Collection of intestinal contents from laboratory reared fish
Total intestinal contents were collected from Río Choy surface fish and
Tinaja, Molino, and Pachón cavefish that were surface sterilized as
embryos and raised on the same recirculating water system and diet as
described below. These fish originated from parents that were bred for
at least five generations in the laboratory of Clifford Tabin at Harvard
Medical School (TMR2, SMR1, PMR1, MMR7). To sterilize the embryos, we
incubated them in 0.3096% sodium hypochlorite for 5 minutes, then 3.2
mM Sodium thiosulfate for 1 minute, then rinsed them in fish ready
water. Sterilized embryos were allowed to hatch in 1L containers at a
density of 20 fish per container. The fish were fed L-type rotifers to
14 days post fertilization, then transferred to 20-gallon tanks on the
same recirculating system at a density of approximately 2 fish per liter
of water. The fish were fed Artemia fransiscana until 60 days
post fertilization, then New Life Spectrum Thera-A Medium Sinking
Pellets. At six-months post fertilization, fish were euthanized in
MS-222 five hours after eating. The fish were weighed, and total
intestinal contents were collected using the same method used for
field-caught fish. Samples were stored at room temperature for 24 hours,
then stored at -20°C until DNA extraction was carried out. The sex of
most surface fish samples was apparent while most cavefish samples did
not have clearly visible gonads. Gut contents from 9 surface (3 male, 4
female, 2 undifferentiated), 9 Pachón (1 male, 8 undifferentiated), 8
Tinaja (8 undifferentiated), 9 Molino (6 male, 3 undifferentiated) were
shipped on dry ice to Microbiome Insights Inc. (Vancouver, BC, Canada)
for DNA isolation and sequencing. The weight and sex of fish used for
sequencing is shown in Figure S1B .