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.

Exogenous Abscisic Acid Confers Salinity Tolerance in Chlamydomonas reinhardtii During Its Life Cycle.

The plant hormone abscisic acid (ABA) coordinates responses to environmental signals with developmental changes and is important for stress resilience and crop yield. However, fundamental questions remain about how this phytohormone affects microalgal growth and stress regulation throughout the different stages of their life cycle. In this study, the effects of ABA on the physiology of the freshwater microalga Chlamydomonas reinhardtii at its different life cycle stages were investigated. Exogenously added ABA enhanced the growth and photosynthesis of C. reinhardtii during the vegetative stage. The hormone also increased the tolerance of this alga to high-salinity stress during gamete formation under nutrient depletion, as well as it extended their survival. We show that the level of reactive oxygen species (ROS) generated in the ABA-treated cells was significantly less than that in the untreated cells under inhibiting NaCl concentrations. Cell size examination showed that ABA prevents cells from forming palmella when exposed to high salinity. All together, these results suggest that ABA can support the vitality and survival of C. reinhardtii under high salt conditions.

Acclimation of thermotolerant algae to light and temperature interaction1.

Here, we explore the responses of photosynthesis and related cellular processes in the thermotolerant microalga Micractinium sp. acclimated to limiting and saturating irradiances combined with elevated temperatures, using a novel computer-controlled multi-sensor system. This system allows for the monitoring of online values of oxygen exchange during photosynthesis and respiration with high accuracy. Micractinium sp. cells showed maximum growth and net oxygen production rates under the optimal temperature of 25°C regardless of the light acclimation conditions. Our results show that the upper thermal threshold for Micractinium sp. photosynthesis and growth ranges between 35°C and 40°C. This microalga exhibited stable photosynthetic efficiency and effective non-photochemical quenching (NPQ) under saturating light, and was more susceptible to temperature change when acclimated to limiting light levels. These results demonstrate that the acclimation of thermotolerant microalgae to saturating light helps to enhance the thermal tolerance of PSII. This feature results from enhanced heat stability of PSII photochemistry and oxygen evolution.

In Vitro Coral Biomineralization under Relevant Aragonite Supersaturation Conditions.

The biomineralization of corals occurs under conditions of high and low supersaturation with respect to aragonite, which corresponds to day- or night-time periods of their growth, respectively. Here, in vitro precipitation of aragonite in artificial seawater was investigated at a high supersaturation, allowing spontaneous nucleation and growth, as well as at low supersaturation conditions, which allowed only the crystal growth on the deliberately introduced aragonite seeds. In either chemical systems, soluble organic matrix (SOM) extracted from Balanophyllia europaea (light sensitive) or Leptopsammia pruvoti (light insensitive) was added. The analyses of the kinetic and thermodynamic data of aragonite precipitation and microscopic observations showed that, at high supersaturation, the SOMs increased the induction time, did not affect the growth rate and were incorporated within aggregates of nanoparticles. At low supersaturation, the SOMs affected the aggregation of overgrowing crystalline units and did not substantially change the growth rate. On the basis of the obtained results we can infer that at high supersaturation conditions the formation of nanoparticles, which is typically observed in the skeleton's early mineralization zone may occur, whereas at low supersaturation the overgrowth on prismatic seeds observed in the skeleton fiber zone is a predominant process. In conclusion, this research brings insight on coral skeletogenesis bridging physicochemical (supersaturation) and biological (role of SOM) models of coral biomineralization and provides a source of inspiration for the precipitation of composite materials under different conditions of supersaturation.

Gene expression profiles during short-term heat stress in the red sea coral Stylophora pistillata.

