Russian dolls of coral reefs: Bacteria within algal endosymbionts within animal hosts.

Symbioses between corals and intracellular algae are essential for the survival of coral reefs and have been at the center of hundreds of studies. Here, we uncovered an additional symbiotic layer by describing the presence of intracellular bacteria, within algal endosymbionts, within corals.

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Coral reefs are fascinating ecosystems. Through the construction of their calcium carbonate skeleton, corals are the master engineers behind the three-dimensional structure of coral reefs (Figure 1A). This enables countless marine organisms to colonize coral reefs, making them some of the most biodiverse and important ecosystems on Earth.

The real stars of coral reefs, however, are endosymbiotic algae of the Symbiodiniaceae family. By translocating products of photosynthesis to their hosts, Symbiodiniaceae provide an essential carbon source for a wide variety of reef organisms, including corals themselves, anemones, jellyfish, and clams (Figure 1B). Without Symbiodiniaceae, all these organisms would die, and coral reef ecosystems would collapse. The natural phenomenon of coral bleaching, whereby corals lose their Symbiodiniaceae because of prolonged heat stress and subsequently die, is testament to the importance of Symbiodiniaceae for coral reefs and shows how climate change is drastically affecting them. Understanding how Symbiodiniaceae function is at the center of coral reef conservation and research efforts aimed at mitigating coral bleaching.

Figure 1. A: Coral reefs are extremely diverse ecosystems, whose complex three-dimensional structure is home to more than 25% of all marine species. B: Corals host algae of the Symbiodiniaceae family that provide them with most of their carbon requirements. Symbiodiniaceae are visible as brown dots (left) and red dots (right, fluorescence microscopy) in the tentacles of the tropical coral Galaxea fascicularis. The green fluorescence is caused by host pigments. Photo credits: A: Justin Maire and Ashley Dungan (University of Melbourne); B: Wing Yan Chan (University of Melbourne).

Many aspects of Symbiodiniaceae functioning remain to be uncovered. In particular, how Symbiodiniaceae interact with other microorganisms, such as bacteria, remains elusive. Using a wide diversity of cultured and freshly isolated (from the host tissues) Symbiodiniaceae, we sought to characterize bacterial communities that associate with these photosymbionts. The first task was to localize the bacteria. Scanning electron microscopy provided us with some marvelous photos of bacteria attached to Symbiodiniaceae cells (Figure 2). We were also particularly interested in exploring whether the algae harbour intracellular bacteria, which we examined through fluorescence in situ hybridization (FISH). The high autofluorescence of Symbiodiniaceae chlorophyll (see Figure 1B – they are quite bright!) made this experiment challenging as the FISH signal was barely discernable. Photobleaching of fixed cells allowed us to drastically reduce autofluorescence, and indeed detect bacteria living within Symbiodiniaceae cells. This was the first time that intracellular bacteria were detected in a wide range of Symbiodiniaceae species.

Figure 2: Scanning electron microscopy visualization of bacteria closely attached to the cell wall of the Symbiodiniaceae Symbiodinium tridacnidorum (left) and Breviolum minutum (right). Photo credit: Justin Maire.

But who are these bacteria? Are extracellular and intracellular bacterial communities different? To determine the taxonomic affiliations of bacteria associated with cultured Symbiodiniaceae, we separated extracellular and intracellular bacteria, and conducted 16S rRNA gene metabarcoding to identify them. Laser-capture microdissection was first attempted to excise the inside of Symbiodiniaceae cells, including the intracellular bacteria, without sampling the cell wall. However, Symbiodiniaceae cells are around 10 µm in diameter, and lasers can only cut so thin: cutting around such a small cell, without destroying the cell itself, was just not feasible. This prompted us to switch to simpler methods: Symbiodiniaceae cells were deposited on a 5-µm strainer, a mesh that would retain Symbiodiniaceae cells but let through the extracellular bacteria, and washed them with sodium hypochlorite to detach even the most closely associated extracellular bacteria. Only Symbiodiniaceae cells and intracellular bacteria remained on the strainer, and these were used for amplicon sequencing.

Data analysis uncovered highly diverse bacterial communities, both at the extra- and intracellular level. Interestingly, while extracellular communities varied across Symbiodiniaceae strains, intracellular communities were largely conserved across 11 cultured Symbiodiniaceae strains encompassing six different genera. This suggests that these intracellular bacteria have conserved, important functions in cultured Symbiodiniaceae. We also detected intracellular bacteria associated with Symbiodiniaceae freshly isolated from the sea anemone Exaiptasia diaphana and the coral Galaxea fascicularis. Determining the functions of Symbiodiniaceae-associated bacteria as well as deciphering the three-way interactions between bacteria, Symbiodiniaceae and cnidarians will be of high interest for future research. While coral bleaching has long been thought of as a coral-Symbiodiniaceae affair, potential bacterial involvement will change the way we view coral bleaching and how we approach mitigation strategies.

Banner photo credit: Justin Maire and Ashley Dungan.

 

Justin Maire

Postdoctoral fellow, The University of Melbourne