β carotene

The cell viability of P. marinus was assessed using the Cell Counting Kit‐8 (Dojindo Laboratories, Kumamoto, Japan) as previously reported (Sakamoto et al. 2016), and always three independent assays were performed in triplicate. For the growth inhibition assay, 100 mM fluridone (Fluka, Riedel‐de Haën, Germany) dissolved in dimethyl sulfoxide (DMSO) was added to each test well in the plate to yield the indicated final concentration. Absorbance of vehicle control at 0 and 3 d were defined as 0 and 100% of parasite growth, respectively. A dose–response curve was fitted for each assay using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA) with the built‐in model “log(inhibitor) vs. response” by constraining the Bottom plateau to 0. The mean IC₅₀ with 95% confidence interval (CI) was then calculated from three independent assays.
For a rescue assay, β‐carotene (Wako Pure Chemical Industries, Osaka, Japan), lycopene (Wako Pure Chemical Industries), and ABA (Sigma‐Aldrich) solutions were added to P. marinus cultures treated with or without 50 μM fluridone to yield the indicated final concentrations. ABA was dissolved in DMSO at 100 mM. lycopene and β‐carotene were dissolved in tetrahydrofuran (THF) (Wako Pure Chemical Industries) at 2 mM immediately before each experiment. The amount of DMSO and THF added to the cells did not exceed 0.1% (v/v) and 0.5% (v/v), respectively.

The Raman microscope we used in this study is a homemade confocal microscopy system with 532 nm continuous-wave laser irradiation. The laser system was Millennia Pro from Spectra-Physics Lasers, Inc. The laser power at the sample was 1 mw. The objective lens used for the study was the CFI S Fluor 40× oil immersion lens with NA 1.30. The spectrometer was the iHR320 model from Horiba, and CCD camera was the liquid nitrogen-cooled Spec-10 2 kV-EV/LN model from Princeton Instruments. The groove density of the grating was 600 grooves/mm and the center wavelength was 500 nm. The spectral resolution of all measurements was 6 cm⁻¹.
As for the standard samples that are used to represent the spectra of carbohydrates, lipids, proteins, and carotenoids: starch (from corn), albumin (from bovine serum, fatty acid free), and β-carotene were purchased from Wako Pure Chemical Industries, Ltd. Oleic acid was purchased from Sigma.

Anhydrous sodium chloride (NaCl) and magnesium sulfate (MgSO₄) for formulations as QuEChERS components were purchased from Fujifilm Wako Pure Chemicals (Tokyo, Japan). β-Carotene, β-cryptoxanthin, campesterol, ferulic acid, stigmasterol, sitosterol, and rutin were purchased from Fujifilm Wako Pure Chemicals. p-Coumaric acid, diosmin, hesperidin, naringin, and narirutin were purchased from Tokyo Chemical Institute (Tokyo, Japan). Pyrogallol and 2,6-di-tert-butyl-4-methylphenol (BHT) used to prevent autooxidation were purchased from Fujifilm Wako Pure Chemicals (Tokyo, Japan).
All organic solvents (acetonitrile, formic acid, tetrahydrofuran, and methanol) and ultrapure HPLC- or LC/MS-grade water were purchased from Fujifilm Wako Pure Chemicals.
Orange juice was purchased from a local market in Tokyo, Japan.