Scientists Discover How Algae Colonized Corals

The reefs scattered throughout the tropics arose only after algae took up full-time residence in coral cells, supplying corals with abundant food and enabling them to build extensive shallow-water communities. But with warming oceans, algae are often abandoning coral - causing what's known as bleaching - and turning reefs that were once teeming with life into ghost towns.

A research team at UC Berkeley has now answered a major question in coral biology: How are algae able to thrive inside the cells of coral? The finding could lead to new insights into why algae and coral are failing to thrive symbiotically and suggest ways to reestablish the connection and save the world's reefs.

The conclusions come from experiments conducted in the lab of Phillip Cleves, a Berkeley assistant professor of molecular and cell biology who has constructed a state-of-the-art saltwater nursery for coral. On Australia's Great Barrier Reef, corals spawn once a year, releasing eggs and sperm in great clouds on a night in October or November around the full moon. This makes it hard to conduct genetic studies to understand the symbiotic relationship between algae and corals and their response to heat stress.

In their nursery, Cleves and his team have been able to raise different groups of corals to spawn throughout the year, giving researchers the opportunity to conduct genetic experiments every few months.

Based on genetic manipulations of these lab-grown corals and related anemones, which also incorporate algae into their cells, the researchers are challenging the assumption that coral long ago absorbed algae into specialized compartments inside their cells called symbiosomes. Symbiosomes resemble mitochondria, the powerhouses of the cell, and chloroplasts, which harbor the photosynthetic machinery in plants. Both are thought to have been independent organisms that were long ago incorporated into cells and are now essential cellular components in all animals, plants and fungi.

Cleves argues instead that algae parasitized coral, having found a way to live unscathed inside a type of lysosome, the cellular organs that normally digest food and invading organisms. In doing so, the algae learned how to absorb carbon from the host cell and release the products of photosynthesis - primarily glucose - to feed the coral. That's a win-win for both algae and coral, and for other animals that harbor symbiotic algae.

In a paper to be published online July 1 in the journal Cell, Cleves and his colleagues report experiments that support this idea that symbiosomes form by fusing with lysosomes, and that algae have somehow evolved to resist the active digestive enzymes in the lysosome.

"Parasites basically trick cells to do what they want. That's what I think is happening here," Cleves said. "The algae are hijacking the nutrient centers of the cells and acting like food that just never gets digested because they can fix carbon and make glucose from photosynthesis. I think about it as an everlasting gobstopper."

He noted that the difference is not always clear between a parasite, which exploits its host without the host benefitting, and a symbiont, which lives mutualistically with its host. The coral-algae relationship could be a new type of symbiosis that involves parasitizing a cellular organelle.

"It's good for the coral, it's good for the algae, so it's symbiosis," he said. "But I think it's basically repurposing the entire cell and taking over that nutrient center."

Promiscuous algae

In genetic experiments led by postdoctoral fellow Shumpei Maruyama of Berkeley and doctoral student Catherine Henderson of Carnegie Science in Baltimore, Cleves and his colleagues identified at least 200 proteins located on the symbiosome in which algae live. One of the proteins transports bicarbonate, which is converted to carbon dioxide in the symbiosome, potentially explaining how the alga gets the carbon dioxide it needs to convert sunlight into sugars, despite being isolated inside a cell inside the stomach of the coral.

"By doing a CRISPR knockout in the coral Galaxea fascicularis, we've shown that this bicarbonate transporter is required for symbiosis," Maruyama said.

Cleves is currently investigating the role played by the other symbiosomal proteins in hopes of identifying how heat stress disrupts this amicable living situation, causing coral to lose their algae. He's also exploring how these proteins are involved in communication between the algae inside the symbiosome and the coral cell.

"Our overall research goal is to understand symbiosis and also to understand the genetic mechanisms for why corals bleach," he said. "We are now showing that the symbiosome is actually a type of phagolysosome, which is profound. We think that this hijacking of the phagolysosome tells us how these algae are so promiscuous at evolving new symbioses with coral, anemones, jellyfish, clams and even flatworms."

