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.