Coral bleaching under thermal stress: putative involvement of host/symbiont recognition mechanisms

<p>Abstract</p> <p>Background</p> <p>Coral bleaching can be defined as the loss of symbiotic zooxanthellae and/or their photosynthetic pigments from their cnidarian host. This major disturbance of reef ecosystems is principally induced by increases in water temperature. Since the beginning of the 1980s and the onset of global climate change, this phenomenon has been occurring at increasing rates and scales, and with increasing severity. Several studies have been undertaken in the last few years to better understand the cellular and molecular mechanisms of coral bleaching but the jigsaw puzzle is far from being complete, especially concerning the early events leading to symbiosis breakdown. The aim of the present study was to find molecular actors involved early in the mechanism leading to symbiosis collapse.</p> <p>Results</p> <p>In our experimental procedure, one set of <it>Pocillopora damicornis </it>nubbins was subjected to a gradual increase of water temperature from 28°C to 32°C over 15 days. A second control set kept at constant temperature (28°C). The differentially expressed mRNA between the stressed states (sampled just before the onset of bleaching) and the non stressed states (control) were isolated by Suppression Subtractive Hybridization. Transcription rates of the most interesting genes (considering their putative function) were quantified by Q-RT-PCR, which revealed a significant decrease in transcription of two candidates six days before bleaching. RACE-PCR experiments showed that one of them (<it>PdC-Lectin</it>) contained a C-Type-Lectin domain specific for mannose. Immunolocalisation demonstrated that this host gene mediates molecular interactions between the host and the symbionts suggesting a putative role in zooxanthellae acquisition and/or sequestration. The second gene corresponds to a gene putatively involved in calcification processes (<it>Pdcyst-rich</it>). Its down-regulation could reflect a trade-off mechanism leading to the arrest of the mineralization process under stress.</p> <p>Conclusion</p> <p>Under thermal stress zooxanthellae photosynthesis leads to intense oxidative stress in the two partners. This endogenous stress can lead to the perception of the symbiont as a toxic partner for the host. Consequently, we propose that the bleaching process is due in part to a decrease in zooxanthellae acquisition and/or sequestration. In addition to a new hypothesis in coral bleaching mechanisms, this study provides promising biomarkers for monitoring coral health.</p

Thermal Stress Triggers Broad Pocillopora damicornis Transcriptomic Remodeling, while Vibrio coralliilyticus Infection Induces a More Targeted Immuno-Suppression Response

Global change and its associated temperature increase has directly or indirectly changed the distributions of hosts and pathogens, and has affected host immunity, pathogen virulence and growth rates. This has resulted in increased disease in natural plant and animal populations worldwide, including scleractinian corals. While the effects of temperature increase on immunity and pathogen virulence have been clearly identified, their interaction, synergy and relative weight during pathogenesis remain poorly documented. We investigated these phenomena in the interaction between the coral Pocillopora damicornis and the bacterium Vibrio coralliilyticus, for which the infection process is temperature-dependent. We developed an experimental model that enabled unraveling the effects of thermal stress, and virulence vs. non-virulence of the bacterium. The physiological impacts of various treatments were quantified at the transcriptome level using a combination of RNA sequencing and targeted approaches. The results showed that thermal stress triggered a general weakening of the coral, making it more prone to infection, non-virulent bacterium induced an ‘efficient’ immune response, whereas virulent bacterium caused immuno-suppression in its host

Genes Related to Ion-Transport and Energy Production Are Upregulated in Response to CO₂-Driven pH Decrease in Corals: New Insights from Transcriptome Analysis

Since the preindustrial era, the average surface ocean pH has declined by 0.1 pH units and is predicted to decline by an additional 0.3 units by the year 2100. Although subtle, this decreasing pH has profound effects on the seawater saturation state of carbonate minerals and is thus predicted to impact on calcifying organisms. Among these are the scleractinian corals, which are the main builders of tropical coral reefs. Several recent studies have evaluated the physiological impact of low pH, particularly in relation to coral growth and calcification. However, very few studies have focused on the impact of low pH at the global molecular level. In this context we investigated global transcriptomic modifications in a scleractinian coral (Pocillopora damicornis) exposed to pH 7.4 compared to pH 8.1 during a 3-week period. The RNAseq approach shows that 16% of our transcriptome was affected by the treatment with 6% of upregulations and 10% of downregulations. A more detailed analysis suggests that the downregulations are less coordinated than the upregulations and allowed the identification of several biological functions of interest. In order to better understand the links between these functions and the pH, transcript abundance of 48 candidate genes was quantified by q-RT-PCR (corals exposed at pH 7.2 and 7.8 for 3 weeks). The combined results of these two approaches suggest that pH ≥ 7.4 induces an upregulation of genes coding for proteins involved in calcium and carbonate transport, conversion of CO₂ into HCO₃⁻ and organic matrix that may sustain calcification. Concomitantly, genes coding for heterotrophic and autotrophic related proteins are upregulated. This can reflect that low pH may increase the coral energy requirements, leading to an increase of energetic metabolism with the mobilization of energy reserves. In addition, the uncoordinated downregulations measured can reflect a general trade-off mechanism that may enable energy reallocation

