Sampling and preparation of gut microbiome samples
Colonies of Cyprinodon pupfishes were collected from two
hypersaline lakes on San Salvador Island, Bahamas (Crescent Pond and
Osprey Lake) and Lake Cunningham, Bahamas in March, 2018 and were reared
in aquaria at the University of California, Berkeley. Additional
generalist populations were collected in May, 2018 from Fort Fisher
Estuary in North Carolina. Cualac tessellatus eggs were provided
by the Zoological Society of London and reared in the lab to produce a
large second generation used for the four samples in this study. All
samples, except for the recently collected NC population, came from
first or second-generation captive-bred individuals reared in aquaria
(40–80 L) according to species and location at 5–10 ppt salinity
(Instant Ocean synthetic sea salt) and between 23 to 30°C. Individuals
used for this study were first fed once daily ad libitum with a
single commercial pellet food (New Life Spectrum Cichlid Formula, New
Life International, Inc., Homestead, FL), containing 34% crude protein,
5% crude fat, and 5% crude fiber, for one month without exposure to
any other food or tankmates. All animal care and experiments were
conducted under approved protocols and guidelines of the University of
California, Berkeley Institutional Animal Care and Use Committee
(AUP-2018-08-11373).
In total, forty fishes were euthanized in an overdose of MS-222 and the
entire intestinal tissue was immediately excised (Cyprinodontidae do not
possess stomachs; Wilson and Castro, 2010) for DNA extraction. Standard
length and gut length were measured for all samples (Table S1). Five
individuals (F2 generation) from each of three species
(C. variegatus , C. brontotheroides , and C.
desquamator ) in both lake populations from San Salvador Island were
sampled (n = 30 total). In addition, we included the following
pupfish species as outgroups to our study: C. laciniatus (F1
generation; Lake Cunningham, New Providence Island, Bahamas; n =
4), C. variegatus (F0 generation; Fort Fisher, North Carolina,
United States; n = 2) plus liver tissue as a tissue control, andCualac tessellatus (long-term captive colony; San Luis Potosí,
Mexico, n = 4).
Each gut was divided into proximal and distal regions for all San
Salvador Island samples to compare microbial composition between these
regions. All outgroup samples used whole intestines. In addition, the
microbial community was isolated from aquaria water in two tanks which
contained F2 individuals of Osprey Lake C. variegatus and
Crescent Pond C. variegatus , and used as controls (n = 2).
The Vincent J. Coates Genomics Sequencing Laboratory at the University
of California, Berkeley also generated three controls, including a
positive control and two no template controls (NTC). Microbial DNA
extractions were performed in batches (stored on ice) immediately after
intestinal dissections with the Zymobiomics DNA Miniprep Kit (Zymo
Research, Irvine, CA).