Reef-building corals farm and feed on their photosynthetic symbionts.

Coral reefs are highly diverse ecosystems that thrive in nutrient-poor waters, a phenomenon frequently referred to as the Darwin paradox1. The energy demand of coral animal hosts can often be fully met by the excess production of carbon-rich photosynthates by their algal symbionts2,3. However, the understanding of mechanisms that enable corals to acquire the vital nutrients nitrogen and phosphorus from their symbionts is incomplete4-9. Here we show, through a series of long-term experiments, that the uptake of dissolved inorganic nitrogen and phosphorus by the symbionts alone is sufficient to sustain rapid coral growth. Next, considering the nitrogen and phosphorus budgets of host and symbionts, we identify that these nutrients are gathered through symbiont 'farming' and are translocated to the host by digestion of excess symbiont cells. Finally, we use a large-scale natural experiment in which seabirds fertilize some reefs but not others, to show that the efficient utilization of dissolved inorganic nutrients by symbiotic corals established in our laboratory experiments has the potential to enhance coral growth in the wild at the ecosystem level. Feeding on symbionts enables coral animals to tap into an important nutrient pool and helps to explain the evolutionary and ecological success of symbiotic corals in nutrient-limited waters.

Green fluorescent protein-like pigments optimise the internal light environment in symbiotic reef-building corals.

Pigments homologous to the green fluorescent protein (GFP) have been proposed to fine-tune the internal light microclimate of corals, facilitating photoacclimation of photosynthetic coral symbionts (Symbiodiniaceae) to life in different reef habitats and environmental conditions. However, direct measurements of the in vivo light conditions inside the coral tissue supporting this conclusion are lacking. Here, we quantified the intra-tissue spectral light environment of corals expressing GFP-like proteins from widely different light regimes. We focus on: (1) photoconvertible red fluorescent proteins (pcRFPs), thought to enhance photosynthesis in mesophotic habitats via wavelength conversion, and (2) chromoproteins (CPs), which provide photoprotection to the symbionts in shallow water via light absorption. Optical microsensor measurements indicated that both pigment groups strongly alter the coral intra-tissue light environment. Estimates derived from light spectra measured in pcRFP-containing corals showed that fluorescence emission can contribute to >50% of orange-red light available to the photosynthetic symbionts at mesophotic depths. We further show that upregulation of pink CPs in shallow-water corals during bleaching leads to a reduction of orange light by 10-20% compared to low-CP tissue. Thus, screening by CPs has an important role in mitigating the light-enhancing effect of coral tissue scattering and skeletal reflection during bleaching. Our results provide the first experimental quantification of the importance of GFP-like proteins in fine-tuning the light microclimate of corals during photoacclimation.

Optical Feedback Loop Involving Dinoflagellate Symbiont and Scleractinian Host Drives Colorful Coral Bleaching.

Coral bleaching, caused by the loss of brownish-colored dinoflagellate photosymbionts from the host tissue of reef-building corals, is a major threat to reef survival. Occasionally, bleached corals become exceptionally colorful rather than white. These colors derive from photoprotective green fluorescent protein (GFP)-like pigments produced by the coral host. There is currently no consensus regarding what causes colorful bleaching events and what the consequences for the corals are. Here, we document that colorful bleaching events are a recurring phenomenon in reef regions around the globe. Our analysis of temperature conditions associated with colorful bleaching events suggests that corals develop extreme coloration within 2 to 3 weeks after exposure to mild or temporary heat stress. We demonstrate that the increase of light fluxes in symbiont-depleted tissue promoted by reflection of the incident light from the coral skeleton induces strong expression of the photoprotective coral host pigments. We describe an optical feedback loop involving both partners of the association, discussing that the mitigation of light stress offered by host pigments could facilitate recolonization of bleached tissue by symbionts. Our data indicate that colorful bleaching has the potential to identify local environmental factors, such as nutrient stress, that can exacerbate the impact of elevated temperatures on corals, to indicate the severity of heat stress experienced by corals and to gauge their post-stress recovery potential. VIDEO ABSTRACT.

