DOI: 10.5281/zenodo.18429390
title: Negative effects of increased ozone concentrations and heat stress on bumblebees are exacerbated by microplastics

description: 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.
description:  
Attribute table for mortality dataset
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Blinded_ID
Number
Colony
Clear_Treatment
Treatment
Analysis
Total_Mortality
Day1_Mortality
Day2_Mortality
Day3_Mortality
Day4_Mortality
Day5_Mortality
Day6_Mortality
Day7_Mortality
Day8_Mortality
Day9_Mortality
Day10_Mortality
Day_of_Death


six-digit random number for blinded handling and analysis
consecutive number of individuals (1-384)
Colony number 9-16 (1-8 were used in first trial with experiment, which was stopped due to a machine malfunctioning)
Abbreviation of Treatment
Letter-Code for treatments orderd by increasing stressor intensity
 
 
 
 
 
 
 
 
 


 
 
 
C = Control
A
Type of analysis (proteome or relative fat body content)
Did individual die throughout the experiment (yes = 1, no = 0)
Did individual die on this day (yes = 1, no = 0)
 
 
 
 
 
 
Day of death (if individual did not die, day of death is day 11)


 
 
 
O3 = Ozone
B
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
H = Heat
C
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
HO3 = Heat+Ozone
D
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
MP =Microplastic
E
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
O3MP = Ozone+Microplastic
F
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
HMP = Heat+Microplastic
G
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
HO3MP = Heat+Ozone+Microplastic
H
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
Attribute table for fat body assay data set
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
Number
Colony
TreatmentAbb
Treatment
WeighingOrder
Bodyweight
Abdomen_with_FB
Abdomen_without_FB
FB
FB_in_mg
Ratio_Abdomen_to_FB
Problems
Comments
 
 
 
 


 
consecutive number of individuals (50 % of values between 1-384)
Colony number (9-16, since 1-8 were already taken for the first trial of experiment which needed to be stopped due to a malfuncitoning of the machines)
Abbreviation of Treatment
Letter-Code for treatments orderd by increasing stressor intensity
order in which animals were weighed
measurements in g
dry weight of abdomen before removal of the fat body
dry weight of abdomen after removal of the fat body
difference between dry weight of abdomen before and after removal of the fat body --> fat body weight
fat body weight in mg
ratio of dry weights of fat body and abdomen before removal of fat body
did there occur any problems throughout the analysis? (yes = 1, no = 0, if problems occured values is excluded from statistical analysis)


 
 
 
C = Control
A
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
O3 = Ozone
B
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
H = Heat
C
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
HO3 = Heat+Ozone
D
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
MP =Microplastic
E
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
O3MP = Ozone+Microplastic
F
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
HMP = Heat+Microplastic
G
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
HO3MP = Heat+Ozone+Microplastic
H
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
Number