Sample Collection

Zooplankton samples were collected using either a 60 cm diameter, 200 μm mesh ring net with a non-filtering cod- end or a 60 cm diameter, 335 μm mesh bongo net with non-filtering codends, lifted vertically from 10 m off the seafloor. Samples were stored in a cooler and air-bubbled for <24 h until actively swimming adult female C. pacificus were manually sorted under a microscope into 1 liter jars filled with 200 μm filtered seawater. The copepods were kept at 14°C and fed a pre-made mixture of five marine microalgae (Isochrysis, Pavlova, Tetraselmis, Thalassiosira weissflogii and T. pseudonana) daily, for no more than 2 wk until they were used in a single laboratory experiment.

Experiment Details

Water treatments of 2 different salinities (29 and 31) were made using Instant Ocean (~36 and ~39 g l−1) and verified with a YSI Pro 2030 salinity probe. Replicate 2-layer water columns with stable haloclines were created by pumping light (low salinity) water into the bottom of the tanks using a peristaltic pump until the water level was 500 mm from the bottom. Then, heavy (high salinity) water was pumped slowly to avoid mixing into the bottom, displacing the light water up- wards until the water columns were 780 mm deep with haloclines located 280 mm above the bottom.

To manipulate pH conditions, bottom water was split into 2 buckets of equal volume to use in treatment and control tanks. Treatment bottom water was bubbled with CO2 until it reached the desired pH. In the first 5 pH experiments, treatment bottom water was bubbled with a 2000 ppm CO2−air mixture for approximately 40 min. The final pH in this treatment was approximately 7.6, as measured by a Star A221 pH meter with a Ross ultra gel pH/ATC electrode that was calibrated daily prior to use. In the last 5 experiments, treatment bottom water was made using a feedback-control system that supplied mixed lab air and pure CO2 gas at 3000 ppm at 4.1 l min−1 through an airstone. The pH was measured with a Sunburst AFT (assumed constant salinity of 31) during bubbling to ensure a pH of 7.4 was maintained.

C. pacificus behaviors and vertical distributions in response to either hypoxic or acidic bottom waters were observed in an array of 4 replicate 1 × 0.1 × 0.1 m acrylic tanks, installed in an environmental chamber set to 14°C. Stressful water layers (or non-stressful controls) were placed at the bottoms of salinity-stratified tanks, modeled after conditions experienced in the field. To start each experiment, 20 animals were gently introduced to the top of each of the 4 tanks. Swimming behavior was then observed for 90 min using 5-megapixel IR USB cameras. Experiments were run during the day in the dark, with the tanks backlit with IR LED strips behind and around the base of each tank. Two front-facing cameras (‘bottom camera’, ‘surface camera’) recorded swimming in the X (left, right) and Z (up, down) directions, observing true vertical motion and projected horizontal motion. An upwards-facing ‘base camera’ was added to each tank in 2020 to improve tracking and behavioral analysis of copepods near the bottom. Base cameras recorded the bottom 2 cm of each tank in the X and Y (front, back) directions and therefore observed true horizontal motion but not vertical motion.

Video output details

Videos were processed with the software Fosica (Wallingford Imaging) to distinguish moving copepods from stationary background and noise and to extract copepod pixel coordinates. Pixel coordinates were converted into physical space units and then assembled into individual swimming paths using the Matlab software package Tracker3D (Chan & Grünbaum 2010), neglecting parallax in the camera field of view. A smoothing spline was applied to remove features changing faster than 6 Hz, which were dominated by frame rate noise. X and Z (or, for the base cameras, X and Y) pixel coordinates for each object and the total projected speed and velocities were calculated at every frame for each swimming path. Swimming paths included only animals actively moving in the tank, excluding motionless (moribund) animals at the bottom of the tank. Copepod swimming paths were used to calculate the mean height from the bottom of the tank, mean number of copepod localizations per frame, and mean swimming speeds.

Our video system could not distinguish between individuals that were dead and those that were lying immobilized on the bottom for extended periods (a behavior leading, at least in hypoxia, to a high likelihood of eventual mortality). Therefore, for 2020 experiments, we developed a video-based metric using the base cameras to classify copepods at the bottom of tanks that were ‘moribund.’ Remaining motionless on the bottom of the tank is an uncommon behavior for C. pacificus, and we conservatively estimated that copepods motionless for a 2 minute threshold were in a ‘moribund’ or stressed state. To quantify moribundity, we calculated the mean brightness values for each pixel from frames in each of the last 1 minute sections (89th and 90th minutes). Because the videos were recorded in a dark field with IR back-lighting, copepods appeared as bright spots in the videos. The brightness of pixels representing a copepod in these 1 min means was a direct function of the number of frames in which it remained stationary. We then used a brightness threshold to classify copepods as moribund if pixel brightness indicated they had not moved during the last 2 minute of video observations.

More details on the methodology can be found here: Wyeth, A.C., Grünbaum D., Keister J.E. (2022). Effects of hypoxia and acidification on Calanus pacificus: behavioral changes in response to stressful environments. Marine Ecology Progress Series, 697: 15-29. https://doi.org/10.3354/meps14142.

926368_v1_zooplankton_acidification_ph_experiment_overview.csv