Azunoru dish

Each 0.05 g (fresh weight) of thalli aeration-cultured at 15 °C was incubated statically in dishes (Azunoru dish; 90 mm diameter × 20 mm height, As One) containing 50 mL of seawater at 28 °C for 7 days or 28 °C for 7 days plus subsequent treatment at 32 °C for 1 to 7 days. The viability of these thalli was examined as described above. Analysis of samples under each treatment condition was repeated three times.

Each 0.05 g sample (fresh weight) of thalli (from aeration cultures grown at 15 °C) was incubated statically in dishes (Azunoru dish; 90 mm diameter × 20 mm height, As One Co., Ltd.) containing 50 mL of seawater for marine species or Contrex^® for freshwater species at 20, 25, 28, 30, 32, and 34 °C for 7 days, while control experiments were performed at 15 °C. The viability of these thalli was visualized daily by staining with artificial seawater containing 0.01% erythrosine (Wako Pure Chemical Industries, Japan) as described by Kishimoto et al. [16 (link)]. In brief, thalli were stained for 5 min at room temperature, gently rinsed with artificial seawater or Contrex^® to remove excess erythrosine, and mounted on a slide with medium. The thalli were observed and photographed under an Olympus IX73 light microscope equipped with an Olympus DP22 camera. Cells stained by the dye were defined as dead cells. Viability was calculated from the number of living and dead cells obtained using micrographs. Analysis of samples under each treatment condition was repeated three times.

Samples (0.05 g fresh weight) of thalli cultured under aeration at 15°C were incubated without agitation in dishes (Azunoru dish, 90 mm diameter × 20 mm height; As One Co., Ltd., Osaka, Japan) containing 50 ml of seawater at 15°C for 7 days to adapt to changes in culture conditions. We previously demonstrated that ‘Bangia’ sp. ESS1 acquires heat stress tolerance by establishing heat stress memory, which is mostly maintained for 2 days after recovery from a 7-day non-lethal stress (Kishimoto et al., 2019 (link)). Thus, the samples were then subjected to one of six different stress treatments (Figure 1A): (1) 28°C for 7 days (priming, P); (2) 32°C for 1 day (lethal high temperature-1, LHT-1); (3) 32°C for 6 days (lethal high temperature-6, LHT-6); (4) 28°C for 7 days and 15°C for 2 days (recovery, R); (5) 28°C for 7 days, 15°C for 2 days, and 32°C for 1 day (triggering-1, T-1); and (6) 28°C for 7 days, 15°C for 2 days, and 32°C for 6 days (triggering-6, T-6). The control condition was incubation at 15°C for 7 days. Samples from all stress treatments from three repeated experiments (three samples per treatment) were harvested, frozen in liquid nitrogen, and stored at −80°C prior to H₂O₂ quantification and gene expression analysis.