Materials and Methods
Animal care
Female and male crayfish, Procambarus spp., were used. They measured 7-10 cm in total body length. The crayfish were procured from Carolina Biological Supply (Burlington, NC), but they were farmed and harvested in Louisiana. They were kept in plastic tubs filled with tap water (up to 3 individuals per tub, for no longer than 1 week), fed dry food daily, and kept on a 12-hour light cycle.
Saline
The saline solution used in this experiment is Standard Saline (modified after van Harreveld, 1936), and its components were: 12.0 g/L NaCl; 0.4 g/L KCl; 1.5 g/L CaCl2 (anhydrous); 0.5 g/L MgCl2• 6H2O; 0.17 g/L NaHCO3. The solution was aerated and mixed thoroughly. Drops of dilute HCl or additional NaHCO3 may have been added to bring the solution to pH 7.4.
Dissection
The crayfish were ice-anesthetized for no less than 1 hour before dissection. After the crayfish were anesthetized, they were sacrificed by an abdomen separation. After the separation, the cephalotorax was put into a trash bin inside the freezer compartment of a refrigerator. As described in Strawn et al., 2000, to gain exposure of the ventral nerve cord with the nerve roots, the midline of the ventral articular membrane was cut. An additional cut lateral to the midline was needed to remove the membrane and thus expose the superficial flexor muscles, the ventral nerve cord, and segmental ganglia to the saline bath solution.
Electrode Construction
Suction electrodes were constructed in-home by using colored wiring with plugs and caps, a silver wire, aluminum tape, and other materials. For step-by-step instructions and a more detailed list of materials, see Johnson et al., 2007.
Recording
Throughout the experiment, the saline added was chilled and kept chilled to avoid having changes in temperature that could interfere with our results. The recordings for the control group were taken from nerve root 1, from possibly different ganglia. In order to obtain a recording, the tip of the electrode lightly sucked the nerve root for which the action potentials were being recorded. The action potentials were recorded by using the hardware PowerLab and an amplifier, as needed, and then shown on a graph using the software LabChart.
Experimental Design
For the control group, the crayfish remained in conditions as similar as possible as those in the treatment group, with the exception of the imidacloprid added to those in the treatment group. For the treatment group, 5 doses of treatments were added to each crayfish, and then the change in action potentials was recorded for the following 60 seconds. Each treatment consisted of 1 mL of an imidacloprid solution with a concentration of 10ng per liter. As a result of the experimental design, the concentration of imidacloprid increased as each treatment was added.