Coral reefs are among the most valuable ecosystems on Earth, providing major benefits to human societies and hosting a wide variety of species. Today, these ecosystems are threatened by a combination of local and global factors that overcome the natural capacity of coral reefs for regeneration. Therefore, active restoration methods have been developed and conducted to help coral reefs recover faster than before. The construction of artificial reefs, which are artificial structures that are deliberately submerged in aquatic environments and whose characteristics mimic those of natural reefs, is a marine restoration strategy. These strategies have the potential to increase the ecological value of degraded sites in both the short term and long term by providing additional habitats for coral growth and acting as future hubs for coral larval dispersal. However, the success of artificial reefs in achieving these objectives depends largely on the materials used to construct them. Concrete, plastics, metals and wood, which are the major materials found in modern reefs, suffer from substantial drawbacks that limit the ability to reach restoration goals. In this short study, we discuss innovations that have been developed to overcome the drawbacks of modern artificial reefs to encourage the creation of a new generation of artificial reefs that can substantially contribute to active coral reef restoration.

Antipatharians (black corals) are major components of mesophotic ecosystems in the Mediterranean Sea. The arborescent species Antipathella subpinnata has received particular attention as it is the most abundant and forms dense forests harbouring high levels of biodiversity. This species is currently categorized as “Near Threatened” in the IUCN Red List, due to increasing fishing pressure and bottom-trawling activities. Yet, the effects of ocean warming have never been investigated for this species, nor for any other antipatharians from temperate regions. Our study aimed at evaluating the effects of increasing seawater temperatures on A. subpinnata, by combining predictive distribution modelling with a physiological tolerance experiment. During the latter, we exposed A. subpinnata for 15 days to different temperature conditions spanning the current seasonal range to forecasted temperatures for 2100, while measuring biological endpoints such as oxygen consumption rates and different signs of stress (tissue necrosis, total antioxidant capacity). Unexpectedly, no stress was found at organism nor cellular level (wide thermal breadth) suggesting low susceptibility of this species to mid-term temperature increase. If the response to the 15-days heat stress is representative of the response to longer-term warming, ocean warming is unlikely to affect A. subpinnata. The species distribution model predicted the presence of A. subpinnata at depths that correspond to temperatures colder than its maximum thermal tolerance (as determined by the physiology experiment). This suggests that the presence of A. subpinnata at shallower depths is not limited by physiological constraints but by other ecological factors including interspecific competition.

Ecosystems worldwide are suffering from climate change. Coral reef ecosystems are globally threatened by increasing sea surface temperatures. However, gene expression plasticity provides the potential for organisms to respond rapidly and effectively to environmental changes, and would be favored in variable environments. In this study, we investigated the thermal stress response in Pocillopora coral colonies from two contrasting environments by exposing them to heat stress. We compared the physiological state, bacterial and Symbiodiniaceae communities (using 16S and ITS2 metabarcoding), and gene expression levels (using RNA-Seq) between control conditions and heat stress (the temperature just below the first signs of compromised health). Colonies from both thermal regimes remained apparently normal and presented open and colored polyps during heat stress, with no change in bacterial and Symbiodiniaceae community composition. In contrast, they differed in their transcriptomic responses. The colonies from Oman displayed a more plastic transcriptome, but some genes had a higher basal expression level (frontloading) compared to the less thermotolerant colonies from New Caledonia. In terms of biological functions, we observed an increase in the expression of stress response genes (including induction of tumor necrosis factor receptors, heat shock proteins, and detoxification of reactive oxygen species), together with a decrease in the expression of genes involved in morpho-anatomical functions. Gene regulation (transcription factors, mobile elements, histone modifications and DNA methylation) appeared to be overrepresented in the Oman colonies, indicating possible epigenetic regulation. These results show that transcriptomic plasticity and frontloading can be co-occurring processes in corals confronted with highly variable thermal regimes.

Unlike most reef fish families, which are broadcast spawners, damselfishes and anemonefishes are benthic spawners. Indeed, as mentioned in Chapter 6, anemonefishes live in the vicinity of their sea anemone host and lay eggs on a substrate nearby.

