Microbiome dynamics of the Coral Madracis auretenra and Coral Probiotic Response to Heat Stress

Corals are threatened by the rapid deterioration of the environmental conditions that affect their health, stability, and maintenance. The variable environment also influences the coral-associated microbial communities, inducing shifts in their structure and leading to the loss of symbiotic, mutualistic, and commensal relationships. However, some corals are resistant and have adapted to the environmental changes. This response is thought to be mediated by the coral-associated microbiome, and studying this interaction and its dynamic could lead to an understanding of how bacteria are involved in the coral host response to environmental stress. In this study, we evaluated the dynamics of the coral Madracis auretenra-associated microbiome under different conditions and manipulated the coral response to heat stress by inoculating probiotic bacteria. First, we assessed the microbiome associated with M. auretenra from Santa Marta, Colombian Caribbean in two contrasting locations, one urban and the other non-urban (or protected), in two years (four-time points across dry and rainy seasons), and based on the health status (healthy and stressed), whether the corals were in contact or not with algae, and by compartments (mucus, tissue, and seawater). Second, we explored the status of coral M. auretenra in both locations and evaluated the presence of probiotic traits in culturable bacteria from the urban M. auretenra. Third, we assessed the physiological and microbiome response of two coral species in a heat-stress experiment and when inoculating an assembled potential probiotic consortium composed of bacteria native to the urban M. auretenra, and displaying different beneficial properties. Using a 16S rRNA V4 region amplicon sequencing-based approach, we observed that the microbiome of M. auretenra showed patterns in alpha diversity that differed among sites, seasons, and between the coral compartments and the seawater, but no differences in the compositional structure were observed indicating similar microbiomes over time and space. The composition of the microbiome varied but reshaped seasonally showing the dominance of Endozoicomonas and a core microbiome composed of this genus and members of the Vibrionaceae and Rhodobacteraceae. We observed that Endozoicomonas abundance is negatively affected during the rainy season and was correlated with changes in temperature, sediments, and nutrients. Family Vibrionaceae remained stable across seasons, locations, and health conditions. These results suggested a balanced and beneficial microbiome. The status of M. auretenra revealed the presence of a resistant or adapted phenotype in the urban location. After assessing ecological attributes, we observed a similar coverage when comparing patches of M. auretenra from both locations. The urban M. auretenra exhibited a high abundance of vibrios in coral tissues and despite observing more frequency of signs of stress compared to the protected M. auretenra, a “good” health index condition was obtained, so we hypothesized the beneficial microbiome is responsible. To test this, we isolated 132 bacterial strains from the healthy urban M. auretenra and screened for probiotic traits such as antioxidant, antagonistic, and nutrient-scavenging activities. Culturable bacteria were affiliated with 11 genera, including Vibrio, Shewanella, Bacillus, Exiguobacterium, Priestia, and Niallia, among others. Vibrio was the most dominant genus in the culturable fraction of this coral. We uncovered the predominant presence of catalase, anti-quorum sensing, and the production of siderophore activities among the bacterial isolates. We proposed a list of 24 bacterial isolates as probiotic precandidates selected for exhibiting three or more of these traits. Most of the precandidates were Vibrio and Bacilli strains. The precandidates were then evaluated for additional traits and seven strains were selected due to rapid growth, non-pathogenicity, and morphological differences. These strains were assembled in a consortium that was tested in a heat-stress experiment. The assembled consortium was composed of two strains of Vibrio, two strains of Bacillus, and one strain of Priestia, Exiguobacterium, and Fictibacillus. All members displayed catalase and anti-quorum sensing activities, among other traits accomplishing functional redundancy. Acclimatized coral fragments of M. auretenra and Porites sp. were subjected to a heat stress regime (30–31°C) for 11 days. Then, the temperature was re-established (24 °C) and fragments were allowed to recover for 20 days. Corals were simultaneously treated with the consortium and a placebo (sterile saline solution), in independent triplicated treatments. We assessed the physiological and microbiome responses by collecting data from symbiont density, chlorophyll a, tissue thickness and color loss (paling and bleaching), and microbiome data for Porites sp. fragments. We additionally sequenced the genome of each member of the consortium and identified putative probiotic functional genes. The inoculation of the consortium generated a delayed appearance of signs of deterioration and attenuated the color loss in both species. Moreover, the consortium treatment significantly reduced symbiont and chlorophyll loss during the heat stress and post-stress time points, although no effect was observed on the tissue thickness parameter. The application of the consortium maintained the microbiome of Porites sp. relatively stable during the stress timepoint and prevented the increase of opportunistic bacteria such as Fusibacter and Rhodobacteraceae which were significantly more abundant in the placebo treatment. The genome-based functional analysis detected the presence of genes involved in the antioxidant defense (sodA, cat, ahpC), osmotic balance (gbs, betA, mtlD), and nutrition through siderophore production (ddc, iucC), vitamin B12 synthesis (btuR, cobP), and sulfur metabolism (gshA), all of them considered as probiotic properties. These findings provided key evidence of a stable microbiome in M. auretenra that reshapes under seasonal rather than local conditions and which dynamics are linked to the dominance of Endozoicomonas, the stability of Vibrionaceae, and the complex interactions that enhance a balanced and beneficial microbiome even under a variable environment. This beneficial microbiome served as a source of probiotic bacteria and here we provide a first insight into inoculating a functionally redundant bacterial consortium composed of non-obligate and unconventional strains with positive effects on manipulating the coral microbiome and the coral physiological response under experimental heat stress which is promising for coral reefs conservation.