheds light on the metamorphosis and larval development of a novel aquaculture species, and the interplay between hormonal and morphological changes throughout a critical period of its rearing. These findings may promote the development of protocols for mass-scale production of orbicular batfish in captivity, which may be particularly beneficial to the aquaculture industries of many Pacific Island countries.

BackgroundEphippidae fish are characterized by a discoid shape with a very small visceral cavity. Among themPlatax orbicularishas a high economic potential due to its flesh quality and flesh to carcass ratio. Nonetheless, the development of its aquaculture is limited by high mortality rates, especially due toTenacibaculum maritimuminfection, occurring one to three weeks after the transfer of fishes from bio-secure land-based aquaculture system to the lagoon cages for growth. Among the lines of defense against this microbial infection, the gastrointestinal tract (GIT) is less studied. The knowledge about the morphofunctional anatomy of this organ inP. orbicularisis still scarce. Therefore, the aims of this study are to characterize the GIT in non-infectedP. orbicularisjuveniles to then investigate the impact ofT. maritimumon this multifunctional organ.MethodsIn the first place, the morpho-anatomy of the GIT in non-infected individuals was characterized using various histological techniques. Then, infected individuals, experimentally challenged byT. maritimumwere analysed and compared to the previously established GIT reference.ResultsThe overlapped shape of the GIT ofP. orbicularisis probably due to its constrained compaction in a narrow visceral cavity. Firstly, the GIT was divided into 10 sections, from the esophagus to the rectum. For each section, the structure of the walls was characterized, with a focus on mucus secretions and the presence of the Na+/K+ATPase pump. An identification key allowing the characterization of the GIT sections usingin totohistology is given. Secondly, individuals challenged withT. maritimumexhibited differences in mucus type and proportion and, modifications in the mucosal and muscle layers. These changes could induce an imbalance in the trade-off between the GIT functions which may be in favour of protection and immunity to the disadvantage of nutrition capacities.

Four new brominated tyrosine metabolites, aplyzanzines C&ndash, F (1&ndash, 4), were isolated from the French Polynesian sponge Pseudoceratina n. sp., along with the two known 2-aminoimidazolic derivatives, purealidin A (5) and 6, previously isolated, respectively, from the sponges Psammaplysilla purpurea and Verongula sp. Their structures were assigned based on the interpretation of their NMR and HRMS data. The compounds exhibited quorum sensing inhibition (QSi) and antifouling activities against several strains of bacteria and microalgae. To our knowledge, the QSi activity of this type of bromotyrosine metabolite is described here for the first time.

We report herein a ring-distortion strategy applied to marine natural substances ilimaquinone and 5-epi-ilimaquinone. A chemically diverse library of molecules was synthesised that included rearrangements of the sesquiterpene moiety and original reorganisations of the quinone ring. Chemoinformatic analyses evaluated the rise of structural diversity and the exploration of chemical space. Some focussed biological activities of this library were also investigated; quorum sensing activity of Vibrio harveyi was envisaged and some of the new compounds were shown to be good quorum sensing inhibitor candidates, whereas others were activators. Toxicities were also evaluated and some products showed micromolar activities against human umbilical vein endothelium, human hepatocellular carcinoma and human lung carcinoma (A549) cells.

In a series of Polynesian pearls collected after short cultivation periods, early post-grafting mineral deposits were characterized by high resolution synchrotron-based X-ray fluorescence with unprecedented accuracy. Morphological patterns and elemental composition are correlated through simultaneous imaging processes. Evidence that aragonite and calcite occur in neighboring units during the earliest biomineralization stages reveals that the grafting process can result in a greater degradation than usually admitted in the widely shared ‘reversed shell’ concept. Compared with ultrastructure of the pristine nacreous tablets, this method enables a precise evaluation of the possible biological changes in the biomineralization mechanism during grafting.

