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 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 Dunaliella 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 (Tf) 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).

This dataset includes unprocessed data and simple data calculations accomplished with Excel.