Collection & Larval Rearing

We collected adult Olympia oysters (Ostrea lurida) from Fidalgo Bay in June 2017 and maintained them in a sea table with continuous flowing seawater heated to 19-20°C at the Shannon Point Marine Center. We fed adult oysters were fed concentrated algae once a day (Shellfish Diet, Reed Mariculture) and utilized banjo-style filters (60-m) attached to the outflow pipes of the sea table to catch released O. lurida larvae. We then collected and reared larvae at 12°C in 3-L jars (2 individuals mL-1). Each jar of larvae received a 50% water change with 0.35-m filtered sea water and were fed Isochrysis galbana algae (50,000 cells mL-1) daily.

Experimental Design

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 Percoll (Mills 1984; Podolskey & 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.

On the day of each experiment, we incubated the experimental treatment columns in clear plexiglass water baths connected to a Fisher Scientific Isotemp recirculating water bath to maintain treatment temperature at 12°C throughout the experiment. 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. Olympia oyster larvae are highly phototactic (personal observations), so we gave the larvae 10 minutes to acclimate in darkness and then recorded their vertical position in the water columns under infrared light. We video recorded the larvae’s vertical position in each column using an infrared uEye camera equipped with Edmund Optics VIS-NIR Lens mounted on a motorized stand. We later counted by eye the number of larvae per centimeter area of each column from the videos.

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 water column and right at the transition layer where the top and bottom layers of water met and 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.