Stress environnementaux chez le corail scléractiniaire Pocillopora damicornis : du modèle expérimental à l'identification de marqueurs fonctionnels du stress.

High biodiversity and productivity, complexity of biotic interactions and trophic networks, wealth and product source, coastal protection are some of the many emblematic characteristics of the coral reef ecosystem. This prodigious diversity is largely due to the particular biology of the principal reef bioconstructors, the hermatypic corals and associated zooxanthellae symbionts. However, over the last decades, most of the reefs have been critically affected by various natural and anthropogenic disturbances which are, at least for some of them, increasing in frequency and intensity in correlation with global climate changes. Among these disturbances, coral bleaching (the loose of zooxanthellae and/or their photosynthetic pigments) and coral diseases have caused high or even massive rates of coral mortality worldwide. Results of the last worldwide coral reefs monitoring programs are particularly alarming and established that 19% of the reefs are completely destroyed, 20% present all symptoms of an imminent threat of collapse, and 20% are at risk for the forthcoming decades. In this context, the objective of this work was to improve our knowledge of enhanced physiological and transcriptomic mechanisms of coral under thermal stress leading to bleaching and bacterial stress. The objective was also to provide strong bases for the use of relevant biomarkers for coral health monitoring and early detection of these disturbances. This work focused on the scleractinian species Pocillopora damicornis, the bacteria Vibrio coralliilyticus, and on the implementation of stress experiments in a controlled but ecologically realistic system. In the first study, P. damicornis colonies were confronted to a gradual water temperature increase (28°C to 32°C) over a 15 days period. mRNAs differentially expressed (between control and thermally stressed conditions) were isolated by subtractive hybridization and the transcription rate of the most promising genes were measured by quantitative-RT-PCR. These approaches revealed 2 candidates that displayed a drastic expression decrease 6 days before the occurrence of the first symptoms of bleaching. RACE-PCR experiments showed that one of them (PdC-Lectin) contains a C-type lectin domain presenting a specificity for recognition and binding of mannose. Immuno-localization experiments have demonstrated that this protein could be the molecular mediator of host/symbionts interaction, which suggest a putative role in the acquisition or the sequestration of symbionts. The second gene (Pdcyst-rich) codes for a protein potentially involved in calcification processes. Its down-regulation could picture a trade-off mechanism leading to calcification failure during thermal stress. In the second study, responses of P. damicornis confronted to its specific pathogen V. coralliilyticus in a virulent (temperature increased) or non-virulent (constant low temperature) state were studied. The infectious process was exained by the combination of electronic microscope observations and quantitative-RT-PCR experiments, and coral health status was evaluated by visual observations and zooxanthellae density measures. Results show that infection only occurs after water temperature was increased. Among several ESTs obtained by subtractive hybridization (between control, bacterially, and thermally plus bacterialy conditions), 6 gene candidates were selected for their putative involvement in the immune response, and their transcription rate was measured by quantitative-RT-PCR all long the kinetics of virulent and non-virulent experiments. Among these genes, 3 belong to the lectin family, 2 code for proteins involved in metal binding and the last one codes for a protease inhibitor. Their expression patterns allowed better understanding the immune response of scleractinian corals as well as the impact of Vibrio on its host. The third publication focus on the characterization of the first antimicrobial peptide (AMP) of scleractinian corals, the damicornin of P. damicornis. Results showed that the damicornin is constituvely transcribed and stocked in an inactive form in ectodermal granular cells. Under immune challenge, damicornin is secreted and activated. Results also showed that after bacterial internalization, Vibrio triggers a down-regulation of the damicornin. In our knowledge this is the first report of the down-regulation of an AMP in a host/Vibrio interaction. Finally, 9 biomarkers of environmental stress were identified and demonstrated to be early regulated, in response of thermal stress for 2 of them, and of bacterial stress for the 7 others. For the last ones, it is possible to differentiate between corals confronted to virulent or non-virulent interaction. After this identification step, a validation stage appears to be necessary. This should focus on the quantification of expression variability of each biomarker at different scales, but also on the definition of an expression baseline, which is essential for biomarkers use for coral health monitoring. This work is particularly promising, and opens perspectives in the field of epigenetic mechanisms involvement in functional adaptation of corals to environmental stress. Another interesting perspective would be the study of the links existing between immunity and symbiosis, which have been put to the fore in the present work by the different involvement of a same gene in both functions (PdC-Lectin). Finally, studying responses of corals to acidic stress would be particularly relevant in the actual context of ocean acidificationBiodiversité élevée, complexité des réseaux trophiques et des interactions biotiques, forte productivité, source de produits et de richesses, protection du littoral ... sont quelques unes des caractéristiques emblématiques