Experimental design

Two types of experiments were carried out in this study: an acute temperature stress and gradual warming. Prior to the exposures animals were pre-acclimated for 3–7 days under the constant aeration in filtered water from their native habitats. Pre-acclimation was conducted at the temperature recorded at the time of sampling (15 °C for both populations) or at 7 °C (as annual average temperature of both waterbodies) in the case of the gradual warming. Amphipods were fed daily with natural food (elm leaves) with addition of commercial food (Tetra-Min, Tetra GmbH, Germany) and potato ad libitum. No mortality was observed during pre-acclimation. Only actively swimming animals were used for experiments. Control animals for all experiments were kept under the same conditions as during the preliminary acclimation.

To determine median lethal times (LT50) that cause 50% mortality during acute heat exposure, 10 individuals from each of the two studied populations were placed in separate tanks filled with 1 L of the filtered water from their respective habitats pre-heated to 30 °C. Nine replicate tanks were used for each of the studied populations, to the total of 90 animals per population. Mortality was monitored every hour until all animals died.

Amphipods were placed in 2.5 L aerated glass tanks (n = 5) with water, pre-heated to 30 °C (according to our observations, the maximum temperature in the littoral zones of lake in Irkutsk can reach up to 30 °C in a scorching summer). After 0.5, 1, 3 and 6 h of exposure, three individuals from each of the five replicate tanks were collected and flash-frozen in liquid nitrogen for subsequent biochemical analyses.

Animals were pre-acclimated for seven days at the temperature of 7 °C in separate glass tanks (2.5 L, n = 7) and subjected to the gradual temperature increase of 1 °C per hour continuing until 100% mortality occurred (modified from Sokolova & Pörtner (2003)). After every 2 degrees of temperature increase (i.e., every 2 h), three specimens were collected from each tank of the seven replicate tanks and shock-frozen in liquid nitrogen.