In Mo’orea, to assess the impact of sea cucumber removal on sediment- and coral-associated microbiomes, as well as how farmerfish turf on the base of corals might affect disease prevalence, we erected thirty-six 50 cm x 50 cm x 12 cm tall cages using 1 cm2 grid metal screening to contain or exclude sea cucumbers and prevent access by coral consumers. Cages were situated in an ~85 m2 sand patch within the fringing reef area utilized in our initial experiment described above and were separated from adjacent cages by ≥60 cm, creating a 6 x 6 grid of enclosures. Each cage was stocked with either zero, one, or two H. atra (12 cages treatment-1) that were approximately 9-14 cm in length, as is typical for individuals at our site. Density treatments were assigned at random. Cages were inspected daily to ensure that density treatments were maintained (they were), and sea cucumbers outside of cages were removed daily from within about 10 m of the 2 m deep area where cages were situated. All cages were brushed every other day to prevent fouling.
Seven days after applying sea cucumber treatments, sediment samples were taken for microbiome analyses by scraping 30-40 mL of surficial sediment from the top ~5 mm of each caged area into a small Whirl-Pak. Samples were immediately placed on ice and stored in a -80°C freezer upon return to shore. Following sediment sampling, three A. pulchra outplants were embedded into the sediment of each cage (108 outplants total) to test the potential effects of (i) sea cucumber density, and (ii) protective effects of farmerfish-cultivated turf algae on coral health and microbiomes (see below). Corals used were approximately 8-10 cm in length and were initially fragmented from numerous A. pulchra thickets adjacent to our study area and outplanted using the methods described above.
Of the three outplants included in each cage, two were fragmented from colonies in the field in such a way as to include farmerfish-generated turf algae at their base, while the third was fragmented so that it lacked turf at its base. These three outplants were embedded into the sediment as follows: (i) coral lacking turf planted in direct contact with benthic sediment (hereafter “no turf”), (ii) coral separated from direct contact with sediment by turf algae growing at its base (hereafter “turf”), or (iii) coral with turf on its base, but embedded more deeply into the sediment so that the living coral tissue was in direct contact with the sediment (hereafter “embedded turf”). Percent coral tissue mortality among outplants was visually estimated daily for 36 days. The microbiomes of all corals and the sediment within a cage were sampled when one or more outplants within that cage exhibited ≥50% tissue mortality or when the experiment was terminated on day 36.
Organism identifiers:
Coral: Acropora pulchra, LSID (urn:lsid:marinespecies.org:taxname:207015)
Cucumber: Holothuria atra, LSID (urn:lsid:marinespecies.org:taxname:1672768)
farmerfish: Stegastes spp, LSID (urn:lsid:marinespecies.org:taxname:203822)