The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in Drosophila melanogaster

Abstract

Carbon dioxide gas is used as an insect anesthetic in many laboratories, despite recent studies which have shown that CO(2) can alter behavior and fitness. We examine the effects of CO(2) and anoxia (N(2)) on cold tolerance, measuring the rapid cold-hardening (RCH) response and chill coma recovery in Drosophila melanogaster. Short exposures to CO(2) or N(2) do not significantly affect RCH, but 60 min of exposure negates RCH. Exposure to CO(2) anesthesia increases chill coma recovery time, but this effect disappears if the flies are given 90 min recovery in air before chill coma induction. Flies treated with N(2) show a similar pattern, but require significantly longer chill coma recovery times even after 90 min of recovery from anoxia. Our results suggest that CO(2) anesthesia is an acceptable way to manipulate flies before cold tolerance experiments (when using RCH or chill coma recovery as a measure), provided exposure duration is minimized and recovery is permitted before chill coma induction. However, we recommend that exposure to N(2) not be used as a method of anesthesia for chill coma studies.

Fig. 1

Survival of 4-day old adult male Drosophila melanogaster after 2 h exposure to a subzero temperature, with or without a 2 h 0 °C RCH pre-treatment. Data points are means ± SE, n = 3 replications of 10 flies.

Fig. 2

Experimental design of the RCH experiment. Adult male flies were either exposed or not to a gas treatment, and then either given a 2 h pre-treatment at 0 °C (RCH) before treatment at −5.2 °C, or transferred directly into −5.2 °C for 2 h. Each treatment group contained four replicates of 10 flies and was repeated on 3 consecutive days.

Fig. 3

Time required for 80% recovery after an exposure to CO₂, N₂ or air. Data points are mean ± SE. n = 10 replicates of 10 flies. N₂ and CO₂ treatments differ significantly where marked with *. See text for details of statistics.

Fig. 4

Survival after a defined length of N₂ or CO₂ exposure and a 2 h treatment at −5.2 °C, with (pre-treatment) or without (direct transfer) a 2 h 0 °C pre-treatment. Data points are means ± SE. n = 4 replicates of 10 flies. Pre-treatments differ from controls where marked with *. See text for details of statistics. Development time refers to time to develop from egg to adult.

Fig. 5

Time required for recovery from chill coma (4 h at 0 °C) after a defined length of CO₂ or N₂ exposure. Data points represent mean ± SE. n = 10 replicates of 10 flies. N₂ and CO₂ treatments differ significantly where marked with *. See text for details of statistics.

Fig. 6

Time required for recovery from chill coma (4 h at 0 °C) after 60 min of gas treatment, with varying amounts of recovery time from anesthesia. Data points represent means ± SE. n = 10 replicates of 10 flies. N₂ and CO₂ treatments differ significantly where marked with *. N₂ and CO₂ treatments differ from the control where marked with † or §, respectively. See text for details of statistics.