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.

Oxygen and carbon isotope variations in Chamelea gallina shells: Environmental influences and vital effects.

Stable isotopes in mollusc shells, together with variable growth rates and other geochemical properties, can register different environmental clues, including seawater temperature, salinity and primary productivity. However, the strict biological control over the construction of biominerals exerted by many calcifying organisms can constrain the use of these organisms for paleoenvironmental reconstructions. Biologically controlled calcification is responsible for the so called vital effects that cause a departure from isotopic equilibrium during shell formation, resulting in lower shell oxygen and carbon compared to the equilibrium value. We investigated shell oxygen and carbon isotopic composition of the bivalve Chamelea gallina in six sites along with a latitudinal gradient on the Adriatic Sea (NE Mediterranean Sea). Seawater δ18 O and δ13 CDIC varied from North to South, reflecting variations in seawater temperature, salinity, and chlorophyll concentration among sites. Shell δ18 O and δ13 C differed among sites and exhibited a wide range of values along with the ~400 km latitudinal gradient, away from isotopic equilibrium for both isotopes. These results hampered the utilization of this bivalve as a proxy for environmental reconstructions, in spite of C. gallina showing promise as a warm temperature proxy. Rigorous calibration studies with a precise insight of environment and shell growth are crucial prior to considering this bivalve as a reliable paleoclimatic archive.

Multiscale analysis on otolith structural features reveals differences in ontogenesis and sex in Merluccius merluccius in the western Adriatic Sea.

Otolith biomineralization results from biochemical processes regulated by the interaction of internal (physiological) and external (environmental) factors which lead to morphological and ultrastructural variability at intra- and interspecific levels. The aim of this study was to conduct a multi-scale analysis of the sagittal otoliths of the Merlucius merlucius (European hake) from the western Adriatic Sea in order to correlate otolith features with fish ontogeny and sex. We show that otoliths of sexually undifferentiated (non-sexed) individuals having a fish body total length (TL) less than 15 cm had faster growth in length, width, area, perimeter, volume and weight and a higher amount of organic matrix compared with otoliths of sexually differentiated individuals (females and males) having a fish size range of 15-50 cm. Most importantly, with increasing fish TL, female saccular otoliths contained a higher number of protuberances and rougher surface compared with male specimens, which showed more uniform mean curvature density. The differences between females and males discovered in this study could be associated with fish hearing adaptation to reproductive behavioural strategies during the spawning season. The outcomes of this research provide insights on how size and sex-related variations in otolith features may be affected by fish ecological and behavioural patterns.

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.

The skeleton of Balanophyllia coral species suggests adaptive traits linked to the onset of mixotrophy.

The diversity in the skeletal features of coral species is an outcome of their evolution, distribution and habitat. Here, we explored, from macro- to nano-scale, the skeletal structural and compositional characteristics of three coral species belonging to the genus Balanophyllia having different trophic strategies. The goal is to address whether the onset of mixotrophy influenced the skeletal features of B. elegans, B. regia, and B. europaea. The macroscale data suggest that the presence of symbiotic algae in B. europaea can lead to a surplus of energy input that increases its growth rate and skeletal bulk density, leading to larger and denser corals compared to the azooxanthellate ones, B. regia and B. elegans. The symbiosis would also explain the higher intra-skeletal organic matrix (OM) content, which is constituted by macromolecules promoting the calcification, in B. europaea compared to the azooxanthellate species. The characterization of the soluble OM also revealed differences between B. europaea and the azooxanthellate species, which may be linked to diverse macromolecular machineries responsible for skeletal biosynthesis and final morphology. Differently, the crystallographic features were homogenous among species, suggesting that the basic building blocks of skeletons remained a conserved trait in these related species, regardless of the trophic strategy. These results show changes in skeletal phenotype that could be triggered by the onset of mixotrophy, as a consequence of the symbiotic association, displaying remarkable plasticity of coral skeletons which repeatedly allowed this coral group to adapt to a range of changing environments throughout its geological history.

Impact of Marine Aquaculture on the Microbiome Associated with Nearby Holobionts: The Case of Patella caerulea Living in Proximity of Sea Bream Aquaculture Cages.

