Challenges to “Loss of Escape Response” and short-term
acclimation
All experimental procedures were conducted with the approval of the UC
Davis IACUC under protocols 20007 and 21846. Two adult Oreochromis
mossambicus female-male pairs (4 adults total) were bred, and
fertilized eggs from the two brood groups incubated in separate tanks.
Following hatch, fish were raised for 6 months before the start of
acclimation experiments, and all acclimations started before fish were
eight months post-hatch. Fish were grown in a 200L tank system in
dechlorinated Davis, California tap water, as previously described
[25] until used for experiments. In all acclimations, two tanks
containing fish from each brood group were used as replicates for a
total of 4 tanks per treatment. Two acclimation protocols were used to
determine CSMAX , and two additional protocols used to
collect samples at relevant salinity-level/duration points before loss
of organismal function, defined as Loss of Escape Response (LER, see
below)(Supplementary figure 1). In each case salinity was increased once
every 24h. To determine CSMAX and impacts of salinity
stress, salinity was raised at five different rates of change per day
(32, 24, 12, 8, and 6g/kg/day). To determine CSMAX over
time at hypersalinity, salinity was increased at a rate of 6g/kg/day to
three target salinity levels (85, 95, and 105g/kg) followed by
continuous exposure at target salinity until LER. To assess
physiological and molecular function at hypersalinity before LER,
salinity was raised to four target levels (21g/kg, 55g/kg, 85g/kg, and
105g/kg), with rate adjusted to reach the target after 14 days and
samples collected following 24 hours at the final salinity. The 14-day
period was chosen such that none of the treatments were impacted by
acute salinity stress from too-rapid salinity increase. Finally, to
assess the long-term impacts of hypersalinity at just below the critical
salinity threshold, fish were acclimated from FW to 75g/kg at 6g/kg/day
over 14 days followed by maintenance at the final salinity for 10 weeks
for a 12 week acclimation in total. The level of 75g/kg for the final
acclimation was determined based on the results from the two
acclimations to CSMAX.
All acclimations except the long-term 75g/kg treatment were conducted by
increasing salinity via adding saturated brine solution (200g/kg) made
from synthetic sea salt (Instant Ocean). Brine was added during daily
10% volume water changes to achieve the appropriate salinity increase
to within 0.1g/kg as determined using a portable conductivity meter on
salinity mode (TWT Cond 3310 meter, TetraCon 325 probe). At salinities
above 60g/kg, 5mL of ammonia/nitrite detoxifier solution (Kordon AmQuel)
was added daily to maintain low ammonia and nitrite levels due to
reduced biological filtration efficiency. Fish were kept in 30L tanks at
27°C ± 1° on three-tiered racks with treatments randomly distributed to
avoid biasing results from increased stress near walking paths [26],
differences in light levels, or order of feed distribution. Controls
were held in equal volume tanks in FW for 14 days and handled in the
same fashion. Fish were fed ad libitum in the morning three hours
after lights-on in the vivarium on a formulated tilapia floating pellet
diet (35% Hi Production Tilapia Food, Star Milling Company), recording
total number of pellets added. Following 30 minutes of undisturbed
feeding time, remaining pellets were retrieved using a dip net and
counted.
For the twelve-week acclimation at 75g/kg salinity, an alternate tank
set-up was needed because nitrifying bacteria efficiency in biological
filters was significantly reduced in salinities above 60g/kg [27]. A
recirculating system was constructed with 30L experimental tanks with
overflow outlets sitting inside a larger (400L) tank and a 200L sump
containing a flow-through bed of ceramic ring filter media (approx. 15L
volume) and protein skimmer (BubbleMagus Curve 7). This allowed for
consistent tank size between all acclimations but with a greatly
expanded water volume (600 L) and biological filter capacity (biofilter
volume of 15L versus 0.5L per tank) which kept ammonia and nitrite
levels below 0.25 ppm without the need for additional detoxifying
solution. To accurately quantify feeding, pellets were weighed before
adding to tanks. Following a 30-minute undisturbed feeding period, feed
was retrieved and oven dried at 105°C until weight became constant
(approx. 3 hours). Natural pellet dissolution was determined for 10g of
feed placed in a 30L tank without fish and treated in the same manner.
Treatment tanks reached the final salinity after two weeks, at which
time three fish from each tank were euthanized after visually selecting
one large, one median, and one small fish and samples acquired to
determine blood osmolality before long term acclimation. To determine
weight gain, the remaining 12 fish were removed using a dip net from
tanks and placed in a pre-weighed 2L pitcher containing water from the
tank, final weight recorded, and fish then returned to the tank. This
process was repeated every two weeks until the end of the experiment.