Negative effects of increased ozone concentrations and heat stress on bumblebees are exacerbated by microplastics

Working question: Does exposure of Bombus terrestris to ozone, heat stress, and microplastic (MP) pollution (individually and in combination) affect bumblebee health on individual level? Are effects more pronounced when individuals are exposed to multiple stressors? Experimental design --> B. terrestris individuals + ozone /+ heat stress /+ LDPE (low density poly ethylene) Experimental animals inidividuals: B. terrestris workers, picked form colonies the day before the experiment (into cages for acclimatization, fed with sugar water --> 1:1, Apiinvert:water) Divided into eight groups of 48 individuals (6 of each colony from 8 colonies, 3 individuals for fat body analyses, 3 individuals for proteome analyses) 384 workers in total (with eight individuals per colony as reserve for acclimatization, à 448 experimental animals) Multiple stressor experiment with individuals: Individuals were exposed to the stressors for 10 days. The individuals were kept in individual Nicot Queen cages, they were fed ad libitum with a ball of food dough (Apifonda + powdered sugar OR Apifonda + LDPE (20 µm - 75 µm) +powdered sugar; 0.8 g ± 0.1 g) attached to the top of the syringe and provided with water from the syringes ad libitum (4 ml). The tips of the syringes were shortened to ensure access to the water. The syringes were refreshed (food dough and water) every three days. To prevent the food dough from drying out, it was sprayed through the cage once a day (after ozone exposure/control exposure) with 2 ml deionised water. The Individuals were kept in climate cabinets (at 27°C or 33 °C, under constant darkness), from which they were taken daily for exposure to ozone/control exposure. After exposure, they were put back in the climate cabinets randomized to guarantee rotation within the cabinets. Exposure to ozone/control exposure was conducted daily for 2 hours in an airtight fumigated glass tank, positioned in an incubator. Prior to the 2 hours of exposure time, the values (RH 50 % - 80 %, T, O3 in ppb) were allowed to stabilize for 30 min (in which the animals were already in the closed glass tank). The 3-D coordintes of where an individual was positioned in the glass tank was noted upon loading of the glass tank. There were 8 Treatments: Control (C): Kept at 27 °C (50-80% relative humidity (RH)), 0 ppb O3, no MP in food dough MP: Kept at 27 °C (50-80% relative humidity (RH)), 0 ppb O3, 8.8 % LDPE in food dough Heat (H): Kept at 33 °C (50-80% relative humidity (RH)), 0 ppb O3, no MP in food dough Ozone (O3): Kept at 27 °C (50-80% relative humidity (RH)), 120 ppb O3, no MP in food dough MP+H: Kept at 33 °C (50-80% relative humidity (RH)), 0 ppb O3, 8.8 % LDPE in food dough MP+O3: Kept at 27 °C (50-80% relative humidity (RH)), 120 ppb O3, 8.8 % LDPE in food dough H+O3: Kept at 33 °C (50-80% relative humidity (RH)), 120 ppb O3, no MP in food dough MP+H+O3: Kept at 33 °C (50-80% relative humidity (RH)), 120 ppb O3, 8.8 % LDPE in food dough For all of the animals the weight was measured upon the end of the experiment . Individuals for fat body assays were euthanized on dry ice (cold + CO2) then frozen and stored at -20 °C.