Correctly delimiting species and populations is a prerequisite for studies of connectivity, adaptation and conservation. Genomic data are particularly useful to test species differentiation for organisms with few informative morphological characters or low discrimination of cytoplasmic markers, as in Scleractinians. Here we applied Restriction site Associated DNA sequencing (RAD-sequencing) to the study of species differentiation and genetic structure in populations of Pocillopora spp. from Oman and French Polynesia, with the objectives to test species hypotheses, and to study the genetic structure among sampling sites within species. We focused here on coral colonies morphologically similar to P. acuta (damicornis type β). We tested the impact of different filtering strategies on the stability of the results. The main genetic differentiation was observed between samples from Oman and French Polynesia. These samples corresponded to different previously defined primary species hypotheses (PSH), i.e., PSHs 12 and 13 in Oman, and PSH 5 in French Polynesia. In Oman, we did not observe any clear differentiation between the two putative species PSH 12 and 13, nor between sampling sites. In French Polynesia, where a single species hypothesis was studied, there was no differentiation between sites. Our analyses allowed the identification of clonal lineages in Oman and French Polynesia. The impact of clonality on genetic diversity is discussed in light of individual-based simulations.

Background: Pocillopora acuta is a hermatypic coral with strong ecological importance. Anthropogenic disturbances and global warming are major threats that can induce coral bleaching, the disruption of the mutualistic symbiosis between the coral host and its endosymbiotic algae. Previous works have shown that somaclonal colonies display different levels of survival depending on the environmental conditions they previously faced. Epigenetic mechanisms are good candidates to explain this phenomenon. However, almost no work had been published on the P. acuta epigenome, especially on histone modifications. In this study, we aim at providing the first insight into chromatin structure of this species. Methods: We aligned the amino acid sequence of P. acuta core histones with histone sequences from various phyla. We developed a centri-filtration on sucrose gradient to separate chromatin from the host and the symbiont. The presence of histone H3 protein and specific histone modifications were then detected by western blot performed on histone extraction done from bleached and healthy corals. Finally, micrococcal nuclease (MNase) digestions were undertaken to study nucleosomal organization. Results: The centri-filtration enabled coral chromatin isolation with less than 2% of contamination by endosymbiont material. Histone sequences alignments with other species show that P. acuta displays on average ~90% of sequence similarities with mice and ~96% with other corals. H3 detection by western blot showed that H3 is clipped in healthy corals while it appeared to be intact in bleached corals. MNase treatment failed to provide the usual mononucleosomal digestion, a feature shared with some cnidarian, but not all; suggesting an unusual chromatin structure. Conclusions: These results provide a first insight into the chromatin, nucleosome and histone structure of P. acuta. The unusual patterns highlighted in this study and partly shared with other cnidarian will need to be further studied to better understand its role in corals.

Background: Pocillopora acuta is a hermatypic coral with strong ecological importance. Anthropogenic disturbances and global warming are major threats that can induce coral bleaching, the disruption of the mutualistic symbiosis between the coral host and its endosymbiotic algae. Previous works have shown that somaclonal colonies display different levels of survival depending on the environmental conditions they previously faced. Epigenetic mechanisms are good candidates to explain this phenomenon. However, almost no work had been published on the P. acuta epigenome, especially on histone modifications. In this study, we aim at providing the first insight into chromatin structure of this species. Methods: We aligned the amino acid sequence of P. acuta core histones with histone sequences from various phyla. We developed a centri-filtration on sucrose gradient to separate chromatin from the host and the symbiont. The presence of histone H3 protein and specific histone modifications were then detected by western blot performed on histone extraction done from bleached and healthy corals. Finally, micrococcal nuclease (MNase) digestions were undertaken to study nucleosomal organization. Results: The centri-filtration enabled coral chromatin isolation with less than 2% of contamination by endosymbiont material. Histone sequences alignments with other species show that P. acuta displays on average ~90% of sequence similarities with mice and ~96% with other corals. H3 detection by western blot showed that H3 is clipped in healthy corals while it appeared to be intact in bleached corals. MNase treatment failed to provide the usual mononucleosomal digestion, a feature shared with some cnidarian, but not all; suggesting an unusual chromatin structure. Conclusions: These results provide a first insight into the chromatin, nucleosome and histone structure of P. acuta. The unusual patterns highlighted in this study and partly shared with other cnidarian will need to be further studied to better understand its role in corals.