Symbiosis: High-Carb Diet of Reef Corals as Seen from Space.

High levels of phytoplankton visible in satellite imagery are correlated with an increased uptake of carbon compounds by corals. This suggests that corals rely less on carbon production by photosynthetic symbionts when other resources are plentiful, and that the changes in the acquisition mode of carbon can be inferred by remote-sensing techniques.

Validation of the binary designation Symbiodinium thermophilum (Dinophyceae).

The binary designation Symbiodinium thermophilum was invalid due to the absence of an illustration as required by Article 44.2 of the ICN. Herein, it is validated. This species is the most common symbiont in reef corals in the southern Persian/Arabian Gulf, the world's hottest body of water sustaining reef coral growth.

The role of floridoside in osmoadaptation of coral-associated algal endosymbionts to high-salinity conditions.

The endosymbiosis between Symbiodinium dinoflagellates and stony corals provides the foundation of coral reef ecosystems. The survival of these ecosystems is under threat at a global scale, and better knowledge is needed to conceive strategies for mitigating future reef loss. Environmental disturbance imposing temperature, salinity, and nutrient stress can lead to the loss of the Symbiodinium partner, causing so-called coral bleaching. Some of the most thermotolerant coral-Symbiodinium associations occur in the Persian/Arabian Gulf and the Red Sea, which also represent the most saline coral habitats. We studied whether Symbiodinium alter their metabolite content in response to high-salinity environments. We found that Symbiodinium cells exposed to high salinity produced high levels of the osmolyte 2-O-glycerol-α-d-galactopyranoside (floridoside), both in vitro and in their coral host animals, thereby increasing their capacity and, putatively, the capacity of the holobiont to cope with the effects of osmotic stress in extreme environments. Given that floridoside has been previously shown to also act as an antioxidant, this osmolyte may serve a dual function: first, to serve as a compatible organic osmolyte accumulated by Symbiodinium in response to elevated salinities and, second, to counter reactive oxygen species produced as a consequence of potential salinity and heat stress.

Genetic structure of coral-Symbiodinium symbioses on the world's warmest reefs.

Corals in the Arabian/Persian Gulf (PAG) survive extreme sea temperatures (summer mean: >34°C), and it is unclear whether these corals have genetically adapted or physiologically acclimated to these conditions. In order to elucidate the processes involved in the thermal tolerance of PAG corals, it is essential to understand the connectivity between reefs within and outside of the PAG. To this end, this study set out to investigate the genetic structure of the coral, Platygyra daedalea, and its symbiotic algae in the PAG and neighbouring Gulf of Oman. Using nuclear markers (the ITS region and an intron of the Pax-C gene), this study demonstrates genetic divergence of P. daedalea on reefs within the thermally extreme PAG compared with those in the neighbouring Gulf of Oman. Isolation by distance of P. daedalea was supported by the ITS dataset but not the Pax-C intron. In addition, the symbiont community within the PAG was dominated by C3 symbionts, while the purportedly thermotolerant clade D was extremely rare and was common only at sites outside of the PAG. Analysis of the psbAncr indicates that the C3 variant hosted by P. daedalea in the PAG belongs to the newly described species, Symbiodinium thermophilum. The structuring of the coral and symbiont populations suggests that both partners of the symbiosis may contribute to the high bleaching thresholds of PAG corals. While limited gene flow has likely played a role in local adaptation within the PAG, it also indicates limited potential for natural export of thermal tolerance traits to reefs elsewhere in the Indian Ocean threatened by climate change.

FRET-Mediated Long-Range Wavelength Transformation by Photoconvertible Fluorescent Proteins as an Efficient Mechanism to Generate Orange-Red Light in Symbiotic Deep Water Corals.

