Dataset: Copepod Aggregate Microscale Interaction Experiments 2020

Included are 33 video files (MP4) from three experiments conducted in 2020. 26 of the videos show interactions between individual tethered copepods and sinking phytoplankton aggregates from above. 7 of the videos are control observations with tethered copepods. 

The filenames of each copepod-aggregate interaction video have the following format:
Exp#_copepod#_aggregate#

The filenames of each control video have the following format:
Exp#_copepod#_control_abbreviation
where the abbreviation describes the type of control that is described further in Table 1 (see Supplemental Files).

Video files are available in file bundles (zip), in the "Data Files" section:
control_copepod_videos.zip
Exp1_copepod_videos.zip
Exp2_copepod_videos.zip
Exp3_copepod_videos_part1.zip
Exp3_copepod_videos_part2.zip

Sampling and analytical procedures:

Three experiments were conducted between October and December of 2020 (Experiment 1 on October 2, Experiment 2 on November 21, and Experiment 3 on December 19), in which individual, tethered Calanus pacificus female copepods were exposed to sinking aggregates in order to determine if copepods are capable of modifying phytoplankton aggregate properties. Each copepod was exposed to 1-4 aggregates in order to increase the chances of observing a direct interaction between female C. pacificus copepods (2.6-3.4 mm prosome length) and phytoplankton aggregates (~ 3-5 mm major axis length, ~2-4 minor axis length).

Aggregates were formed in the lab from non-axenic cultures of Thalassiosira weisflogii grown in f/2 media at room temperature. Cylindrical acrylic tanks (volume 2.2 L) were prepared with these cultures at concentrations of 30,000 cells per mL. These cylindrical tanks were placed on a roller table and allowed to rotate at 3.3 revolutions per minute for 48 hours to form aggregates. Aggregate formation occurred in the dark to arrest phytoplankton growth. Aggregates were always formed during the exponential growth phase of the cultures and new cultures were used for each experiment.

C. pacificus copepods were collected with a 300 µm mesh plankton net (0.5 m diameter mouth) using a small boat near Scripps Canyon in La Jolla, CA (32° 51’ 23.8” N, 117° 16’ 00.1” W) 6-7 days before each experiment. 5-6 oblique tows were taken per sampling trip to a depth of at least 40 m and for a duration of 3 to 5 minutes. Zooplankton samples were sorted in the lab to isolate adult C. pacificus. Copepods were maintained with regular water changes in an incubator in the dark at 18 degC until the experiment and fed a diet of Thalassiosira weissflogii.

Copepods were starved for 24 hours prior to each experiment by transferring 20-25 C. pacificus individuals (adult females) into 1-2 200 mL beakers (depending on copepods available for the experiment). Each beaker was wrapped in aluminum foil and placed in a dark room to maintain darkness, and was kept at room temperature.

Copepods were pipetted out of the 200 mL one at a time 10-20 minutes before the experiment and tethered to a 3-4 cm hair strand glued to a plastic straw on one end (taking the shape of a garden pick). To tether the copepod to the far end of the hair strand, an individual copepod was placed on a Petri dish with the least possible amount of water to limit the copepod movement. The far end of the hair strand was dipped into a drop of cyanoacrylate glue and immediately attached to the back of the copepod prosome (as if poking the copepod). Once tethered, the copepod was submerged in filtered seawater at room temperature and was observed for a few minutes to ensure that the copepod was still alive and no appendages had been damaged (typically the long antennules can break during this process). If the copepod was in good shape and still active, it was used for the experiment. This mode of tethering allows the copepod to still perform forward escape jumps or other movements since all the appendages (sensory, locomotory and feeding appendages) were free of movement.           

Two experimental tanks were used for this experiment. One tank had a square base (10 cm × 10 cm) and a height of 50 cm, and a second tank had a rectangular base (16 cm x 8 cm) and a height of 30.5 cm. For the experiments, a tank filled with filtered natural seawater was set up on a table. At the top of the tank there was an adjustable rig used to attach and suspend the straw with the tethered copepod. This allowed us to set the copepod at a consistent distance away from the walls and directly under a small funnel used to introduce individual aggregates, one at a time. Importantly, the tether was used to constrain the movement of the copepod so that it remained in the camera's field of view (FOV).

A high-speed camera (Photron FASTCAM Mini AX50) was placed facing a side of the tank in a way that the copepod appeared within the camera's FOV (~2 cm x 2 cm) and on a sideview body position so the camera could capture all the copepod appendages (antennule, feeding and swimming appendages). This allowed us to capture the copepod appendages when it was still as well as when it performed escape jumps. These jumps were limited by the hair tether, but constrained within the FOV to observe when the copepod made contact with the aggregate passing by on its downward trajectory. A 730 nanometer near-infrared light-emitting diode (M730L4 730 nm, 515 mW Mounted LED, Thorlabs) was set up facing straight into the lens but on the opposite side of the tank; this created a shadowgraph-type image. A near-infrared light was used because copepods, like most crustaceans and marine animals, are not sensitive to this wavelength. This was done to avoid light-induced responses by the copepods. All recordings were filmed at 2000 frames per second (fps), but the videos were compressed to 30 fps for visual processing. All experiments were conducted inside a dark room.

Before switching to a new copepod for recording, the tank was emptied and replaced with fresh filtered seawater to remove any residual aggregate material from the tank. Video recordings were only kept if the aggregate successfully sank within the field of the view of the camera (thus allowing a possible interaction to occur). So, for example, in Experiment 1, there is a recording for Copepod #3, Aggregate #3, but no other aggregates, indicating that Aggregate #1, #2, and #4 did not successfully sink within the field of view of the camera.