The ability to survive freezing comes naturally to a select group of insects. These cold-adapted insect species live in areas where they might experience sub-zero temperatures for at least some of the year. But Drosophila melanogaster, the favourite fly of researchers from several life sciences disciplines, is better known for its preference for the warmth of human kitchens and is injured by cold – even at temperatures above freezing. Scientists would love to know how to successfully put these flies into suspended animation using cryopreservation to maintain their valuable stocks of laboratory-modified D. melanogaster lines. In recent work published in Proceedings of the National Academy of Sciences, Vladimir Koštál and colleagues from the Czech Republic show that with a few simple tricks picked up from a freeze-tolerant cousin, it is possible to convert the chill-susceptible D. melanogaster into a fly that can survive freezing.
Earlier work by Koštál and his colleagues showed that Chymomyza costata, a drosophilid fly closely related to D. melanogaster, has two requirements to survive freezing: (1) it must be in developmental arrest (called diapause) during an overwintering stage, and (2) it must accumulate large quantities of the free amino acid proline. The authors thought a similar protocol might work for D. melanogaster. First, the team reared larvae at either room temperature or a relatively low temperature for D. melanogaster (15°C) until they reached the final larval stage. Then, they subjected the larvae to temperatures that fluctuated between 6°C and 11°C for 3 days to induce a type of diapause. In addition, some of the insects were fed diets rich in known cryoprotectants: glycerol, proline or trehalose. Finally, the team slowly cooled the flies to –5°C and held them there for over an hour before allowing the insects to resume development.
The researchers found that feeding the larvae diets rich in cryoprotectants or subjecting them to fluctuating temperatures both increased the larvae's survival of freezing, but only for a short time after the stress. However, the combination of the proline-rich diet in particular with the fluctuating temperatures had a synergistic effect, producing larvae with almost a 10% chance of surviving to reproduce successfully after being frozen at –5°C for over an hour – which is long enough to convert half of their body water to ice.
To investigate how these treatments protected the diapausing larvae from freezing to death, the authors measured the concentration of several of the larvae's metabolites after consuming their cryoprotectant-supplemented diet, using mass spectrometry. They found that the larvae fed on the supplemented diets all accumulated extra cryoprotectant, although the proline-supplemented diet had the largest effect on cryoprotectant concentration. The authors thought that perhaps the larvae do not control their proline levels as tightly as they do other metabolites, which might have contributed to the success of proline in producing freeze tolerance.
While Koštál and colleagues plan further studies into the mechanisms of proline's effects, this first report of inducing freeze tolerance in a tropical insect like D. melanogaster contributes to the elucidation of the mechanisms of natural freeze tolerance, and may lead to an end of the labour-intensive work of maintaining laboratory fly stocks.
