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. 2023 Feb 14;11(1):e0246422.
doi: 10.1128/spectrum.02464-22. Epub 2023 Jan 18.

Mutualistic Interactions between Dinoflagellates and Pigmented Bacteria Mitigate Environmental Stress

Affiliations

Mutualistic Interactions between Dinoflagellates and Pigmented Bacteria Mitigate Environmental Stress

Toshiyuki Takagi et al. Microbiol Spectr. .

Abstract

Scleractinian corals form symbiotic relationships with a variety of microorganisms, including endosymbiotic dinoflagellates of the family Symbiodiniaceae, and with bacteria, which are collectively termed coral holobionts. Interactions between hosts and their symbionts are critical to the physiological status of corals. Coral-microorganism interactions have been studied extensively, but dinoflagellate-bacterial interactions remain largely unexplored. Here, we developed a microbiome manipulation method employing KAS-antibiotic treatment (kanamycin, ampicillin, and streptomycin) to favor pigmented bacteria residing on cultured Cladocopium and Durusdinium, major endosymbionts of corals, and isolated several carotenoid-producing bacteria from cell surfaces of the microalgae. Following KAS-antibiotic treatment of Cladocopium sp. strain NIES-4077, pigmented bacteria increased 8-fold based on colony-forming assays from the parental strain, and 100% of bacterial sequences retrieved through 16S rRNA amplicon sequencing were affiliated with the genus Maribacter. Microbiome manipulation enabled host microalgae to maintain higher maximum quantum yield of photosystem II (variable fluorescence divided by maximum fluorescence [Fv/Fm]) under light-stress conditions, compared to the parental strain. Furthermore, by combining culture-dependent and -independent techniques, we demonstrated that species of the family Symbiodiniaceae and pigmented bacteria form strong interactions. Dinoflagellates protected bacteria from antibiotics, while pigmented bacteria protected microalgal cells from light stress via carotenoid production. Here, we describe for the first time a symbiotic relationship in which dinoflagellates and bacteria mutually reduce environmental stress. Investigations of microalgal-bacterial interactions further document bacterial contributions to coral holobionts and may facilitate development of novel techniques for microbiome-mediated coral reef conservation. IMPORTANCE Coral reefs cover less than 0.1% of the ocean floor, but about 25% of all marine species depend on coral reefs at some point in their life cycles. However, rising ocean temperatures associated with global climate change are a serious threat to coral reefs, causing dysfunction of the photosynthetic apparatus of endosymbiotic microalgae of corals, and overproducing reactive oxygen species harmful to corals. We manipulated the microbiome using an antibiotic treatment to favor pigmented bacteria, enabling their symbiotic microalgal partners to maintain higher photosynthetic function under insolation stress. Furthermore, we investigated mechanisms underlying microalgal-bacterial interactions, describing for the first time a symbiotic relationship in which the two symbionts mutually reduce environmental stress. Our findings extend current insights about microalgal-bacterial interactions, enabling better understanding of bacterial contributions to coral holobionts under stressful conditions and offering hope of reducing the adverse impacts of global warming on coral reefs.

Keywords: Symbiodiniaceae; carotenoid; coral holobiont; dinoflagellate; microbiome manipulation; pigmented bacteria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Colony-forming assays of bacteria in microalgal cultures after antibiotic treatment. Colony-forming assays were performed to evaluate bacterial survival after antibiotic treatment in F/2 agar medium. One thousand harvested cells of established cultures (Abx-4077, Abx-2466, and SGF) were resuspended in 100 μL of filtered seawater, and serial dilutions of samples were spread on marine agar plates without antibiotics. Numbers of colonies were counted after incubation at 25°C for 7 days.
FIG 2
FIG 2
Bacterial community analysis of microalgal cultures before and after antibiotic treatment. Bacterial community composition (relative abundance %) of microalgal cultures before and after antibiotic treatment, based on extracted total DNA and 16S rRNA gene amplicon sequencing (Illumina MiSeq). Circle sizes represent relative abundances. Data are provided as means of relative abundances from three biological replicates. Abx-4077 and Abx-2466 were established on F/2 agar plates containing antibiotics (50 μg/mL kanamycin, 100 μg/mL ampicillin, and 50 μg/mL streptomycin) from NIES-4077 and CCMP2466, respectively. Durusdinium sp. strain SGF was isolated directly from the coral Galaxea fascicularis with F/2 agar plates supplemented with antibiotics. Bacterial taxa with >0.5% relative abundance in all three biological replicates in at least one microalgal culture were adopted.
FIG 3
FIG 3
Antibiotic susceptibility tests of pigmented bacteria on marine agar plates. Pigmented strains (GF1, C-4077, C-2466, and DU1) were spread on marine agar (MB) plates with antibiotics (50 μg/mL of kanamycin [Kan], 100 μg/mL of ampicillin [Amp], or 50 μg/mL of streptomycin [Sm]) at 24°C for 4 to 6 days.
FIG 4
FIG 4
Fluorescence in situ hybridization (FISH) analysis of pigmented bacteria in microalgal cultures. DAPI (4′,6-diamidino-2-phenylindole) and cyamine 3 (Cy3) fluorescence were detected using a DAPI filter (excitation: 360/40 nm; emission: 460/50 nm; dichroic: 400 nm) and a TRITC (tetramethylrhodamine) filter (ex: 545/25 nm; em: 605/70 nm; dichroic: 565 nm), respectively. Microalgal samples were hybridized with CF319a/b (left panel) and NonEUB338 (right panel) probes labeled with Cy3. Scale bars = 10 μm. S, Symbiodiniaceae.
FIG 5
FIG 5
Analysis of carotenoids produced by bacterial isolates. (a) Extracts of pigmented bacteria and their TLC analyses. Cell pellets were extracted with acetone by vortexing. Reverse-phase thin-layer chromatography separation of synthetic carotenoid standards (lane 1) and pigments extracted from the pigmented bacteria listed below (lanes 2 to 5). Lane 2, GF1; lane 3, C-4077; lane 4, C-2466; lane 5, DU1. Absorption spectra of extracts of (b) GF1, (c) C-4077, (d) C-2466, (e) DU1, and (f) 0.1 mg/mL zeaxanthin standard.
FIG 6
FIG 6
Effect of bacterial communities on the light tolerance of cultured Symbiodiniaceae. Maximum quantum yield of photosystem II (PSII) (variable fluorescence divided by maximum fluorescence [Fv/Fm]) in cultured Symbiodiniaceae under non-stressful lighting (24°C, 50 μmol photons m−2 s−1) and stressful lighting (24°C, 400 μmol photons m−2 s−1). To analyze the effects of light stress on dinoflagellate cells, we compared the Fv/Fm of NIES-4077 and Abx-4077 cells under stressful and non-stressful lighting at the same time. All data are given as means ± standard error of the mean (n =3). Statistical analysis was conducted using one-way analysis of variance followed by Tukey’s post hoc test for multiple-comparison tests. P values less than 0.05 were considered significant. Significant differences are represented by different letters. Colors of letters correspond to each sample (gray; Abx-4077 [Control], red; Abx-4077 [light stress], black; NIES-4077 [Control], yellow; NIES-4077 [light stress]). Abx-4077 was dominated by sequences affiliated with Maribacter, with a mean abundance of 100%, which had a relative mean abundance of 1.2% in the parent culture of NIES-4077.

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