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= In Vivo Assessment of Cold Adaptation in Insect Larvae by Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy =

Authors: Daniel Mietchen, Bertram Manz, Frank Volke, Kenneth Storey

Background
Temperatures below the freezing point of water and the ensuing ice crystal formation pose serious challenges to cell structure and function. Consequently, species living in seasonally cold environments have evolved a multitude of strategies to reorganize their cellular architecture and metabolism, and the underlying mechanisms are crucial to our understanding of life. In multicellular organisms, and poikilotherm animals in particular, our knowledge about these processes is almost exclusively due to invasive studies, thereby limiting the range of conclusions that can be drawn about intact living systems.

Methodology
Given that non-destructive techniques like 1H MR imaging and spectroscopy exhibit an enormous signal loss upon freezing and a corresponding signal increase upon thawing and have proven useful for in vivo investigations of a wide range of biological systems, we aimed at evaluating their potential to observe cold adaptations in living insect larvae. Specifically, we chose two cold-hardy insect species that frequently serve as cryobiological model systems and share the same habitat (stem galls on goldenrod plants, genus Solidago) but use different overwintering strategies–the freeze-avoiding gall moth Epiblema scudderiana and the freeze-tolerant gall fly Eurosta solidaginis.

CHESS imaging has previously been used to investigate insect larval development in a non-cryobiological context, and technological developments in the MR field have recently seen in vivo spatial resolution in non-frozen samples reach the size range relevant for entomological and subcellular investigations. The present study combined these two fields of investigation by demonstrating the feasibility of high-resolution MR imaging of insect larvae in a cryobiological context.

Results
In vivo MR images were acquired from autumn-collected larvae at temperatures between 0°C and about −70°C and at spatial resolutions down to 27 µm. These images revealed three-dimensional (3D) larval anatomy at a level of detail currently not in reach of other in vivo techniques. Furthermore, they allowed visualization of the 3D distribution of the remaining liquid water and of the endogenous cryoprotectants at subzero temperatures, and temperature-weighted images of these distributions could be derived. Finally, individual fat body cells and their nuclei could be identified in intact frozen Eurosta larvae.

Conclusions
These findings suggest that high resolution MR techniques provide for interesting methodological options in comparative cryobiological investigations, especially in vivo.

Citation: Mietchen D, Manz B, Volke F, Storey K (2008) In Vivo Assessment of Cold Adaptation in Insect Larvae by Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy. PLoS ONE 3(12): e3826. doi:10.1371/journal.pone.0003826. Editor: Brent Sinclair

Funding: The study was supported by an IBMT-internal grant. Competing interests: The authors have declared that no competing interests exist.