CO2 \u00d7 temperature manipulations and measurements:
\nFor 2 \u00d7 2 and 3 \u00d7 2 factorial designs, replicate rearing containers (20 L) were placed into large temperature-controlled water baths. Elevated CO2 levels were achieved via gas proportioners (ColeParmer\u00ae) mixing air with 100% CO2 (bone dry grade) that was delivered continuously to the bottom of each replicate rearing container via airstone. To counteract metabolic CO2 accumulation, control CO2 conditions were achieved by forcing compressed laboratory air through a series of CO2 stripping units containing granular soda lime (AirGas\u00ae), a particle filter (1 \u00b5m), and then to each replicate via airstone. Target pH levels were monitored daily using a handheld pH probes (Orion Ross Ultra pH/ATC Triode with Orion Star A121 pH Portable Meter; Intellical PHC281 pH Electrode with Hach\u00ae HQ11D Handheld pH/ORP Meter) calibrated bi-weekly with 2-point pHNBS references. Continuous bubbling maintained dissolved oxygen saturation (>8 mg/ DO) in rearing vessels. Target treatment temperatures were controlled by thermostats (Aqualogic\u00ae) which powered chillers (DeltaStar\u00ae) or glass submersible heaters to maintain water bath temperatures. For 3 \u00d7 3 factorial experiments, we developed an automated acidification system composed of nine discrete recirculation units designed for larval fish rearing. We designed a LabView (National Instruments\u00ae) based program to fully automate the control of seawater chemistry. The software interfaces with the recirculating units via a data-acquisition module (NI cDAQ-9184, National Instruments\u00ae), which controls nine sampling pumps (one per tank) and a series of gas and water solenoid valves, while receiving input from a central pH electrode (Hach pHD\u00ae digital electrode calibrated weekly using 2-point pHNBS references) and dissolved oxygen probe (Hach LDO\u00ae Model 2). The software sequentially assesses the pH conditions in each rearing unit (each tank once per hour) by pumping water for ~450 seconds through the housing of the central pH probe, comparing measured pH levels to set-points and then adjusting levels by bubbling standardized amounts 100% CO2 (bone dry grade, AirGas\u00ae) or CO2-stripped air into the sump of each tank. The software also maintains DO saturation (>8 mg/l) by bubbling in CO2-stripped air. LabView logs current pH, temperature, and DO conditions before cycling to the next unit. Temperatures were controlled by thermostats (Aqualogic\u00ae) that powered submersible heaters or in-line chillers (DeltaStar\u00ae).
Actual treatment CO2 levels were determined based on measurements of pH, temperature, salinity, and total alkalinity (AT). Treatment tanks were sampled three times per experiment for measurements of AT (\u03bcmol kg-1). Seawater was siphoned and filtered (to 10 \u00b5m) into 300 ml borosilicate bottles. Salinity was measured at the time of sampling using a refractometer. Bottles were stored at 3\u02daC and measured for AT within two weeks of sampling using an endpoint titration (Mettler Toledo\u00ae\u00a0G20 Potentiometric Titrator). Methodological accuracy (within \u00b11%) of alkalinity titrations were verified and calibrated using Dr. Andrew Dickson\u2019s (University of California San Diego, Scripps Institution of Oceanography) certified reference material for AT in seawater. The partial pressure of CO2 (pCO2,; \u03bcatm) was calculated in CO2SYS (V2.1, http://cdiac.ornl.gov/ftp/co2sys) based on measured AT, pHNBS, temperature, and salinity using K1 and K2 constants from Mehrbach et al. (1973)\u00a0 refit by Dickson and Millero (1987)\u00a0 and Dickson (1990) for KHSO4.
\nField sampling and experimental designs:
\nCollections of wild, spawning ripe Atlantic silversides were made during high tide 1-3 days prior to full or new moons during the species spawning season. Adults were caught with a 30 m \u00d7 2 m beach seine from local salt marshes and transported live to our laboratory facilities. Ripe adults were held overnight at 20\u00b0C in well aerated tanks at low densities with no food and strip spawned the next day.
For each experiment, eggs from 20+ running-ripe females were gently mixed into shallow plastic dishes lined with 1 mm plastic window screening. 20+ males were stripped-spawned together into 500 ml glass beakers, mixed with seawater, stirred, then gently poured into spawning dishes and mixed with eggs for ~15 minutes. Screens were rinsed with seawater to remove unfertilized eggs and then soaked in a 100 ppm buffered iodine (Ovadine\u00ae) solution for 15 minutes to prevent fungal infection. Experiments were initiated within two hours of fertilization when replicate rearing vessels received precisely 100 embryos. Vessels were filled with clean seawater (filtered to 1 \u00b5m and UV sterilized). Optimal salinity (27-31) and light conditions (15 h light:9 h dark) for rearing M. menidia were maintained across experiments. Upon hatching larvae were immediately provided ad libitum rations of newly hatched brine shrimp nauplii (Artemia salina, San Francisco strain, brineshrimpdirect.com) and equal rations of powdered weaning diet (Otohime Marine Fish Diet, size A1, Reed Mariculture\u00ae). To quantify hatching survival, one day post first hatch larvae were counted by gently scooping small groups into replacement rearing vessels. For initial hatch measurements, random sub-samples (N = 10) from each replicate were preserved in 5% formaldehyde/freshwater solution buffered with saturated sodium tetraborate. All experiments were terminated when larvae reached ~10 mm standard length (SL). At termination, all survivors were counted and measured for standard length (SL, nearest 0.01 mm) via calibrated digital images (Image Pro Premier\u00ae V9.0).
CO2 x temperature specific early life survival and growth assessed by 5 factorial experiments.
\nThese data are published in: Murray, C.S. & Baumann, H. 2018 You better repeat it: complex temperature \u00d7 CO2 effects in Atlantic silverside offspring revealed by serial experimentation. Diversity 10, 1-19. doi:10.3390/d10030069
BCO-DMO Processing:
\n- modified parameter names to conform with BCO-DMO naming conventions\u00a0(replaced spaces with underscores);
\n- changed date format from mm/dd/yyyy to yyyy/mm/dd;
\n- replaced \"n/a\" with \"nd\";
\n- replaced spaces with underscores in columns: species, adult_collection_site;
\n- removed commas from adult_collection_site field;
\n- replaced original lat/lon values with decimal degree values provided by PI.