Chloride Treatments
To test for changes in foraging behavior as a result of exposure to chloride, crayfish were stored in small exposure areas within artificial streams. Diamond Crystal’s Solar Naturals salt was used to mimic the use the road salt and perform the acute exposures. Three crayfish per stream were exposed to their respective treatments for 6 h. Exposure areas were separated by two sets of grid screens, which were staggered to prevent crayfish from moving between areas, reducing aggression and competition for sheleter. Exposure areas had the same gravel substrate as that in the Y-maze arena. Water samples were taken a total of three times throughout experimental trials for chloride testing. Chloride concentrations were determined on a ThermoFisher/Dionex Integrion ion chromatography system.
Experimental Design
Crayfish were exposed for 6 h to two sublethal concentrations of chloride (Cl): 6-8 ppm (control), 20-30 ppm (low), and 50-62 ppm (medium). Concentrations varied due to variations in flow rates of inputs of saline water. Each crayfish was only used for one trial, as chloride could potentially damage the chemoreceptors. The control treatment was determined by the concentration of chloride present in the East Branch of the Maple River, as that was the natural stream water that was being pumped into the artificial streams. The sublethal concentrations were chosen based on mean chloride concentrations in other bodies of water during the winter months due to road-salt runoff. The low concentration was chosen to mimic the mean and median concentrations of chloride in streams near less urbanized parts of Indiana and chloride concentrations in the Mohawk River in New York (Gardner and Royer 2010; Godwin, Hafner, and Buff 2003). Most concentrations in freshwater lakes and rivers do not exceed 20 mg/L in most parts of the country (“TRB Document: Special Report 235” 2016). The high concentration was chosen by using the mean and median concentrations of streams near more urbanized parts of Indiana as well as maximum concentrations of chloride in bodies of water adjacent to ones that received large road-salt runoffs (Gardner and Royer 2010; “TRB Document: Special Report 235” 2016).
Experimental Exposure
All crayfish were placed in the exposure areas of the artificial streams, which were constructed at the University of Michigan Biological Station’s stream research facility. Each artificial stream had dimensions of 0.855 m (w) x 2.03 m (l) x 0.406 m (h) and were constructed using cinderblocks and clear plastic lining. Each artificial stream had a PVC pipe at the head of the stream, providing stream water from the East Branch of the Maple River. Each stream contained two mesh screens with tubing on the sides at the head of the stream to help stabilize flow. The firsts mesh screen was placed such that the end of the mesh screen was 4 in from the head of the stream while the second mesh screen was placed such that the beginning of the mesh screen was 7 in from the head of the stream. After the second mesh screen, the Y-maze was built into the stream. Downstream of the Y-maze, the exposure area was built. The exposure area was constructed from two staggered grid screens, with two sets of two staggered smaller grid screens evenly spaced in order to create three equal sized exposure areas. Each exposure area had dimensions of 0.2032 m (w) x 0.4061 m (l). Exposure areas had a plexiglass ceiling to prevent crayfish from escaping. Exposure areas were closed off by a piece of wood with mesh screen covered holes to allow for outflow. Gravel was used as a substrate in the entire stream. Water depth was maintained at 5.5 ± 0.5 in in each stream for all trials.
The design of the artificial streams is of flowing water to mimic a real stream. Crayfish were placed within the exposure areas for an acclimation period for at least 12 h. After acclimation, saline water was added into the inflow pipes by connecting a 55 L barrel containing water with added NaCl to the inflow pipes using hose pipes and syringe piping and a hole in the PVC pipes. Inflow pipes that did not have an addition of saline water a plug inserted into the hole in the inflow PVC pipe to help standardize flow.
Behavioral Trials
Behavior trials were performed in the Y-mazes of the artificial streams [neutral zone = 29.5 x 24 in2, arm = 25 x 12 in2 (length, width respectively)]. Before testing, Fluorescein dye was used to confirm that water was flowing downstream and food odors would be flushed out within a thirty minute timespan. Behavioral trials were staggered to account for differences in time of day and thus, available shade. Staggered trials also allowed for food odors to be flushed out of the system. Concentrations were staggered to account for possible physical differences and bias in the streams themselves. After 6 h of exposure, foraging assays were conducted on the animals under the same chloride concentrations. After 6 h of exposure, one animal was moved from the exposure area into the neutral zone and held under a gridded cage before being allowed to explore the arena for 15 minutes. After the 15 minute exploration period, crayfish were moved back to the neutral zone and placed under the gridded cage. Food stimulus was placed in an arm, randomly determined by a coin flip. Behavioral tests began after removing the gridded cage and lasted fifteen minutes. This was repeated for each animal.
Food Stimuli
Fish gelatin was prepared as the food source for the behavioral analysis. The fish gelatin was made by mixing 45 g of sardines, 28 g of unflavored gelatin and 0.71 L of boiling water in a high-speed blender until homogenized. The gelatin was then placed into plastic water bottle caps, covered in plastic paraffin film and refrigerated until solidified. The plastic water bottle caps serve to keep the food stimuli submerged during the behavioral trial. Each cap contained approximately 5 g of fish gelatin and was not used 72 h after preparation.