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 dialyzed 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 counted the vertical distribution of larvae in each water column. Using a small hand-held flashlight, we 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.

At the end of the experiment we collected water from bottom layer (1-2cm above bottom), top layer (18-20cm from the bottom), and the transition point (visually determined based on color of where two water layers met) and measured pH with a pH probe.

Sampling and analytical procedures:

Carefully collected water with a syringe and pipet from the top 1-3cm of the column, the bottom 1-3cm of the column, and at the transition layer where the top and bottom layers of water met, which was visible by the blue dye in the bottom layer of water. The water from the syringe was carefully transferred to a clean 2 ml microcentrifuge tube and pH was measured directly using a pH probe (Micro PerpHect Ross Ross® Combination pH electrode) and read with a Thermo Scientific Orion Star pH meter.