INNER WORKINGS

Amicroscopic mystery at the heart of mass-coralbleachingAmy McDermott, Science Writer

In the summer of 2017, a small plane hummed overAustralia’s Great Barrier Reef. Corals far below gleamedpale white in the sunlight, a stark contrast to the ceruleansea. The scene might have been gorgeous, if it wasn’tso bleak.

Aerial surveys by the Australian Research CouncilCentre of Excellence for Coral Reef Studies in Towns-ville, Australia, revealed that two-thirds of the GreatBarrier Reef had severely paled in 2016 and 2017,“bleaching” under the extreme stress of marine heatwaves that can kill corals (1, 2). Summer 2017 markedthe finale of the worst mass-bleaching event on recordworldwide, three consecutive years of feverish oceantemperatures, driven by climate change, which affectedmore than 75% of reefs (3, 4).

Newspaper headlines frequently reference bleach-ing events. It’s no secret that reefs are in trouble. Butfor all the attention to bleaching, researchers are

still puzzling over the cellular mechanisms thatcause it.

What’s clear is that bleaching is the breakup of thetenuous relationship between a coral and the photo-synthetic algae that live inside its cells. Heat stress candisrupt this relationship, causing the coral to expel itsalgae and to pale. New research suggests that algae,too, can be disruptive, turning on their hosts at hightemperatures by switching from symbionts to para-sites, which may also lead to bleaching. Coral algaehave a reputation “as this friendly, only do-good kindof hero,” because they provision the coral with nutri-ents, says reef ecologist David Baker at the Universityof Hong Kong. “And I think that’s a misguidedsentiment.”

New efforts in genomics are helping to furtherexplicate the basic biology of bleaching. In 2018,researchers even used CRISPR/Cas9 to edit coral

At least two-thirds of the Great Barrier Reef has been bleached under the extreme stress of marine heat waves. Imagecredit: The Ocean Agency/XL Catlin Seaview Survey.

Published under the PNAS license.

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genes for the first time, potentially leading to moretargeted efforts to understand the cellular mecha-nisms of bleaching. The mysterious roots of thisecological disaster are starting to come into fullview.

Feeling FeverishResearchers do have some idea how the bleachingprocess works. Corals bleach when heat stress disruptstheir relationship with symbiotic algae (4, 5). Coral maylook like rocks, but up close they are a living skin ofsoft, tentacled polyps, each anchored at its base by ahard calcium carbonate skeleton, covering over theskeletons of generations of polyps-past. Each livingpolyp is a sac of translucent tissue, crowned by a whorlof tentacles surrounding a central mouth, which opensto the polyp’s gut. Polyp tissue is colored by somecoral pigments, as well as splotches of ruddy brownalgae that live inside vacuoles in coral cells. The algaemake sugars from sunlight, most of which they pass tothe coral, in exchange for carbon for photosynthesisand some nitrogen in the host’s waste.

The symbiosis breaks down during stressful marineheat waves, when the coral rejects its colorful algae,bleaching white as its skeleton becomes visible throughits now-translucent tissues. Bleaching can kill a coral,starving it to death, unless conditions normalize and thealgae return.

Past studies suggest that one mechanism causingbleaching is damage to the algae’s photosyntheticapparatus, leading the host to reject the algae througha stress response (6). The gears of this stress mechanismbegin to turn long before a coral ever bleaches, explainsecophysiologist Clint Oakley, a postdoctoral researcherat Victoria University of Wellington, New Zealand. Pro-longed elevated temperatures damage the algae’sphotosynthetic machinery, so that it can’t process lightefficiently, and produces reactive oxygen and nitrogenspecies, such as hydrogen peroxide, as a damagingbyproduct (7). These molecules are “little bombs ba-sically, and whatever they contact, often a protein,they will damage it,” Oakley says. The little bombsthen presumably escape out of the algal cell into thecoral host cell, he says (8). To defend itself, the coralpolyp rejects its algae, destroying them inside coralcells, or ejecting them back into the gut and thenexpelling them through the mouth (9).