Aquaculture plays a major role in the coastal economy of the Mediterranean Sea. This raises the issue of the impact of fish cages on the surrounding environment. Here, we explore the impact of aquaculture on the composition of the digestive gland microbiome of a representative locally dwelling wild holobiont, the grazer gastropod Patella caerulea, at an aquaculture facility located in Southern Sicily, Italy. The microbiome was assessed in individuals collected on sea bream aquaculture cages and on a rocky coastal tract located about 1.2 km from the cages, as the control site. Patella caerulea microbiome variations were explained in the broad marine metacommunity context, assessing the water and sediment microbiome composition at both sites, and characterizing the microbiome associated with the farmed sea bream. The P. caerulea digestive gland microbiome at the aquaculture site was characterized by a lower diversity, the loss of microorganisms sensitive to heavy metal contamination, and by the acquisition of fish pathogens and parasites. However, we also observed possible adaptive responses of the P. caerulea digestive gland microbiome at the aquaculture site, including the acquisition of putative bacteria able to deal with metal and sulfide accumulation, highlighting the inherent microbiome potential to drive the host acclimation to stressful conditions.

Climate variation during the Holocene influenced the skeletal properties of Chamelea gallina shells in the North Adriatic Sea (Italy).

Understanding how marine taxa will respond to near-future climate changes is one of the main challenges for management of coastal ecosystem services. Ecological studies that investigate relationships between the environment and shell properties of commercially important marine species are commonly restricted to latitudinal gradients or small-scale laboratory experiments. This paper aimed to explore the variations in shell features and growth of the edible bivalve Chamelea gallina from the Holocene sedimentary succession to present-day thanatocoenosis of the Po Plain-Adriatic Sea system (Italy). Comparing the Holocene sub-fossil record to modern thanatocoenoses allowed obtaining an insight of shell variations dynamics on a millennial temporal scale. Five shoreface-related assemblages rich in C. gallina were considered: two from the Middle Holocene, when regional sea surface temperatures were higher than today, representing a possible analogue for the near-future global warming, one from the Late Holocene and two from the present-day. We investigated shell biometry and skeletal properties in relation to the valve length of C. gallina. Juveniles were found to be more porous than adults in all horizons. This suggested that C. gallina promoted an accelerated shell accretion with a higher porosity and lower density at the expense of mechanically fragile shells. A positive correlation between sea surface temperature and both micro-density and bulk density were found, with modern specimens being less dense, likely due to lower aragonite saturation state at lower temperature, which could ultimately increase the energetic costs of shell formation. Since no variation was observed in shell CaCO3 polymorphism (100% aragonite) or in compositional parameters among the analyzed horizons, the observed dynamics in skeletal parameters are likely not driven by a diagenetic recrystallization of the shell mineral phase. This study contributes to understand the response of C. gallina to climate-driven environmental shifts and offers insights for assessing anthropogenic impacts on this economic relevant species.

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.

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.

Evaluating bivalve cytoprotective responses and their regulatory pathways in a climate change scenario.

Temperature is a relevant abiotic factor affecting physiological performance and distribution of marine animals in natural environments. The changes in global seawater temperatures make it necessary to understand how molecular mechanisms operate under the cumulative effects of global climate change and chemical pollution to promote/hamper environmental acclimatization. Marine mussels are excellent model organisms to infer the impacts of those anthropogenic threats on coastal ecosystems. In this study, Mediterranean mussels (Mytilus galloprovincialis) were exposed to different concentrations of the metal copper (Cu as CuCl2: 2.5, 5, 10, 20, 40 µg/L) or the antibiotic oxytetracycline (OTC: 0.1, 1, 10, 100, 1000 µg/L) at increasing seawater temperatures (16 °C, 20 °C, 24 °C). Transcriptional modulation of a 70-kDa heat shock protein (HSP70) and of the ABC transporter P-glycoprotein (P-gp, encoded by the ABCB gene) was assessed along with the cAMP/PKA signaling pathway regulating both gene expressions. At the physiological temperature of mussels (16 °C), Cu and OTC induced bimodal changes of cAMP levels and PKA activities in gills of exposed animals. A correlation between OTC- or Cu- induced changes of PKA activity and expression of hsp70 and ABCB was observed. Temperature increases (up to 24 °C) altered ABCB and hsp70 responses to the pollutants and disrupted their relationship with cAMP/PKA modulation, leading to loss of correlation between the biological endpoints. On the whole, the results indicate that temperature may impair the effects of inorganic and organic chemicals on the cAMP/PKA signaling pathway of mussels, in turn altering key molecular mediators of physiological plasticity and cytoprotection.

Microplastics impair the feeding performance of a Mediterranean habitat-forming coral.

