Nature, source and function of pigments in tardigrades: in vivo raman imaging of carotenoids in Echiniscus blumi

Abstract

Tardigrades are microscopic aquatic animals with remarkable abilities to withstand harsh physical conditions such as dehydration or exposure to harmful highly energetic radiation. The mechanisms responsible for such robustness are presently little known, but protection against oxidative stresses is thought to play a role. Despite the fact that many tardigrade species are variously pigmented, scarce information is available about this characteristic. By applying Raman micro-spectroscopy on living specimens, pigments in the tardigrade Echiniscus blumi are identified as carotenoids, and their distribution within the animal body is visualized. The dietary origin of these pigments is demonstrated, as well as their presence in the eggs and in eye-spots of these animals, together with their absence in the outer layer of the animal (i.e., cuticle and epidermis). Using in-vivo semi-quantitative Raman micro-spectroscopy, a decrease in carotenoid content is detected after inducing oxidative stress, demonstrating that this approach can be used for studying the role of carotenoids in oxidative stress-related processes in tardigrades. This approach could be thus used in further investigations to test several hypotheses concerning the function of these carotenoids in tardigrades as photo-protective pigments against ionizing radiations or as antioxidants defending these organisms against the oxidative stress occurring during desiccation processes.

Figure 1. Raman maps of carotenoid pigments in E. blumi.

(A) Bright field micrograph and (B,C) intensity Raman maps of a living E. blumi; Raman map C covers the anterior region of the tardigrade (white rectangle in map B). (D) Bright field micrograph and (E) intensity Raman map of an exuvium containing three eggs of E. blumi. (F) Average Raman spectra (black) and intensity standard deviation (grey) of Raman maps B and E; spectral intensity was re-scaled for better comparison. All Raman maps depict the relative concentration of carotenoid species, measured as the integrated Raman intensity between 1501 cm⁻¹ and 1541 cm⁻¹, corresponding to the intense ν₁ C = C stretching vibration band of carotenoids. In C, white arrows indicate spots with high carotenoid concentration corresponding to the eye-spots. The color scale bar on the right of each Raman map has units of total photon counts/Δcm⁻¹ for the Raman shift interval considered.

Figure 2. Variety of spectral characteristics for carotenoids within a E. blumi specimen.

(A) Image depicting the ν₁/ν₂ band intensity ratio (“IR”, color scale bar on the right) for the Raman map shown in Fig. 2B. (B) Average normalized intensity of the two subsets of spectra having the lowest and the highest ν₁/ν₂ ratio, in blue (IR<1.2, 26 spectra) and red (IR>1.45, 21 spectra), respectively; for each subset, the intensity of standard deviation is reported in grey. (B’) Inset with detail of ν₁ bands.

Figure 3. Food origin of carotenoids.

(A) Photoluminescence emission spectrum from the gut of a living E. blumi (excitation at 514.5 nm). Raman bands due to carotenoids (dotted box) are observed together with the chlorophyll fluorescence emission bands at 670 and 737 nm; (A’) inset with fluorescence intensity map (emission at 670 nm); black bar is 50 µm, grey scale bar on the bottom of the Raman map has units of counts at 670 nm. (B) Bottom spectrum: average normalized spectrum (black) and intensity standard deviation (grey) of a set of 300 Raman spectra collected from the moss leaves of G. orbicularis; top spectrum: Raman spectrum from tardigrade’s gut (dotted box in A).

Figure 4. Effect of induced oxidative stress on the carotenoid content.

(A–B, D–E) Histograms of the integrated Raman intensity in the 1460–1570 cm⁻¹ region from Raman maps of two living E. blumi specimens before any treatment (A, D) and after exposure to 25 mM hydrogen peroxide (B, treated) or water (E, control) for 15 min. For each histogram, the corresponding intensity Raman map depicting the carotenoids distribution (i.e. the intensity at 1521 cm⁻¹) is shown as inset. White scale bars = 200 µm, color scale bars have units of counts. (C, F) Average spectra of the Raman maps before (red) and after (blue) exposure to hydrogen peroxide (maps in A, B) or water (maps in C, D).