Preparation of extracts: Coral samples of orange and red colonies of Montipora capitata were collected in Kaneohe Bay, Oahu. Frozen samples were extracted in Millipore® water, which was replaced daily for 3 d. Extracts were filtered, lyophilized, and weighed. We tested extracts at concentrations approximating those naturally found in the coral tissues (Gochfeld & Aeby 2008). To determine the volumetric concentrations of extracts, the surface area of each piece of coral was calculated using the wax technique (Gochfeld 1991). Tissue volume was determined by multiplying surface area by tissue depth measured from replicate decalcified pieces of M. capitata. Extract concentrations were determined as g dried extract ml–1 of coral tissue. Extracts were re-suspended to these concentrations in Millipore® water for use in bacterial growth assays.

Bacterial strains tested: The strains used for our antibacterial assays were selected as model systems to represent a range of potential bacterial pathogens from the marine environment, including known coral pathogens (Aurantimonas coralicida, Serratia marcescens, Vibrio coraliilyticus, Vibrio shiloi) and human enteric bacteria that have the potential to enter near-shore waters and can survive in the marine environment (Yersinia enterocolitica), and strains previously isolated from the surfaces of Hawaiian corals (Klebsiella pneumonia, Vibrio agarivorans) (Gochfeld & Aeby 2008). In addition, bacterial strains isolated from surfaces of Montipora capitata (OCN001, OCN002, OCN003, OCN008, O1-1, O2-8, O2-12, R1-13, R5-5, R5-13, R5-29) by Dr. Sean Callahan’s lab at University of Hawaii were also used. Two of these, OCN002 and OCN008, are pathogens associated with Montipora White Syndrome (Ushijima et al. 2012, 2014).

Growth inhibition assay (Gochfeld & Aeby 2008): Assays were run in triplicate in 96 well plates. Plates contained 3 wells of bacteria + each extract (n = 10 replicate corals of 2 color morphs), along with the following controls: bacteria only, media only, and extract solution to control for the coloration of extracts. Aqueous extracts were dissolved in Millipore® water. Twenty-four hour bacterial cultures in exponential growth were diluted to optical density (OD) 600, and 100 μl were added to each well. 10 μl of extract were added to the experimental wells. Wells were mixed by carefully pipetting their contents, and an initial reading (time 0) of OD was made on a BioTek Synergy™ HT Multi-Detection Microplate Reader. Plates were then covered with foil and incubated on a shaker for 24 h and a final reading was made. Prior to each reading, wells were mixed again to resuspend any settled material.

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