Scleractinian corals are of great ecological interest as ecosystem engineer species. Accordingly, there is a wealth of studies on their adaptive abilities facing climate change. Such studies should rely on precise species and population delimitation. Nevertheless species delimitation in corals can be hindered by the lack of adequate genetic markers, by hybridization, and by morphological plasticity. Here we applied RAD sequencing to the study of species delimitation and genetic structure in populations of Pocillopora spp. from Oman and French Polynesia with the objectives to test primary species hypotheses based on mitochondrial DNA sequencing, and to study the genetic structure among sampling sites inside species. Regarding the varying levels of missing data observed among samples we tested different filtering strategy. The main genetic differentiation was observed between samples from Oman and French Polynesia, which also corresponded to different mitochondrial lineages and species hypotheses. In Oman, we did not observe any clear differentiation according to the main mitochondrial lineages considered here, nor between sampling sites. In French Polynesia where a single mitochondrial lineage was studied, we did not evidence any differentiation according to sampling sites. These results provide an additional example of the importance of using independent nuclear markers for the study of species delimitation. Our analyses also allowed the identification of clonal lineages in our samples, and to take them into account in our interpretations. We used simulations to study the impact of clonal reproduction on the distribution of statistics of genetic diversity and genetic structure among loci.

As interest increases in ecological, evolutionary, and developmental biology (Eco-Evo-Devo), wild species are increasingly used as experimental models. However, we are still lacking a suitable model for marine fish species, as well as coral reef fishes that can be reared at laboratory scales. Extensive knowledge of the life cycle of anemonefishes, and the peculiarities of their biology, make them relevant marine fish models for developmental biology, ecology, and evolutionary sciences. Here, we present standard methods to maintain breeding pairs of the anemonefish Amphiprion ocellaris in captivity, obtain regular good quality spawning, and protocols to ensure larval survival throughout rearing. We provide a detailed description of the anemonefish husbandry system and life prey culturing protocols. Finally, a "low-volume" rearing protocol useful for the pharmacological treatment of larvae is presented. Such methods are important as strict requirements for large volumes in rearing tanks often inhibit continuous treatments with expensive or rare compounds.

Cnidarians are known to undergo reverse development as a survival mechanism against adverse environmental conditions. Polyp bail-out consists in the polyps’ detachment from the mother colony due to stressful conditions, followed by a complete tissue and cells rearrangement and in some cases in a regression into a simple, ciliated form. Here we describe a massive polyp bail-out event occurred in the mesophotic black coral Antipathella subpinnata in reared conditions. This is the first report of a bail-out event in this species providing new insights into the life cycle and ecology of black corals.

Pocillopora acuta is a hermatypic coral with a worldwide distribution and a strong ecological importance. Anthropogenic disturbances and global warming threaten it. Thermal stress can induce coral bleaching, a phenomenon in which the mutualistic symbiosis between the coral polyps host and its endosymbiotic unicellular algae is disrupted, and can lead to the death of entire colonies. Previous works have shown that soma clonal colonies display different levels of survival depending on the environmental conditions they previously faced. Epigenetic mechanisms are good candidates to explain this phenomenon. The clonal nature of a colony and the possibility of generating genetically identical colonies through propagation make corals an attractive model to study the impact of the environment on the epigenome. However, until now, no work had been published on the P. acuta epigenome. One of the main problems is caused by the intracellular location of Symbiodinium, which makes it complicated to isolate coral chromatin free of contamination by endiosymbiotic biological material. Here, (i) we describe a simple method to purify P. acuta chromatin, (ii) we provide the first description of a coral methylome, with a mosaic pattern of cytosine methylation principally in a CpG context (4% of all CpG), and (iii) we show that P. acuta , but not all corals, has an unusual chromatin structure, and displays histone H3 clipping.

BACKGROUND:The clownfish Amphiprion ocellaris is one of the rare coral reef fish species that can be reared in aquaria. With relatively short embryonic and larval development, it could be used as a model species to study the impact of global changes such as temperature rise or anthropogenic threats (eg, pollution) on the postembryonic development at molecular and endocrinological levels. Establishing a developmental table allows us to standardize sampling for the scientific community willing to conduct experiments on this species on different areas: ecology, evolution, and developmental biology.RESULTS:Here, we describe the postembryonic developmental stages for the clownfish A. ocellaris from hatching to juvenile stages (30 days posthatching). We quantitatively followed the postembryonic growth and described qualitative traits: head, paired and unpaired fins, notochord flexion, and pigmentation changes. The occurrence of these changes over time allowed us to define seven stages, for which we provide precise descriptions.CONCLUSIONS:Our work gives an easy system to determine A. ocellaris postembryonic stages allowing, thus, to develop this species as a model species for coral reef fishes. In light of global warming, the access to the full postembryonic development stages of coral reef fish is important to determine stressors that can affect such processes.

