2014 Study: 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. 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 500 ml jars of filtered (5 μm) seawater at a concentration of 30 females/L. Jars of adult females were given a 75% water change daily and were fed pCO2 acclimated prey twice daily. Experiment 1 (EP1, EP2, EP3) was fed Prorocentrum micans and Experiment 2 (EP4, EP5, EP6) was fed Ditylum brightwellii.

During Experiment 2 (Ditylum) in addition to feeding every 12 hours, jars were also stirred 6 hours after each feeding to resuspend cells that had settled out of the water column. Females were acclimated to pCO2 conditions and pCO2-acclimated prey for 7 days.

The direct effects of pCO2 on C. pacificus egg production, hatching success, and naupliar development were tested at the beginning of each experiment before adults were acclimated to pCO2 levels and fed pCO2-acclimated prey. Immediately after collection from the field, twenty females were placed individually in 250 ml containers of pCO2-equilibrated seawater and incubated overnight (female Calanus are known to spawn between 00:00 and 07:00 Runge 1985). The following morning females were removed and measured and any eggs they spawned were counted and left in the jars (with water change) for eggs to hatch and nauplii to develop for 4 days (EP1, direct effects, was 5 days), at which time approximately 50% of the nauplii had reached the nauplius 3 stage. Because nauplius 3 is the first feeding stage nauplii were not fed. After 4 days the jars were checked for naupliar survival and all nauplii were preserved in 5% buffered formalin/seawater for counting and staging. Hatching success was calculated from the number of hatched nauplii and unhatched eggs found at the end of the experiment; naupliar survival was calculated from the proportion of hatched nauplii that were alive at the end of the experiment; development was calculated as the proportion of hatched nauplii that reached nauplius 3.

After seven days of acclimation, twenty females from each treatment were again placed individually in 250 ml containers of pCO2-equilibrated seawater and incubated overnight. The eggs and nauplii were treated the same way as with the direct effects.

2015 Study: During Exp 1 (12˚C) Acartia hudsonica were maintained at three target pCO2 levels, 400, 800, 1200 μatm, and were fed Rhodomonas salina cultured at the same pCO2 levels. We assessed the reproductive output and larval development of A. hudsonica at each pCO2 level before and after the acclimation period. At the beginning of the experiment females were 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 at target pCO2 levels 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 3000 cells/mL while allowing for a maximum grazing rate of 6000 cells/hr/female.

In Exp 1 (12˚C) the acclimation period was five days. On day 3 some females were added to jars of males at a ratio of 1 female: 2 males to ensure they would be fertilized for egg production, hatching, and development experiments. On day 6 of the experiment females that were incubated with males were used for egg production and subsequent hatching and naupliar development tests. Egg production, hatching, and naupliar development before and after the acclimation period were both tested in two separate trials in Exp 1, however, due to logistical constraints, the 800 μatm pCO2 target treatment was only included in one trial.

In all egg production, hatching, and naupliar development tests, adult females were incubated individually inside mesh-bottom egg production chambers within 250 ml containers of target treatment pCO2-equilibrated seawater. After 24 hours the females were removed, measured, and the eggs were placed back into the atmospheric simulation chambers to develop. Nauplii were fed R. salina once per day at 25% the amount calculated for adults (described above). During tests of the direct effects of pCO2 on A. hudsonica naupliar development the nauplii were fed stock R. salina that was not pCO2 acclimated; during tests at the end of the acclimation period nauplii were fed R. salina that was cultured at the corresponding target pCO2 treatment. At the conclusion of the naupliar development tests, 10% of the jars were checked for naupliar survival and all were preserved in 5% buffered formalin/seawater solution for counting and staging. Hatching success was calculated from the number of hatched nauplii and unhatched eggs found at the end of the experiment; naupliar development was calculated by the proportion of hatched nauplii that reached the Nauplius IV stage (N IV). In Exp 1 (12˚C) eggs and nauplii were allowed to develop for 8 days, and were fed pCO2-acclimated R. salina starting on day 4.