The impact of plastic debris, and in particular of microplastics (here referred as particles smaller than 5 mm) on aquatic environments has now become a topic of raising concern. Microplastics are particularly abundant in the Mediterranean Sea, potentially exerting substantial pressures on marine organisms at different levels of organization. Ingestion of microplastics has been observed in a large number of marine species. The aim of this work is to test if microplastics produce a feeding impairment in Astroides calycularis, a shallow water, habitat-forming coral endemic to the Mediterranean Sea. Our findings suggest a lack of any avoidance mechanism allowing the polyps to discern between food items and microplastics when occurring simultaneously. Moreover, polyps spend a considerable amount of time on handling microplastic particles. As a consequence, microplastics impair the feeding efficiency in A. calycularis, since polyps may not be fully able to profit from the drifting plankton aggregations. Therefore, we suggest that microplastics can cause a reduction of fitness in A. calycularis, and presumably also in other species characterized by suspension feeding strategy.

Anthropogenic impact is negatively related to coral health in Sicily (Mediterranean Sea).

Shallow-water marine organisms are among the first to suffer from combined effects of natural and anthropogenic drivers. The orange coral Astroides calycularis is a shallow-water bioconstructor species endemic to the Mediterranean Sea. Although raising conservation interest, also given its special position within the Dendrophylliidae, information about the threats to its health is scant. We investigated the health status of A. calycularis at five locations in northwestern Sicily along a gradient of cumulative human impact and the most probable origin of the threats to this species, including anthropogenic land-based and sea-based threats. Cumulative human impact appeared inversely related to the performance of A. calycularis at population, colony, and polyp levels. Sea-based human impacts appeared among the most likely causes of the variation observed. The reduction in polyp length can limit the reproductive performance of A. calycularis, while the decrease of percent cover and colony area is expected to impair its peculiar feeding behaviour by limiting the exploitable dimensional range of prey and, ultimately, reef functioning. This endangered habitat-forming species appeared susceptible to anthropogenic pressures, suggesting the need to re-assess its vulnerability status. Creating microprotected areas with specific restrictions to sea-based human impacts could be the best practice preserve these bioconstructions.

Environmental influence on calcification of the bivalve Chamelea gallina along a latitudinal gradient in the Adriatic Sea.

Environmental factors are encoded in shells of marine bivalves in the form of geochemical properties, shell microstructure and shell growth rate. Few studies have investigated how shell growth is affected by habitat conditions in natural populations of the commercial clam Chamelea gallina. Here, skeletal parameters (micro-density and apparent porosity) and growth parameters (bulk density, linear extension and net calcification rates) were investigated in relation to shell sizes and environmental parameters along a latitudinal gradient in the Adriatic Sea (400 km). Net calcification rates increased with increasing solar radiation, sea surface temperature and salinity and decreasing Chlorophyll concentration in immature and mature shells. In immature shells, which are generally more porous than mature shells, enhanced calcification was due to an increase in bulk density, while in mature shells was due to an increase in linear extension rates. The presence of the Po river in the Northern Adriatic Sea was likely the main driver of the fluctuations observed in environmental parameters, especially salinity and Chlorophyll concentration, and seemed to negatively affect the growth of C. gallina.

Mediterranean Bioconstructions Along the Italian Coast.

Marine bioconstructions are biodiversity-rich, three-dimensional biogenic structures, regulating key ecological functions of benthic ecosystems worldwide. Tropical coral reefs are outstanding for their beauty, diversity and complexity, but analogous types of bioconstructions are also present in temperate seas. The main bioconstructions in the Mediterranean Sea are represented by coralligenous formations, vermetid reefs, deep-sea cold-water corals, Lithophyllum byssoides trottoirs, coral banks formed by the shallow-water corals Cladocora caespitosa or Astroides calycularis, and sabellariid or serpulid worm reefs. Bioconstructions change the morphological and chemicophysical features of primary substrates and create new habitats for a large variety of organisms, playing pivotal roles in ecosystem functioning. In spite of their importance, Mediterranean bioconstructions have not received the same attention that tropical coral reefs have, and the knowledge of their biology, ecology and distribution is still fragmentary. All existing data about the spatial distribution of Italian bioconstructions have been collected, together with information about their growth patterns, dynamics and connectivity. The degradation of these habitats as a consequence of anthropogenic pressures (pollution, organic enrichment, fishery, coastal development, direct physical disturbance), climate change and the spread of invasive species was also investigated. The study of bioconstructions requires a holistic approach leading to a better understanding of their ecology and the application of more insightful management and conservation measures at basin scale, within ecologically coherent units based on connectivity: the cells of ecosystem functioning.

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.