Eight years of community structure monitoring through recreational citizen science at the "SS Thistlegorm" wreck (Red Sea).

Large artificial coral reef communities, such as those thriving on sunken shipwrecks, tend to mirror those of nearby natural coral reefs and their long-term dynamics may help future reef resilience to environmental change. We examined the community structure of the world-renown "SS Thistlegorm" wreck in the northern Red Sea from 2007 through 2014, analyzing data collected during the recreational citizen science Red Sea monitoring project "Scuba Tourism for the Environment". Volunteer divers collected data on 6 different diving parameters which included the date of the dive, maximum depth, average depth, temperature, dive time, hour of dive, and gave an abundance estimation of sighted taxa from a list of 72 target taxa. Although yearly variations in community structure were significant, there was no clear temporal trend, and 71 of all 72 target taxa were sighted throughout the 8 years. The 5 main taxa driving variations among year clusters in taxa presence/absence (Soft Tree Coral-Dendronephthya spp., Giant Moray-Gymnothorax javanicus, Squirrel Fish-Sargocentron spp., Humpback Batfish-Platax spp., and Caranxes-Carangidae) and taxa abundance (Soft Tree Coral, Giant Moray, Red Sea Clownfish-Amphiprion bicinctus, Napoleon Wrasse-Cheilinus undulatus, and Caranxes) data were determined. The "SS Thistlegorm" provides a compelling example of how artificial coral reefs can sustain a well-established community structure similar to those of their natural counterparts.

Acclimatization of a coral-dinoflagellate mutualism at a CO2 vent.

Ocean acidification caused by shifts in ocean carbonate chemistry resulting from increased atmospheric CO2 concentrations is threatening many calcifying organisms, including corals. Here we assessed autotrophy vs heterotrophy shifts in the Mediterranean zooxanthellate scleractinian coral Balanophyllia europaea acclimatized to low pH/high pCO2 conditions at a CO2 vent off Panarea Island (Italy). Dinoflagellate endosymbiont densities were higher at lowest pH Sites where changes in the distribution of distinct haplotypes of a host-specific symbiont species, Philozoon balanophyllum, were observed. An increase in symbiont C/N ratios was observed at low pH, likely as a result of increased C fixation by higher symbiont cell densities. δ13C values of the symbionts and host tissue reached similar values at the lowest pH Site, suggesting an increased influence of autotrophy with increasing acidification. Host tissue δ15N values of 0‰ strongly suggest that diazotroph N2 fixation is occurring within the coral tissue/mucus at the low pH Sites, likely explaining the decrease in host tissue C/N ratios with acidification. Overall, our findings show an acclimatization of this coral-dinoflagellate mutualism through trophic adjustment and symbiont haplotype differences with increasing acidification, highlighting that some corals are capable of acclimatizing to ocean acidification predicted under end-of-century scenarios.

Metagenomic shifts in mucus, tissue and skeleton of the coral Balanophyllia europaea living along a natural CO2 gradient.

Using the Mediterranean coral Balanophyllia europaea naturally growing along a pH gradient close to Panarea island (Italy) as a model, we explored the role of host-associated microbiomes in coral acclimatization to ocean acidification (OA). Coral samples were collected at three sites along the gradient, mimicking seawater conditions projected for 2100 under different IPCC (The Intergovernmental Panel on Climate Change) scenarios, and mucus, soft tissue and skeleton associated microbiomes were characterized by shotgun metagenomics. According to our findings, OA induced functional changes in the microbiomes genetic potential that could mitigate the sub-optimal environmental conditions at three levels: i. selection of bacteria genetically equipped with functions related to stress resistance; ii. shifts in microbial carbohydrate metabolism from energy production to maintenance of cell membranes and walls integrity; iii. gain of functions able to respond to variations in nitrogen needs at the holobiont level, such as genes devoted to organic nitrogen mobilization. We hence provided hypotheses about the functional role of the coral associated microbiome in favoring host acclimatation to OA, remarking on the importance of considering the crosstalk among all the components of the holobiont to unveil how and to what extent corals will maintain their functionality under forthcoming ocean conditions.

Coral micro- and macro-morphological skeletal properties in response to life-long acclimatization at CO2 vents in Papua New Guinea.