des écosystèmes coralliens. Cette prodigieuse diversité est largement liée à la biologie particulière des principaux bioconstructeurs du récif, les coraux hermatypiques et leurs zooxanthelles symbiotiques. Mais depuis quelques décennies, la plupart des récifs sont durement affectés par diverses perturbations d'origine naturelle ou anthropique, dont l'intensité et la fréquence augmentent, en lien avec le changement climatique global pour certaines d'entre elles. Parmi ces perturbations, le blanchissement corallien (perte de zooxanthelles symbiotiques et/ou de leurs pigments photosynthétiques) et les maladies coralliennes ont engendré une mortalité importante, parfois massive, des coraux. Le dernier bilan de l'état de santé des récifs à l'échelle planétaire réalisé en 2008 est particulièrement préoccupant : 19% des récifs sont complètement détruits, 20% présentent tous les symptômes d'une destruction imminente, et 20% sont considérés comme menacés dans les décennies à venir. Dans ce contexte, l'ambition de ce travail est d'améliorer les connaissances sur les méchanismes physiologiques et transcriptomiques des coraux soumis à des stress thermiques conduisant au blanchissement, et à des stress biotiques d'origine bactérienne. L'objectif est également de fournir des bases solides pour la mise en oeuvre de biomarqueurs pertinents pour le suivi de l'état de santé des coraux et la prédiction précoce des perturbations. Ce travail a porté sur le corail scléractiniaire Pocillopora damicornis et la bactérie Vibrio coralliilyticus, et a mis en oeuvre des expérimentations "écologiquement réalistes" de stress en milieu contrôlé. Dans une première étude, des colonies de P. damicornis ont été confrontées à une montée graduelle de la température (28 à 32°C) sur une période de 15 jours. Les ARNms différentiellement exprimés (entre conditions stressées et non stressées) ont été isolés par hybridation soustractive et les taux de transcription des gènes les plus intéressants ont été quantifiés par RT-PCR-quantitative. Ces approches ont révélé 2 candidats présentant une répression drastique de leur expression 6 jours avant les premiers symptômes visibles du blanchissement. Des expériences de RACE-PCR ont montré que l'un d'entre eux (PdC-Lectin) contient un domaine lectine de type C présentant une spécificité de reconnaissance pour le mannose. Les expériences d'immuno-localisation ont démontré que cette protéine hôte était potentiellement le médiateur moléculaire des interactions hôte/symbiote, suggérant un rôle dans l'acquisition ou la séquestration des symbiotes. Le second gène (Pdcyst-rich) code une protéine potentiellement impliquée dans les mécanismes de calcification. Sa répression pourrait être le reflet d'un mécanisme de type trade off conduisant à l'arrêt de la croissance durant le stress. La seconde étude a examiné les réponses de P. damicornis confronté à son pathogène spécifique V. coralliilyticus, dans un état virulent (augmentation de la température) et non virulent (température stable). Le processus infectieux a été examiné par microscopie électronique et RT-PCR-quantitative alors que l'état général des coraux a été évalué par des observations visuelles et des mesures de la densité en zooxanthelles. Les résultats montrent que l'infection ne s'est développée qu'après augmentation de la témpérature. Au sein de nombreuses EST obtenues par hybridations soustractives, 6 gènes candidats ont été sélectionnés pour leur implication potentielle dans les mécanismes de la réponse immunitaire, et leur expression a été mesurée tout au long des cinétiques d'interactions virulences et avirulentes (RT-PCR-quantitative). Parmi ces gènes, 3 appartiennent à la famille des lectines, 2 codes des protéines fixant des éléments métalliques et le dernier code un inhibiteur de protéase. L'analyse de leurs patterns de transcription a permis de mieux comprendre la réponse immunitaire du corail, mais aussi l'impact du vibrio sur son hôte. Enfin, la dernière étude a spécifiquement porté sur la caractérisation du premier peptide antimicrobien (AMP) de scléractiniaires, la damicronin de P. damicornis. Nos résultats montrent que la damicornin est constitutivement transcrite dans des cellules granulaires de l'ectoderme oral et stockée sous forme inactive dans les granules. Lors d'un stimulus immun, la damicornin est sécrétée et activée. Nos résultats montrent également que la transcription de la damicornin est réprimée par V. coralliilyticus lorsque ce dernier s'installe dans les tissus coralliens, ce qui semble être le premier exemple de répression d'un AMP lors d'une interaction hôte/Vibrio. Finalement ce sont donc 9 biomarqueurs de stress environnementaux qui ont été identifiés, 2 présentent une répression précoce de leur expression lors d'un stress thermique inducteur de blanchissement et 7 répondent à des stress bactériens. Pour ces derniers, selon l'association de biomarqueurs utilisée il est possible de distinguer des coraux confrontés à des interactions de type avirulente ou virulente. Maintenant que ces biomarqueurs sont identifiés, il apparaît nécessaire de passer aux étapes de validations. Ces dernières auront pour but de quantifier la variabilityé d'expression de ces biomarqueurs à différentes échelles, mais aussi de définir une ligne de base, indispensable pour suivre l'état de santé des coraux à l'aide de ces biomarqueurs. Au terme de ce travail, il apparaît particulièrement judicieux de se pencher sur l'implication des mécanismes épigénétiques dans l'adaptation fonctionnelle des coraux en réponse aux stress environnementaux. L'étude des liens entre l'immunité et la symbiose, marquée ici par les implications différentes d'un même gène (PdC-Lectin) dans ces fonctions, nous paraît également une voie de recherche prometteuse. Enfin, il conviendrait d'étudier l'effet d'un stress acide, ce qui serait particulièrement judicieux dans le contexte actuel d'acidification des océans

