A feces addition experiment was conducted three times (Final N = 11 replicates) using fragments of multiple species of Pocillopora in Moorea, French Polynesia. An initial experimental iteration was performed in October 2020 (four replicates); two additional iterations took place in June 2021 (5 replicates each). Coral colonies were collected from the fore reef on the north shore of Mo'orea on Scuba at 4-8 m depth and immediately returned to the research station and placed into flow-through seawater tables. After 24 h, all corals were cut into five ~10 cm long fragments and left to acclimate for 48 hours, and any macrosymbionts (e.g., Trapezia crabs, shrimp) were removed. All fragments were transferred to glass jars containing 500 ml of 0.2 μm-filtered (sterile) seawater with bubblers and air stones to promote aeration and water circulation, and photographed under a dissection microscope with 3.5-180X zoom (AmScope SM-1TSZZ-144S-10M).
We then blindly assigned each jar containing a coral fragment to one of the following five treatments (such that one fragment from each coral colony was assigned to each treatment, 4 replicate colonies x 5 treatments = 20 coral fragments in separate jars): fresh feces from a corallivorous butterflyfish (FC); fresh feces from a grazer/detritivore (FG); sterilized feces from a corallivorous butterflyfish (SC); sterilized feces from a grazer/detritivore (SG); no-feces control (C). For the fresh feces treatments (FC, FG), we applied 100 µl of fresh feces isolated from the hindgut of the butterflyfish Chaetodon ornatissimus (FC) or the grazer/detritivore Ctenochaetus striatus (FG) directly onto each coral fragment. Fish were collected on the north shore of Mo'orea using a Hawaiian sling and immediately transported on ice to the laboratory. Feces were isolated from the hindgut using sterile tools by making an incision from the anus to the pelvic fin and removing the intestinal tract; feces were then squeezed from the hindgut into sterile collection tubes using sterile tweezers and pipetted onto coral fragments using 1 ml filter tips that were modified to widen the tip opening using a sterilized razor blade. For the sterilized feces treatments (SC, SG), fecal pellets were sterilized in a pressure cooker for 40 minutes at 120 °C and then applied in the same manner as fresh feces. The fresh feces used in the experiment were subsampled for DNA extractions by preservation in DNA/RNA Shield (Zymo Research, CA). No manipulation was conducted on the no-feces control fragments. The experiment ran for ~22 hours; this treatment duration is reflective of time periods over which corals may sometimes be in direct contact with fish feces in situ (up to 48 hours; Ezzat et al., 2019; Ezzat et al., 2021).
Minor modifications were incorporated into the design of the second and third experimental iterations: for these iterations, five replicate colonies were used instead of four. Additionally, fecal treatments in these iterations were composed of feces from two individuals per fish species that were mixed prior to application (instead of using feces from a single fish individual per treatment as in the first experimental iteration).
To test how microbial communities in fish feces affected coral health, we quantified the frequencies and sizes of coral lesions caused by fecal treatments (in addition to measuring the photosynthetic efficiency of each fragment). In brief, fecal pellets were removed from each fragment and photographs were taken using a dissection microscope.