Photoconvertible fluorescent proteins (pcRFPs) are a group of fluorophores that undergo an irreversible green-to-red shift in emission colour upon irradiation with near-ultraviolet (near-UV) light. Despite their wide application in biotechnology, the high-level expression of pcRFPs in mesophotic and depth-generalist coral species currently lacks a biological explanation. Additionally, reduced penetration of near-UV wavelengths in water poses the question whether light-driven photoconversion is relevant in the mesophotic zone, or whether a different mechanism is involved in the post-translational pigment modification in vivo. Here, we show in a long-term mesocosm experiment that photoconversion in vivo is entirely dependent on near-UV wavelengths. However, a near-UV intensity equivalent to the mesophotic underwater light field at 80 m depth is sufficient to drive the process in vitro, suggesting that photoconversion can occur near the lower distribution limits of these corals. Furthermore, live coral colonies showed evidence of efficient Förster Resonance Energy Transfer (FRET). Our simulated mesophotic light field maintained the pcRFP pool in a partially photoconverted state in vivo, maximising intra-tetrameric FRET and creating a long-range wavelength conversion system with higher quantum yield than other native RFPs. We hypothesise that efficient conversion of blue wavelengths, abundant at depth, into orange-red light could constitute an adaptation of corals to life in light-limited environments.

Phosphate deficiency promotes coral bleaching and is reflected by the ultrastructure of symbiotic dinoflagellates.

Enrichment of reef environments with dissolved inorganic nutrients is considered a major threat to the survival of corals living in symbiosis with dinoflagellates (Symbiodinium sp.). We argue, however, that the direct negative effects on the symbiosis are not necessarily caused by the nutrient enrichment itself but by the phosphorus starvation of the algal symbionts that can be caused by skewed nitrogen (N) to phosphorus (P) ratios. We exposed corals to imbalanced N:P ratios in long-term experiments and found that the undersupply of phosphate severely disturbed the symbiosis, indicated by the loss of coral biomass, malfunctioning of algal photosynthesis and bleaching of the corals. In contrast, the corals tolerated an undersupply with nitrogen at high phosphate concentrations without negative effects on symbiont photosynthesis, suggesting a better adaptation to nitrogen limitation. Transmission electron microscopy analysis revealed that the signatures of ultrastructural biomarkers represent versatile tools for the classification of nutrient stress in symbiotic algae. Notably, high N:P ratios in the water were clearly identified by the accumulation of uric acid crystals.

Ancestral genetic diversity associated with the rapid spread of stress-tolerant coral symbionts in response to Holocene climate change.

Coral communities in the Persian/Arabian Gulf (PAG) withstand unusually high salinity levels and regular summer temperature maxima of up to ∼35 °C that kill conspecifics elsewhere. Due to the recent formation of the PAG and its subsequent shift to a hot climate, these corals have had only <6,000 y to adapt to these extreme conditions and can therefore inform on how coral reefs may respond to global warming. One key to coral survival in the world's warmest reefs are symbioses with a newly discovered alga,Symbiodinium thermophilum Currently, it is unknown whether this symbiont originated elsewhere or emerged from unexpectedly fast evolution catalyzed by the extreme environment. Analyzing genetic diversity of symbiotic algae across >5,000 km of the PAG, the Gulf of Oman, and the Red Sea coastline, we show thatS. thermophilumis a member of a highly diverse, ancient group of symbionts cryptically distributed outside the PAG. We argue that the adjustment to temperature extremes by PAG corals was facilitated by the positive selection of preadapted symbionts. Our findings suggest that maintaining the largest possible pool of potentially stress-tolerant genotypes by protecting existing biodiversity is crucial to promote rapid adaptation to present-day climate change, not only for coral reefs, but for ecosystems in general.

Local bleaching thresholds established by remote sensing techniques vary among reefs with deviating bleaching patterns during the 2012 event in the Arabian/Persian Gulf.

