Loss of righting reflex metodology

To study the dependence of T_H and T_C on the temperature of a prolonged acclimation T_A, fishes Danio rerio (size 20–30 mm) were acclimated at various temperatures ranging from 9 to 38 °C for 7–10 days. The daylight duration was 12 h. Then the fishes were placed into an actively aerated transparent cylindrical 600 mL vessel with a diameter of about 10 cm. Air bubbles created a vertical flow of water along the axis of the cylinder. Water temperature was set significantly higher or lower than the temperature of the prolonged acclimation. The aeration intensity was adjusted so that in the normal conditions (that is, at any temperature not inducing loss of righting reflex) the fishes could maintain stability in the water flow created by the air bubbles. The temperature, at which the loss of righting reflex was observed within one minute after a sharp change of the ambient temperature, was defined as T_H or T_C [30]. The recording time (1 min) was chosen on the basis of the fact that within one minute the temperature gradients between water in the test chamber and the fish internal organs disappeared, while the cells' ionic composition and, the more so, the membrane lipid composition remained unchanged. Indeed, in our experiments, at a maximal temperature difference between the test and acclimation chambers of about 10 °C and a fish body width of 1.5–2.0 mm, the temperature gradients disappeared within tens of seconds. Regardless of the testing results, after the lapse of 1 min the fish was relocated back from the test chamber to the acclimation chamber with T_A, the righting reflex recovered within seconds. If the fish retained the mobility in the water flow within 1 min, the temperature in the test chamber was increased by 1 °C, if the heat shock (T_H) was tested, or lowered by 1 °C, if the cold shock (T_C) was tested. In each experiment, 20 to 50 fishes were used.

In addition, the kinetics of adaptation of fishes to different temperatures was studied. The adaptation kinetics was evaluated by changes in T_H and T_C after a sharp change of T_A. For this purpose, after a prolonged acclimation at fixed temperatures for 5–10 days, fishes were transferred to another acclimation chamber with different temperature. All chambers were aerated. The dependencies of temperatures T_H and T_C on the time of adaptation to a new temperature was recorded in the range from 5 min to 5–7 days. Each fish was used in the experiment no more than once a day.

The values of T_H and T_C and the measurement errors were determined using the Microsoft Office Excel 2007 software on the basis of a mean value and half width of the obtained distribution of the experimental temperatures.