Impaired climbing and flight behaviour in Drosophila melanogaster following carbon dioxide anaesthesia

Abstract

Laboratories that study Drosophila melanogaster or other insects commonly use carbon dioxide (CO2) anaesthesia for sorting or other work. Unfortunately, the use of CO2 has potential unwanted physiological effects, including altered respiratory and muscle physiology, which impact motor function behaviours. The effects of CO2 at different levels and exposure times were examined on the subsequent recovery of motor function as assessed by climbing and flight assays. With as little as a five minute exposure to 100% CO2, D. melanogaster exhibited climbing deficits up to 24 hours after exposure. Any exposure length over five minutes produced climbing deficits that lasted for days. Flight behaviour was also impaired following CO2 exposure. Overall, there was a positive correlation between CO2 exposure length and recovery time for both behaviours. Furthermore, exposure to as little as 65% CO2 affected the motor capability of D. melanogaster. These negative effects are due to both a CO2-specific mechanism and an anoxic effect. These results indicate a heretofore unconsidered impact of CO2 anaesthesia on subsequent behavioural tests revealing the importance of monitoring and accounting for CO2 exposure when performing physiological or behavioural studies in insects.

Figure 1. Drosophila melanogaster climbing and flight abilities are reduced following a 10 minute exposure to 100% CO₂.

Flies were exposed to 100% CO₂ for 10 minutes and allowed to recover for varying times and assayed for the ability to (a) climb and (b) fly. CO₂ inhibited climbing at all recovery time points assayed while flight was only reduced through 8 hours. Data points represent the mean ± SEM of the percentage of flies able to climb or fly. Statistical analysis was performed by Student’s t-tests. a = P < 0.001, b = P < 0.01.

Figure 2. Drosophila melanogaster climbing and flight abilities are reduced by 100% CO₂ in a dose-dependent manner.

Flies were exposed to 100% CO₂ for the times (minutes) noted in the legend and allowed to recover for varying times and assayed for the ability to climb and fly. (a) CO₂ exposure inhibited climbing at all recovery time points assayed. Compared to the flies that were exposed for 10 minutes, the five minute exposure flies climbed better at all recovery time points, while the 30 minute exposed flies performed worse at the one and two hour recovery time points. (b) Flight was reduced in flies exposed to 100% CO₂ for five minutes for up to four hours, and for 8 hours in flies exposed for 10 minutes. Flies that were exposed for 15 or 30 minutes had reduced flight at all recovery time points assayed. Compared to the flies that were exposed for 10 minutes, the 30 minute exposure flies performed worse at all recovery time points, while the 15 minute exposed flies flew less only at the 24 hour recovery time point. The Air-exposed points represent the average of all controls performed for that time point; however, the statistical analysis (Student’s t-test) was performed with the individual control flies for each exposure length. The data points represent the mean ± SEM of the percentage of flies able to climb or fly. a = P < 0.01 vs. Air control flies, b = P < 0.05 vs. 10 minute CO₂ flies.

Figure 3. A 10 minute exposure to 100% N₂ reduces D. melanogaster flight but not climbing, which is rescued by 1% O₂.

Flies were exposed to 100% N₂ or 99% N₂/1% O₂ for 10 minutes and recovered for one hour prior to being assayed for climbing or flight. (a) Climbing ability is unaffected by the N₂ exposure; however, (b) flight is reduced. (c) Climbing remains unaffected by 99% N₂ exposure, while (d) the flight deficit is rescued by the addition of 1% O₂. Columns represent the mean ± SEM of the percentage of flies able to climb or fly. Statistical analysis was performed by Student’s t-tests. a = P < 0.001.

Figure 4. Two different ranges of CO₂ levels adversely affect D. melanogaster climbing ability.

Flies were exposed to varying levels of CO₂ for 10 minutes prior to a one hour recovery. The CO₂ mixes were initially offset by O₂ up to 20% and then with N₂. Bars represent the mean ± SEM of the percentage of flies able to climb. The Air-exposed control bar represents the average of all controls performed for this experiment, while the statistical analysis (Student’s t-test) was performed between the specific CO₂ level and their individual air-exposed flies. a = P < 0.01.

Figure 5. CO₂ flow rate through a CO₂ pad reduces D. melanogaster climbing ability in a flow rate-dependent manner.

Flies were exposed to varying levels of CO₂ on a CO₂ pad with varying flow rates and allowed to recover for one hour prior to being assayed for climbing. 2.4 L/minute is the minimum CO₂ flow rate found to anaesthetize flies and has no effect on subsequent climbing ability. However, increasing the flow to 3.0 and 5.0 L/minute decreased the ability to climb in a flow rate-dependent manner. Bars represent the mean ± SEM of the percentage of flies able to climb. The Air-exposed control bar represents the average of all controls performed for this experiment. Statistical analysis was performed by Student’s t-tests between the individual Air control flies and each CO₂ flow rate. a = P < 0.001 vs. the Air controls and b = P < 0.001 vs. the 3.0 L/minute CO₂ flies.

Figure 6. Exposure to 99% CO₂ reduces D. melanogaster climbing ability for an extended period of time in a dose-dependent manner.

Flies that were exposed to 99% CO₂/1% O₂ in the exposure apparatus had reduced climbing abilities at various recovery time points. Flies exposed for 10 minutes had reduced climbing through 8 hours, while the climbing of flies that were exposed for 30 minutes was reduced through 16 hours. The 30 minute exposure inhibited D. melanogaster climbing more than the 10 minute exposure. The Air-exposed points represent the average of all controls performed for that time point; however, the statistical analysis (Student’s t-test) was performed with the individual control flies for each exposure length. The data points represent the mean ± SEM of the percentage of flies able to climb. a = P < 0.01 vs. Air control flies, b = P < 0.01 vs. 10 minute CO₂ flies.