During the summer of 2018, two experiments were conducted to investigate the effect of marine snow distribution on the ingestion rage of the copepod Calanus pacificus: Experiment 1 was conducted on July 2 and Experiment 2 was conducted on July 13. Each experiment consisted of three treatments: a control (in which copepods were placed in a tank with no marine snow), a layer treatment (in which copepods were placed in a tank with a layer of marine snow in the middle), and a homogenous treatment (in which copepods were placed in a tank with a roughly homogenous distribution of marine snow).
C. pacificus was collected for both experiments using a small boat near Scripps Canyon in La Jolla, CA (32° 51.720' N, 117° 16.816' W) on June 28th, 2021 with a 333 µm mesh plankton net (0.5 m diameter mouth). Samples were sorted in the lab to isolate individuals of the species C. pacificus. Copepods were maintained with regular water changes in an incubator in the dark at 18°C until the experiment and fed Thalassiosira weissflogii. Copepods were starved for 24 hours prior to each experiment by transferring copepods into beakers with filtered seawater which were wrapped in aluminum foil to maintain darkness and kept at room temperature.
Prior to each experiment, less dense filtered seawater was mixed by diluting filtered seawater with distilled (DI) water to be used as the top layer fluid in the control treatment and layer treatment (both of which had a density gradient). The density of the top layer fluid was 0.004 g/cm3 less than the bottom layer fluid. Densities of the different seawaters were measured using a handheld density meter (DMA 35, Anton Paar), and were kept roughly consistent for the two experiments.
Also before each experiment, two phytoplankton cultures of the species T. weissflogii were started in 2L flasks and were grown in f/2 media at room temperature under 24 hour light for 6 days corresponding to the exponential growth phase. Three days before each experiment, the cultures were diluted to 32,500 cell/mL to form aggregates. The filtered seawater used to dilute these cultures had different densities depending on the treatment: aggregates for the layer treatment were rolled in the top layer fluid (with a density 0.004 g/cm3 less than the experimental bottom layer fluid) and aggregates for the homogenous treatment were rolled in seawater with a density 0.001 g/cm3 less than the experimental bottom layer fluid. The diluted cultures were then added to cylindrical acrylic tanks (each with a volume of 2.2 L) and these cylindrical tanks were allowed to rotate in the dark on roller table for 3 days at a rate of 3.3 rpm to form aggregates.
The experimental tank had a square base (10 cm × 10 cm) and a height of 50 cm. For the stratified treatments (control treatment and layer treatment) density gradients were established by filling the tank with ~2.5 L of bottom layer fluid, followed by ~2.5 L of less dense top layer fluid. Top layer fluid was slowly pumped on top of the bottom layer fluid through a diffuser to minimizing mixing. The non-stratified homogenous treatment was set up by pouring ~5 L of filtered seawater/bottom layer fluid into the tank.
Experimental treatments were done in succession, with the control treatment first, followed by the layer treatment, and then the homogenous treatment. Once each tank was set up, aggregates were pipetted into the top of the tank (except for the control treatment). For the layer treatment, ~5 mL of aggregates were pipetted into the top of the tank at once, allowing to settle and form a layer at the density gradient. For the homogenous treatment, a similar total volume of aggregates were pipetted into the tank but were pipetted individually evenly across the surface of the tank and throughout the duration of the experiment. For each treatment, 20-25 copepods (a mixture of adult males, adult females, and copepodite-V stage copepods) were carefully pipetted into the top of the tank. For the layer treatment, copepods were added after the majority of aggregates had settled at the density gradient. For the homogenous treatment, copepods were added after about half of the aggregates had been added to the tank and the first aggregates had settled about halfway through the tank.
Copepods were allowed to forage in the tank until aggregates in the layer and homogenous treatment started reaching the bottom of the tank (since we wanted to avoid measuring any bottom feeding). The total time that the copepods were in the tank for these treatments was between 4-7 minutes. The total time the copepods were in the tank for the control treatment was about 9-10 minutes.
The copepods were removed from the experimental tank with gentle suctioning of water onto a sieve. For gut pigment analysis, two copepods were placed in 8-10 amber vials (depending on the total number of copepods recovered), which contained 3 mL of 90% acetone. A sonicator was used to break up the copepods at 40% amplitude for 5 seconds and release their gut content into the acetone solution. In addition, after each experiment water from each experimental tank was evenly mixed, and three subsamples of 25 mL of tank water was filtered onto a GF/F filter and placed into 5 mL of acetone. After about a day in a -20°C freezer, the copepod and tank water samples were analyzed using a Trilogy Laboratory Fluorometer (Turner Designs) to measure the concentration of chlorophyll and pheophytin in the acetone solution. Tank fluorometer measurements are given in the attached Supplemental File "Tank_Fluorescence_Measurements.csv".
©2020 Biological and Chemical Oceanography Data Management Office.