Microbial Comparison of Healthy and Bleached Corals from a Spontaneous Coral Bleaching Event off the Coast of Puerto Rico Kylen Tromblay Microbiology Undergraduate Vega Thurber Laboratory Oregon State University Multiple Factors Cause Coral Bleaching Microbes play a Role in the Health of the Corals Study Aimed to Compare Bacteria Present on and in Healthy and Bleached Corals Corals from the Caribbean near Puerto Rico Coral Species Diploria labyrinthiformis Siderastrea siderea Colpophyllia natans Orbicella franksi Process Bioinformatical Analysis Extract Microbial DNA from Coral Tissue and Skeleton DNA Sequencing 1st Step PCR of 16S rRNA Agarose Gel Agarose Gel Electrophoresis 2nd Step PCR of Electrophoresis and Extraction of 16S rRNA and DNA Clean 16S Band Up Extraction Collected Coral Tissue and Skeleton Samples using Used DNA Extraction Bone Cutters to Create Beads to Obtain Microbial Small Samples DNA from the Coral 1st Step PCR 1st Step PCR using Tough Taq, Extraction of 16S 515F and 806R Primers rRNA Band 16S 16S 12S 12S 2nd Step PCR and Clean Up 2nd Step PCR Using Cleaned Up DNA using Primers with Barcodes Magnetic Beads 16S 12S 16S illumina MiSeq was used to Sequence the Data Analyzed the Data Using MOTHUR and PRIMER Beta Diversity of Diploria labyrinthiformis 100% Bacteria_unclassified Clostridiales_unclassified Alphaproteobacteria_unclassified Gammaproteobacteria_unclassified Corynebacterium Planctomycetaceae_unclassified Staphylococcus Acinetobacter 90% unknown_unclassified Rhizobiales_unclassified Proteobacteria_unclassified Bacteroidetes_unclassified Sphingobacteriales_unclassified Blastopirellula Rhodospirillaceae_unclassified Rhodobacteraceae_unclassified 80% Rhodospirillales_unclassified Actinobacteria_unclassified Firmicutes_unclassified Desulfarculus Deltaproteobacteria_unclassified Pseudomonadaceae_unclassified Gp4 Phyllobacteriaceae_unclassified 70% Chloroflexi_unclassified Pseudomonas Desulfovibrionales_unclassified Actinomycetales_unclassified Prosthecochloris Microbulbifer Enterobacteriaceae_unclassified Ilumatobacter 60% Flavobacteriaceae_unclassified Clostridia_unclassified Rhodopirellula Flammeovirgaceae_unclassified Acidimicrobiales_unclassified Hoeflea Paenibacillaceae_1_unclassified Pelagibius 50% Myxococcales_unclassified Bacillales_unclassified Sphingomonas Endozoicomonas Anaerococcus Streptococcus Planctomyces Lactococcus 40% Escherichia_Shigella Methylobacterium Betaproteobacteria_unclassified Kineococcus Rothia Verrucomicrobiaceae_unclassified Anoxybacillus Veillonella 30% Oxalobacteraceae_unclassified Tumebacillus Gp10 Sphingomonadaceae_unclassified Leuconostoc Alishewanella Weissella Desulfopila 20% Stenotrophomonas Ruminococcaceae_unclassified Oceanicola Massilia Pseudoalteromonas Chryseobacterium Pseudoxanthomonas Xanthomonadaceae_unclassified 10% Cardiobacterium Veillonellaceae_unclassified Vibrionaceae_unclassified Paenibacillus Vagococcus Bacillaceae_1_unclassified Empedobacter Propionibacterium 0% Burkholderiales_unclassified Listonella DLB.1 DLB.2 DLB.3 DLB.4 DLB.5 DLH.1 DLH.2 DLH.3 DLH.4 DLH.5 DLH.6 Cloacibacterium Petrimonas Thermicanus Capnocytophaga Diploria labyrinthiformis NMDS DL AMOVA B-H AMONG WITHIN TOTAL SS 0.656289 2.2464 2.90269 DF 1 9 10 MS 0.656289 0.2496 FS 2.62936 p-value 0.024 Diploria labyrinthiformis ANOSIM Diploria labyrinthiformis SIMPER Health Status Percent Similarity Bleached 