Acclimation to moderate temperatures can have strong negative impacts on heat tolerance of arctic arthropods

Microhabitat temperatures Temperature loggers were placed at the sites where the animals were collected. At Ny Ålesund, two SmartButton loggers (SmartButton, ACR System Inc.) were placed in and on gravel at the beach to record the thermal environment (every 5 minutes) where individuals of N. littoralis were caught, and at a protected and an exposed tundra area, representative. At Narsarsuaq, air temperature and ground-level temperature at the collection site were continuously recorded (every 15 minutes) using TMS-4 dataloggers (TMS-4, TOMST, Czech Republic) to record the thermal environment in which the individuals were caught. Thermal tolerance Two assays were used to measure heat tolerance in the present study: 1) critical thermal maximum temperature (CTmax), where the specimens were exposed to gradually increasing temperatures and the temperature at which they entered heat coma was recorded, and 2) heat knockdown time (HKDT), where the specimens were exposed to a constant, stressful temperature, and the time until heat coma was recorded. Thermal maximum temperature (CTmax) CTmax was established for the Svalbard species (N. littoralis and M. arctica) using Elara 3.0 (IoTherm, Laramie, WY), a portable fully programmable heating/cooling anodized aluminum stage designed to maintain precision temperatures (Oyen et al., 2016). Individuals were individually placed in a 24-well plate drilled in 5mm acryl (each well had a diameter of 15 mm), with a bottom made of 1mm aluminum, and a lid made of 5mm acryl. The plate was subsequently placed on a thermoelectric plate of Elara 3.0. The plate was insulated from room air within a polystyrene box (16 (W)X 18.5 (L) X 19 (H) cm and 2.5 cm thick). Using a metal frame, a camera with an 8mm lens (Basler USB 3.0) (Basler Inc, Exton, PA) was mounted on top of the cooler allowing recording of the behaviour of animals during thermal exposure using the Basler camera Pylon Viewer (version 5.1.0.12681) software. The individuals were exposed to the following temperature profile: 10 min at 10°C and subsequently the temperature was increased by 0.2 ± 0.01°C min−1. Heat tolerance estimates were based on manual observations of behaviour using video recordings and the time of the last movement was assumed to constitute their CTmax. During each run, a temperature logger (SmartButton, ACR System Inc.) was placed on the thermoelectric plate of the Elara 3.0 and used to calculate CTmax. Heat knockdown time (HKDT) For the Greenlandic species (N. groenlandicus, N. flavomarginatus, and P. lividellus), 5 ml vials containing field-collected individuals were mounted to a rack and submerged into a temperature-controlled water bath (PolyScience MX Immersion Circulator: MX-CA12E) maintained at 48.3°C for N. groenlandicus, at 44.4°C for N. flavomarginatus, and at 46.1°C for P. lividellus. The individuals were then observed and stimulated with flashes of light and gentle tapping on the vial caps with a metal rod. The time until movement ceased was recorded for each individual. We used heat knockdown temperatures that resulted in HKDT lower than 60 minutes for each species. For Svalbard species (N. littoralis and M. arctica), HKDT was obtained using Elara 3.0 (IoTherm, Laramie, WY), where N. littoralis was exposed to a constant temperature of 40.5°C and M. arctica at 30.0°C. The heat tolerance estimates were based on manual observations of behaviour using video recordings and the time of the last movement to constitute their HKDT. During each run, a SmartButton temperature logger (SmartButton, ACR System Inc.) was placed on the thermoelectric plate of the Elara 3.0 to establish realised heat knockdown temperatures.