During the past several decades, corals worldwide have been affected by severe bleaching events leading to wide-spread coral mortality triggered by global warming. The symbiotic Red Sea coral Stylophora pistillata from the Gulf of Eilat is considered an opportunistic 'r' strategist. It can thrive in relatively unstable environments and is considered a stress-tolerant species. Here, we used a S. pistillata custom microarray to examine gene expression patterns and cellular pathways during short-term (13-day) heat stress. The results allowed us to identify a two-step reaction to heat stress, which intensified significantly as the temperature was raised to a 32 °C threshold, beyond which, coping strategies failed at 34 °C. We identified potential 'early warning genes' and 'severe heat-related genes'. Our findings suggest that during short-term heat stress, S. pistillata may divert cellular energy into mechanisms such as the ER-unfolded protein response (UPR) and ER-associated degradation (ERAD) at the expense of growth and biomineralization processes in an effort to survive and subsequently recover from the stress. We suggest a mechanistic theory for the heat stress responses that may explain the success of some species which can thrive under a wider range of temperatures relative to others.

Analytical pyrolysis-based study on intra-skeletal organic matrices from Mediterranean corals.

Off-line analytical pyrolysis combined with gas chromatography­mass spectroscopy (GC­MS), directly or after trimethylsilylation, along with infrared spectroscopy and amino acid analysis was applied for the first time to the characterization of the intra-skeletal organic matrix (OM) extracted from four Mediterranean hard corals. They were diverse in growth form and trophic strategy namely Balanophyllia europaea and Leptopsammia pruvoti­solitary corals, only the first having zooxanthelle­and Cladocora caespitosa and Astroides calycularis­colonial corals, only the first with zooxanthelle. Pyrolysis products evolved from OM could be assigned to lipid (e.g. fatty acids, fatty alcohols, monoacylglicerols), protein (e.g. 2,5-diketopiperazines, DKPs) and polysaccharide (e.g. anhydrosugars) precursors. Their quantitative distribution showed for all the species a low protein content with respect to lipids and polysaccharides. A chemometric approach using principal component analysis (PCA) and clustering analysis was applied on OM mean amino acidic compositions. The small compositional diversity across coral species was tentatively related with coral growth form. The presence of N-acetyl glucosamine markers suggested a functional link with other calcified tissues containing chitin. The protein fraction was further investigated using novel DKP markers tentatively identified from analytical pyrolysis of model polar linear dipeptides. Again, no correlation was observed in relation to coral ecology. These analytical results revealed that the bulk structure and composition of OMs among studied corals are similar, as it is the textural organization of the skeleton mineralized units. Therefore, they suggest that coral's biomineralization is governed by similar macromolecules, and probably mechanisms, independently from their ecology.

Control of aragonite deposition in colonial corals by intra-skeletal macromolecules.

Scleractinian coral skeletons are composed mainly of aragonite in which a small percentage of organic matrix (OM) molecules is entrapped. It is well known that in corals the mineral deposition occurs in a biological confined nucleation site, but it is still unclear to what extent the calcification is controlled by OM molecules. Hence, the shape, size and organization of skeletal crystals from the fiber level through the colony architecture, were also attributed to factors as diverse as nucleation site mineral supersaturation and environmental factors in the habitat. In this work the OMs were extracted from the skeleton of three colonial corals, Acropora digitifera, Lophelia pertusa and Montipora caliculata. A. digitifera has a higher calcification rate than the other two species. OM molecules were characterized and their CaCO3 mineralization activity was evaluated by experiments of overgrowth on coral skeletons and of precipitation from solutions containing OM soluble and insoluble fractions and magnesium ions. The precipitates were characterized by spectroscopic and microscopic techniques. The results showed that the OM molecules of the three coral share similar features, but differ from those associated with mollusk shells. However, A. digitifera OM shows peculiarities from those from L. pertusa and M. caliculata. The CaCO3 overgrowth and precipitation experiments confirm the singularity of A. digitifera OM molecules as mineralizers. Moreover, their comparison indicates that only specific molecules are involved in the polymorphism control and suggests that when the whole extracted materials are used the OM's main effect is on the control of particles' shape and morphology.

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.

A Poly-D-lysine-Coated Coralline Matrix Promotes Hippocampal Neural Precursor Cells' Differentiation into GFAP-Positive Astrocytes.

