Vibrio coralliilyticus infection triggers a behavioural response and perturbs nutritional exchange and tissue integrity in a symbiotic coral.

Under homoeostatic conditions, the relationship between the coral Pocillopora damicornis and Vibrio coralliilyticus is commensal. An increase in temperature, or in the abundance of V. coralliilyticus, can turn this association pathogenic, causing tissue lysis, expulsion of the corals' symbiotic algae (genus Symbiodinium), and eventually coral death. Using a combination of microfluidics, fluorescence microscopy, stable isotopes, electron microscopy and NanoSIMS isotopic imaging, we provide insights into the onset and progression of V. coralliilyticus infection in the daytime and at night, at the tissue and (sub-)cellular level. The objective of our study was to connect the macro-scale behavioural response of the coral to the micro-scale nutritional interactions that occur between the host and its symbiont. In the daytime, polyps enhanced their mucus production, and actively spewed pathogens. Vibrio infection primarily resulted in the formation of tissue lesions in the coenosarc. NanoSIMS analysis revealed infection reduced 13C-assimilation in Symbiodinium, but increased 13C-assimilation in the host. In the night incubations, no mucus spewing was observed, and a mucus film was formed on the coral surface. Vibrio inoculation and infection at night showed reduced 13C-turnover in Symbiodinium, but did not impact host 13C-turnover. Our results show that both the nutritional interactions that occur between the two symbiotic partners and the behavioural response of the host organism play key roles in determining the progression and severity of host-pathogen interactions. More generally, our approach provides a new means of studying interactions (ranging from behavioural to metabolic scales) between partners involved in complex holobiont systems, under both homoeostatic and pathogenic conditions.

Using NanoSIMS coupled with microfluidics to visualize the early stages of coral infection by Vibrio coralliilyticus.

BACKGROUND: Global warming has triggered an increase in the prevalence and severity of coral disease, yet little is known about coral/pathogen interactions in the early stages of infection. The point of entry of the pathogen and the route that they take once inside the polyp is currently unknown, as is the coral's capacity to respond to infection. To address these questions, we developed a novel method that combines stable isotope labelling and microfluidics with transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS), to monitor the infection process between Pocillopora damicornis and Vibrio coralliilyticus under elevated temperature. RESULTS: Three coral fragments were inoculated with 15N-labeled V. coralliilyticus and then fixed at 2.5, 6 and 22 h post-inoculation (hpi) according to the virulence of the infection. Correlative TEM/NanoSIMS imaging was subsequently used to visualize the penetration and dispersal of V. coralliilyticus and their degradation or secretion products. Most of the V. coralliilyticus cells we observed were located in the oral epidermis of the fragment that experienced the most virulent infection (2.5 hpi). In some cases, these bacteria were enclosed within electron dense host-derived intracellular vesicles. 15N-enriched pathogen-derived breakdown products were visible in all tissue layers of the coral polyp (oral epidermis, oral gastrodermis, aboral gastrodermis), at all time points, although the relative 15N-enrichment depended on the time at which the corals were fixed. Tissues in the mesentery filaments had the highest density of 15N-enriched hotspots, suggesting these tissues act as a "collection and digestion" site for pathogenic bacteria. Closer examination of the sub-cellular structures associated with these 15N-hotspots revealed these to be host phagosomal and secretory cells/vesicles. CONCLUSIONS: This study provides a novel method for tracking bacterial infection dynamics at the levels of the tissue and single cell and takes the first steps towards understanding the complexities of infection at the microscale, which is a crucial step towards understanding how corals will fare under global warming.

Substrate and electron donor limitation induce phenotypic heterogeneity in different metabolic activities in a green sulphur bacterium.

Populations of genetically identical cells can display marked variation in phenotypic traits; such variation is termed phenotypic heterogeneity. Here, we investigate the effect of substrate and electron donor limitation on phenotypic heterogeneity in N2 and CO2 fixation in the green sulphur bacterium Chlorobium phaeobacteroides. We grew populations in chemostats and batch cultures and used stable isotope labelling combined with nanometer-scale secondary ion mass spectrometry (NanoSIMS) to quantify phenotypic heterogeneity. Experiments in H2 S (i.e. electron donor) limited chemostats show that varying levels of NH4+ limitation induce heterogeneity in N2 fixation. Comparison of phenotypic heterogeneity between chemostats and batch (unlimited for H2 S) populations indicates that electron donor limitation drives heterogeneity in N2 and CO2 fixation. Our results demonstrate that phenotypic heterogeneity in a certain metabolic activity can be driven by different modes of limitation and that heterogeneity can emerge in different metabolic processes upon the same mode of limitation. In conclusion, our data suggest that limitation is a general driver of phenotypic heterogeneity in microbial populations.

