Long-Term Cold Acclimation Extends Survival Time at 0°C and Modifies the Metabolomic Profiles of the Larvae of the Fruit Fly Drosophila melanogaster

Abstract

Background Drosophila melanogaster is a chill-susceptible insect. Previous studies on this fly focused on acute direct chilling injury during cold shock and showed that lower lethal temperature (LLT, approximately −5°C) exhibits relatively low plasticity and that acclimations, both rapid cold hardening (RCH) and long-term cold acclimation, shift the LLT by only a few degrees at the maximum.Principal FindingsWe found that long-term cold acclimation considerably improved cold tolerance in fully grown third-instar larvae of D. melanogaster. A comparison of the larvae acclimated at constant 25°C with those acclimated at constant 15°C followed by constant 6°C for 2 d (15°C→6°C) showed that long-term cold acclimation extended the lethal time for 50% of the population (Lt50) during exposure to constant 0°C as much as 630-fold (from 0.137 h to 86.658 h). Such marked physiological plasticity in Lt50 (in contrast to LLT) suggested that chronic indirect chilling injury at 0°C differs from that caused by cold shock. Long-term cold acclimation modified the metabolomic profiles of the larvae. Accumulations of proline (up to 17.7 mM) and trehalose (up to 36.5 mM) were the two most prominent responses. In addition, restructuring of the glycerophospholipid composition of biological membranes was observed. The relative proportion of glycerophosphoethanolamines (especially those with linoleic acid at the sn-2 position) increased at the expense of glycerophosphocholines.ConclusionThird-instar larvae of D. melanogaster improved their cold tolerance in response to long-term cold acclimation and showed metabolic potential for the accumulation of proline and trehalose and for membrane restructuring.

Conversion of the chill susceptible fruit fly larva ( Drosophila melanogaster ) to a freeze tolerant organism

“…Because diapause in the fruit fly is weakly expressed in the adult stage, and no capacity for diapause exists in the larval stage (21, 22), we exposed larvae to low temperatures in order to achieve two aims at once: (i) slowing down larval development and/or bringing larvae into a state of dormancy (quiescence), and (ii) allowing the process of long-term cold acclimation (5). The development from the egg stage to puparium formation takes approximately 5 d at 25°C (treatment no.…”

“…2 shows that no larva survived when acclimated at constant 25°C or 15°C on a standard diet. Thus, absolutely no capacity for freeze tolerance at −5°C was detected, although these larvae can survive when supercooled at the same temperature (2,5,6). FTR-quiescence resulted in the occurrence of freeze tolerance in some individuals; 6.3% of larvae (18 of 284) showed signs of activity at the cellular level, and one (0.35% of total) could continue developing to the adult stage.…”

“…Osmolality and SCPs were measured as described earlier (5). The SCPs were recorded in: (i) the dry state (larvae were directly attached to thermocouples using thermally conductive paste or surrounded with dry cellulose); and (ii) the wet state (a similar arrangement as in our freeze-tolerance assays) in order to verify the occurrence of ice-nucleation of larval body fluids with surrounding ice crystals between −1°C and −5°C.…”

“…Thermal analyses of whole larvae were conducted on a DSC4000 calorimeter (Perkin Elmer) as described earlier (5). Two different analyses, I. and II.…”

“…The ancestral members of this genus were adapted to warm temperatures (2), and most of the extant species still have tropical and/or subtropical distributions and are chill susceptible (3). The immature development of D. melanogaster halts at temperatures below 10°C (4), chill injury occurs below 6°C (5), and all developmental stages die when chilled (supercooled) below −5°C for just a few hours (6). The ability to tolerate freezing; i.e., formation of ice crystals in body fluids, is unknown in any Drosophila species (2,5).…”