A major goal of regenerative medicine of the central nervous system is to accelerate the regeneration of nerve tissue, where astrocytes, despite their positive and negative roles, play a critical role. Thus, scaffolds capable of producing astrocytes from neural precursor cells (NPCs) are most desirable. Our study shows that NPCs are converted into reactive astrocytes upon cultivation on coralline-derived calcium carbonate coated with poly-D-lysine (PDL-CS). As shown via nuclei staining, the adhesion of neurospheres containing hundreds of hippocampal neural cells to PDL-CS resulted in disaggregation of the cell cluster as well as the radial migration of dozens of cells away from the neurosphere core. Migrating cells per neurosphere averaged 100 on PDL-CS, significantly higher than on uncoated CS (28), PDL-coated glass (65), or uncoated glass (20). After 3 days of culture on PDL-CS, cell migration plateaued and remained stable for four more days. In addition, NPCs expressing nestin underwent continuous morphological changes from round to spiky, extending and elongating their processes, resembling activated astrocytes. The extension of the process increased continuously during the maturation of the culture and doubled after 7 days compared to day 1, whereas bifurcation increased by twofold during the first 3 days before plateauing. In addition, nestin positive cells' shape, measured through the opposite circularity level correlation, decreased approximately twofold after three days, indicating spiky transformation. Moreover, nestin-positive cells co-expressing GFAP increased by 2.2 from day 1 to 7, reaching 40% of the NPC population on day 7. In this way, PDL-CS promotes NPC differentiation into reactive astrocytes, which could accelerate the repair of neural tissue.

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.

Inferred calcification rate of a Mediterranean azooxanthellate coral is uncoupled with sea surface temperature along an 8° latitudinal gradient.

INTRODUCTION: Correlations between sea surface temperature (SST) and growth parameters of the solitary azooxanthellate Dendrophylliid Leptopsammia pruvoti were assessed along an 8° latitudinal gradient on western Italian coasts (Mediterranean Sea), to check for possible negative effects of increasing temperature as the ones reported for a closely related, sympatric but zooxanthellate species. RESULTS: Calcification rate was correlated with skeletal density but not with linear extension rate, indicating that calcium carbonate deposition was preferentially allocated to keep a constant skeletal density. Unlike most studies on both temperate and tropical zooxanthellate corals, where calcification rate is strongly related to environmental parameters such as SST, in the present study calcification rate was not correlated with SST. CONCLUSIONS: The lower sensitivity of L. pruvoti to SST with respect to other sympatric zooxanthellate corals, such as Balanophyllia europaea, may rely on the absence of a temperature induced inhibition of photosynthesis, and thus the absence of an inhibition of the calcification process. This study is the first field investigation of the relationship between SST and the three growth parameters of an azooxanthellate coral. Increasing research effort on determining the effects of temperature on biological traits of the poorly studied azooxanthellate scleractinians may help to predict the possible species assemblage shifts that are likely to occur in the immediate future as a consequence of global climatic change.

Photosynthetic energy storage efficiency in Chlamydomonas reinhardtii, based on microsecond photoacoustics.

Using a novel, pulsed micro-second time-resolved photoacoustic (PA) instrument, we measured thermal dissipation and energy storage (ES) in the intact cells of wild type (WT) Chlamydomonas reinhardtii, and mutants lacking either PSI or PSII reaction centers (RCs). On this time scale, the kinetic contributions of the thermal expansion component due to heat dissipation of absorbed energy and the negative volume change due to electrostriction induced by charge separation in each of the photosystems could be readily distinguished. Kinetic analysis revealed that PSI and PSII RCs exhibit strikingly different PA signals where PSI is characterized by a strong electrostriction signal and a weak thermal expansion component while PSII has a small electrostriction component and large thermal expansion. The calculated ES efficiencies at ~10 µs were estimated to be 80 ± 5 and 50 ± 13% for PSII-deficient mutants and PSI-deficient mutants, respectively, and 67 ± 2% for WT. The overall ES efficiency was positively correlated with the ratio of PSI to PSI + PSII. Our results suggest that the shallow excitonic trap in PSII limits the efficiency of ES as a result of an evolutionary frozen metabolic framework of two photosystems in all oxygenic photoautotrophs.

