Fig. 6.

HPLC analysis of the retinoid (A) and carotenoid (B) content of Oikopleura dioica samples from different stages, including unfertilized eggs, 7-hpf embryos, day-4 nonmature adults, and day-5 mature males and day-5 mature females. (A) Oikopleura dioica extracts analyzed in normal phase HPLC. Chromatogram extracted at 350 nm. No atRA or its 9-cis-, 11-cis-, or 13-cis-isomers were detected. Unidentified peak 2 eluted at 14.2 min, may represent an endogenous retinoid, while peak 1 eluted at 5.0 min, is an unrelated compound (see text and supplementary file S4, Supplementary Material online, for details). (B) Analysis of carotenoid content in O. dioica by reverse phase HPLC. Chromatograms extracted at 450 nm showed four peaks eluted at 25.8, 28.3, 31.3, and 33.8 min in most O. dioica samples. None of the peaks appeared to be β-carotene standard. Peaks 2,3 and 4 have absorbance spectra typical for carotenoids, while peak 1 represents a different compound. Carotenoids (peaks 2, 3, and 4) likely have a dietary origin (see supplementary file S5, Supplementary Material online, for the carotenoid content of the four microalgae species used in the O. dioica diet). The inverse relative abundance of peak 1 and the carotenoid peaks at different stages suggested that peak 1 might be derived from carotenoids and that O. dioica might have the ability to actively store carotenoids in eggs, and to metabolize them throughout their life cycle. The presence of β-carotene in the dietary algae (supplementary file S5, Supplementary Material online) suggests that the absence of atRA in O. dioica was not due to a dietary deficiency of β-carotene. The absence of a β-carotene in O. dioica samples could be explained by its transformation into astaxanthin, which appears to be the one of the major carotenoids found in larvaceans (Mojib et al. 2014).