2.2 Animals and sample collection
Samples from adult sea cucumbers Holothuria leucospilota and their surrounding environments (water and sediments) were collected in December 2019 from each of the field sites. Sea cucumbers (5 animals per site, separated at least by 10m) were isolated in Ziploc bags filled with water from the collection site. In addition, surface sediment samples of the top 3 cm (n = 3 per site) were collected around the sampled animals using sterile 15 mL falcon tubes. In parallel, 2L seawater samples (n = 3 per site) were also collected using sterile plastic bottles wrapped with aluminium foil. All samples were immediately stored in ice for transportation and processed immediately in the laboratory at the University of Hong Kong.
Sea cucumbers were dissected to obtain microbial samples from the body surface and guts. For this, animals were rinsed under sterile seawater twice to remove particulates such as sand, algae and other loose organic matter loosely attached to the surface. Then, a sterile cotton swab was rolled over the whole outer body surface to collect microbial samples from the skin. For the gut microbiome collection, the outer surface of the sea cucumbers was first sterilized with 70 % ethanol to reduce contamination. After that, the ventral part of the animal was cut open with a sterile scalpel. A 2 cm segment of the midgut including the luminal epithelium was then collected from each animal. All skin and gut samples were individually placed into sterile 1.5 mL microcentrifuge tubes, snap-frozen in liquid nitrogen, and stored at -80 ℃ until required for further analysis. For the seawater samples, a two-step filtering process was conducted. First, samples were passed through Millipore membrane filters of 0.45µm pore size to remove large particles, and water was then filtered again using Millipore membrane filters of 0.22 µm pore size. These last filters were transferred to 15 mL falcon tubes. Sediment samples were centrifuged (4,000 g x 5 minutes), and the supernatant of excess seawater was removed without disturbing the sediment. Sediment and water samples were stored at -80 ℃ until required for further analysis.