Illustrator v24

Triplicate 10 µL aliquots of preserved pyrosome samples were analyzed using compound light microscopy and environmental scanning electron microscopy (eSEM). A Nikon Epifluorescence microscope equipped with a Canon EOS 5D camera was used to capture images of cells greater than 10 µm in diameter. A FEI Quanta eSEM microscope was used to capture images of cells < 10 µm. Images of whole cells were identified by morphology and size.^{50 (link)–52} Cells were identified into broad groupings: diatoms (single vs. chain, pennate vs. centric), flagellates, nanozooplankton, mixed assemblages, and unidentified. Cells captured using eSEM were also identified to the genus level where visibly distinct. Brightness and contrast of images were adjusted using ImageJ v1.52k or Adobe Illustrator v24.1.

The sample size was determined a priori based on effect sizes of previous studies^{6 (link),10 (link),11 (link),106 (link),107 (link)} or by post hoc analyses to ensure >80% power was achieved to reduce type II error (for example, heart histology, developmental milestone achievement and cognition in adults). All statistical analyses were conducted in GraphPad Prism v8.0.1 or R v4.0.1 (refs. ^{108 ,109} ) and are listed with sample sizes in the corresponding figure legends. Preprocessing, statistical analysis and initial plot generation for all datasets were carried out using R v4.0.1. Method schematics were generated using Biorender.com. All figures were finalized in Adobe Illustrator v24.1.

ROS imaging data were analyzed with EasyRatioPro (PTI, HORIBA Scientific) software and further processed with Excel (Microsoft, Redmond, WA, USA) and Igor Pro v8.0 (Wavemetrics, Lake Oswego, OR, USA) software. Protoplast images were processed with ImageJ (NIH). Figures were prepared with Origin Pro v2020 (Originlab, Northampton, MA, USA) and Adobe Illustrator v24.1 (Adobe, San Jose, CA, USA). Averaged data are presented as means ± SEM (N = number of protoplasts from 3–5 independent measurements). For comparisons with two groups such as basal ROS levels and ROS levels from DF M. sexta OS and tomato PF M. sexta OS, we used the non-parametric Mann-Whitney U test. For comparison with three groups, as depicted in Figure 4, for basal OS/tbH₂O₂ and NAC, we used a non-parametric Kruskal-Wallis test followed by Dunn’s pairwise post hoc comparisons. Non-parametric tests were used since data failed to meet normality assumptions after transformations. For all analyses, data from extractions were pooled to attain a sample size of 66–124 protoplasts and were repeated for at least three replications. All analyses were carried out using GraphPad Prism v9.0 (La Jolla, CA, USA).