Sampling
All fish were terminally sampled after the ten-day trial (November 3,
2020). The fish were not fed on the day of sampling. The final mean mass
of the fish was 23.3 g (±7.2 SE) across all families and treatments (no
treatment effect on fish body weight was detected). Three fish were dip
netted from each tank and humanely euthanized immediately in an overdose
solution of clove oil (Toews et al., 2019). Of the 72 tanks, four tanks
(control) had 100 % mortality and those replicates were excluded from
the study, bringing the total number of samples to 204 fish (72
probiotic treated fish, 72 antibiotic treated fish, 60 control fish) and
68 water samples (one per tank)). The sampled fish were immediately
weighed and dissected, with the entire GI tract placed in a 50 mL tube
with 35 mL of a highly concentrated salt buffer (ammonium sulfate, 1 M
sodium citrate, 0.5 M EDTA, H2SO4 to
bring the pH to 5.2) for preservation for later RNA and DNA extraction.
Additionally, 500 mL water samples were collected from each of the tanks
(N=68) before sampling the fish and filtered immediately using
0.22-micron pore size, 47 mm diameter polycarbonate filters (Isopore™,
Millipore, MA). All samples (tissue in preservative and the filters)
were stored at –20 °C, until used for DNA or RNA extraction.