Spawning and fertilization

We collected adult sand dollars (D. excentricus) from Semiahmoo Bay, WA, on July 7, 2017, and maintained them in 14°C continuous flowing seawater at the Shannon Point Marine Center. On July 12, 2017, we induced twelve individuals to spawn by injecting 1-mL of 0.5-M KCl into the coelom following methods outlined by Strathmann (1987). We then collected and mixed concentrated gametes of four males and four females for fertilization. We added five drops of sperm to 500-mL of filtered seawater and 5-mL of eggs. We placed the fertilized eggs in 12°C incubator and bubbled them with ambient pCO2 condition for 12-hrs before dividing the embryos into pCO2 treatment conditions before gastrulation. We then counted and transferred the larvae into jars with 1.5 L of nanopore filtered seawater at densities of 1-2 individuals mL^-1.

Grazing experiment

To assess the interactive effects of high temperature and pCO₂ on Dunaliella excentricus feeding behavior, our experimental design had six treatments with four experimental jars (replicates) in each. The treatments combined three levels of CO₂: 400 ppmv (ambient atmospheric level), 800 ppmv (moderate atmoshpheric level) and 1,500 ppmv (high atmospheric level), and two temperatures: 12°C (ambient temperature) and 17°C (high temperature). We fed D. tertiolecta at approximately 6,000 cells ml^-1 to six-arm stage larvae to evaluate feeding rates at each treatment condition.

For each replicate, a corresponding 150-mL control bottle containing only D. tertiolecta was also prepared. Feeding rate was estimated as ingestion rate by measuring the algal concentration (cells ml^-1) at the beginning (T₀) and after 24 hours (T_f) in control bottles and experimental jars using a Sedgewick Rafter Chamber (Stumpp et al., 2011). Ingestion rate (cells ind^-1 hr^-1) was calculated as I = (Clearance rate) x (time-average algae concentration).

Lipid storage analysis

At the end of the long-term experiment, larval lipid index was assessed using a procedure adapted from Talmage et. al (2010). First, we randomly selected D. excentricus larvae from each treatment and stained them with Nile Red dissolved in acetone. Nile Red stains intracellular lipid droplets bright yellow. Larvae were exposed to the stain for ∼1.5 h, after which they were photographed under an epi-fluorescent microscope (Leica 80i) within 4 hours of being stained (Ko et al., 2014). The lipid areas of approximately 5−15 larvae per sample were measured using the ImageJ software. The lipid index was calculated by dividing the area of the larva stomach containing the fluorescing lipids by the total stomach area (Talmage & Gobler, 2010).