International audience; Trophic conditions and water temperature strongly influence bivalve physiological processes and metabolism. In black-lip pearl oyster Pinctada margaritifera, these parameters have been shown to affect shell biomineralization. The present study investigated the effect of preoperative food level (i.e., microalgal concentration) and temperature on pearl biomineralization. Donor and recipient oysters were conditioned at different levels of food and temperature during the preoperative phase to evaluate the influence of these factors on 1) pearl retention rate (grafting success), 2) expression of genes involved in biomineralization in the mantle and pearl sac and 3) pearl quality traits. Our study confirmed the influence of both microalgal concentration and temperature on shell growth. Food level of donor oysters was decisive for pearl biomineralization, with donors that had been fed at a high microalgal concentration producing pearl sacs with significantly higher biomineralization capabilities and faster nacre establishment during early stages of pearl formation. However, food level showed no effects on quality traits of the pearls harvested 12 months postgrafting, while preoperative temperature only influenced the relative expression of two genes in pearl sacs at 12 months postgrafting. No significant effects of the preoperative conditioning of recipient oysters were detected in either experiment considering gene expression measurements and pearl quality traits. However, mortality was significantly lower in grafted recipient oysters fed at an intermediate trophic level. Finally, pearl weight was shown to be positively correlated with recipient oyster growth.

The purpose of our study is to investigate (1) the influence of phenotype plasticity and local adaptation on pearl-oyster physiology by testing the persistence of growth differentiation of two pearl oyster populations (Arutua and Mangareva) in common garden experiment; (2) to quantify the influence of biofouling development on the growth of each pearl oyster population. According to our observations, the growth rate in terms of total oyster weight suggested better growth performance of the pearl oyster Pinctada margaritifera in Mangareva (0.21–0.24 ± 0.01 g.day−1) than Arutua (0.14–0.15 ± 0.01 g.day−1). However, similar growth performances are observed at the Vairao common garden site for oyster stocks from Mangareva (0.07 ± 0.01 mm.day−1 or 0.15 ± 0.01 g.day−1) and Arutua (0.07 ± 0.01 mm.day−1 or 0.13 ± 0.01 g.day−1). Our results suggest that growth performance variability observed at the pearl farming sites of Arutua and Mangareva is due more to phenotypic plasticity than to local adaptation. This result thus accords a major importance to site selection for the pearl farming optimization process. Biofouling dynamics on Pinctada margaritifera shells differed radically between Arutua and Mangareva sites. In Arutua, biofouling colonization was relatively slow (0.016 g.oyster−1.month−1) and was mainly composed of sponges and bivalves. On Mangareva, the colonization process appeared faster and more continuous over the study period (0.15–0.18 g.oyster−1.month−1) and the biofouling community was dominated by tunicates. On the basis of our results obtained on growth performance between cleaned and uncleaned stock in Arutua and Mangareva, biofouling development after 14–15 months of colonization does not appear to have any negative effect on P. margaritifera growth. Due to the high cost of biofouling management in pearl farming facilities, our results suggest once more that reconsideration of the pearl farming management process is needed.

International audience; The possible consequences of acidification on pearl farming are disruption of oyster metabolism and change in growth. In the laboratory, we studied the impact of pCO2 (3540, 1338 and 541μatm) on the physiology of pearl oysters exposed for 100 days. This experiment was repeated after an interval of one year. Several physiological compartments were examined in pearl oysters: the scope for growth by measuring ingestion, assimilation and oxygen consumption, gametogenesis by means of histological observations, shell growth by measurement and observation by optical and electronic microscopy, and at molecular level by measuring the expression of nine genes of mantle cells implied in the biomineralisation process. Results from both experiments showed that high pCO2 had no effect on scope for growth and gametogenesis. High pCO2 (3540 μatm) significantly slowed down the shell deposit rate at the ventral side and SEM observations of the inside of the shell found signs of chemical dissolution. Of the nine examined genes high pCO2significantly decreased the expression level of one gene (Pmarg-PUSP 6). This study showed that shell growth of the pearl oyster would be slowed down without threatening the species since the management of energy and reproduction functions appeared to be preserved. Further investigations should be conducted on the response of offspring to acidification.

