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.

Larval Rearing

We reared D. excentricus larvae (2 individuals mL-1) at 12°C in eight 3-L jars that were individually bubbled with CO2 to achieve four replicates of ambient (400ppm) and acidic (1500ppm) pCO2 conditions. Each jar of larvae received a water change with pre-equilibrated 0.35-m filtered ambient and acidic seawater and fed the larvae D. tertiolecta (6,000 cells ml-1) daily. Pre-equilibrated ambient and acidic water was held in tanks within the same 12°C incubator as the rearing jars.

Experimental Design

We conducted two behavioral experiments; one when the larvae were 4-arm pleutei and one when the larvae were 6-armed pleutei.

To measure the effect of pH conditions on the vertical distribution of larvae we established three experimental pycnocline treatments within clear plexiglass water columns (2.5cm x 2.5cm x 30cm): (1) ambient water (400ppm) in the top layer and acidic water in the bottom layer (1500ppm), (2) ambient water (400ppm) in both top and bottom layers, and (3) acidic water (1500ppm) in the top layer and ambient water (400ppm) in the bottom layer. Each water layer was 60-mL of water and filled the column 10-cm high, so when each experimental treatment was established it filled the column to 20-cm. We established the experimental treatments by increasing the density of seawater in the bottom layer by 0.003-0.005 g ml-1 using PercollTM GE Healthcare (Podolsky & Emlet 1993). Experimental treatment water was kept at 12°C and pre-equilibrated to the desired pCO2 level and density. We also included blue food coloring (1 drop per 100-mL) to the dense bottom layer to more easily visualize the density layers while establishing experimental treatments. We set-up four replicate columns for each experimental treatment making twelve columns total per experiment.

Columns were positioned in a randomized order along the table of a walk-in incubator set to 12°C. Once columns were in position and treatments were established, we carefully injected 150 larvae by syringe into the bottom 2-cm of each column with no more than 2-mL of their culture water. Larvae were given 10 minutes in darkness to acclimate before we performed the first count of vertical positions of larvae in each water column. At 30 minutes of acclimation, we performed a second count of vertical positions of larvae in each column. For each larval count, we used a small hand-held flashlight and counted by eye the number of larvae occupying each centimeter of the water column beginning at the bottom and moving up to the top. We did these counts in the dark, so only one column received direct light from our small flashlight at a time to reduce the influence of light on the larvae’s behavior.