Beyond that, how exactly the symbiosis falls apartat the cellular level remains unclear. Understandingthe cellular mechanisms that drive the symbiosis break-down could help predict, and perhaps even prevent,future bleaching events.

Fair-Weather FriendsFor starters, those reactive oxygen bombs may onlybe part of the story. Studies in the last 10 years haveshown that bleaching is possible even without heatstress or reactive oxygen, says environmental genomicistChristian Voolstra at the University of Konstanz inGermany. Out in nature, when one cell invades another,their relationship is usually parasitic, he notes. Coral andalgae had been held up as this ideal, mutually beneficial

relationship. But building on several years of work, re-searchers have begun to turn a more critical eye to thesymbiosis, especially the algae.

Back in 2009, Scott Wooldridge, then a researcherat the Australian Institute ofMarine Science in Townsville,first suggested that algae might shift from good sharersto selfish partners in unusually warm water (10). Underthe right circumstances, he suspected that algae mayleech off the host without sharing nutrients.

A 2018 study in The ISME Journal provided ex-perimental evidence for Wooldridge’s theory (11). Thework found that algae don’t share well in unusuallywarm water, even though they keep taking resourcesfrom their star coral hosts. “The symbionts are doinggood business when the water is warm,” explains Baker,who led the work. “But they are not sharing that wealthwith their landlord.”

Baker used isotope tracers to follow the flow ofnutrients between the algae and the host underwarming conditions. He focused on corals fringing thesmall Belizean island of Carrie Bow Cay, a brushstrokeof white sand in a swirl of turquoise water. Baker collectedfragments of healthy star corals, Orbicella faveolata,from the reefs around the island, and plunked them intoshallow trays of seawater on the beach, gradually heat-ing some to create a microcosm of warming condi-tions, while holding other, control tanks at normalambient temperature.

He then took a few coral fragments from each tankand put them in Nalgene bottles full of seawater, withdissolved isotope tracers. Tracers are labeled com-pounds, such as nitrate, that the algae convert into abiologically available form for the coral. After incu-bating the coral fragments with the tracers for about10 hours, Baker tested the concentrations of thesetracers in the tissues of the algae and the coral, to see howmuch of each compound the algae kept for them-selves, and how much they shared with their host. He

Tiny algal symbionts (brown specks) dot Acropora tenuis coral polyps. Imagecredit: Katarina Damjanovic (Australian Institute of Marine Science, Townsville,Australia).

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found that the algae’s metabolism went into overdrivein the heat, absorbing high concentrations of thetracers. But the algae didn’t share a proportionateincrease in these resources with the coral. “The part-nership didn’t rise together,” Baker says. “One part-ner accelerated, and the other was fixed.”

Nutrient availability could help explain this selfishtendency, Voolstra notes. Normally, the coral hoardsmost nitrogen for itself, leaving the algae nitrogen-limited and unable to metabolize all of their photo-synthetic sugars, which they release into the coral cell.“This is not a friendly service by the algae,” Voolstrasays, but rather a trade-off to live inside the coral.

The trade-off ends in warm water when nitrogen-fixing bacteria become more active, hence makingmore nitrogen available to the coral–algae symbiosis.Now the algae can metabolize their sugars rather thanreleasing them, Voolstra explains. Unpublished datafrom his lab suggest that algae may limit sugar transfer

at high temperatures because of their higher energyneeds. “Essentially coral is acting in its own interest,and the symbiont is too,” Voolstra says.

Exactly how “selfish” algae might trigger bleachingis the next open question. Some researchers, includingBaker, speculate that selfish algae force the coral todraw on its own carbon reserves, which eventually runout. Then the coral can’t supply carbon to the algae,forcing these ancient ocean plants to switch fromcarbon-based photosynthesis to oxygen-based pho-torespiration, which produces the destructive littlebombs of reactive oxygen molecules that damage thecoral cell and can induce bleaching. The finding could

be “a missing piece of the puzzle” in the mechanismof bleaching Baker says.