Clownfishes and sea anemones form an intriguing long-term association, but the mechanism underlying this symbiosis is not well understood. Since clownfishes seem to cover themselves with sea anemone mucus, we investigated the microbiomes of the two partners to search for possible shifts in their compositions. We used a 16S rRNA gene sequencing strategy to study the dynamics of the microbiota during the association between the clownfish Amphiprion ocellaris and its host Heteractis magnifica under laboratory conditions. The experiment conducted in aquaria revealed that both clownfish and sea anemone mucus had specific signatures compared to artificial sea water. The microbiomes of both species were highly dynamic during the initiation of the symbiosis and for up to seven days after contact. Three families of bacteria ( Haliangiaceae, Pseudoalteromonadacae, Saprospiracae ) were shared between the two organisms after symbiosis. Once the symbiosis had been formed, the clownfishes and sea anemone then shared some communities of their mucus microbiota. This study paves the way for further investigations to determine if similar microbial signatures exist in natural environments, whether such microbial sharing can be beneficial for both organisms, and whether the microbiota is implicated in the mechanisms that protect the clownfish from sea anemone stinging.

Octocrylene (OC) is an ingredient used in many sunscreens and cosmetics worldwide. Our group evaluated the toxicity of OC in corals. Adult Pocillopora damicornis coral was treated with OC at concentrations of 5, 50, 300, and 1000 μg/L. Most polyps were closed at concentrations of 300 μg/L and higher. Further, metabolomic profiling provided crucial information regarding OC accumulation in coral tissues and OC toxicity. First, we demonstrated that OC was transformed into fatty acid conjugates via oxidation of the ethylhexyl chain, yielding very lipophilic OC analogues that accumulate in coral tissues. Second, the differential analysis of coral profiles revealed higher levels of 15 acylcarnitines, suggesting abnormal fatty acid metabolism related to mitochondrial dysfunction. The formation of OC analogues suggests that OC concentrations measured in the environment, and organisms may have been largely underestimated. Overall, these results call for an in-depth evaluation of OC toxicity and the reevaluation of the actual OC accumulation rate in the ocean's food chain, including OC-fatty acid conjugates.

In this study, we tested the tolerance of tissue balls (TBs, 100-400 μm in diameter) from the coral Pocillopora damicornis produced using mechanical excision to exposure to cryoprotectant (CPA) solutions. TBs were treated for 20 min at room temperature with individual, binary, ternary or quaternary CPA solutions with a total molarity from 2.0 to 5.0M. Four CPAs were used: ethylene glycol (EG), dimethylsulfoxide (Me2SO), methanol (Met) and glycerol (Gly). In some experiments, the molarity of the CPA solutions was increased and decreased in a stepwise manner. The tolerance of TBs following CPA treatment was evaluated using two parameters. The Tissue Ball Regression (expressed in μm/h) measured the diameter regression of TBs over time. The % Undamaged TBs quantified the proportion of TBs, which remained intact over time after the CPA treatment. TBs tolerated exposure to binary solutions with a total molarity of 4.0 M containing 2.0 M EG+2.0 M Met and 2.0 MEG+2.0 M Gly. TBs displayed tolerance to ternary solutions with a total molarity up to 3.0 M, containing each CPA at 1.0 M. Quaternary solutions with a total molarity of 4.0M containing each CPA at 1.0 M were not tolerated by TBs. When the molarity of the CPA solutions was increased and decreased in a stepwise manner, TBs withstood exposure to a CPA solution with a total molarity of 4.5 M, containing 1.5 M EG+1.5 M Gly+1.5 M Me(2)SO. This study confirmed the interest of using TBs to test CPA solutions, with the objective of developing a vitrification-based cryopreservation protocol.

Numerous studies have highlighted the diversity of fish inner ear morphology. However, the function of the shape, size and orientation of the different structures remains poorly understood. The saccule (otolithic endorgan) is considered to be the principal hearing organ in fishes and it has been hypothesized that sagitta (saccular otolith) shape and size affect hearing capacities: large sagittae are thought to increase sensitivity. The sagittae of many ophidiids and carapids occupy a large volume inside the neurocranium. Hence they are a good structure with which to test the size hypothesis. The main aim of this study was to investigate hearing capacities and inner ear morphology in two ophidiiform species: Ophidion rochei and Carapus acus. We used a multidisciplinary approach that combines dissections, μCT-scan examinations and auditory evoked potential techniques. Carapus acus and O. rochei sagittae have similar maximal diameters; both species have larger otoliths than many non-ophidiiform species, especially compared with the intra-neurocranium volume. Both species are sensitive to sounds up to 2100 Hz. Relative to the skull, O. rochei has smaller sagittae than the carapid, but better hearing capacities from 300 to 900 Hz and similar sensitivities at 150 Hz and from 1200 to 2100 Hz. Results show that hearing capacities of a fish species cannot be predicted only based on sagitta size. Larger otoliths (in size relative to the skull) may have evolved mainly for performing vestibular functions in fishes, especially those species that need to execute precise and complex movements.

