Subcellular investigation of photosynthesis-driven carbon assimilation in the symbiotic reef coral Pocillopora damicornis.

Reef-building corals form essential, mutualistic endosymbiotic associations with photosynthetic Symbiodinium dinoflagellates, providing their animal host partner with photosynthetically derived nutrients that allow the coral to thrive in oligotrophic waters. However, little is known about the dynamics of these nutritional interactions at the (sub)cellular level. Here, we visualize with submicrometer spatial resolution the carbon and nitrogen fluxes in the intact coral-dinoflagellate association from the reef coral Pocillopora damicornis by combining nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy with pulse-chase isotopic labeling using [(13)C]bicarbonate and [(15)N]nitrate. This allows us to observe that (i) through light-driven photosynthesis, dinoflagellates rapidly assimilate inorganic bicarbonate and nitrate, temporarily storing carbon within lipid droplets and starch granules for remobilization in nighttime, along with carbon and nitrogen incorporation into other subcellular compartments for dinoflagellate growth and maintenance, (ii) carbon-containing photosynthates are translocated to all four coral tissue layers, where they accumulate after only 15 min in coral lipid droplets from the oral gastroderm and within 6 h in glycogen granules from the oral epiderm, and (iii) the translocation of nitrogen-containing photosynthates is delayed by 3 h. IMPORTANCE: Our results provide detailed in situ subcellular visualization of the fate of photosynthesis-derived carbon and nitrogen in the coral-dinoflagellate endosymbiosis. We directly demonstrate that lipid droplets and glycogen granules in the coral tissue are sinks for translocated carbon photosynthates by dinoflagellates and confirm their key role in the trophic interactions within the coral-dinoflagellate association

Pulsed 86Sr-labeling and NanoSIMS imaging to study coral biomineralization at ultra-structural length scales

A method to label marine biocarbonates is developed based on a concentration enrichment of a minor stable isotope of a trace element that is a natural component of seawater, resulting in the formation of biocarbonate with corresponding isotopic enrichments. This biocarbonate is subsequently imaged with a NanoSIMS ion microprobe to visualize the locations of the isotopic marker on sub-micrometric length scales, permitting resolution of all ultra-structural details. In this study, a scleractinian coral, Pocillopora damicornis, was labeled 3 times with 86Sr-enhanced seawater for a period of 48h with 5days under normal seawater conditions separating each labeling event. Two non-specific cellular stress biomarkers, glutathione-S-transferase activity and porphyrin concentration plus carbonic anhydrase, an enzymatic marker involved in the physiology of carbonate biomineralization, as well as unchanged levels of zooxanthellae photosynthesis efficiency indicate that coral physiological processes are not affected by the 86Sr-enhancement. NanoSIMS images of the 86Sr/44Ca ratio in skeleton formed during the experiment allow for a determination of the average extension rate of the two major ultra-structural components of the coral skeleton: Rapid Accretion Deposits are found to form on average about 4.5 times faster than Thickening Deposits. The method opens up new horizons in the study of biocarbonate formation because it holds the potential to observe growth of calcareous structures such as skeletons, shells, tests, spines formed by a wide range of organisms under essentially unperturbed physiological condition

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

Metabolic interactions with endosymbiotic photosynthetic dinoflagellate Symbiodinium spp. are fundamental to reefbuilding 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 15N 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. © 2013 Kopp et al

Coral photosymbiosis: Linking phylogenetic identity to single cell metabolic activity in mixed symbiont populations

Tropical reef-building corals live in symbiosis with a wide range of micro-organisms, including unicellular Symbiodinium dinoflagellates also called zooxanthellae. These photosynthetic endosymbionts live inside the coral gastroderm cells. Although corals can feed on planktonic preys, the dinoflagellates significantly contribute to the nutrition of their host by transferring a large fraction (up to 90%) of photosynthates that are produced through the fixation of dissolved inorganic carbon (DIC) and nitrogen (nitrate or ammonium). Nine major clades (A-I) of Symbiodinium have been identified by molecular genetic analyses. Each clade presents distinct physiological features and the specific association between coral species and Symbiodinium clade determines the phenotype of the holobiont. Differences in the photosynthetic response to irradiance, rates of carbon fixation, and thermal tolerance can be attributed to symbiont clade. Several Symbiodinium clades can simultaneously exist within a single coral and the host can dynamically modify the proportion between dominant and background clades to adapt to changing environmental conditions. Investigating at the cellular level the metabolic exchanges between coral and Symbiodinium is of great interest to understand e.g. the bleaching process (loss of zooxanthellae) which often leads to coral death. We are aiming at quantitatively image the differential metabolic activity between symbiont clades in the same host, in the intact symbiosis. Previous studies have used mass bulk techniques to investigate the metabolism of symbionts at the colony scale. However, such studies cannot determine the specific contribution of the individual cells, as a function of their distribution in the coral host tissue. We have developed a SIMS-ISH method combining nanoscale secondary ion mass spectrometry (NanoSIMS) and in situ hybridization (ISH) for the simultaneous in situ identification of Symbiodinium genotype, and visualization of symbiont-host metabolic exchange at the level of individual cell. We focus on two reef-building coral species Pocillopora damicornis and Stylophora pistillata for which a large amount of complementary metabolic data exists. We designed specific fluorescent DNA probes to identify clade C Symbiodinium in P. damicornis and clade A in S. pistillata, and in mixed cultures. We combined the probes with pulse-chase experiments using isotopically labeled seawater (13C-bicarbonate and 15N-nitrate) to attribute a particular metabolism to a specific clade. This combined method enables us to phylogenetically identify metabolically active cells from a NanoSIMS isotopic/elemental image. The precise correlation between TEM and the NanoSIMS isotopic maps allows us to follow the turnover and translocation of metabolites with sub-cellular precision in both the symbionts and the host. Due to the complex nature of the coral symbiosis, the ability to discriminate the phylogenetic identity and metabolic role of specific Symbiodinium populations in situ is crucial to understand the effects of environmental stress on the coral holobiont plasticity. Moreover, analyzing symbiotic associations in situ provides a unique insight into the spatio-temporal patterns of metabolic interactions in holobionts. This analytical breakthrough promises to open entirely new areas of research focused on understanding the dynamics of interactions between animals and the microbial world

Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo

Super-resolution light microscopy, correlative light and electron microscopy, and volume electron microscopy are revolutionising the way in which biological samples are examined and understood. Here, we combine these approaches to deliver super-accurate correlation of fluorescent proteins to cellular structures. We show that YFP and GFP have enhanced blinking properties when embedded in acrylic resin and imaged under partial vacuum, enabling in vacuo single molecule localisation microscopy. In conventional section-based correlative microscopy experiments, the specimen must be moved between imaging systems and/or further manipulated for optimal viewing. These steps can introduce undesirable alterations in the specimen, and complicate correlation between imaging modalities. We avoided these issues by using a scanning electron microscope with integrated optical microscope to acquire both localisation and electron microscopy images, which could then be precisely correlated. Collecting data from ultrathin sections also improved the axial resolution and signal-to-noise ratio of the raw localisation microscopy data. Expanding data collection across an array of sections will allow 3-dimensional correlation over unprecedented volumes. The performance of this technique is demonstrated on vaccinia virus (with YFP) and diacylglycerol in cellular membranes (with GFP)

Cell proliferation and migration during early development of a symbiotic scleractinian coral

In scleractinian reef-building corals, patterns of cell self-renewal, migration and death remain virtually unknown, limiting our understanding of cellular mechanisms underlying initiation of calcification, and ontogenesis of the endosymbiotic dinoflagellate relationship. In this study, we pulse-labelled the coral Stylophora pistillata for 24 h with BrdU at four life stages (planula, early metamorphosis, primary polyp and adult colony) to investigate coral and endosymbiont cell proliferation during development, while simultaneously recording TUNEL-positive (i.e. apoptotic) nuclei. In the primary polyp, the fate of BrdU-labelled cells was tracked during a 3-day chase. The pharynx and gastrodermis were identified as the most proliferative tissues in the developing polyp, and BrdU-labelled cells accumulated in the surface pseudostratified epithelium and the skeletogenic calicodermis during the chase, revealing cell migration to these epithelia. Surprisingly, the lowest cell turnover was recorded in the calicodermis at all stages, despite active, ongoing skeletal deposition. In dinoflagellate symbionts, DNA synthesis was systematically higher than coral host gastrodermis, especially in planula and early metamorphosis. The symbiont to host cell ratio remained constant, however, indicating successive post-mitotic control mechanisms by the host of its dinoflagellate density in early life stages, increasingly shifting to apoptosis in the growing primary polyp

Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure

Nutritional input from dinoflagellate symbionts in reef-building corals is minimal during planula larval life stage

Dispersion of larval offspring is of fundamental ecological importance to sessile marine organisms. Photosymbiotic planulae emitted by many reef-forming corals may travel over large distances before settling to form a new colony. It is not clear whether the metabolic requirements of these planula larvae are met exclusively with lipid and protein reservoirs inherited from the mother colony or when metabolic inputs from their endosymbiotic dinoflagellates become important. Pulse-chase experiments using [C-13] bicarbonate and [N-15] nitrate, combined with subcellular structural and isotopic imaging of freshly emitted symbiotic larvae from the coral Pocillopora damicornis, show that metabolic input from the dinoflagellates is minimal in the planulae compared with adult colonies. The larvae are essentially lecithotrophic upon emission, indicating that a marked shift in metabolic interaction between the symbiotic partners takes place later during ontogeny. Understanding the cellular processes that trigger and control this metabolic shift, and how climate change might influence it, is a key challenge in coral biology