A severe bleaching event affected coral communities off the coast of Abu Dhabi, UAE in August/September, 2012. In Saadiyat and Ras Ghanada reefs ~40% of the corals showed signs of bleaching. In contrast, only 15% of the corals were affected on Delma reef. Bleaching threshold temperatures for these sites were established using remotely sensed sea surface temperature (SST) data recorded by MODIS-Aqua. The calculated threshold temperatures varied between locations (34.48 °C, 34.55 °C, 35.05 °C), resulting in site-specific deviations in the numbers of days during which these thresholds were exceeded. Hence, the less severe bleaching of Delma reef might be explained by the lower relative heat stress experienced by this coral community. However, the dominance of Porites spp. that is associated with the long-term exposure of Delma reef to elevated temperatures, as well as the more pristine setting may have additionally contributed to the higher coral bleaching threshold for this site.

Spectral Diversity and Regulation of Coral Fluorescence in a Mesophotic Reef Habitat in the Red Sea.

The phenomenon of coral fluorescence in mesophotic reefs, although well described for shallow waters, remains largely unstudied. We found that representatives of many scleractinian species are brightly fluorescent at depths of 50-60 m at the Interuniversity Institute for Marine Sciences (IUI) reef in Eilat, Israel. Some of these fluorescent species have distribution maxima at mesophotic depths (40-100 m). Several individuals from these depths displayed yellow or orange-red fluorescence, the latter being essentially absent in corals from the shallowest parts of this reef. We demonstrate experimentally that in some cases the production of fluorescent pigments is independent of the exposure to light; while in others, the fluorescence signature is altered or lost when the animals are kept in darkness. Furthermore, we show that green-to-red photoconversion of fluorescent pigments mediated by short-wavelength light can occur also at depths where ultraviolet wavelengths are absent from the underwater light field. Intraspecific colour polymorphisms regarding the colour of the tissue fluorescence, common among shallow water corals, were also observed for mesophotic species. Our results suggest that fluorescent pigments in mesophotic reefs fulfil a distinct biological function and offer promising application potential for coral-reef monitoring and biomedical imaging.

Local adaptation constrains the distribution potential of heat-tolerant Symbiodinium from the Persian/Arabian Gulf.

The symbiotic association of corals and unicellular algae of the genus Symbiodinium in the southern Persian/Arabian Gulf (PAG) display an exceptional heat tolerance, enduring summer peak temperatures of up to 36 °C. As yet, it is not clear whether this resilience is related to the presence of specific symbiont types that are exclusively found in this region. Therefore, we used molecular markers to identify the symbiotic algae of three Porites species along >1000 km of coastline in the PAG and the Gulf of Oman and found that a recently described species, Symbiodinium thermophilum, is integral to coral survival in the southern PAG, the world's hottest sea. Despite the geographic isolation of the PAG, we discovered that representatives of the S. thermophilum group can also be found in the adjacent Gulf of Oman providing a potential source of thermotolerant symbionts that might facilitate the adaptation of Indian Ocean populations to the higher water temperatures expected for the future. However, corals from the PAG associated with S. thermophilum show strong local adaptation not only to high temperatures but also to the exceptionally high salinity of their habitat. We show that their superior heat tolerance can be lost when these corals are exposed to reduced salinity levels common for oceanic environments elsewhere. Consequently, the salinity prevailing in most reefs outside the PAG might represent a distribution barrier for extreme temperature-tolerant coral/Symbiodinium associations from the PAG.

Nitrogen cycling in corals: the key to understanding holobiont functioning?