Burial-induced oxygen-isotope re-equilibration of fossil foraminifera explains ocean paleotemperature paradoxes.

Oxygen-isotope compositions of fossilised planktonic and benthic foraminifera tests are used as proxies for surface- and deep-ocean paleotemperatures, providing a continuous benthic record for the past 115 Ma. However, visually imperceptible processes can alter these proxies during sediment burial. Here, we investigate the diffusion-controlled re-equilibration process with experiments exposing foraminifera tests to elevated pressures and temperatures in isotopically heavy artificial seawater (H218O), followed by scanning electron microscopy and quantitative NanoSIMS imaging: oxygen-isotope compositions changed heterogeneously at submicrometer length scales without any observable modifications of the test ultrastructures. In parallel, numerical modelling of diffusion during burial shows that oxygen-isotope re-equilibration of fossil foraminifera tests can cause significant overestimations of ocean paleotemperatures on a time scale of 107 years under natural conditions. Our results suggest that the late Cretaceous and Paleogene deep-ocean and high-latitude surface-ocean temperatures were significantly lower than is generally accepted, thereby explaining the paradox of the low equator-to-pole surface-ocean thermal gradient inferred for these periods.

MALDI-MS and NanoSIMS imaging techniques to study cnidarian-dinoflagellate symbioses.

Cnidarian-dinoflagellate photosynthetic symbioses are fundamental to biologically diverse and productive coral reef ecosystems. The hallmark of this symbiotic relationship is the ability of dinoflagellate symbionts to supply their cnidarian host with a wide range of nutrients. Many aspects of this association nevertheless remain poorly characterized, including the exact identity of the transferred metabolic compounds, the mechanisms that control their exchange across the host-symbiont interface, and the precise subcellular fate of the translocated materials in cnidarian tissues. This lack of knowledge is mainly attributed to difficulties in investigating such metabolic interactions both in situ, i.e. on intact symbiotic associations, and at high spatial resolution. To address these issues, we illustrate the application of two in situ and high spatial resolution molecular and ion imaging techniques-matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and the nano-scale secondary-ion mass spectrometry (NanoSIMS) ion microprobe. These imaging techniques provide important new opportunities for the detailed investigation of many aspects of cnidarian-dinoflagellate associations, including the dynamics of cellular interactions.

Simultaneous extension of both basic microstructural components in scleractinian coral skeleton during night and daytime, visualized by in situ 86Sr pulse labeling.

Using in situ (12 h) pulse-labeling of scleractinian coral aragonitic skeleton with stable 86Sr isotope, the diel pattern of skeletal extension was investigated in the massive Porites lobata species, grown at 5 m depth in the Gulf of Eilat. Several microstructural aspects of coral biomineralization were elucidated, among which the most significant is simultaneous extension of the two basic microstructural components Rapid Accretion Deposits (RAD; also called Centers of Calcification) and Thickening Deposits (TD; also called fibers), both at night and during daytime. Increased thickness of the 86Sr-labeled growth-front in the RADs compared to the adjacent TDs revealed that in this species RADs extend on average twice as fast as TDs. At the level of the individual corallite, skeletal extension is spatially highly heterogeneous, with sporadic slowing or cessation depending on growth directions and skeletal structure morphology. Daytime photosynthesis by symbiotic dinoflagellates is widely acknowledged to substantially increase calcification rates at the colony and the corallite level in reef-building corals. However, in our study, the average night-time extension rate (visualized in three successive 12 h pulses) was similar to the average daytime extension (visualized in the initial 12 h pulse), in all growth directions and skeletal structures. This research provides a platform for further investigations into the temporal calibration of coral skeletal extension via cyclic growth increment deposition, which is a hallmark of coral biomineralization.

Scleractinian coral cell proliferation is reduced in primary culture of suspended multicellular aggregates compared to polyps.

