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Microbial aggregates can be found in many different animals and plants. These aggregates play important roles in their host with various ecological functions, like symbiosis or parasitism. Although several reports have claimed the finding of microbial aggregates reside in corals; however, the knowledge of their physiology, biochemistry, morphology, genetics, ecological functions… is barely studied.

In this research, we intensively studied different characteristics of microbial aggregates within the coral, Stylophora pistillata, covering from their distribution in different geographic area and coral anatomy to bacteria composition, genomics, and elements variation in the coral-associated microbial aggregate (CAMA).

Combing fluorescence in situ hybridization (FISH) technique with confocal microscopy and lightsheet microscopy, we were able to determine the distribution, dimension, abundance, and bacteria cell number of each individual CAMA.

The results turned out that CAMAs in Kenting, Taiwan and Okinawa, Japan exhibited variation in certain features such as bacteria composition, cell density, and size, indicating that there is biogeographic specificity among CAMAs in S. pistillata at the two sites. Most of the CAMA distributed only around the tentacles of S. pistillata in the both sampling sites and every coral polyp contained average 670,000 bacterial cells in the form of CAMA. This is the first time in the world to estimate bacterial cell number in individual CAMA or microbial aggregates (Figure 1).

Furthermore, 16S rRNA gene amplicon sequencing results suggested that CAMAs were mainly dominated by Endozoicomonas sp. which is commonly believed to be a critical beneficial bacterium, functionally related to coral health. The CAMA-forming Endozoicomonas sp. were classified into a new phylogenetic group under the genus Endozoicomonas. Meta-genomic analysis result indicated these Endozoicomonas may have the metabolic potential to accumulate phosphate and transport nutrients.

In order to further prove this assumption, we coupled the FISH technique with Nanoscale secondary ion mass spectrometry (NanoSIM), labelled CAMA by FISH and detected element composition of CAMA and the surrounding coral’s or symbiotic algal cells by NanoSIM. The result showed significantly high phosphorus signal inside CAMA, but very low phosphorus signal in nearby coral and Symbiodiniaceae cells. The result suggested CAMA may participate the phosphate cycling in the coral, which supports the genomic analysis result.

Endosymbiotic microbial aggregates are crucial for many organisms to adapt to environment or to overcome environmental stress. Previous studies showed CAMA widely prevalent among different geographic area and different coral specie, suggesting an intimate and close relation between CAMA and coral.

Our research, for the first time, uncovered that CAMA can be dominated by single or multiple bacteria strains and its sizes, abundance, cell density, and distribution may vary different geographic regions. Through genomic analysis and NanoSIMS results, we proved that CAMA may take part in the phosphate cycling to their host and help to regulate photosynthesis efficient of the symbiotic algae.

The research team comprised Dr. Sen-Lin Tang at Biodiversity Research Center, Academia Sinica, the core facility nanometer-scale secondary ion mass spectrometry (NanoSIMS) maintained by Institute of Astronomy and Astrophysics and Institute of Earth Science, Dr. Bi-Chang Chen at Research Center for Applied Sciences, Academia Sinica, and Prof. David Bourne from James Cook University, Australia. Our great gratitude is to the first author Dr. Naohisa Wada at Biodiversity Research Center, Academia Sinica; most of the work was mainly established and conducted by Dr. Wada. This study has been published in Science Advance in July 7, 2022.

Figure 1. Three-dimensional image of CAMAs (red) within a single coral polyp (a) using a specific FISH probe for all bacteria, and cross-sectional images of CAMA from Kenting and Okinawa using two specific FISH probes for the multiple bacterial phylotypes (b-d, red and green indicates Endo-group A and B, respectively). The results indicate that multiple bacterial phylotypes coexist in corals from Kenting (b and c) but a single phylotype detected in corals from Okinawa (d).