But other studies (7) show increased reactive oxy-gen species in response to heat stress, notes VirginiaWeis at Oregon State University in Corvallis. Weisdoesn’t necessary dispute Baker’s hypothesis as asecond possible model of reactive oxygen production,but she’d like to see more evidence. Voolstra addsthat some researchers even question whether re-active oxygen molecules always drive bleaching. Hethinks imbalanced nutrient-sharing might contributeindependently of oxygen radicals.

Forward ThinkingGenomics, proteomics, and transcriptomics are nowbeing brought to bear to further elucidate the cellularpathways underlying the symbiosis breakup.

A July 2019 study found that 292 cnidarian genesin transcriptome analysis of the symbiotic sea anemoneExaiptasia diaphana, a close relative and emergingmodel organism of corals, changed their expressionlevels at bleaching-threshold temperatures (12). Someof the most affected genes are involved in metabo-lizing sugars, which the algae normally provide to thecoral. That these genes are strongly affected by hightemperatures suggests that sugar metabolism maybe a key step to maintain the stable symbiosis, sayscoauthor Shinichiro Maruyama, an evolutionary bi-ologist at Tohoku University in Sendai, Japan. Al-though not the first study to look at temperature-sensitive genes, the work supports a central rolefor nutrient exchange in bleaching, as in the parasitichypothesis.

To directly decipher coral gene function, geneti-cists from Stanford University in Stanford, CA, alongwith collaborators, used CRISPR/Cas9 to geneticallyedit corals for the first time in 2018 (13). The re-searchers successfully mutated three genes as proof-of-concept, none of which is suspected to play majorroles in bleaching. In a follow-up study, Stanford post-doctoral molecular geneticist and study coauthor PhillipCleves repeated the process for bleaching-relatedgenes. He used CRISPR to knock out a gene suspected

Researchers used a microinjection instrument to injectcoral eggs with CRISPR/Cas9 components, seekingtargeted mutations in the coral genome in hopes ofunderstanding coral gene function. Image credit: LornaMitchison-Field and Amanda Tinoco (StanfordUniversity, Stanford, CA).

Rescuing Corals with the Right BacteriaClarifying the relative roles of algae and coral in the bleaching processcould offer researchers new ways to protect reefs (16).

Madeleine van Oppen, a coral geneticist at the University of Mel-bourne and Australian Institute of Marine Science, is culturing many strainsof the bacteria that naturally live on corals. In unpublished research, she hasfound that some strains are good at mopping up reactive oxygen mole-cules. Now, van Oppen is inoculating a model anemone with some of themost efficient strains, but she says initial results have been inconclusive. A2018 study showed promising early results, however, using bacterialprobiotics to ease coral bleaching (17).

If researchers accumulate more evidence that algae become parasiticand trigger bleaching through disrupted nutrient sharing, similar studiesmight inoculate coral with bacteria to scavenge reactive oxygen, or offercritical nutrients when sharing breaks down in the symbiosis.

Such targeted interventions require an understanding of the cell bi-ology that results in bleaching. “It’s really important to know what themechanism is,” van Oppen says. “We need to do something to try andhelp corals survive climate change this century.”

“It’s really important to know what the mechanism is.We need to do something to try and help corals surviveclimate change this century.”

—Madeleine van Oppen

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to be involved in heat tolerance, confirming in still-unpublished experiments in 2018 that corals needthe gene to survive heat.

Cleves’ motivation is to uncover the basic biologyof why corals bleach, but other researchers point outthat similar work could lead to more active interven-tions on reefs. “Once we know which [genes] arecritically important in thermal tolerance, then we couldthink about making corals more tolerant through ge-netic engineering,” says Madeleine van Oppen, a coralgeneticist at the University ofMelbourne, Australia, andAustralian Institute of Marine Science. Indeed, a recentreport from the National Academies of Sciences, En-gineering, and Medicine in Washington, DC, summarizesmore than 20 genetic, ecological, and environmentalinterventions to help reefs survive and become moreresilient into the future. A follow-up report offersguidance to coral managers choosing between these

many possible interventions, to build a local decision-making strategy (14, 15).

