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.

NanoSIMS isotopic imaging - Deciphering the nature of coral-symbiont metabolic interactions

Isabelle Domart-Coulon, Maoz Fine, Christophe Kopp, Thomas Krüger, Anders Meibom

The ability of endosymbiotic Symbiodinium to fix inorganic nutrients and their translocation to the coral host is considered a key element for the growth of coral reefs in tropical coastal waters and has been a fundamental research topic for over 50 years. With pulse-chase experiments using stable isotopes and combined TEM and NanoSIMS ultrastructural and isotopic analyses it is now possible to visualize and quantify the fixation, translocation, and turnover of essential elements such as carbon and nitrogen with unprecedented subcellular resolution. With these techniques we have monitored the assimilation of 15N nitrate, 15N ammonium, and 13C bicarbonate in the coral Pocillopora damicornis. These studies have revealed dynamic intra-symbiont storage of assimilated nitrate and ammonium in the form of uric acid crystals, and the formation of lipid droplets and starch granules in the symbiont and their subsequent remobilization. Carbon translocation toward the coral gastrodermal lipid droplets was detected within 15 min. Moreover, glycogen granules in the coral tissue were found to be an important sink for translocated carbon. This work has now been extended to the related pocilloporid species, Stylophora pistillata, with the important addition of heterotrophy (feeding the coral with dual-labelled brine shrimps) to contrast the fate and timeframes of heterotrophic vs. autotrophic (15N nitrate/13C bicarbonate) nutrient uptake under natural conditions and environmental stress due to elevated water temperatures. The in situ spatial data provided by NanoSIMS allow us for the first time to address questions that cannot be answered by traditional bulk measurements, such as 1) the influence of local symbiont densities on individual Symbiodinium assimilation rates, 2) metabolic capabilities of different symbiont ITS2 types in hospite, and (3) the effect of changes in intracellular host and symbiosome pH on individual symbiont productivity.

2015

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

Isabelle Domart-Coulon, Maoz Fine, Béatrice Gaume, Anders Meibom

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.

2017

Short-Term Thermal Acclimation Modifies the Metabolic Condition of the Coral Holobiont

Julia Marie Jeanne Yvonne Bodin, Emma Mary Gibbin, Thomas Krüger, Anders Meibom

The nutritional symbiosis between coral hosts and photosynthetic dinoflagellates is fundamental to the functioning of coral reefs. Rising seawater temperatures destabilize this relationship, resulting in drastic declines in world-wide coral cover. Thermal history is thought to play an important role in shaping a coral's response to subsequent thermal stress. Here, we exposed Pocillopora damicornis to two thermal acclimation regimes (ambient vs. warm) and compared the effect that acclimation had on the coral holobiont's response to a subsequent seven day heat stress event. We conducted daily physiological measurements at the holobiont level (gross photosynthesis, respiration, host protein content, symbiont density and chlorophyll content) throughout the heating event, as well as cellular-level imaging of 13C-bicarbonate and 15N-nitrate assimilation (using NanoSIMS) at the end of the heat stress event. Thermal acclimation history had a negligible effect on the measurements conducted at the holobiont level during the heat stress event. No differences were observed in the O2-budget between ambient and warm-acclimated corals and only small fluctuations in host protein, symbiont density and chlorophyll content were detected. In contrast, this lack of differential response, was not mirrored at the cellular level. Warm-acclimated corals had substantially higher 13C enrichment in the host gastrodermis and lipid bodies, but significantly lower 15N-nitrate assimilation in the symbionts and the host tissue layers, relative to the ambient-acclimated corals. We discuss potential reasons for the disconnect that occurred between symbiont bicarbonate and nitrate assimilation (in the absence of photosynthetic breakdown) in the warm-acclimated corals. We suggest this represents either a shift in nitrogen utilization, or supply limitation by the host. Our findings raise several interesting hypotheses regarding the role that nitrogen metabolism plays in thermal stress, which will warrant further investigation if we are to understand the acclimatization capacity of the coral holobiont.

2018