Cell cultures from reef-building scleractinian corals are being developed to study the response of these ecologically important organisms to environmental stress and diseases. Despite the importance of cell division to support propagation, cell proliferation in polyps and in vitro is under-investigated. In this study, suspended multicellular aggregates (tissue balls) were obtained after collagenase dissociation of Pocillopora damicornis coral, with varying yields between enzyme types and brands. Ultrastructure and cell type distribution were characterized in the tissue balls (TBs) compared to the polyp. Morphological evidence of cellular metabolic activity in their ciliated cortex and autophagy in their central mass suggests involvement of active tissue reorganization processes. DNA synthesis was evaluated in the forming multicellular aggregates and in the four cell layers of the polyp, using BrdU labeling of nuclei over a 24 h period. The distribution of BrdU-labeled coral cells was spatially heterogeneous and their proportion was very low in tissue balls (0.2 ± 0.1 %), indicating that suspended multicellular aggregate formation does not involve significant cell division. In polyps, DNA synthesis was significantly lower in the calicoderm (<1 %) compared to both oral and aboral gastroderm (about 10 %) and to the pseudostratified oral epithelium (15-25 % at tip of tentacle). DNA synthesis in the endosymbiotic dinoflagellates dropped in the forming tissue balls (2.7 ± 1.2 %) compared to the polyp (14 ± 3.4 %) where it was not different from the host gastroderm (10.3 ± 1.2 %). A transient (24 h) increase was observed in the cell-specific density of dinoflagellates in individually dissociated coral cell cultures. These results suggest disruption of coral cell proliferation processes upon establishment in primary culture.

Highly dynamic cellular-level response of symbiotic coral to a sudden increase in environmental nitrogen.

UNLABELLED: Metabolic interactions with endosymbiotic photosynthetic dinoflagellate Symbiodinium spp. are fundamental to reef-building corals (Scleractinia) thriving in nutrient-poor tropical seas. Yet, detailed understanding at the single-cell level of nutrient assimilation, translocation, and utilization within this fundamental symbiosis is lacking. Using pulse-chase (15)N labeling and quantitative ion microprobe isotopic imaging (NanoSIMS; nanoscale secondary-ion mass spectrometry), we visualized these dynamic processes in tissues of the symbiotic coral Pocillopora damicornis at the subcellular level. Assimilation of ammonium, nitrate, and aspartic acid resulted in rapid incorporation of nitrogen into uric acid crystals (after ~45 min), forming temporary N storage sites within the dinoflagellate endosymbionts. Subsequent intracellular remobilization of this metabolite was accompanied by translocation of nitrogenous compounds to the coral host, starting at ~6 h. Within the coral tissue, nitrogen is utilized in specific cellular compartments in all four epithelia, including mucus chambers, Golgi bodies, and vesicles in calicoblastic cells. Our study shows how nitrogen-limited symbiotic corals take advantage of sudden changes in nitrogen availability; this opens new perspectives for functional studies of nutrient storage and remobilization in microbial symbioses in changing reef environments. IMPORTANCE: The methodology applied, combining transmission electron microscopy with nanoscale secondary-ion mass spectrometry (NanoSIMS) imaging of coral tissue labeled with stable isotope tracers, allows quantification and submicrometric localization of metabolic fluxes in an intact symbiosis. This study opens the way for investigations of physiological adaptations of symbiotic systems to nutrient availability and for increasing knowledge of global nitrogen and carbon biogeochemical cycling.

Micro- to nanostructure and geochemistry of extant crinoidal echinoderm skeletons.

This paper reports the results of micro- to nanostructural and geochemical analyses of calcitic skeletons from extant deep-sea stalked crinoids. Fine-scale (SEM, FESEM, AFM) observations show that the crinoid skeleton is composed of carbonate nanograins, about 20-100 nm in diameter, which are partly separated by what appears to be a few nm thick organic layers. Sub-micrometre-scale geochemical mapping of crinoid ossicles using a NanoSIMS ion microprobe, combined with synchrotron high-spatial-resolution X-ray micro-fluorescence (µ-XRF) maps and X-ray absorption near-edge structure spectroscopy (XANES) show that high Mg concentration in the central region of the stereom bars correlates with the distribution of S-sulphate, which is often associated with sulphated polysaccharides in biocarbonates. These data are consistent with biomineralization models suggesting a close association between organic components (including sulphated polysaccharides) and Mg ions. Additionally, geochemical analyses (NanoSIMS, energy dispersive spectroscopy) reveal that significant variations in Mg occur at many levels: within a single stereom trabecula, within a single ossicle and within a skeleton of a single animal. Together, these data suggest that physiological factors play an important role in controlling Mg content in crinoid skeletons and that great care should be taken when using their skeletons to reconstruct, for example, palaeotemperatures and Mg/Ca palaeo-variations of the ocean.