32.73% Healthy 40.51% Health Status Percent Dissimilarity Bleached vs Healthy 72.34% Diploria labyrinthiformis Chao1 Average Chao1 100 94.99991524 90 80 70 59.95711177 60 50 Average Chao1 Average 40 30 20 10 0 Healthy Bleached Health Status Beta Diversity of Siderastrea siderea 100% Chlamydiales_unclassified Nannocystaceae_unclassified Thalassomonas Marinifilum Ilumatobacter Paramoritella Simkania Rhizobium 90% Lewinella Moraxella Acidimicrobiales_unclassified Prosthecochloris Desulfobacteraceae_unclassified Bacteriovorax Paenibacillus Gp22 80% Chloroflexi_unclassified Rhodospirillales_unclassified Nocardioides Cryomorphaceae_unclassified Gp10 Undibacterium Hyphomicrobiaceae_unclassified Propionibacterium 70% Intrasporangiaceae_unclassified Erythrobacteraceae_unclassified Flavobacteriales_unclassified Veillonellaceae_unclassifie Pasteurellaceae_unclassified Ignavibacterium Clostridiales_unclassified Ferrimonas 60% Gardnerella Lactobacillus Escherichia_Shigella Sphingomonadaceae_unclassified Desulforhopalus Pseudomonas Verrucomicrobiaceae_unclassified Blastopirellula 50% Pseudoalteromonas Rubritalea Sphaerotilus Enterobacteriaceae_unclassified Actinobacteria_unclassified Verrucomicrobiales_unclassified Pseudoxanthomonas Haliea 40% Photobacterium Lactococcus Neisseria Dermacoccus Wautersiella Myxococcales_unclassified Exiguobacterium Vibrionaceae_unclassified 30% Deltaproteobacteria_unclassifie Pseudomonadaceae_unclassified Streptococcus Fusobacterium Saprospiraceae_unclassified Planctomycetaceae_unclassified Rhodococcus Rhodospirillaceae_unclassified 20% Actinomyces Endozoicomonas Rhizobiales_unclassified Gemella Flammeovirgaceae_unclassified Bacillales_unclassified Staphylococcus Sphingobacteriales_unclassified Veillonella Flavobacteriaceae_unclassified 10% Sphingomonas Labrenzia Campylobacter Dyella Rhodobacteraceae_unclassified Paenibacillaceae_1_unclassified Proteobacteria_unclassified Acinetobacter 0% Alphaproteobacteria_unclassified Xanthomonadaceae_unclassified Gammaproteobacteria_unclassified Bacteroidetes_unclassified Bacteria_unclassified Siderastrea siderea NMDS Siderastrea siderea ANOSIM Siderastrea siderea SIMPER Health Status Percent Similarity Bleached 36.70% Healthy 30.83% Health Status Percent Dissimilarity Bleached vs Healthy 66.90% Siderastrea siderea Chao1 Average Chao1 350 333.5178855 300 250 201.2862981 200 150 Average Chao1 Average 100 50 0 healthy bleached Health Status Future Plans • Compare OTU identification using Greengenes instead of Silva database • Analyze alpha diversity of each coral sample • PCOA (mothur) • Multiple corrections (indi taxa) • Read paper • Check dispersion (permanova package) Acknowledgments • Dr. Rebecca Vega Thurber • Dr. Ernesto Weil • Emily Schmeltzer, Stephanie Rosales, Adriana Messyasz, Becca Maher, Grace Klinges, Ryan McMinds • Dr. Ryan Mueller • Oregon State University Center for Genome Research and Biocomputing References • National Ocean Service. 2018. What is coral Bleaching? Ocean Facts • Svoboda E. 2018. A cure for Coral Reefs? bioGraphic • Peixoto R, Rosado P, de Assis Leite DC, Rosado A, Bourne D. 2017. Beneficial Microorganisms for Corals (BMC): Proposed Mechanisms for Coral Health and Resilience. Front. Microbiol 8: 341 • Illumina. 2011. High-Speed, Multiplexed 16S Microbial Sequencing on the MiSeq System • Schloss P.D. 2009. Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75(23):7537-41.