Researchers in the X-ray hutch. Left to right: Wah-Keat Lee (Argonne National Laboratory), Brent Sinclair, Steve Roberts (now of Central Michigan University), Arun Rajamohan (now of North Dakota State University), Allen Gibbs (University of Nevada at Las Vegas) and Jake Socha (Virginia Tech).
Why can some insects survive freezing, while others can’t? The answer seems to be related to how freezing occurs, according to research conducted at the University of Western Ontario (Canada) using images obtained with the Advanced Photon Source (APS) from Argonne National Laboratory (PLoS ONE 4(12): e8259; doi:10.1371/journal.pone.0008259).
To investigate the ways that various flies freeze, the researchers filmed the formation and spread of ice in real time as the flies (as larvae) froze using high-energy X-rays from the APS. They compared Chymomyza amoena, an insect native to Ontario that survives freezing, with the fruit fly often used for research, Drosophila melanogaster.
Brent Sinclair, an assistant professor in the university’s biology department, has been interested in insects that can survive internal ice formation since he was an undergraduate. He says, “It is really neat that something can be frozen solid, yet thaw out and be up and about hours after thawing!”
Fly larva in the process of freezing. (Left) Phase-contrast X-ray image. (Right) Composite of subtraction images of the frames where the gut appeared to freeze (orange) and the initial freezing frame (green).
From a practical standpoint, figuring out the mechanism could have immediate applications, since fruit flies are used in thousands of labs. Maintaining something like 250,000 different fly populations is a hassle. Also, the reproductive speed that makes flies an attractive research animal becomes a problem as spontaneous mutations can creep into the line of flies. Sinclair says, “They are not as stable a resource as we would like.” It would be more convenient if the flies could be stored in a freezer when they weren’t needed.
The current model for how insects survive freezing suggests that ice starts to form between cells. All the other things in solution—ions, sugars, proteins, etc.—are pushed out from the ice lattice, forming increasingly concentrated pockets of brine. The brine, against cell walls, draws water out of the cells. The theory, says Sinclair, is that “at a cellular level, insects are turning this into a dehydration problem.”
The Western Ontario group wanted to watch ice form inside the insects in real time and see if there is anything fundamentally different about how ice forms in larvae that can survive freezing and those that can’t. The flies that survive consistently freeze at higher temperatures than those that don’t—which suggests that the insects have some control over when and where ice starts crystallizing.
Other techniques (such as MRI and confocal imaging) exist for looking inside frozen insects, but those don’t give the temporal resolution that the researchers needed for watching changes every few seconds. The APS provided this as well as parallel 15-keV X-rays, chosen to optimize image clarity. The larvae were attached to a thermocouple, and the temperature was reduced by about half a degree per minute. The X-rays didn’t appear to perturb the process. (See video of the process attached to the PLoS ONE paper or at www.youtube.com/watch?v=m07CKU1XGdk.)
Sinclair would like to find ways to use contrast agents to tell different sorts of soft tissues apart. Since the images depend on density differences, the imagery shows only the difference between “air in the tracheal systems, wet stuff and ice,” he explains. For now, Sinclair is continuing to investigate the biochemistry of freezing—and thawing.
