Three hundred copepods were collected in April of 2018 from eastern Long Island Sound, Connecticut, USA (41.3°N, 72.0°W) and raised for one year (~12 generations) (14 degrees Celsius (°C), 400 microatmospheres (μatm) CO2, 30 ‰ salinity, 12:12 hours light:dark) as stock cultures to limit maternal effects (Falconer, 1989, Introduction to Quantitative Genetics). Three resulting stock cultures with >2,000 individuals each were combined and then split evenly into three groups for each of the four treatments. Groups were acclimatized within a generation to temperature (15°C or 13°C, 1°C per day) and pCO2 (1000 μatm, 100 μatm per day, OA treatments only). Groups seeded the F0 individuals for 7-10 days yielding ~15,000 eggs per treatment. Resulting F0 eggs and nauplii were combined for each treatment, redistributed among three replicate cultures, and returned to their respective experimental conditions. The experimental environmental conditions were: 1) Ambient control (AM): 13°C, 400 µatm CO2, pH = 8.2; 2) Ocean Acidification (OA): 13°C, 1000 µatm CO2, pH = 7.85; 3) Ocean Warming (OW): 15°C, 400 μatm CO2, pH = 8.2; 4) Combined warming and acidification (OWA): 15°C, 1000 μatm CO2, pH = 7.85. Copepods were fed equal proportions of the live phytoplankters Tetraselmis sp., Rhodomonas sp., and Thalassiosira weissflogii every 48-72 hours to achieve food-replete conditions (≥600 micrograms (μg) Carbon per liter (L)) (Feinberg and Dam, 1998. Marine Ecology Progress Series), deliberately raised under ambient conditions to avoid confounding effects of possible food quality changes.

Body size was measured as prosome length at C1 and C6 stages using Image-J (https://imagej.nih.gov/ij/) for individuals grown in 250-milliliter (mL) beakers alongside survivorship experiments. Ten individuals per replicate and treatment (i.e. 10 C1, 10 males, and 10 females) were preserved in non-acid Lugol's solution each generation for life-history trait measurements. Individuals were isolated in a drop of filtered seawater and photographed using a Lumenera Infinity5-5 camera (Teledyne Lumenera, Ottawa, ON, CAN) attached to an inverted microscope (Olympus IX70, Olympus, Waltham, MA, USA) after the water droplet had been removed. Full methods on replication can be found in deMayo, et al. 2023.