This study investigates the effects of long-term exposure to OA on skeletal parameters of four tropical zooxanthellate corals naturally living at CO2 seeps and adjacent control sites from two locations (Dobu and Upa Upasina) in the Papua New Guinea underwater volcanic vent system. The seeps are characterized by seawater pH values ranging from 8.0 to about 7.7. The skeletal porosity of Galaxea fascicularis, Acropora millepora, massive Porites, and Pocillopora damicornis was higher (up to ~ 40%, depending on the species) at the seep sites compared to the control sites. Pocillopora damicornis also showed a decrease of micro-density (up to ~ 7%). Thus, further investigations conducted on this species showed an increase of the volume fraction of the larger pores (up to ~ 7%), a decrease of the intraskeletal organic matrix content (up to ~ 15%), and an increase of the intraskeletal water content (up to ~ 59%) at the seep sites. The organic matrix related strain and crystallite size did not vary between seep and control sites. This multi-species study showed a common phenotypic response among different zooxanthellate corals subjected to the same environmental pressures, leading to the development of a more porous skeletal phenotype under OA.

Decreasing pH impairs sexual reproduction in a Mediterranean coral transplanted at a CO2 vent.

Ocean acidification, due to the increase of carbon dioxide (CO2) concentration in the atmosphere and its absorption by the oceans, affects many aspects of marine calcifying organisms' biology, including reproduction. Most of the available studies on low pH effects on coral reproduction have been conducted on tropical species under controlled conditions, while little information is reported for either tropical or temperate species in the field. This study describes the influence of decreasing pH on sexual reproduction of the temperate non-zooxanthellate colonial scleractinian Astroides calycularis, transplanted in four sites along a natural pH gradient at the underwater volcanic crater of Panarea Island (Tyrrhenian Sea, Italy). The average pH values of each site (range: pHTS 8.07-7.40) match different scenarios of the Intergovernmental Panel on Climate Change (IPCC) for the end of the century. After 3 months under experimental conditions, the reproductive parameters of both oocytes and spermaries (abundance, gonadal index, and diameters) seem to be unaffected by low pH. However, a delay in spermary development in the pre-fertilization period and a persistence of mature oocytes in the fertilization period were observed in the most acidic site. Furthermore, no embryos were found in colonies from the two most acidic sites, suggesting a delay or an interruption of the fertilization process due to acidified conditions. These findings suggest a negative effect of low pH on A. calycularis sexual reproduction. However, long-term experiments, including the synergistic impact of pH and temperature, are needed to predict if this species will be able to adapt to climate change over the next century.

Ocean acidification causes variable trait-shifts in a coral species.

High pCO2 habitats and their populations provide an unparalleled opportunity to assess how species may survive under future ocean acidification conditions, and help to reveal the traits that confer tolerance. Here we utilize a unique CO2 vent system to study the effects of exposure to elevated pCO2 on trait-shifts observed throughout natural populations of Astroides calycularis, an azooxanthellate scleractinian coral endemic to the Mediterranean. Unexpected shifts in skeletal and growth patterns were found. Colonies shifted to a skeletal phenotype characterized by encrusting morphology, smaller size, reduced coenosarc tissue, fewer polyps, and less porous and denser skeletons at low pH. Interestingly, while individual polyps calcified more and extended faster at low pH, whole colonies found at low pH site calcified and extended their skeleton at the same rate as did those at ambient pH sites. Transcriptomic data revealed strong genetic differentiation among local populations of this warm water species whose distribution range is currently expanding northward. We found excess differentiation in the CO2 vent population for genes central to calcification, including genes for calcium management (calmodulin, calcium-binding proteins), pH regulation (V-type proton ATPase), and inorganic carbon regulation (carbonic anhydrase). Combined, our results demonstrate how coral populations can persist in high pCO2 environments, making this system a powerful candidate for investigating acclimatization and local adaptation of organisms to global environmental change.

Accumulation of PAHs in the tissues and algal symbionts of a common Mediterranean coral: Skeletal storage relates to population age structure.