Extreme deuterium excesses in ultracarbonaceous micrometeorites from central Antarctic snow.

Primitive interplanetary dust is expected to contain the earliest solar system components, including minerals and organic matter. We have recovered, from central Antarctic snow, ultracarbonaceous micrometeorites whose organic matter contains extreme deuterium (D) excesses (10 to 30 times terrestrial values), extending over hundreds of square micrometers. We identified crystalline minerals embedded in the micrometeorite organic matter, which suggests that this organic matter reservoir could have formed within the solar system itself rather than having direct interstellar heritage. The high D/H ratios, the high organic matter content, and the associated minerals favor an origin from the cold regions of the protoplanetary disk. The masses of the particles range from a few tenths of a microgram to a few micrograms, exceeding by more than an order of magnitude those of the dust fragments from comet 81P/Wild 2 returned by the Stardust mission.

A layered structure in the organic envelopes of the prismatic layer of the shell of the pearl oyster Pinctada margaritifera (Mollusca, Bivalvia).

The organic interprismatic layers of the mollusc Pinctada margaritifera are studied using a variety of highly spatially-resolved techniques to establish their composition and structure. Our results show that both the interlamellar sheets of the nacre and interprismatic envelopes form layered structures. Additionally, these organic layers are neither homogeneous in composition, nor continuous in their structure. Both structures play a major role in the biomineralization process and act as a boundary between mineral units.

H2-rich fluids from serpentinization: geochemical and biotic implications.

Metamorphic hydration and oxidation of ultramafic rocks produces serpentinites, composed of serpentine group minerals and varying amounts of brucite, magnetite, and/or FeNi alloys. These minerals buffer metamorphic fluids to extremely reducing conditions that are capable of producing hydrogen gas. Awaruite, FeNi3, forms early in this process when the serpentinite minerals are Fe-rich. Olivine with the current mantle Fe/Mg ratio was oxidized during serpentinization after the Moon-forming impact. This process formed some of the ferric iron in the Earth's mantle. For the rest of Earth's history, serpentinites covered only a small fraction of the Earth's surface but were an important prebiotic and biotic environment. Extant methanogens react H2 with CO2 to form methane. This is a likely habitable environment on large silicate planets. The catalytic properties of FeNi3 allow complex organic compounds to form within serpentinite and, when mixed with atmospherically produced complex organic matter and waters that circulated through basalts, constitutes an attractive prebiotic substrate. Conversely, inorganic catalysis of methane by FeNi3 competes with nascent and extant life.

A new astrophysical setting for chondrule formation.

Chondrules in the metal-rich meteorites Hammadah al Hamra 237 and QUE 94411 have recorded highly energetic thermal events that resulted in complete vaporization of a dusty region of the solar nebula (dust/gas ratio of about 10 to 50 times solar). These chondrules formed under oxidizing conditions before condensation of iron-nickel metal, at temperatures greater than or equal to 1500 K, and were isolated from the cooling gas before condensation of moderately volatile elements such as manganese, sodium, potassium, and sulfur. This astrophysical environment is fundamentally different from conventional models for chondrule formation by localized, brief, repetitive heating events that resulted in incomplete melting of solid precursors initially residing at ambient temperatures below approximately 650 K.

Large-scale thermal events in the solar nebula: evidence from Fe,Ni metal grains in primitive meteorites

Chemical zoning patterns in some iron, nickel metal grains from CH carbonaceous chondrites imply formation at temperatures from 1370 to 1270 kelvin by condensation from a solar nebular gas cooling at a rate of approximately 0.2 kelvin per hour. This cooling rate requires a large-scale thermal event in the nebula, in contrast to the localized, transient heating events inferred for chondrule formation. In our model, mass accretion through the protoplanetary disk caused large-scale evaporation of precursor dust near its midplane inside of a few astronomical units. Gas convectively moved from the midplane to cooler regions above it, and the metal grains condensed in these parcels of rising gas.