Réponse, acclimatation et adaptation des invertébrés marins aux changements environnementaux biotiques et abiotiques

Pour de très nombreuses populations d’organismes, l’aire anthropocène est synonyme de perturbations croissantes et diversifiées. Ces changements peuvent donc impliquer des modifications environnementales biotiques, telle que l’émergence de pathogène, ou abiotiques telle que l’augmentation des températures. Quelle que soit leur origine et leur nature, ces perturbations conduiront dans un premier temps au développement d’une réponse de stress puis d’un processus éventuel d’acclimatation et enfin à l’échelle populationnelle et transgénérationnelle à l’adaptation. C’est à l’étude et la compréhension de ces mécanismes de réponse, d’acclimatation et enfin d’adaptation rapides que je me suis intéressé depuis le début de ma carrière. Cette recherche s’est focalisée sur deux grands modèles d’étude : i) les coraux constructeurs de récifs et ii) les bivalves marins confrontés à des pressions de sélection fortes et soudaines induites par l’émergence de pathogènes, le réchauffement climatique ou encore l’acidification des océans. Afin d’appréhender ces mécanismes j’ai développé des approches expérimentales écologiquement réalistes aussi bien en milieu contrôlé qu’en milieu naturel et à différentes échelles spatiales et temporelles. Par la suite, j’ai étudié les réponses, les processus d’acclimatation et d’adaptation de ces populations et organismes par des approches d’omics intégratives et pluridisciplinaires en appréhendant notamment le triptyque génome, épigénome, phénome et leurs interactions avec l’environnement.L’objectif finalisé de ma recherche est de transformer cette compréhension des mécanismes d’adaptation (sensus lato) rapides en des outils de gestion des changements en cours que ce soit dans un contexte d’aquaculture ou de protection et de restauration des écosystème

Response of coral assemblages to thermal stress: Are spatial and taxonomic patterns in bleaching consistent between events?

