Localization and symbiotic status of probiotics in the coral holobiont

Abstract

Corals establish symbiotic relationships with microorganisms, especially endosymbiotic photosynthetic algae. Although other microbes have been commonly detected in coral tissues, their identity and beneficial functions for their host are unclear. Here, we confirm the beneficial outcomes of the inoculation of bacteria selected as probiotics and use fluorescence in situ hybridization (FISH) to define their localization in the coral Pocillopora damicornis. Our results show the first evidence of the inherent presence of Halomonas sp. and Cobetia sp. in native coral tissues, even before their inoculation. Furthermore, the relative enrichment of these coral tissue-associated bacteria through their inoculation in corals correlates with health improvements, such as increases in photosynthetic potential, and productivity. Our study suggests the symbiotic status of Halomonas sp. and Cobetia sp. in corals by indicating their localization within coral gastrodermis and epidermis and correlating their increased relative abundance through active inoculation with beneficial outcomes for the holobiont. This knowledge is crucial to facilitate the screening and application of probiotics that may not be transient members of the coral microbiome.

Importance: Despite the promising results indicating the beneficial outcomes associated with the application of probiotics in corals and some scarce knowledge regarding the identity of bacterial cells found within the coral tissue, the correlation between these two aspects is still missing. This gap limits our understanding of the actual diversity of coral-associated bacteria and whether these symbionts are beneficial. Some researchers, for example, have been suggesting that probiotic screening should only focus on the very few known tissue-associated bacteria, such as Endozoicomonas sp., assuming that the currently tested probiotics are not tissue-associated. Here, we provide specific FISH probes for Halomonas sp. and Cobetia sp., expand our knowledge of the identity of coral-associated bacteria and confirm the probiotic status of the tested probiotics. The presence of these beneficial microorganisms for corals (BMCs) inside host tissues and gastric cavities also supports the notion that direct interactions with the host may underpin their probiotic role. This is a new breakthrough; these results argue against the possibility that the positive effects of BMCs are due to factors that are not related to a direct symbiotic interaction, for example, that the host simply feeds on inoculated bacteria or that the bacteria change the water quality.

Keywords: BMC; Cobetia sp.; FISH; Halomonas sp.; coral; coral-associated microbes; location; probiotics; tissue.

Fig 1

(A) Experimental design for temperature treatment and inoculation group. (B) Mesocosm experimental design, detailing inoculation frequency and temperature regime.

Fig 2

Relative abundances of Cobetia sp. (A) and Halomonas sp. (B) found in P. damicornis fragments, during the four timepoints of the experiment: T0 (day 1), T1 (day 10), T2 (day 26), and T3 (day 35), n = 5. Jitter points represent values from individual replicates, crosses represent the mean values of each group, and box edges represent quartiles, whereas middle horizontal bars represent median values.

Fig 3

Fluorescence in situ hybridization images show the specific bacterial symbionts associated with tissue samples of P. damicornis of the experimental groups treated only with saline solution (placebo) and subjected to heat treatment on the 26th day of the experiment (T2). The first column shows samples hybridized with a probe specifically designed for Cobetia sp. (in green), the middle column shows samples hybridized with a probe specifically designed for Halomonas sp. (in green), and the last column shows samples hybridized with a general Eub-338 probe (in green). All hybridizations were performed with a NonEub-338 probe (in red) to account for unspecific staining, and all sections were also stained with DAPI (in blue). Sym = Symbiodiniaceae cells; epi = epidermis; gast = gastrodermis; sp = spyrocyst; arrowhead = granular gland cell; arrow = bacteria. Scale bars in first four lines = 20 µm, scale bars at last line = 5 µm.

Fig 4

Fluorescence in situ hybridization images show the specific bacterial symbionts associated with tissue samples of P. damicornis incubated with BMC and subjected to heat treatment on the 26th day of the experiment (T2). The first column shows samples hybridized with a probe specifically designed for Cobetia sp. (in green), the middle column shows samples hybridized with a probe specifically designed for Halomonas sp. (in green), and the last column shows samples hybridized with a general Eub-338 probe (in green). All hybridizations were performed with a NonEub-338 probe (in red) to account for unspecific staining, and all sections were also stained with DAPI (in blue). Sym = Symbiodiniaceae cells; epi = epidermis; gast = gastrodermis; mes = mesoglea; arrowhead = bacterial aggregates; arrow = bacteria cells. Scale bars in first four lines = 20 µm, scale bars at last line = 5 µm.

Fig 5

Comparative photographs of P. damicornis coral fragments used in the aquarium experiments before (day 1) and after (day 35) exposure to heat stress and treatment with the probiotic consortium. Colored squares indicate colors found in the coral chart for each replicate, and red crosses represent dead coral fragments.

Fig 6

F_v/F_m values of coral fragments treated with a placebo or the BMC consortium and exposed to heat stress or maintained at a constant temperature of 26°C (n = 5). Gray dots represent the specific F_v/F_m values found for each replicate during different timepoints of the experiment. Red lines represent the fit of a mixed-effect linear model.

Fig 7

Calcification (A) and net primary productivity (B) values in P. damicornis fragments during the four timepoints of the experiment: T0 (day 1), T1 (day 10), T2 (day 26), and T3 (day 35), n = 4. Jitter points represent values from individual replicates, crosses represent mean values of each group, box edges represent quartiles, whereas middle horizontal bars represent median values.