The objective of this study was to observe the impact of temperature on pearl formation using an integrative approach describing the rotation of the pearls, the rate of nacre deposition, the thickness of the aragonite tablets and the biomineralizing potential of the pearl sac tissue though the expression level of some key genes. Fifty pearl oysters were grafted with magnetized nuclei to allow the rotation of the pearls to be described. Four months later, 32 of these pearl oysters were exposed to four temperatures (22, 26, 30 and 34°C) for 2 weeks. Results showed that the rotation speed differed according to the movement direction: pearls with axial movement had a significantly higher rotation speed than those with random movement. Pearl growth rate was influenced by temperature, with a maximum between 26 and 30°C but almost no growth at 34°C. Lastly, among the nine genes implicated in the biomineralization process, only

International audience; Environmental parameters, such as food level and water temperature, have been shown to be major factors influencing pearl oyster shell growth and molecular mechanisms involved in this biomineralization process. The present study investigates the effect of food level (i.e., microalgal concentration) and water temperature, in laboratory controlled conditions, on the last stages of pearl mineralization in order to assess their impact on pearl quality. To this end, grafted pearl oysters were fed at different levels of food and subjected to different water temperatures one month prior to harvest to evaluate the effect of these factors on 1) pearl and shell deposition rate, 2) expression of genes involved in biomineralization in pearl sacs, 3) nacre ultrastructure (tablet thickness and number of tablets deposited per day) and 4) pearl quality traits. Our results revealed that high water temperature stimulates both shell and pearl deposition rates. However, low water temperature led to thinner nacre tablets, a lower number of tablets deposited per day and impacted pearl quality with better luster and fewer defects. Conversely, the two tested food level had no significant effects on shell and pearl growth, pearl nacre ultrastructure or pearl quality. However, one gene, Aspein, was significantly downregulated in high food levels. These results will be helpful for the pearl industry. A wise strategy to increase pearl quality would be to rear pearl oysters at a high water temperature to increase pearl growth and consequently pearl size; and to harvest pearls after a period of low water temperature to enhance luster and to reduce the number of defects.

To enable the rapid and accurate identification of Vibrio splendidus-related and V. aestuarianus strains associated with Pacific cupped oyster Crassostrea gigas mortality, we developed a duplex Taqman real-time PCR assay and evaluated its efficacy. This technique proved to be rapid, sensitive, and specific and will be particularly valuable for epidemiologic studies.

La perle est une production conchylicole originale qui, en Polynesie francaise, se place au 2e rang economique. Cette industrie s’appuie fortement sur la recherche pour introduire du progres dans les pratiques. Cet article a pour ambition de presenter les bases techniques de sa production en detaillant les travaux menes en lien avec les enjeux de developpement durable de la perliculture. Les recherches sont principalement axees sur l’amelioration de la qualite des perles via la selection genetique. Cela necessite de comprendre les processus physiologiques et moleculaires en jeu. Nous concluons sur les mises au point, les developpements scientifiques et techniques en cours ou restant a mener.

Plants have been reported to produce various effects such as antistress, growth promotion, appetite stimulation, immunostimulation, aphrodisiac and to have antipathogen properties in fish and shrimp aquaculture due to their varied active principles such as alkaloids, terpenoids, tannins, saponins and flavonoids. To date, most scientific studies on the use of medicinal plants in aquaculture have focused on identification of biological activity rather than natural product determination. The plant species that have displayed the highest potential for use in aquaculture are garlic (Allium sativum), pomegranate (Punica granatum), bermuda grass (Cynodon dactylon), Indian ginseng (Whitania somnifera) and ginger (Zingiber officinale). Algae are considered to be a rich source of original bioactive molecules which display multiple bioactivities. In aquaculture, several recent studies have showed the potential of algae for the treatment of pathogens or to improve fish fitness.

