* NOTE: Units for cell counts for the two different years vary: 2014 are raw settled counts while the 2015 counts are cells/ml from the coulter counter.

2014 Study:

This study consisted of two separate experiments in which Calanus pacificus was maintained at three target pCO2 levels, 400, 800, 1200 μatm at a temperature of 12˚C and was fed either Prorocentrum micans or Ditylum brighwellii cultured at the same pCO2 and temperature levels. Copepods and phytoplankton were acclimated to treatment pCO2 for 7 or 8 days. After this acclimation period the phytoplankton were sampled for Chlorophyll content, cell size and Carbon and Nitrogen content.

Adult female Calanus pacificus were collected with a 1-m diameter, 335-μm mesh, ring net lifted vertically from 200m to the surface in Rosario Strait, Washington State. During the morning (0900-1130 7/14/14; 0800-11:45 8/4/14) copepods were transported back to Shannon Point Marine Center (SPMC) where mature adult females were separated under the microscope and placed in 1 L jars of filtered (5 μm) seawater containing respective treatment pCO2 levels. Jars of adult females (5 per liter) were given a 75% water change daily with pre-equilibrated pCO2 seawater and were fed either phytoplankton acclimated to the same pCO2 as the copepods (Grazing 1 and 2) or prey held at pCO2 400 for all treatments (Grazing 3) twice daily. P. micans was used in experiment 1 (Grazing 1) at 150 cells per ml and D. brightwellii was used in experiment 2 (Grazing 2 and 3) at 120 cells/ml. In all experiments, the number of prey cells added equaled saturating food concentrations of 400 µg C per liter. Jars were covered in foil and kept in the dark and allowed to sit in the climate controlled room for 90 minutes prior to sampling the T=0 time point. During Experiment 2 (Ditylum) in addition to feeding every 12 hours, jars were also stirred 6 hours after each feeding to re-suspend cells that had settled out of the water column. After the T=0 time point, treatment bottles were placed in the environmental chambers supplied with CO2 gas of equal concentration to respective treatment.

Samples were fixed in alkaline Lugol’s at each time point. Aliquots between 1 and 5 milliliters were later settled in 6 well culture plates and counted with an inverted microscope.

2015 Study:

This study consisted of two separate experiments in which Acartia hudsonica was maintained at three target pCO2 levels, 400, 800, 1200 μatm at two different temperatures (12˚C and 17˚C) and was fed Rhodomonas salina cultured at the same pCO2 and temperature levels. Copepods and R. salina were acclimated to treatment pCO2 for 5 and 4 days for the 12˚C and 17˚C experiments, respectively. After this acclimation period the physiology and biochemistry of R. salina was characterized.

A. hudsonica was attained from Michael Finiguerra (University of Connecticut) and was maintained at approximately 20˚C and fed R. salina ad libitum. Adult copepods for experiments were picked out of the culture over a two-day period. A subset of females was set up for initial egg production, hatching success, and naupliar development tests (described below) and the rest of the adults were divided into 45 adults per 500 mL jar of pCO2-equilibrated seawater and held in the Atmospheric Carbon Control Simulator (ACCS) for the duration of the acclimation period. During the acclimation period jars of adult females were given a 75% water change daily and fed pCO2 acclimated R. salina every 12 hours to maintain a concentration above the saturation feeding density of 400 µg C L-1. At the end of the acclimation period, grazing rates, carbon, nitrogen, and fatty acid content of the adult females (described below), as well as carbon, nitrogen, and fatty acid content of R. salina were measured.

The direct and indirect effects of R. salina cultured under different pCO2 levels on A. hudsonica grazing were each evaluated once per experiment. The direct effects were measured by comparing the grazing rates of acclimated A. hudsonica to R. salina cultured under the same pCO2 treatment. Indirect effects compared the grazing on R. salina cultured under ambient (400 μatm) pCO2, regardless of the copepod pCO2 acclimation treatment. Grazing rate tests were done in 250 ml bottles with 15 female A. hudsonica per bottle, with three replicates and two controls per treatment. Bottles were covered in foil and incubated for 24 hours. R. salina cell concentrations were counted before and after the incubation using a Beckman Z2 coulter counter, and compared to R. salina growth in the control bottles with no copepods.