Unconventional high-value products from microalgae: A review.

Microalgae have gained significant importance in biotechnology development, providing valuable goods and services in multiple applications. Although there is a rising market for most of these applications, the incorporation and introduction of microalgae into new venues will extend in the near future. These advances are due to the vast biodiversity of microalgal species, recent genetic engineering tools, and culture techniques. There are three main possible approaches for novel algal compounds from: (1) recently isolated yet less known microalgae; (2) selectively stressed conditions; and (3) enzymatically adjusted compounds from conventional molecules. All these approaches can be combined in a specific manner. This review discusses the opportunities, potential and limitations of introducing novel microalgae-based products, and how the recent technologies can be deployed to make these products financially viable. To give an outlook to the future, an analysis of the developments and predicted future market that further enlarge the promise of cultivating microalgae for commercial purposes are considered.

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.

Deep neural network recognition of shallow water corals in the Gulf of Eilat (Aqaba).

We describe the application of the computerized deep learning methodology to the recognition of corals in a shallow reef in the Gulf of Eilat, Red Sea. This project is aimed at applying deep neural network analysis, based on thousands of underwater images, to the automatic recognition of some common species among the 100 species reported to be found in the Eilat coral reefs. This is a challenging task, since even in the same colony, corals exhibit significant within-species morphological variability, in terms of age, depth, current, light, geographic location, and inter-specific competition. Since deep learning procedures are based on photographic images, the task is further challenged by image quality, distance from the object, angle of view, and light conditions. We produced a large dataset of over 5,000 coral images that were classified into 11 species in the present automated deep learning classification scheme. We demonstrate the efficiency and reliability of the method, as compared to painstaking manual classification. Specifically, we demonstrated that this method is readily adaptable to include additional species, thereby providing an excellent tool for future studies in the region, that would allow for real time monitoring the detrimental effects of global climate change and anthropogenic impacts on the coral reefs of the Gulf of Eilat and elsewhere, and that would help assess the success of various bioremediation efforts.

Aragonite-Polylysine: Neuro-Regenerative Scaffolds with Diverse Effects on Astrogliosis.

Biomaterials, especially when coated with adhesive polymers, are a key tool for restorative medicine, being biocompatible and supportive for cell adherence, growth, and function. Aragonite skeletons of corals are biomaterials that support survival and growth of a range of cell types, including neurons and glia. However, it is not known if this scaffold affects neural cell migration or elongation of neuronal and astrocytic processes, prerequisites for initiating repair of damage in the nervous system. To address this, hippocampal cells were aggregated into neurospheres and cultivated on aragonite skeleton of the coral Trachyphyllia geoffroyi (Coral Skeleton (CS)), on naturally occurring aragonite (Geological Aragonite (GA)), and on glass, all pre-coated with the oligomer poly-D-lysine (PDL). The two aragonite matrices promoted equally strong cell migration (4.8 and 4.3-fold above glass-PDL, respectively) and axonal sprouting (1.96 and 1.95-fold above glass-PDL, respectively). However, CS-PDL had a stronger effect than GA-PDL on the promotion of astrocytic processes elongation (1.7 vs. 1.2-fold above glass-PDL, respectively) and expression of the glial fibrillary acidic protein (3.8 vs. and 1.8-fold above glass-PDL, respectively). These differences are likely to emerge from a reaction of astrocytes to the degree of roughness of the surface of the scaffold, which is higher on CS than on GA. Hence, CS-PDL and GA-PDL are scaffolds of strong capacity to derive neural cell movements and growth required for regeneration, while controlling the extent of astrocytic involvement. As such, implants of PDL-aragonites have significant potential as tools for damage repair and the reduction of scar formation in the brain following trauma or disease.

Gliosis of astrocytes cultivated on coral skeleton is regulated by the matrix surface topography.