In this study, we investigated the tolerance of Pocillopora damicornis apexes to treatments with solutions containing penetrating and non-penetrating cryoprotective agents (CPAs). CPAs were employed individually or in binary, tertiary or quaternary solutions. In some experiments apexes were treated successively with two CPA solutions with increasing total concentration. P. damicornis apexes withstood exposure for up to 30 min to solutions containing 0.6-0.8 M sucrose (Suc) or trehalose (Tre). When apexes were treated with binary cryoprotectant solutions containing Suc and ethylene glycol (EG), methanol (Meth), dimethyl sulfoxide (Me(2)SO) or glycerol (Gly), the CPAs employed in combination with Suc could be ranked in the following order of decreasing tolerance: EG>Meth>Me(2)SO>Gly. P. damicornis apexes tolerated exposure to complex CPA solutions containing Suc, Me(2)SO, EG and/or Meth with a total molarity of 2.45 M. In experiments where two successive CPA solutions were employed, apexes withstood treatment with the second, more concentrated solution at 0°C for up to 10 min. These preliminary results pave the way to the development of a cryopreservation protocol for P. damicornis apexes.

In situ growth patterns of two-species of reef-building cold-water corals were investigated for the first time at different temporal scales, based on the redeployment of coral nubbins in their natural environment. Lophelia pertusa and Madrepora oculata were collected in November 2010 and May 2011 from the Lacaze-Duthiers canyon in the northwestern Mediterranean Sea (520m depth). Three in situ growth experiments were performed from November 2010 to May 2011, May to September 2011 and November 2010 to September 2011. For comparison, aquaria experiments over comparable lengths of time were conducted with coral colonies collected in November 2010. In the canyon, new polyps from M. oculata and L. pertusa displayed similar growth rates in summer (5.8±3.8 and 7.3±1.7mmyr−1 respectively), but M. oculata growed significantly slower during winter/spring than L. pertusa (4.1±1.8 and 8.4±2.7mmyr−1 respectively). Budding rates however (the rate of new polyp addition per mother polyp per year) were similar between both species in winter/spring (45±40% for M. oculata and 48±72% for L. pertusa), but were significantly lower in summer for M. oculata (14±19%) compared to L. pertusa (58±94%). This seasonal difference in the growth between L. pertusa and M. oculata might reflect differences in species-specific physiology (such as reproduction) or feeding strategy, or a higher sensitivity of M. oculata to the variability of food supply in the Lacaze-Duthiers canyon resulting from periodic cascading events. The comparison of in situ and aquaria growth experiments showed no significant differences for budding and new polyp growth rates, which supports the validity of aquaria experiments for these kinds of investigations. However the budding rates observed were consistently lower in coral maintained in aquaria than those in in situ conditions, a finding which is to be considered when extrapolating laboratory based investigation results to the naturally occurring coral ecosystem.

In this study, the tolerance of tissue balls (TBs, 100-300 μm in diameter) from the coral Pocillopora damicornis produced using mechanical excision to exposure to cryoprotectant (CPA) solutions was tested. TBs were treated for 20 min at room temperature with solutions of ethylene glycol (EG), methanol (Met), glycerol (Gly) or dimethyl sulfoxide (Me2SO) at concentrations between 1.0 and 4.5M. Two parameters were used to evaluate the survival of TBs following CPA treatment. The Undamaged Duration of Tissue Balls (expressed in h) corresponded to the time period during which the membrane surface of TBs remained smooth and their motility was preserved. Tissue Ball Regression (expressed in μm/h) corresponded to the size reduction of TBs over time. TBs tolerated exposure to all CPAs tested at the three lower concentrations employed (1.0 M, 1.5 M and 2.0 M). No survival was achieved following exposure to a 4.5 M CPA solution. At concentrations of 3.0 and 4.0 M, higher Undamaged Duration of Tissue Balls and lower Tissue Ball Regression were obtained following treatment with EG compared to the other three CPAs. Our experiments show that TBs constitute a good experimental material to evaluate CPA toxicity on corals using large numbers of samples. Performing preliminary experiments with TBs may allow reducing the number of tests carried out with less easily available coral forms such as planulae, thereby preserving larval stocks.