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. A preliminary experiment conducted with six individuals acclimated using a continuous increase of 6 g/kg/day indicated that loss of equilibrium (LOE), a common endpoint for stress tolerance, did not precede more severe impacts including pronounced lethargy, cessation of feeding, and death. Therefore, we aimed to establish a more humane and consistent endpoint indicative of severe stress. 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 many fish appear to move less frequently under salinity stress, they still approach food pellets and avoid nets. When O. mossambicus no longer evades the net, this was the most consistent indicator that they will very soon become moribund, and for this reason we introduce the term “Loss of Escape Response” (LER) as an alternative and more humane endpoint to LOE. Two fish in the preliminary experiment which reached LER were rescued, showing that this state is not terminal if the salinity stress is ameliorated. In trials with an LER endpoint, constant fish monitoring allowed for capture of fish before death as this was necessary for acquiring usable serum and tissues for proteomics.
High rates of salinity increase induced acute effects, resulting in lower CSMAX than rates below 12g/kg/day (Table 1). Based on the CSMAX­ for the 8 g/kg/day and 6 g/kg/day rates the acclimatory CSMAX is approximately 115g/kg for this population and experimental set-up. This result defines the upper end of the pessimum salinity range. For long-term challenges, 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 LER at these salinity levels were as follows: 263 hours at 105g/kg, 326 hours at 95g/kg, and 559 hours at 85g/kg (Table 1). 79% of individuals survived at 75g/kg for 10 weeks.