Extracellular products (ECPs) of the French V. tubiashii strain 07/118 T2 were previously reported to be toxic for the Pacific oyster Crassostrea gigas. In this study we now assessed host cellular immune responses and bacterial potential effectors by which these ECPs can be associated with host damages. The adhesion capacity (28% inhibition) and phagocytosis ability (56% inhibition) of oyster hemocytes were the main functions affected following in vitro contact between hemocytes and V. tubiashii ECPs. This may be linked to the demonstration of the capability of ECPs to cleave various cellular substrates as oyster collagen. Moreover, a strong metalloproteolytic activity was recorded with general (azocasein) and specific (ADAM) substrates and characterized by the use of standard inhibitors and metal ions. The addition of 1, 10-phenanthroline and Zn2+ decreased proteolytic activity by about 80% and 50% respectively, confirming the presence of zinc metalloproteolytic activity in the ECPs. Mass spectrometry analyses of crude ECPs identified an extracellular zinc metalloprotease encoded by a gene with an open reading frame of 1821 bp (606 aa). Consensus zinc-binding motifs specific to thermolysin family and some glycosylation and phosphorylation sites were located on the deduced protein sequence. Even if taken together, our results let us wonder if this (these) zinc metalloprotease(s) could be involved in the impairment of hemocyte immunological functions, the direct implication of this (these) protein(s) in ECPs toxicity still has to be demonstrated.

International audience; The adaptative bleaching hypothesis (ABH) states that, depending on the symbiotic flexibility of coral hosts (i.e., the ability of corals to ''switch'' or ''shuffle'' their algal symbionts), coral bleaching can lead to a change in the composition of their associated Symbiodinium community and, thus, contribute to the coral's overall survival. In order to determine the flexibility of corals, molecular tools are required to provide accurate species delineations and to detect low levels of coral-associated Symbiodinium. Here, we used highly sensitive quantitative (real-time) PCR (qPCR) technology to analyse five common coral species from Moorea (French Polynesia), previously screened using only traditional molecular methods, to assess the presence of low-abundance (background) Symbiodinium spp. Similar to other studies, each coral species exhibited a strong specificity to a particular clade, irrespective of the environment. In addition, however, each of the five species harboured at least one additional Symbiodinium clade, among clades A–D, at background levels. Unexpectedly, and for the first time in French Polynesia, clade B was detected as a coral symbiont. These results increase the number of known coral-Symbiodinium associations from corals found in French Polynesia, and likely indicate an underestimation of the ability of the corals in this region to associate with and/or ''shuffle'' different Symbiodinium clades. Altogether our data suggest that corals from French Polynesia may favor a trade-off between optimizing symbioses with a specific Symbiodinium clade(s), maintaining associations with particular background clades that may play a role in the ability of corals to respond to environmental change. How to cite this article Rouzé et al. (2017), An updated assessment of Symbiodinium spp. that associate with common scleractinian corals from Moorea (French Polynesia) reveals high diversity among background symbionts and a novel finding of clade B.

International audience; The pearl culture is one of the most lucrative aquacultures worldwide. In many South Pacific areas, it depends on the exploitation of the pearl oyster Pinctada margaritifera and relies entirely on the environmental conditions encountered in the lagoon. In this context, assessing the impact of climatic stressors, such as global warming and ocean acidification, on the functionality of the resource in terms of renewal and exploitation is fundamental. In this study, we experimentally addressed the impact of temperature (22, 26, 30 and 34 °C) and partial pressure of carbon dioxide pCO2 (294, 763 and 2485 μatm) on the biomineralization and metabolic capabilities of pearl oysters. While the energy metabolism was strongly dependent on temperature, results showed its independence from pCO2 levels; no interaction between temperature and pCO2 was revealed. The energy metabolism, ingestion, oxygen consumption and, hence, the scope for growth (SFG) were maximised at 30 °C and dramatically fell at 34 °C. Biomineralization was examined through the expression measurement of nine mantle's genes coding for shell matrix proteins involved in the formation of calcitic prisms and/or nacreous shell structures; significant changes were recorded for four of the nine (Pmarg-Nacrein A1, Pmarg-MRNP34, Pmarg-Prismalin 14 and Pmarg-Aspein). These changes showed that the maximum and minimum expression of these genes was at 26 and 34 °C, respectively. Surprisingly, the modelled thermal optimum for biomineralization (ranging between 21.5 and 26.5 °C) and somatic growth and reproduction (28.7 °C) appeared to be significantly different. Finally, the responses to high temperatures were contextualised with the Intergovernmental Panel on Climate Change (IPCC) projections, which highlighted that pearl oyster stocks and cultures would be severely threatened in the next decade