Polycyclic aromatic hydrocarbons (PAHs) are widespread and harmful environmental pollutants that threaten marine ecosystems. Assessing their level and source is crucial to estimate the potential risks for marine organisms, as PAHs represent an additional threat to organism resilience under ongoing climatic change. Here we applied the QuEChERS extraction method to quantify four PAHs (i.e. acenaphthene, fluorene, fluoranthene, and pyrene) in three biological compartments (i.e. skeleton, tissue, and zooxanthellae symbiotic algae) of adult and old specimens of a scleractinian coral species (Balanophyllia europaea) that is widespread throughout the Mediterranean Sea. A higher concentration of all four investigated PAHs was observed in the zooxanthellae, followed by the coral tissue, with lowest concentration in the skeleton, consistently with previous studies on tropical species. In all the three biological compartments, the concentration of low molecular weight PAHs was higher with respect to high-molecular weight PAHs, in agreement with their bioaccumulation capabilities. PAH concentration was unrelated to skeletal age. Observed PAHs were of petrogenic origin, reflecting the pollution sources of the sampling area. By coupling PAH data with population age structure data measured in the field, the amount of PAHs stored in the long term (i.e. up to 20 years) in coral skeletons was quantified and resulted in 53.6 ng m-2 of acenaphthene, 69.4 ng m-2 of fluorene, 2.7 ng m-2 of fluoranthene, and 11.7 ng m-2 of pyrene. This estimate provides the basis for further assessments of long-term sequestration of PAHs from the marine environment in the whole Mediterranean, given the widespread distribution of the investigated coral species.

Patterns in microbiome composition differ with ocean acidification in anatomic compartments of the Mediterranean coral Astroides calycularis living at CO2 vents.

Coral microbiomes, the complex microbial communities associated with the different anatomic compartments of the coral, provide important functions for the host's survival, such as nutrient cycling at the host's surface, prevention of pathogens colonization, and promotion of nutrient uptake. Microbiomes are generally referred to as plastic entities, able to adapt their composition and functionality in response to environmental change, with a possible impact on coral acclimatization to phenomena related to climate change, such as ocean acidification. Ocean sites characterized by natural gradients of pCO2 provide models for investigating the ability of marine organisms to acclimatize to decreasing seawater pH. Here we compared the microbiome of the temperate, shallow water, non-symbiotic solitary coral Astroides calycularis that naturally lives at a volcanic CO2 vent in Ischia Island (Naples, Italy), with that of corals living in non-acidified sites at the same island. Bacterial DNA associated with the different anatomic compartments (mucus, tissue and skeleton) of A. calycularis was differentially extracted and a total of 68 samples were analyzed by 16S rRNA gene sequencing. In terms of phylogenetic composition, the microbiomes associated with the different coral anatomic compartments were different from each other and from the microbial communities of the surrounding seawater. Of all the anatomic compartments, the mucus-associated microbiome differed the most between the control and acidified sites. The differences detected in the microbial communities associated to the three anatomic compartments included a general increase in subdominant bacterial groups, some of which are known to be involved in different stages of the nitrogen cycle, such as potential nitrogen fixing bacteria and bacteria able to degrade organic nitrogen. Our data therefore suggests a potential increase of nitrogen fixation and recycling in A. calycularis living close to the CO2 vent system.

Transcriptional response of the heat shock gene hsp70 aligns with differences in stress susceptibility of shallow-water corals from the Mediterranean Sea.

Shallow-water corals of the Mediterranean Sea are facing a dramatic increase in water temperature due to climate change, predicted to increase the frequency of bleaching and mass mortality events. However, supposedly not all corals are affected equally, as they show differences in stress susceptibility, as suggested by physiological outputs of corals along temperature gradients and under controlled conditions in terms of reproduction, demography, growth, calcification, and photosynthetic efficiency. In this study, gene expression and induction of a 70-kDa heat shock protein (HSP70) was analyzed in five common shallow-water hard corals in the Mediterranean Sea, namely Astroides calycularis, Balanophyllia europaea, Caryophyllia inornata, Cladocora caespitosa, and Leptopsammia pruvoti. The main aim was to assess the contribution of this evolutionary conserved cytoprotective mechanism to the physiological plasticity of these species that possess different growth modes (solitary vs colonial) and trophic strategies (zooxanthellate vs azooxanthellate). Using quantitative real-time PCR, in situ hsp70 baseline levels and expression profiles after a heat-shock exposure were assessed. Levels of hsp70 and heat stress induction were higher in zooxanthellate than in azooxanthellate species, and different heat stress transcriptional profiles were observed between colonial and solitary zooxanthellate corals. On the whole, the hsp70 transcriptional response to heat stress aligns with stress susceptibility of the species and suggests a contribution of trophic strategy and morphology in shaping coral resilience to stress. Understanding these molecular processes may contribute to assess the potential effects and relative resilience of Mediterranean corals under climate change.

Reproduction of an azooxanthellate coral is unaffected by ocean acidification.