A coral nursery

In the basement of Berkeley's Koshland Hall, Cleves proudly showed off the one-of-a-kind coral nursery that he and lab manager Natalie Swinhoe designed down to the last pipe. Online since January, when Cleves arrived on campus, it consists of osmotic filters that purify city drinking water that is then mixed in three large vats with evaporated salt from the Red Sea. This reconstituted seawater is then piped into brightly-lit trays of coral - Galaxea fascicularis, a reef-building species from the Pacific Ocean - and smaller basins housing Aiptasia, small anemones. The lab is the only one on campus with seawater on tap.

Following years of experimentation, including during the past five years at Carnegie Science, Cleves perfected a technique for getting coral populations in his lab to spawn throughout the year. Because they spawn only once a year in their native reefs, he originally had to travel to Australia for once-a-year genetic experiments on coral. After other researchers learned how to raise coral in the lab, Cleves decided to establish his own lab nursery. Through experimentation, he discovered that, by slowly altering the light cycle and temperature, he could get corals to shift their yearly spawning cycle. Over a two-year period, the corals thoroughly adjust to this jetlag. His Berkeley lab now has six different tanks of coral that should spawn sequentially, providing six opportunities each year to conduct genetic experiments on coral eggs.

"Our big advance here is that we're coupling the technology to spawn the animals with our ability to genetically engineer them," he said. "It really allows us to study gene function for the first time, to study coral-algal symbiosis, bleaching and heat tolerance. We have developed a variety of genetic tools: we can do CRISPR, we can do plasmid transgenesis, we can do RNAi knockdown. The paper is the first time we're showcasing the powerful insights that can be made with these new technologies."

While spawning every few months is frequent enough to let him test ideas on his lab corals, Cleves' everyday experimental model is Aiptasia, which spawns every week and like many cnidarians - the group that includes jellyfish, corals and anemones - also sequesters algae.

Membrane trafficking

The researchers first isolated the symbiosome membrane in Aiptasia and identified the number and types of proteins in it. They discovered that there are about 200 proteins, many of them also found in the lysosome of the anemone. By knocking down some of these proteins using RNA interference (RNAi), Cleves and his colleagues showed that many of the lysosomal proteins in the symbiosome are required for algae to live inside these organelles. Among these are proteins that shuttle molecules in and out of the symbiosome and degrade other proteins.

"There are several vesicle trafficking proteins that we think are actually how the animal and the algae communicate, because they are trafficking cargo on and off the symbiosome, bringing stuff to the algae," Cleves said. "We found a huge diversity of transporters that are predicted to transport everything from lipids to ammonium to neurotransmitters to histidine, as well as bicarbonate. And the most paradoxical thing of all was that we found a huge diversity of lysosomal proteases - these proteins usually eat up the contents of the lysosome to scavenge food to fuel the (coral) cell."

The two lead authors subsequently used CRISPR to mutate the bicarbonate transporter in coral and found that this interfered with algal symbiosis, demonstrating its importance in supplying coral algae with carbon.

To Cleves, the ability of one group of algae - dinoflagellates that are now grouped into a single family of symbionts, Symbiodiniaceae - to survive inside the stomachs of cells explains their success in colonizing a broad range of marine creatures. Because lysosomes are cellular compartments found in all animals and conserved through evolution, the symbiont should be able to hijack these compartments in many hosts to evolve new symbioses.

"If evolving a new symbiosis is as simple as resisting lysosomal digestion, we should be able to have the algae persist inside lysosomes in other organisms, because much of the machinery is conserved," he said.

While this work sheds light on how corals and algae work together during good times, it may also tell biologists something about coral bleaching.

"We think dysfunction of the symbiosomal proteins might be related to the breakdown of symbiosis, so we're now analyzing how the proteome shifts during heat stress to try to understand how this organelle changes during bleaching," Cleves said.

Cleves is supported by the Gordon and Betty Moore Foundation (12187), National Science Foundation (2128073), a Revive and Restore Grant (2023-069) and a Pew Biomedical and Marine Fellow Award (00036631). Other co-authors of the paper are Swinhoe, Emily Meier and Ty Engelke of Berkeley and Griffin Kowalewski of Carnegie Science.