Corals are animals that form close mutualistic associations with endosymbiotic photosynthetic algae of the genus Symbiodinium. Together they provide the calcium carbonate framework of coral reef ecosystems. The importance of the microbiome (i.e., bacteria, archaea, fungi, and viruses) to holobiont functioning has only recently been recognized. Given that growth and density of Symbiodinium within the coral host is highly dependent on nitrogen availability, nitrogen-cycling microbes may be of fundamental importance to the stability of the coral-algae symbiosis and holobiont functioning, in particular under nutrient-enriched and -depleted scenarios. We summarize what is known about nitrogen cycling in corals and conclude that disturbance of microbial nitrogen cycling may be tightly linked to coral bleaching and disease.

Polymersomes prepared from thermoresponsive fluorescent protein-polymer bioconjugates: capture of and report on drug and protein payloads.

Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein-polymer bioconjugate PNIPAM-b-amilFP497 composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and a green-fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co-)encapsulate the fluorescent drug doxorubicin and the fluorescent light-harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM-FRET), we can distinguish the co-encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane.

Fluorescent protein-mediated colour polymorphism in reef corals: multicopy genes extend the adaptation/acclimatization potential to variable light environments.

The genomic framework that enables corals to adjust to unfavourable conditions is crucial for coral reef survival in a rapidly changing climate. We have explored the striking intraspecific variability in the expression of coral pigments from the green fluorescent protein (GFP) family to elucidate the genomic basis for the plasticity of stress responses among reef corals. We show that multicopy genes can greatly increase the dynamic range over which corals can modulate transcript levels in response to the light environment. Using the red fluorescent protein amilFP597 in the coral Acropora millepora as a model, we demonstrate that its expression increases with light intensity, but both the minimal and maximal gene transcript levels vary markedly among colour morphs. The pigment concentration in the tissue of different morphs is strongly correlated with the number of gene copies with a particular promoter type. These findings indicate that colour polymorphism in reef corals can be caused by the environmentally regulated expression of multicopy genes. High-level expression of amilFP597 is correlated with reduced photodamage of zooxanthellae under acute light stress, supporting a photoprotective function of this pigment. The cluster of light-regulated pigment genes can enable corals to invest either in expensive high-level pigmentation, offering benefits under light stress, or to rely on low tissue pigment concentrations and use the conserved resources for other purposes, which is preferable in less light-exposed environments. The genomic framework described here allows corals to pursue different strategies to succeed in habitats with highly variable light stress levels. In summary, our results suggest that the intraspecific plasticity of reef corals' stress responses is larger than previously thought.

One-pot synthesis of high molecular weight synthetic heteroprotein dimers driven by charge complementarity electrostatic interactions.

Despite the importance of protein dimers and dimerization in biology, the formation of protein dimers through synthetic covalent chemistry has not found widespread use. In the case of maleimide-cysteine-based dimerization of proteins, we show here that when the proteins have the same charge, dimerization appears to be inherently difficult with yields around 1% or less, regardless of the nature of the spacer used or whether homo- or heteroprotein dimers are targeted. In contrast, if the proteins have opposing (complementary) charges, the formation of heteroprotein dimers proceeds much more readily, and in the case of one high molecular weight (>80 kDa) synthetic dimer between cytochrome c and bovine serum albumin, a 30% yield of the purified, isolated dimer was achieved. This represents at least a 30-fold increase in yield for protein dimers formed from proteins with complementary charges, compared to when the proteins have the same charge, under otherwise similar conditions. These results illustrate the role of ionic supramolecular interactions in controlling the reactivity of proteins toward bis-functionalized spacers. The strategy here for effective synthetic dimerization of proteins could be very useful for developing novel approaches to study the important role of protein-protein interactions in chemical biology.

Improving FRET dynamic range with bright green and red fluorescent proteins.

A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.

Induced polymersome formation from a diblock PS-b-PAA polymer via encapsulation of positively charged proteins and peptides.

In contrast to simple salts or negatively charged macromolecules, positively charged proteins and peptides including cytochrome c (yeast) and poly-L-lysine are efficiently encapsulated while inducing the formation of polymersomes from polystyrene(140)-b-poly(acrylic acid)(48) (PS(140)-b-PAA(48)).