Species and population genomic differentiation in Pocillopora corals (Cnidaria, Hexacorallia).

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 ß). 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.

A star is born again: Methods for larval rearing of an emerging model organism, the False clownfish Amphiprion ocellaris.

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.

The tropical coral Pocillopora acuta displays an unusual chromatin structure and shows histone H3 clipping plasticity upon bleaching.

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.

Sea anemone and clownfish microbiota diversity and variation during the initial steps of symbiosis.

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.

Staging and normal table of postembryonic development of the clownfish (Amphiprion ocellaris).

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.

Metabolomics Reveal That Octocrylene Accumulates in Pocillopora damicornis Tissues as Fatty Acid Conjugates and Triggers Coral Cell Mitochondrial Dysfunction.

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.

Towards a vitrification-based cryopreservation protocol for the coral Pocillopora damicornis L.: Tolerance of tissue balls to 4.5 M cryoprotectant solutions.

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.

Survival of tissue balls from the coral Pocillopora damicornis L. exposed to cryoprotectant solutions.

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.

Hearing capacities and otolith size in two ophidiiform species (Ophidion rochei and Carapus acus).

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.

Tolerance of apexes of coral Pocillopora damicornis L. to cryoprotectant solutions.

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.