Anthropogenic carbon dioxide (CO2) emissions and consequent ocean acidification (OA) are projected to have extensive consequences on marine calcifying organisms, including corals. While the effects of OA on coral calcification are well documented, the response of reproduction is still poorly understood since no information are reported for temperate corals. Here we investigate for the first time the influence of OA on sexual reproduction of the temperate azooxanthellate solitary scleractinian Leptopsammia pruvoti transplanted along a natural pCO2 gradient at a Mediterranean CO2 vent. After 3 months, future projection of pH levels did not influence the germ cell production, gametogenesis and embryogenesis in this azooxanthellate coral. These findings suggest that reproductive potential may be quite tolerant to decreasing pH, with implications for ecosystem function and services in a changing ocean.

Ecological relevance of skeletal fatty acid concentration and composition in Mediterranean scleractinian corals.

The intra-skeletal fatty acid concentration and composition of four Mediterranean coral species, namely Cladocora caespitosa, Balanophyllia europaea, Astroides calycularis and Leptopsammia pruvoti, were examined in young and old individuals living in three different locations of the Mediterranean Sea. These species are characterized by diverse levels of organization (solitary or colonial) and trophic strategies (symbiotic or non-symbiotic). Fatty acids have manifold fundamental roles comprehensive of membrane structure fluidity, cell signaling and energy storage. For all species, except for B. europaea, the intra-skeletal fatty acid concentration was significantly higher in young individuals than in old ones. Moreover, fatty acid concentration was higher in colonial corals than in solitary ones and in the symbiotic corals compared to non-symbiotic ones. Analysis by gas chromatography-mass spectrometry (GC-MS) revealed that palmitic acid (16:0) was the most abundant fatty acid, followed by stearic (18:0) in order of concentration. Oleic acid (18:1) was detected as the third main component only in skeletons from symbiotic corals. These results suggest that, in the limits of the studied species, intra-skeletal fatty acid composition and concentration may be used for specific cases as a proxy of level of organization and trophic strategy, and eventually coral age.

Mediterranean versus Red sea corals facing climate change, a transcriptome analysis.

The anthropogenic increase in atmospheric CO2 that drives global warming and ocean acidification raises serious concerns regarding the future of corals, the main carbonate biomineralizers. Here we used transcriptome analysis to study the effect of long-term gradual temperature increase (annual rate), combined with lowered pH values, on a sub-tropical Red Sea coral, Stylophora pistillata, and on a temperate Mediterranean symbiotic coral Balanophyllia europaea. The gene expression profiles revealed a strong effect of both temperature increase and pH decrease implying for synergism response. The temperate coral, exposed to a twice as high range of seasonal temperature fluctuations than the Red Sea species, faced stress more effectively. The compensatory strategy for coping apparently involves deviating cellular resources into a massive up-regulation of genes in general, and specifically of genes involved in the generation of metabolic energy. Our results imply that sub-lethal, prolonged exposure to stress can stimulate evolutionary increase in stress resilience.

Negative response of photosynthesis to natural and projected high seawater temperatures estimated by pulse amplitude modulation fluorometry in a temperate coral.

Balanophyllia europaea is a shallow water solitary zooxanthellate coral, endemic to the Mediterranean Sea. Extensive field studies across a latitudinal temperature gradient highlight detrimental effects of rising temperatures on its growth, demography, and skeletal characteristics, suggesting that depression of photosynthesis at high temperatures might cause these negative effects. Here we test this hypothesis by analyzing, by means of pulse amplitude modulation fluorometry, the photosynthetic efficiency of B. europaea specimens exposed in aquaria to the annual range of temperatures experienced in the field (13, 18, and 28°C), and two extreme temperatures expected for 2100 as a consequence of global warming (29 and 32°C). The indicators of photosynthetic performance analyzed (maximum and effective quantum yield) showed that maximum efficiency was reached at 20.0-21.6°C, slightly higher than the annual mean temperature in the field (18°C). Photosynthetic efficiency decreased from 20.0 to 13°C and even more strongly from 21.6 to 32°C. An unusual form of bleaching was observed, with a maximum zooxanthellae density at 18°C that strongly decreased from 18 to 32°C. Chlorophyll a concentration per zooxanthellae cell showed an opposite trend as it was minimal at 18°C and increased from 18 to 32°C. Since the areal chlorophyll concentration is the product of the zooxanthellae density and its cellular content, these trends resulted in a homogeneous chlorophyll concentration per coral surface across temperature treatments. This confirms that B. europaea photosynthesis is progressively depressed at temperatures >21.6°C, supporting previous hypotheses raised by the studies on growth and demography of this species. This study also confirms the threats posed to this species by the ongoing seawater warming.

Gains and losses of coral skeletal porosity changes with ocean acidification acclimation.