The adaptative bleaching hypothesis (ABH) states that depending on the symbiotic flexibility of coral hosts (i.e., the ability of corals to “switch” or “shuffle” their algal symbionts), coral bleaching can lead to a change in the composition of their associated Symbiodinium community, and, thus, contribute to the coral’s overall survival. In order to determine the flexibility of corals, molecular tools are required to provide accurate species delineations, and to detect low levels of coral-associated Symbiodinium. Here, we used highly sensitive quantitative (real-time) PCR (qPCR) technology to analyse five common coral species from Moorea (French Polynesia), previously screened using only traditional conventional molecular methods, to assess the presence of low-abundance (background) Symbiodinium. Similar to other studies, each coral species exhibited a strong specificity to a particular clade, irrespective of the environment. In addition, however, each of the five species harboured at least one additional Symbiodinium clade, among clades A-D, at background levels. Unexpectedly, and for the first time in French Polynesia, clade B was detected as a coral symbiont. These results increase the number of known coral-Symbiodinium associations from corals found in French Polynesia, and likely indicate an underestimation of the ability of the corals in this region to associate with and/or “shuffle” different Symbiodinium clades. Altogether our data suggest that corals from French Polynesia may manage a trade-off between optimizing symbioses with a specific Symbiodinium clade(s), and maintaining associations with particular background clades that may play a role in the ability of corals to respond to environmental change.

International audience; Background: Biofloc technology (BFT), a rearing method with little or no water exchange, is gaining popularity in aquaculture. In the water column, such systems develop conglomerates of microbes, algae and protozoa, together with detritus and dead organic particles. The intensive microbial community presents in these systems can be used as a pond water quality treatment system, and the microbial protein can serve as a feed additive. The current problem with BFT is the difficulty of controlling its bacterial community composition for both optimal water quality and optimal shrimp health. The main objective of the present study was to investigate microbial diversity of samples obtained from different culture environments (Biofloc technology and clear seawater) as well as from the intestines of shrimp reared in both environments through high-throughput sequencing technology. Results: Analyses of the bacterial community identified in water from BFT and " clear seawater " (CW) systems (control) containing the shrimp Litopenaeus stylirostris revealed large differences in the frequency distribution of operational taxonomic units (OTUs). Four out of the five most dominant bacterial communities were different in both culture methods. Bacteria found in great abundance in BFT have two principal characteristics: the need for an organic substrate or nitrogen sources to grow and the capacity to attach to surfaces and co-aggregate. A correlation was found between bacteria groups and physicochemical and biological parameters measured in rearing tanks. Moreover, rearing-water bacterial communities influenced the microbiota of shrimp. Indeed, the biofloc environment modified the shrimp intestine microbiota, as the low level (27 %) of similarity between intestinal bacterial communities from the two treatments. Conclusion: This study provides the first information describing the complex biofloc microbial community, which can help to understand the environment-microbiota-host relationship in this rearing system.