International audienceMass bleaching events resulting in coral mortality are among the greatest threats to coral reefs, and are projected to increase in frequency and intensity with global warming. Achieving a better understanding of the consistency of the response of coral assemblages to thermal stress, both spatially and temporally, is essential to determine which reefs are more able to tolerate climate change. We compared variations in spatial and taxonomic patterns between two bleaching events at the scale of an island (Moorea Island, French Polynesia). Despite similar thermal stress and light conditions, bleaching intensity was significantly lower in 2007 (approximately 37 % of colonies showed signs of bleaching) than in 2002, when 55 % of the colonies bleached. Variations in the spatial patterns of bleaching intensity were consistent between the two events. Among nine sampling stations at three locations and three depths, the stations at which the bleaching response was lowest in 2002 were those that showed the lowest levels of bleaching in 2007. The taxonomic patterns of susceptibility to bleaching were also consistent between the two events. These findings have important implications for conservation because they indicate that corals are capable of acclimatization and/or adaptation and that, even at small spatial scales, some areas are consistently more susceptible to bleaching than others

Sequencing, de novo assembly and annotation of the genome of the scleractinian coral, Pocillopora acuta

Coral reefs are the most divers marine ecosystem. However, under the pressure of global changes and anthropogenic disturbances corals and coral reefs are declining worldwide. In order to better predict and understand the future of these organisms all the tools of modern biology are needed today. However, many NGS based approaches are not feasible in corals because of the lack of reference genomes. Therefore we have sequenced, de novo assembled, and annotated, the draft genome of one of the most studied coral species, Pocillopora acuta (ex damicornis ). The sequencing strategy was based on four libraries with complementary insert size and sequencing depth (180pb, 100x; 3Kb, 25x; 8kb, 12x and 20 kb, 12x). The de novo assembly was performed with Platanus (352 Mb; 25,553 scaffolds; N50 171,375 bp). 36,140 genes were annotated by RNA-seq data and 64,558 by AUGUSTUS (Hidden-Markov model). Gene functions were predicted through Blast and orthology based approaches. This new genomic resource will enable the development of a large array of genome wide studies but also shows that the de novo assembly of a coral genome is now technically feasible and economically realistic

Colour plasticity in the shells and pearls of animal graft model Pinctada margaritifera assessed by HSV colour quantification

The bivalve Pinctada margaritifera has the capacity to produce the most varied and colourful pearls in the world. Colour expression in the inner shell is under combined genetic and environmental control and is correlated with the colour of pearls produced when the same individual is used as a graft donor. One major limitation when studying colour phenotypes is grader subjectivity, which leads to inconsistent colour qualification and quantification. Through the use of HSV (Hue Saturation Value) colour space, we created an R package named ‘ImaginR’ to characterise inner shell colour variations in P. margaritifera. Using a machine-learning protocol with a training dataset, ImaginR was able to reassign individual oysters and pearls to predefined human-based phenotype categories. We then tested the package on samples obtained in an experiment testing the effects of donor conditioning depth on the colour of the donor inner shell and colour of the pearls harvested from recipients following grafting and 20 months of culture in situ. These analyses successfully detected donor shell colour modifications due to depth-related plasticity and the maintenance of these modifications through to the harvested pearls. Besides its potential interest for standardization in the pearl industry, this new method is relevant to other research projects using biological models

Identifying genes associated with genetic control of color polymorphism in the pearl oyster Pinctada margaritifera var. cumingii (Linnaeus 1758) using a comparative whole genome pool‐sequencing approach

International audienceFor hundreds of years, the color diversity of Mollusca shells has been a topic of interest for humanity. However, the genetic control underlying color expression is still poorly understood in mollusks. The pearl oyster Pinctada margaritifera is increasingly becoming a biological model to study this process due to its ability to produce a large range of colors. Previous breeding experiments demonstrated that color phenotypes were partly under genetic control, and while a few genes were found in comparative transcriptomics and epigenetic experiments, genetic variants associated to the phenotypes have not yet been investigated. Here, we used a pooled-sequencing approach on 172 individuals to investigate color-associated variants on three color phenotypes of economic interest for pearl farming, in three wild and one hatchery populations. While our results uncovered SNPs targeting pigment-related genes already identified in previous studies, such as PBGD, tyrosinases, GST, or FECH, we also identified new colorrelated genes occurring in the same pathways, like CYP4F8, CYP3A4 and CYP2R1. Moreover, we identified new genes involved in novel pathways unknown to be involved in shell coloration for P. margaritifera, like the carotenoid pathway, BCO1. These findings are essential to possibly implement future breeding programs focused on individual selection for specific color production in pearl oysters and improve the footprint of perliculture on Polynesian lagoon by producing less, but with a better quality