Induction of rapid cold hardening by cooling at ecologically relevant rates in Drosophila melanogaster

Abstract

Over a decade ago it was hypothesized that the rapid cold hardening process allows an organism's overall cold tolerance to track changes in environmental temperature, as would occur in nature during diurnal thermal cycles. Although a number of studies have since focused on characterizing the rapid cold hardening process and on elucidating the physiological mechanisms upon which it is based, the ecological relevance of this phenomenon has received little attention. We present evidence that in Drosophila melanogaster rapid cold hardening can be induced during cooling at rates which occur naturally, and that the protection afforded in such a manner benefits the organism at ecologically relevant temperatures. Drosophila melanogaster cooled at natural rates (0.05 and 0.1°C min⁻¹) exhibited significantly higher survival after one hour of exposure to −7 and −8°C than did those directly transferred to these temperatures or those cooled at 0.5, or 1.0°C min⁻¹. Protection accrued throughout the cooling process (e.g., flies cooled to 0°C were more cold tolerant than those cooled to 11°C). Whereas D. melanogaster cooled at 1.0°C min⁻¹ had a critical thermal minimum (i.e., the temperature at which torpor occurred) of 6.5±0.6°C, those cooled at an ecologically relevant rate of 0.1°C min⁻¹ had a significantly lower value of 3.9±0.9°C.

Introduction

Aside from the relatively few species that possess the capacity to survive freezing, most insects are killed by internal ice formation and thus are referred to as freeze-intolerant. For these species, the temperature at which ice forms within their tissues (termed the temperature of crystallization, or T_c) represents the lowest temperature at which they could potentially survive. However, the utility of this value as a general measure of an insect's ability to survive chilling is limited by the fact that many species are susceptible to chilling injury or death in the absence of ice formation (Lee and Denlinger, 1985, Knight et al., 1986, Bale, 1987, Lee et al., 1987). One form of non-freezing injury, termed cold shock or direct chilling injury, is caused by a brief exposure to low temperatures above an organism's T_c (Chen et al., 1987, Watson and Morris, 1987).

The mechanisms by which organisms increase their cold hardiness have long been a central theme in the field of thermal biology. Historically, most research on insect cold tolerance has focused on seasonal acclimation, especially that associated with the acquisition of overwintering cold-hardiness. These processes occur over periods of days, weeks or months and are often linked with the organism's entry into a state of quiescence or diapause (Denlinger, 1991). The mechanisms underlying cold hardening include the accumulation, to sometimes multi-molar levels, of sugars and polyhydric alcohols such as glycerol, replacement of cellular proteins with isoforms adapted to low temperature, and modification of membrane lipids (Hochachka and Somero, 1984, Lee, 1991).

In contrast to overwintering cold hardiness, and most examples of thermal acclimation, which require extended periods to fully develop, a rapid cold-hardening process has been described that is induced by brief exposure to moderately low temperature (Lee et al., 1987). Within minutes, this process affords significant protection against subsequent exposure to otherwise injurious low temperature. For instance, although adult Drosophila melanogaster can be chilled to −17°C before the water in their tissues freezes, these flies are killed when exposed to −5°C for 1 h (Czajka and Lee, 1990). However, if first chilled at 0°C for 1 h, most survive this otherwise lethal cold exposure. This form of protection has been identified in a number of diverse insect orders including Coleoptera, Diptera, Hemiptera, Homoptera, and Thysanoptera (Lee et al., 1987, Pullin and Bale, 1988, Lee, 1991). As with overwintering cold hardiness, it has been hypothesized that the protection afforded by rapid cold hardening is produced by cryoprotective substances such as glycerol, the concentration of which may increase as much as 300% to 81.4±3.0 mM after two hours of exposure to 0°C (Chen et al., 1987).

Although well described in the laboratory, little is known of the importance of rapid cold hardening in nature (Coulson and Bale, 1990). Lee et al. (1987) hypothesized that the rapid cold hardening response acts to prevent cold shock injury caused by a sudden decline in temperature. In this scenario, rapid environmental cooling, as often occurs during diurnal cycles, induces a corresponding increase in the exposed insect's cold hardiness. Later, as environmental temperature rises, the insect's cold tolerance decreases as it readjusts to higher temperatures.

Although proposed over a decade ago, the adaptive hypothesis proposed by Lee and colleagues has yet to be tested. Rather, most research to characterize the rapid cold hardening process has focused on its underlying mechanisms using protocols involving the direct transfer of organisms from their rearing temperature to protection-inducing temperatures, and then to potentially injurious temperatures. While useful in extending our understanding of the physiological basis of the protective process, these studies subjected organisms to cooling rates and thermal extremes that would rarely, if ever, occur in nature. In insects and other poikilotherms, less severe chilling (and thus more ecologically relevant) often induces more subtle, yet significant deleterious effects. For instance, when chilled to moderate temperatures, well above those which directly cause death, many organisms enter a state of cold torpor. At or below the temperature at which this occurs (the critical thermal minimum or CT_min), they are unable to seek refugia or food, or to avoid predation (Layne et al., 1985, Rome et al., 1992).

In this study, we determined whether rapid cold hardening could be induced in D. melanogaster during cooling at rates which often occur during diurnal cycles. We found that fruit flies cooled at natural rates exhibited higher survival after exposure to subzero temperatures, than did those directly transferred to these temperatures or those cooled at higher rates. Furthermore, flies cooled at ecologically relevant rates exhibited significantly lower CT_min than did those cooled at higher rates.