Ocean acidification is predicted to impact ecosystems reliant on calcifying organisms, potentially reducing the socioeconomic benefits these habitats provide. Here we investigate the acclimation potential of stony corals living along a pH gradient caused by a Mediterranean CO2 vent that serves as a natural long-term experimental setting. We show that in response to reduced skeletal mineralization at lower pH, corals increase their skeletal macroporosity (features >10 µm) in order to maintain constant linear extension rate, an important criterion for reproductive output. At the nanoscale, the coral skeleton's structural features are not altered. However, higher skeletal porosity, and reduced bulk density and stiffness may contribute to reduce population density and increase damage susceptibility under low pH conditions. Based on these observations, the almost universally employed measure of coral biomineralization, the rate of linear extension, might not be a reliable metric for assessing coral health and resilience in a warming and acidifying ocean.

Isotropic microscale mechanical properties of coral skeletons.

Scleractinian corals are a major source of biogenic calcium carbonate, yet the relationship between their skeletal microstructure and mechanical properties has been scarcely studied. In this work, the skeletons of two coral species:solitary Balanophyllia europaea and colonial Stylophora pistillata, were investigated by nanoindentation. The hardness HIT and Young's modulus E(IT) were determined from the analysis of several load-depth data on two perpendicular sections of the skeletons: longitudinal (parallel to the main growth axis) and transverse. Within the experimental and statistical uncertainty,the average values of the mechanical parameters are independent on the section's orientation. The hydration state of the skeletons did not affect the mechanical properties. The measured values, EIT in the 76-77 GPa range, and H(IT) in the 4.9­5.1 GPa range, are close to the ones expected for polycrystalline pure aragonite. Notably, a small difference in H(IT) is observed between the species. Different from corals, single-crystal aragonite and the nacreous layer of the seashell Atrina rigida exhibit clearly orientation-dependent mechanical properties. The homogeneous and isotropic mechanical behaviour of the coral skeletons at the microscale is correlated with the microstructure,observed by electron microscopy and atomic force microscopy, and with the X-ray diffraction patterns of the longitudinal and transverse sections.

Biomineralization control related to population density under ocean acidification.

Anthropogenic CO2 is a major driver of current environmental change in most ecosystems1, and the related ocean acidification (OA) is threatening marine biota2. With increasing pCO2, calcification rates of several species decrease3, although cases of up-regulation are observed4. Here, we show that biological control over mineralization relates to species abundance along a natural pH gradient. As pCO2 increased, the mineralogy of a scleractinian coral (Balanophyllia europaea) and a mollusc (Vermetus triqueter) did not change. In contrast, two calcifying algae (Padina pavonica and Acetabularia acetabulum) reduced and changed mineralization with increasing pCO2, from aragonite to the less soluble calcium sulphates and whewellite, respectively. As pCO2 increased, the coral and mollusc abundance was severely reduced, with both species disappearing at pH < 7.8. Conversely, the two calcifying and a non-calcifying algae (Lobophora variegata) showed less severe or no reductions with increasing pCO2, and were all found at the lowest pH site. The mineralization response to decreasing pH suggests a link with the degree of control over the biomineralization process by the organism, as only species with lower control managed to thrive in the lowest pH.

A time-domain nuclear magnetic resonance study of Mediterranean scleractinian corals reveals skeletal-porosity sensitivity to environmental changes.

Mediterranean corals are a natural model for studying global warming, as the Mediterranean basin is expected to be one of the most affected regions and the increase in temperature is one of the greatest threats for coral survival. We have analyzed for the first time with time-domain nuclear magnetic resonance (TD-NMR) the porosity and pore-space structure, important aspects of coral skeletons, of two scleractinian corals, Balanophyllia europaea (zooxanthellate) and Leptopsammia pruvoti (nonzooxanthellate), taken from three different sites on the western Italian coast along a temperature gradient. Comparisons have been made with mercury intrusion porosimetry and scanning electron microscopy images. TD-NMR parameters are sensitive to changes in the pore structure of the two coral species. A parameter, related to the porosity, is larger for L. pruvoti than for B. europaea, confirming previous non-NMR results. Another parameter representing the fraction of the pore volume with pore sizes of less than 10-20 µm is inversely related, with a high degree of statistical significance, to the mass of the specimen and, for B. europaea, to the temperature of the growing site. This effect in the zooxanthellate species, which could reduce its resistance to mechanical stresses, may depend on an inhibition of the photosynthetic process at elevated temperatures and could have particular consequences in determining the effects of global warming on these species.