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.