Pearl oyster grafting is a complex surgical operation that should lead to pearl formation after approximately eighteen months. Although this technique has been used for many years in French Polynesia, the grafting process is still not standardised. While studies have been carried out in order to improve graft performance and yield, these remain highly variable due to post-grafting mortality, nucleus rejection and unreliable pearl quality, all of which constrain pearl farm profitability. The present study uses histological analysis to monitor oysters that either rejected or retained their nuclei. Both groups of oysters are compared in terms of evolution of the graft, which could influence retention, and the development of a pearl sac in cases where grafting was successful. Data show that rejection phenomena are linked to a number of causes, notably an inflammatory reaction in the "receiving" oyster, the presence of numerous tissue lesions and the quality of the grafted tissue. These results suggest that study is needed on the different concomitant elements of the grafting process: the graft "donor" oysters, the nucleus and the "receiving" oyster and their interactions.

International audience; The present study investigated the effect of electrolysis on the biomineralization capacities of juveniles of the mollusk Pinctada argaritifera for the first time. Size-selected individuals from two groups, “Medium” and “Large”, from a multi-parental family produced in a hatchery system were subjected to electrolysis under a low voltage current over a nine-week experimental period. The growth of the juveniles was individually monitored and assessed weekly by wet weight and shell height measurements. At the end of the experiment, mantle tissue was sampled for biomineralization-related gene expression analysis. Electrolysis significantly increased pearl oyster growth in terms of shell height and wet weight for Large juveniles from the 5th and the 2nd week, respectively, until the end of the experiment. However, differences were only significant for Medium individuals from the 7th week for shell height and from the 9th week for wet weight. Furthermore, transcriptional analysis of six known biomineralization genes coding for shell matrix proteins of calcitic prisms and/or nacreous shell structures revealed that five were significantly overexpressed in the mantle mineralizing tissue under electrolysis: three in common between the two size class groups and two that were expressed exclusively in one or the other group. Finally, we found no statistical difference of the shell thickness ratio between individuals undergoing electrolysis and control conditions. Taken together, our results indicate, for the first time in a calcifying marine organism, that electrolysis influences molecular mechanisms involved in biomineralization and may stimulate some parameters of pearl oyster growth rate.

The emerging orbicular batfish (Platax orbicularis) aquaculture is the most important fish aquaculture industry in French Polynesia. However, bacterial infections are causing severe mortality episodes. Therefore, there is an urgent need to find an effective management solution. Besides the supplying difficulty and high costs of veterinary drugs in French Polynesia, batfish aquaculture takes place close to the coral reef, where use of synthetic persistent drugs should be restricted. Medicinal plants and bioactive algae are emerging as a cheaper and more sustainable alternative to chemical drugs. We have studied the effect of local Polynesian plants and the local opportunistic algae Asparagopsis taxiformis on batfish when orally administered. Weight gain and expression of two immune-related genes (lysozyme g e Lys G and transforming growth factor beta - TGF-b1) were studied to analyze immunostimulant activity of plants on P. orbicularis. Results showed that several plants increased Lys G and TGF-b1 expression on orbicular batfish after 2 and 3 weeks of oral administration. A. taxiformis was the plant displaying the most promising results, promoting a weight gain of 24% after 3 weeks of oral administration and significantly increasing the relative amount of both Lys G and TGF-b1 transcripts in kidney and spleen of P. orbicularis.

International audience; Coral disease outbreaks have increased over the last three decades, but their causal agents remain mostly unclear (e.g., bacteria, viruses, fungi, protists). This study details a 14-month-long survey of coral colonies in which observations of the development of disease was observed in nearly half of the sampled colonies. A bimonthly qPCR method was used to quantitatively and qualitatively evaluate Symbiodinium assemblages of tagged colonies, and to detect the presence of Vibrio spp. Firstly, our data showed that predisposition to disease development in general, and, more specifically, infection by Vibrio spp. in Acropora cytherea depended on which clades of Symbiodinium were harbored. In both cases, harboring clade D rather than A was beneficial to the coral host. Secondly, the detection of Vibrio spp. in only colonies that developed disease strongly suggests opportunistic traits of the bacteria. Finally, even if sporadic cases of switching and probably shuffling were observed, this long-term survey does not suggest specific-clade recruitment in response to stressors. Altogether, our results demonstrate that the fitness of the coral holobiont depends on its initial consortium of Symbiodinium, which is distinct among colonies, rather than a temporary adaptation achieved through acquiring different Symbiodinium clades.

Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir -dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the P BAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm , deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.

Background Color polymorphism in the nacre of pteriomorphian bivalves is of great interest for the pearl culture industry. The nacreous layer of the Polynesian black-lipped pearl oyster Pinctada margaritifera exhibits a large array of color variation among individuals including reflections of blue, green, yellow and pink in all possible gradients. Although the heritability of nacre color variation patterns has been demonstrated by experimental crossing, little is known about the genes involved in these patterns. In this study, we identify a set of genes differentially expressed among extreme color phenotypes of P. margaritifera using a suppressive and subtractive hybridization (SSH) method comparing black phenotypes with full and half albino individuals. Results Out of the 358 and 346 expressed sequence tags (ESTs) obtained by conducting two SSH libraries respectively, the expression patterns of 37 genes were tested with a real-time quantitative PCR (RT-qPCR) approach by pooling five individuals of each phenotype. The expression of 11 genes was subsequently estimated for each individual in order to detect inter-individual variation. Our results suggest that the color of the nacre is partially under the influence of genes involved in the biomineralization of the calcitic layer. A few genes involved in the formation of the aragonite tablets of the nacre layer and in the biosynthesis chain of melanin also showed differential expression patterns. Finally, high variability in gene expression levels were observed within the black phenotypes. Conclusions Our results revealed that three main genetic processes were involved in color polymorphisms: the biomineralization of the nacreous and calcitic layers and the synthesis of pigments such as melanin, suggesting that color polymorphism takes place at different levels in the shell structure. The high variability of gene expression found within black phenotypes suggests that the present work should serve as a basis for future studies exploring more thoroughly the expression patterns of candidate genes within black phenotypes with different dominant iridescent colors. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1776-x) contains supplementary material, which is available to authorized users.

International audience; Vibrio tubiashii is a marine pathogen isolated from larval and juvenile bivalve molluscs that causes bacillary necrosis. Recent studies demonstrated the isolation of this species in a French experimental hatchery/nursery affecting Crassostrea gigas spat in 2007. Here, using larvae of C. gigas as an interaction model, we showed that the French V. tubiashii is virulent to larvae and can cause bacillary necrosis symptoms with an LD50 of about 2.3x10(3) c.f.u. ml(-1) after 24 h. Moreover, complete or gel permeation HPLC fractionated extracellular products (ECPs) of this strain appeared toxic to larvae. MS-MS analysis of the different ECP fractions revealed the existence of an extracellular metalloprotease and other suspected virulence factors. This observation is also supported by the expression level of some potential virulence factors. The overall results suggest that the pathology caused by the French V. tubiashii in C. gigas oysters is caused by a group of toxic factors and not only the metalloprotease.

The pathogenicity of Vibrio penaeicida Strains KH-1 and AM101, their culture-free supernatant (CFS), and their protein fraction obtained by 40% of ammonium sulfate precipitation (PFs40) were assessed in experimental challenges against juvenile Litopenaeus vannamei. Live Vibrio cells, CFS, and PFs40 from the AM101 strain produced a significantly higher mortality (p < 0.05) compared to the KH-1 strain. Toxicity and median lethal doses (LD50) of Fast Protein Liquid Chromatography (FPLC) products were evaluated on L. vannamei. The first FPLC fraction sample (A) from PFs40 of the AM101 strain displayed LD50 values of 1.68 and 5.61 mu g protein ind.(-1), respectively. The second FPLC process from Fraction A showed a peak (A1) also with toxic effects to shrimp. PFs40, Fraction A, and Peak At showed a 38.5 kDa molecular band (SDS-PAGE), with activity on a gelatin protease zymogram. The lethal effect of PFs40 and Fraction A was inhibited by Proteinase K, CuCl2, E-64, and heat (60 and 100 degrees C) treatments, but was not inhibited by EDTA-Na-2, aprotinin, and soy trypsin treatments. These results and the zymogram inhibition test suggest the presence of a cysteine protease-like proteinaceous exotoxin as a dominant protease, secreted by V. penaeicida Strain AM101.

