Dissecting chill coma recovery as a measure of cold resistance: evidence for a biphasic response in Drosophila melanogaster

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Abstract

Cold resistance in insects has traditionally been measured in terms of survival following a stress, but alternative methods are increasingly being used because of their relevance to the ecology of organisms and their utility in characterizing variation among species, populations and individuals. One such method capable of discriminating among Drosophila species and conspecific Drosophila populations from different environments is adult chill coma recovery time, the time taken for adults to become active again after being knocked down by a cold stress. Here we characterized the chill coma response of D. melanogaster in detail. Adults were exposed to a range of temperatures and stressful periods prior to measuring recovery. Recovery from chill coma in D. melanogaster was biphasic; as flies were stressed under cooler temperatures, recovery times leveled off and then decreased before sharply increasing again as mortality starts to occur. This biphasic response has previously been observed in D. subobscura where it has a somewhat different shape. A second mechanism therefore acts at relatively lower temperatures to ameliorate the effects of the cold stress. When D. melanogaster were reared at 19 and 25 °C for two generations, the shape of the curve relating temperature to recovery time was similar, but flies from the warmer temperature had longer recovery times and showed responses that leveled off and then decreased at relatively higher temperatures. As exposure time to cold stress was increased, recovery times also increased except at mild stress levels. Chill coma recovery in D. melanogaster is a complex trait and likely to reflect multiple underlying components.

Introduction

Insect responses to cold temperature have been widely studied because of the importance of lower temperature limits on insect distributions (Chown, 2001). There is substantial information on the mechanisms that underlie cold resistance including mechanisms that allow insects to cope with conditions well below 0 °C as well as those important near this point (Tauber et al., 1986, Leather et al., 1993). In understanding responses of insects to cold stress, mortality assays are commonly used. However, these assays are difficult to apply when characterizing individual variation in cold responses, which requires simple and rapid tests that can be applied to a large number of organisms (Hori and Kimura, 1998, Gibert et al., 2001, Hoffmann et al., 2003).

A simple assay of cold response that can be used to investigate variability involves exposing individuals to short periods of cold stress (0 °C or lower) and then measuring recovery time (i.e., ability to stand upright) after transfer to room temperature. This method was applied to Drosophila melanogaster by David et al. (1998) and has subsequently been used to investigate species variation (Gibert et al., 2001) and geographic variation (Gibert and Huey, 2001, Hallas et al., 2002, Hoffmann et al., 2002) in cold response.

Recently, this assay was applied by David et al. (2003) to investigate variation in D. subobscura, a species that is resistant to cold relative to other Drosophila species. By exposing flies to a range of temperatures between 1 and −11 °C, the authors showed that there was a biphasic, non-linear relationship between recovery time and exposure temperature, due to a plateau between −6 and −4 °C when the recovery times were fairly similar. While the physiological basis of this plateau was not investigated, the authors interpreted the plateau in terms of two sets of physiological mechanisms, one set governing processes at very low temperatures, and a second overlapping set at less extreme temperatures.

Here we demonstrate that a biphasic pattern of cold response is also evident in D. melanogaster, a species that is more sensitive to cold than D. subobscura. By characterizing chill coma recovery between −5 and 8 °C, we show that there is a ‘plateau’ at −2 to 2 °C; in fact, 0 °C was more stressful than temperatures on either side when flies were raised at 19 °C. This pattern was evident for a range of different exposure times to the cold stress. We also found that flies reared at a higher temperature (25 °C) had slower recovery times, but the biphasic pattern was still evident even though the plateau had shifted to a milder temperature. The different mechanisms underlying the biphasic pattern therefore responded to acclimation induced by rearing temperature.

Section snippets

Materials and methods

The study used a mass bred population of D. melanogaster from Coffs Harbour, which lies in the sub-tropical region on the Australian East Coast at latitude 30.30° S. This population had been established from the offspring of 10 inseminated females, and maintained at a census size of at least 500 flies. Tests were completed when the population had been reared at 19 °C under continuous light for 27–30 generations in the laboratory. Flies for experiments were reared at either 19±1 or 25±1 °C for

Results

Mortality (flies that failed to recover after 3 h or longer) depended on rearing temperature. For flies reared at 19 °C, there was no mortality when flies were exposed to a temperature of >−4 °C regardless of exposure time, whereas for the 25 °C reared flies there was some mortality even at −2 °C when flies were exposed for more than 6 h (Fig. 1). For flies reared at 25 °C, 100% mortality was reached after 6 h at −4 °C, whereas for flies reared at 19 °C only around half had died after 8 h.

For

Discussion

In D. subobscura, David et al. (2003) proposed two mechanisms underlying a plateau in the association between cold knockdown recovery and temperature. They suggested that if the first mechanism influenced resistance at low stress levels and a second mechanism was effective at increased levels of stress, a plateau in the relationship between temperature and recovery times would eventuate. In the present data, the relationship between temperature and recovery times is even more complex than in D.

Acknowledgements

We thank the Australian Research Council for support via their Special Research Centre scheme.

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