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CONFERENCE PROGRAM ABSTRACT BOOKLET www.tardigrada2015.it
13th International Symposium on Tardigrada Modena, Italy 23-26 June, 2015
WATER BEARS GOING NANO: ATOMIC FORCE MICROSCOPY ALLOWS HIGH RESOLUTION IN VIVO IMAGING OF TARDIGRADE CUTICLE 1
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Department of Biology, Faculty of Science, University of Zagreb, Croatia Laboratory for bioelectrochemistry and surface imaging, Rudjer Boskovic Institute, Zagreb, Croatia
Besides taxonomical relevance, cuticular design greatly determines the extent of transpirational water loss and overall anhydrobiotic ability of tardigrades. As conventional light and electron microscopy protocols often suffer from specimen degradation and artifacts caused by fixation and dehydration, we investigated capabilities of atomic force microscopy (AFM) for studying integument of heterotardigrade Echiniscus testudo. This novel approach requires virtually no sample preparation, and allowed us to visualize dry and hydrated dorsal cuticle in its native state using contact mode of AFM operation. In addition, we probed and compared nanomechanical properties of live tardigrade, empty cuticle as well as mucous coat by measuring force-distance curves. Surface of dry cuticle was found to be covered with mucous coat forming irregular aggregates localized mostly in epicuticular depressions (pores). Conversely, imaging of hydrated cuticle shows even distribution of mucous coat hiding pores beneath. We thus hypothesize that latter behaves as a hydrogel: in presence of water it swells facilitating gas exchange and collapses upon dehydration sealing thinner regions of dorsal epicuticle. Supported with known composition of mucus and characteristic patterns observed on force-distance curves, our results are coherent with earlier ideas on protective role of mucous coat. Indenting live tardigrade and exuvium with AFM probe revealed that intact animals are much stiffer due to hydrostatic pressure of body fluid. At present, atomic force microscopy is the only bioimaging technique capable of simultaneous visualization of surface topography on micro- and nanoscale as well as probing nanomechanical properties of tardigrade cuticle. Despite some difficulties to immobilize animals in fluid cell during scanning, overall success promises a powerful new tool for studying cryptobiosis and morphology of these intriguing invertebrates in real-time at ambient conditions.