The present study reports for the first time the involvement of an antimicrobial peptide in the defense reactions of a shrimp infected by a pathogenic Vibrio, Vibrio penaeicida. New members of the penaeidin family were characterized in the shrimp Litopenaeus stylirostris by RT-PCR and RACE-PCR from hemocyte total RNAs, and by mass spectrometry detection and immunolocalization of mature peptides in shrimp hemocytes. In infected shrimps, bacteria and penaeidin distribution colocalized in the gills and the lymphoid organ that represented the main infected sites. Moreover, the shrimp immune response to infection involved massive hemocyte recruitment to infection sites where released penaeidin may participate in the isolation and elimination of the bacteria, We show that the ability of the shrimps to circumvent shrimp infections is closely related to a recovery phase based on the hematopoietic process.

Aquaculture production in French Polynesia and New Caledonia relies upon domesticated broodstock of two introduced species (the shrimp Litopenaeus stylirostris , and the fish Lates calcarifer ) and on natural stock of the local pearl oyster Pinctada margaritifera . For L. stylirostris , refrigeration of shrimp sperm lowers the sanitary risk linked to the re-introduction of variability in Tahiti that is proved necessary by microsatellite markers. They also allow paternity controls that will improve future genetic management. Selective breeding programs for shrimp are underway in Tahiti and under assessment at pilot scale in New Caledonia. Conservation strategies are under elaboration for local populations of P. margaritifera that have been proven genetically differentiated. The recent development of pearl oyster hatcheries is the first step towards genetic improvement of this species. Thanks to geographical isolation and sanitary regulations, French Polynesia and New Caledonia are not affected by the viral outbreaks affecting shrimp elsewhere in the world (e.g., white spot and Taura syndrome viruses) and the pearl oyster in Japan (Akoya virus). Furthermore, a zoosanitary surveillance network of pearl oyster stocks is coming up in order to detect the emergence of any pathogen. Recently, alternative methods for antibiotherapy in shrimp larval rearing have been tested using probiotic bacteria, and could find an application in pearl oyster larval rearing.

An association between vitellogenesis and the immune system was suggested in crustaceans from studies on plasma lipoproteins. The present research studies the effect of an experimentally induced bacterial infection on vitellogenesis in females of the shrimp Penaeus indicus, as a model for penaeid species. Pre-vitellogenic and vitellogenic P. indicus females were experimentally infected with an extremely pathogenic bacterium, Vibrio penaeicida. The peak in mortality occurred earlier in pre-vitellogenic animals than in vitellogenic ones, although the final mortality level ( approximately 64-74%) 52h post-infection was nearly the same for the two groups. Twenty hours after infection, the total number of haemocytes was significantly reduced in vitellogenic females while there was no change in the pre-vitellogenic group. Protein synthesis in ovaries was not significantly affected by infection, at the two stages of ovarian development. No differences were found in mRNA levels of shrimp ovarian peritrophin protein (SOP), but preliminary results showed that mRNA expression of vitellin (VT) was reduced in a heavily infected vitellogenic female. The total amount of lipids in the haemolymph of vitellogenic females was almost twice higher than that of pre-vitellogenic ones. However, there was no change in the total content of lipids, lipid classes and fatty acid distribution in haemolymph or hepatopancreas following infection. Although vitellogenic and pre-vitellogenic females probably respond differently to a lethal bacterial infection, physiological differences may be concealed by the rapid onset of mortality.