Given the scale of threat to reefs, it’s tempting toskip the basic research and dive straight for solutions,Weis says. Could a better understanding of the finerdetails actually help researchers tackle the bleachingproblem?

Maybe. Genetically modified corals, for example,although incredibly complex and difficult to make,could serve as one potential conservation tool thatrequires basic research insights. Researchers could, inprinciple, target genes responsible for the breakup ofthe symbiosis caused by heat stress and parasiticalgae. Van Oppen knows that genetically modifiedorganisms stir controversy, and that releasing modifiedcorals onto reefs would take much greater public ac-ceptance and regulatory approval than exists today.“But,” she says, “that doesn’t mean we shouldn’t de-velop the tools.”

1 H. Harrison et al., Back-to-back coral bleaching events on isolated atolls in the Coral Sea. Coral Reefs 38, 713–719 (2019).2 C. Mooney, ‘An enormous loss’: 900 miles of the Great Barrier Reef have bleached severely since 2016. Washington Post (2017).https://www.washingtonpost.com/news/energy-environment/wp/2017/04/09/for-the-second-year-in-a-row-severe-coral-bleaching-has-struck-the-great-barrier-reef/. Accessed October 29, 2019.

3 T. P. Hughes et al., Spatial and temporal patterns of mass bleaching of corals in the Anthropocene. Science 359, 80–83 (2018).4 National Oceanic and Atmospheric Association, El Nino prolongs longest global coral bleaching event (2016). https://www.noaa.gov/media-release/el-ni-o-prolongs-longest-global-coral-bleaching-event. Accessed October 29, 2019

5 P. W. Glynn, Widespread coral mortality and the 1982-83 El Nino warming event. Environ. Conserv. 11, 133–146 (1984).6 V. M. Weis, Cellular mechanisms of Cnidarian bleaching: Stress causes the collapse of symbiosis. J. Exp. Biol. 211, 3059–3066 (2008).7 M. P. Lesser, Oxidative stress causes coral bleaching during exposure to elevated temperatures. Coral Reefs 16, 187–192 (1997).8 R. Hajiboland, Oxidative Damage to Plants (Academic Press, Cambridge, MA, 2014), chap. 1.9 B. E. Brown, Coral bleaching: Causes and consequences. Coral Reefs 16, S129–S138 (1997).

10 S. A. Wooldridge, A new conceptual model for the warm-water breakdown of the coral–algae endosymbiosis. Mar. Freshw. Res. 60,483–496 (2009).

11 D. M. Baker, C. J. Freeman, J. C. Y. Wong, M. L. Fogel, N. Knowlton, Climate change promotes parasitism in a coral symbiosis. ISME J.12, 921–930 (2018).

12 Y. Ishii et al., Global shifts in gene expression profiles accompanied with environmental changes in Cnidarian-Dinoflagellateendosymbiosis. G3 (Bethesda) 9, 2337–2347 (2019).

13 P. A. Cleves, M. E. Strader, L. K. Bay, J. R. Pringle, M. V. Matz, CRISPR/Cas9-mediated genome editing in a reef-building coral. Proc.Natl. Acad. Sci. U.S.A. 115, 5235–5240 (2018).

14 National Academies of Sciences, Engineering, and Medicine, A Research Review of Interventions to Increase the Persistence andResilience of Coral Reefs (The National Academies Press, Washington, DC, 2019).

15 National Academies of Sciences, Engineering, and Medicine, A Decision Framework for Interventions to Increase the Persistence andResilience of Coral Reefs (The National Academies Press, Washington, DC, 2019).

16 V. M. Weis, Cell biology of coral symbiosis: Foundational study can inform solutions to the coral reef crisis. Integr. Comp. Biol. 59,845–855 (2019).

17 P. M. Rosado et al., Marine probiotics: Increasing coral resistance to bleaching through microbiome manipulation. ISME J. 13, 921–936 (2019).

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