Salinity Tolerance
Non-lethal endpoints indicative of a fish approaching a moribund state are necessary to avoid unnecessary physical distress to animals and for the collection of useable tissue samples. Aberrant swimming and movements are an indicator of this approaching state, and it was initially assumed based on previous experiments determining the maximum critical salinity (CSMAX) that the appropriate endpoint was the inability to maintain upright position, or loss of equilibrium (LOE). Preliminary experiments conducted with six individuals acclimated using a continuous increase of 6 g/kg indicated that this behavior did not precede more severe impacts including pronounced lethargy and cessation of feeding, and that a different endpoint was necessary. Fish approaching a moribund state exhibited several behaviors including: constant fin movement, inability to maintain position in the water column, sustained darkened coloration, and highly reduced feeding. Unfortunately, none of these were consistently observed in all fish. The most consistent behavior was found to be non-reactivity to a potential threat, specifically a dip-net. Normally functioning fish will evade capture in a net, and though fish appear to move less frequently under salinity stress, they still will approach food pellets and avoid nets. When a fish no longer evades the net, this appears to be the most consistent response and indicator that they will become moribund before the following monitoring timepoint, and for this reason we have used the term “morbidity point” (MP). This behavioral response is presented as an alternative endpoint when other responses such as LOE are not applicable.
Two fish in the preliminary experiments which had reached MP were rescued by reducing salinity, suggesting that this state not terminal if the salinity stress is ameliorated. In trials with an MP endpoint, constant fish monitoring allowed for capture of fish before death in most cases as this was necessary for acquiring usable serum and tissues for proteomics. Nonetheless, some individuals were found dead and MP estimated to the closest hour since the previous monitoring timepoint for determining maximum salinity tolerance. Experimental mortalities were not used for proteomic analysis. No premature deaths were recorded in the 14-day acclimation trials to target salinity.
In experiments with continuous gradual salinity increase at variable rates, the mean salinity at MP were as follows: 32.0g/kg at 32g/kg/day, 69.0g/kg at 24g/kg/day, 106.8g/kg at 12g/kg/day, 117.0g/kg at 8g/kg/day, and 114.6g/kg at 6g/kg/day (Figure 1A). Rate of salinity increase was clearly important in determining CSMAX, but lower rates avoid acute affects and resulted in relatively consistent CSMAX. Finding the mean of the CSMAX­for the 8 g/kg/day and 6 g/kg/day rates indicates an acclimatory CSMAX of approximately 115g/kg for this population and experimental set-up. This result is not meant to represent the CSMAX for the species generally, but rather to define the pessimum salinity range for this population so that appropriate samples representative of stages of upper range salinity tolerance could be chosen for later proteomic analysis. For extended exposure to constant salinity experiments, decreasing increments of 10g/kg lower than the CSMAX (115g/kg) were used until a salinity was found in which fish were able to survive indefinitely to determine the critical salinity threshold. The mean times until MP at these salinity levels were as follows: 263 hours at 105g/kg, 326 hours at 95g/kg, and 559 hours at 85g/kg (Figure 1B). At 75g/kg, 79% of individuals survived for 10 weeks, which was the endpoint the experiment.