Astrogilosis is the response of astrocytes to brain trauma which manifest opposite roles on brain injury repair. On the one hand, astrocytes undergoing astrogliosis inhibit tissue regeneration by forming scar tissue, but, on the other hand, they enhance damage repair through secretion of neuro-protecting and neurotrophic factors. Therefore, identifying means that regulate astrogliosis can provide a control over progression and repair of brain damage. We have previously shown that the calcium carbonate skeleton of corals upregulates two manifestations of astrogliosis in astrocytes in culture-expression of the Glial Fibrillay Acidic Protein (GFAP), and shape conversion from non-spiky to reactive spiky cell morphology. Here, we investigated if the surface topography of the coralline skeleton plays a role in GFAP expression and the morphogenesis of reactive astrocytes. To address that, we utilized the non-porous exoskeleton of the coral Trachyphyllia geoffroyi, having three topographies of distinct heights on its surface: rough surface (made of <30 µm height bumps), protrusions (50-250 µm) and ridges (>250 µm). We observed that astrocytes reacted similarly to all three structures in terms of adhesion, acquisition of a spiky morphology and organization in networks. By contrast, the extent by which these cells expressed GFAP was structure-dependent. The expression was 2-fold higher on protrusions and ridges than on the rough surface and acquired. Accordingly, the distribution pattern of the GFAP overexpressing astrocytes followed that of the protrusions and ridges. Hence, fabricating coralline scaffolds with designed flatness/protrusions/ridges ratios can serve to control astrogliosis-derived regeneration in TBI wounds, and as a result improve the capacity to repair brain damage.

Phylogenetic diversity of bacteria associated with the mucus of Red Sea corals.

Coral reefs are the most biodiverse and biologically productive of all marine ecosystems. Corals harbor diverse and abundant prokaryotic communities. However, little is known about the diversity of coral-associated bacterial communities. Mucus is a characteristic product of all corals, forming a coating over their polyps. The coral mucus is a rich substrate for microorganisms. Mucus was collected with a procedure using sterile cotton swabs that minimized contamination of the coral mucus by surrounding seawater. We used molecular techniques to characterize and compare the bacterial assemblages associated with the mucus of the solitary coral Fungia scutaria and the massive coral Platygyra lamellina from the Gulf of Eilat, northern Red Sea. The bacterial communities of the corals F. scutaria and P. lamellina were found to be diverse, with representatives within the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria and Epsilonproteobacteria, as well as the Actinobacteria, Cytophaga-Flavobacter/Flexibacter-Bacteroides group, Firmicutes, Planctomyces, and several unclassified bacteria. However, the total bacterial assemblage of these two corals was different. In contrast to the bacterial communities of corals analyzed in previous studies by culture-based and culture-independent approaches, we found that the bacterial clone libraries of the coral species included a substantial proportion of Actinobacteria. The current study further supports the finding that bacterial communities of coral mucus are diverse.

Optimizing photon dose and frequency to enhance lipid productivity of thermophilic algae for biofuel production.

The aim of this work was to examine the potential of the thermophilic green microalga Micractinium sp. to accumulate triacylglycerols (TAGs) and to develop a light strategy to increase TAG productivity in this alga. To this end, dense cultures of Micractinium sp. were grown at 37 °C under nitrogen (N) starvation and exposed to a light intensity of 1000 µmol photons m-2 s-1 of different light regimes. The highest per-biomass TAG-content and maximal volumetric productivities of TAG were displayed by the cultures grown under flashing light of 5 Hz with 50% duty cycle. Based on the results, a sufficiently high-starting culture density should be combined with a high irradiance delivered by an appropriate light regime to enhance the production of biomass enriched TAGs.

Structure and Function of Stony Coral Intraskeletal Polysaccharides.

Polysaccharides represent a main weight fraction of the intraskeletal organic matrix of corals, but their structure, as well as their function in the calcification process, has been poorly investigated. This communication shows by a combination of techniques (nuclear magnetic resonance, Fourier transform infrared, and monosaccharide composition) that their key component is a 1→3 ß-d glucuronic acid polymer and evidences its influence in vitro in the calcification process.