Challenges to Morbidity Point and two-week 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 were bred, and fertilized eggs incubated in separate tanks. Following hatch, fish were raised for 6 months before the start of acclimation experiments, and all acclimations were begun before the fish were eight months post-hatch. Fish were kept in a 200L tank system in dechlorinated Davis, California tap water, which has been previously described (Fiol et al., 2011) and has a salinity less than 0.5g/kg. Four distinct acclimation protocols were used in this study including; (1) gradual increase in salinity from FW with increases occurring once every 24 hours up to the Morbidity Point (MP) at five different rates of change per day (32, 24, 12, 8, and 6g/kg/day), (2) increase from FW to target salinity level (85, 95, and 105g/kg) at a rate of 6g/kg/day followed by continuous exposure at constant salinity until MP, (3) acclimation from FW to the target salinity at different rates to achieve the target salinity after 14 days with four target salinities (21g/kg at 1.5g/kg/day, 55g/kg at 4g/kg/day, 85g/kg at 6g/kg/day, and 105 at 7.5g/kg/day), with an endpoint following 24 hours at the final salinity, and (4) long term acclimation from FW to 75g/kg at 6g/kg/day followed by maintenance at the final salinity for 10 weeks. Salinity increase in each case was achieved by the addition of a saturated brine solution (200g/kg) made from premixed SW ion blend made for aquariums (Instant Ocean). Brine was added during 10% total water volume daily water changes to achieve the appropriate salinity increase to within 0.1g/kg as determined using a portable conductivity meter set 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. Acclimations 1-3 were conducted in water at 27°C ± 1° in 30L tanks kept on one three-tiered rack with treatments randomly distributed to avoid biasing results based on increased stress on individuals near walking paths (Speare et al., 1995), differences in light levels, or order of feed distribution. Controls for acclimations 1-3 were transferred to tanks prepared in the same way as experimental tanks and held in FW for 14 days with daily 10% water change following feeding. Fish were fed once daily 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), and the total number of pellets added to each tank was recorded. Following 30 minutes of feeding time with no human interaction, all remaining pellets were retrieved using a dip net and counted. In acclimation 4, the twelve-week acclimation to 75g/kg salinity, fish were placed in two replicate tanks from each brood population (n = 4 tanks each with 15 fish) for a total of 60 fish for the 75g/kg treatment and for controls. Because nitrifying bacteria efficiency in biological filters is significantly reduced in salinities above 60g/kg (Cui et al., 2016), alternate tank set-up was needed relative to the other three acclimations. A recirculating system was constructed with 30L experimental tanks sitting inside a larger (400L) tank with overflow outlets 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. In this acclimation, feed was weighed before adding to tanks for increased accuracy of measurment. Following the 30-minute undisturbed feeding period, feed was retrieved and placed in labelled plastic bags. Retrieved feed was then 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 and used as a modifier of final pellet weight for each tank. Total feed consumed was calculated by food retrieved multiplied by the dissolution factor and subtracted from feed distributed. by total weight of fish from biweekly weighing and multiplying by 100 to produce determine percentage of body weight consumed daily. Treatment tanks reached the final salinity after two weeks, at which point 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 to determine weight change.