Raman imaging study on living tardigrades: origin, nature and function of pigments in Echiniscus blumi

Tardigrades exhibit a variety of pigmentations. Several species are translucent or whitish, but many others are markedly colored with red-orange, brown, green and yellow pigments. These pigments may be found in the body cavity, storage cells, epidermis, eye-spots and, according to literature, in cuticle. The genus Echiniscus is composed of many species. Despite occurrence of pigments is well documented for many of them, the chemical nature, source and function (especially in relation to their resistance to harsh physical and chemical conditions) of these pigments remain unknown. Some hypotheses were formulated about them, but to the best of our knowledge no direct and conclusive experimental proof has been reported to date. Therefore, we are taking this opportunity to attempt a resolution for these open questions using Raman spectroscopy on living individuals of Echiniscus blumi. Raman spectroscopy is a non-destructive and (semi)-quantitative analytical technique based on scattering laser radiation by vibrating molecules, which proved to be an ideal tool for studying living cells and biological tissues. Moreover, by incorporating Raman micro-spectroscopy it is possible to obtain an image of the spatial distribution of the main biochemical constituents of a biological sample (i.e. Raman mapping or imaging). In our analyses, pigments in E. blumi are identified as carotenoids. Their spectra well match those available in literature for β-carotene, zeaxanthin and β-cryptoxanthin, all having 11 conjugated C=C bonds, but not that of lutein, which has only 10. Previously reported chemical reactivity data of Echiniscus pigments suggest β-carotene as the most likely candidate pigment in the genus. Moreover, pigment distribution within the animal body cavity is imaged with Raman mapping. The dietary origin of the pigments (from the moss, Grimmia orbicularis) is demonstrated, as well as their presence in the eggs and in eye-spots, and their absence in the animal cuticle. Using in-vivo Raman imaging, a decrease in carotenoid content is detected after the induction of oxidative stress on animals, supporting the hypothesis of an antioxidant function of these pigments during anhydrobiosis. Considering the lack of methods to directly study antioxidant function of carotenoids in vivo, pigmented tardigrades, investigated with Raman imaging, could be used as model organisms for this purpose opening new perspectives of research in living organisms.