Antisera were raised against the C-terminal amino acid sequences of the two rhodopsins ADY17806 and AEA49880 of Oxyrrhis marina. The antisera and affinity-purified antibodies thereof were used in western immunoblotting experiments of total cell protein fractions from cultures grown either in darkness or in white, red, green, or blue light. Furthermore, the rhodopsin abundances were profiled in cultures fed with yeast or the prasinophyte Pyramimonas grossii. The immunosignals of ADY17806 and AEA49880 were similar when O. marina was grown in white, green, or blue light. Signal intensities were lower under conditions of red light and lowest in darkness. Higher amounts were registered for both rhodopsins when O. marina was fed with yeast compared to P. grossii. Furthermore, total cell protein of cultures of O. marina grown under all cultivation conditions was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by tryptic in-gel digestion and mass spectrometric analysis of the 25-kDa protein bands. The rhodopsin ADY17809 was detected in all samples of the light quality experiments and in 14 of the 16 samples of the prey quality experiments. The rhodopsin ABV22427 was not detected in one sample of the light quality experiments. It was detected in 15 of the 16 samples of the prey quality experiments. Peptide fragments of the other rhodopsins were detected less often, and no clear distribution pattern was evident with respect to the applied light quality or offered prey, indicating that none of them was exclusively formed under a distinct light regime or when feeding on yeast or the prasinophyte. Fluorescence light microscopy using the affinity-purified antibodies revealed significant labeling of the cell periphery and cell internal structures, which resembled vacuoles, tiny vesicles, and rather compact structures. Immunolabeling electron microscopy strengthened these results and showed that the cytoplasmic membrane, putative lysosome membranes, membranes encircling the food vacuole, and birefringent bodies became labeled.
Keywords: Dinoflagellate; Immunolabeling; MS analysis; Oxyrrhis marina; Rhodopsins; Western immunoblotting.
© 2022. The Author(s).