Methodology from Snyder, J.T.*, Murray, C.S.*, and Baumann, H. (2017) Potential for maternal effects on offspring CO2-sensitivities in a coastal marine fish. Journal of Experimental Marine Biology and Ecology (in press).

Five randomly selected females were strip-spawned onto cutout sections of window screen (1-mm mesh) that were placed into separate seawater-filled spawning dishes (Murray et al., 2014). To ensure full fertilization success and randomize potential paternal effects, eggs were fertilized with a mixture of milt from 22 males, thus producing full-sib and maternal half-sib embryos from each female. Adults were measured for total length (TL; mean TLmale = 9.14 cm, mean TLfemale = 10.4 cm) and frozen for later analysis of FA. Mesh screens with attached embryos were subsequently cut into smaller sections to allow precise enumeration, and within 2-hr post-fertilization 100 embryos were placed into each of three replicate rearing containers (20 L) per female and CO2 treatment (i.e., 600 embryos for each of five females, 3 × 100 in ambient and 3 × 100 in acidified treatments). Rearing containers were filled with 1-um filtered, UV-sterilized seawater (~30 psu) from Long Island Sound and placed in temperature-controlled water baths set to 24 deg C, the known thermal optimum for survival and growth in this species (Middaugh et al., 1987). Offspring were reared for 24 d post fertilization under a 15h light:9h dark lighting regime. After hatch, larvae were fed ad libitum rations of newly hatched brine shrimp nauplii Artemia salina (brineshrimpdirect.com), and 50% of water was replaced every 5 d to ensure safe ammonia levels (< 0.25 ppm). Hatched larvae were counted and subsampled (n = 10 per replicate) at 1 d post hatch (dph) by gently scooping them into identical 20 L containers, and final samples were taken at 16 dph. All samples were preserved in 5% buffered formalin for later measurements of larval standard length (SL, 0.01 mm) via calibrated digital images (ImagePro Premier, MediaCybernetics). The experiment thus quantified three related survival and three size traits for each replicate, female, and CO2 treatment: embryo survival (fertilization to 1 dph), larval survival (1 to 16 dph), overall survival (fertilization to 16 dph), size (SL) at hatch (1 dph), SL at 16 dph, and larval growth rate (GR = (SL16dph – SL1dph)/15).

CO2 regime:

Offspring were reared at ambient (~ 400 uatm, pHNBS = 8.18) and acidified CO2 conditions (~2,300 uatm, pHNBS = 7.50). The higher value was set to a level commonly used in OA research (consistent with projections of future pCO2 values for open oceans over in the next 200 yr (IPCC, 2007)) and represents current conditions experienced during seasonal extremes by this species in nature (Murray et al., 2014). Ambient conditions were achieved by bubbling partially CO2-stripped air into each rearing container, thereby offsetting metabolic CO2 accumulation. Acidified conditions were achieved via gas proportioners (Cole Parmer®) that mixed CO2 stripped air with 100% bone-dry CO2 delivered to the bottom of each rearing container via air stones. Target pH and temperature were monitored daily via a handheld pH probe (Hach® HQ40d portable meter with a PHC201 standard pH-probe) calibrated regularly via two-point National Bureau of Standards (NBS) pH buffers (electronic supplementary material, Fig.S1). To characterize actual pCO2 levels and related water chemistry parameters, water was sampled from four randomly chosen rearing containers per treatment three times over the course of the experiment and immediately measured for total alkalinity (AT) via endpoint titration (Mettler Toledo™ G20 Potentiometric Titrator). The instrument has previously been shown to quantify AT in Dr. Andrew Dickson’s reference material (batch 147, AT= 2231.39 umol kg seawater-1) with an average error of 0.6%. Actual levels of total dissolved inorganic carbon (CT), partial pressure of CO2 (pCO2), fugacity of CO2 (fCO2), and carbonate ion concentration were calculated in CO2SYS (http://cdiac.ornl.gov/ftp/co2sys) based on measured AT, pH (NBS), temperature, and salinity using K1 and K2 constants from Mehrbach et al. (1973) refit by Dickson and Millero (1987) and Dickson (1990) for KHSO4 (Table 1).

Fatty acid analysis:

Gas chromatography was used to quantify the absolute (mg g dry weight-1) and relative concentrations (% of total) of 27 FAs for each of the 5 females (whole individual) and their unfertilized eggs (~ 1 ml) following the methods of Faulk and Holt (2005) as recently used for this species in Murray et al. (2016). Briefly, frozen samples were shipped on dry ice to the Fisheries and Mariculture Laboratory (University of Texas, Marine Science Institute), where they were first dried and then homogenized in a solution of chloroform-methanol (2:1 v/v) and tricosanoic acid (23:0) as an internal standard for quantification of mg g−1 dry mass of fatty acids. Lipids were cold-extracted from approximately 50 mg of dry mass. Fatty-acid methyl esters were prepared by saponification in potassium hydroxide, followed by transesterification with 14% boron trifluoride in methanol. A Shimadzu GC-2014 gas chromatograph set with a Phenomenex ZB-WAX plus capillary column (30 m long; 0.53 mm ID; 1.0 um thick) was used to quantify FAs, and individual FAs were identified by comparison to commercial standards (Supelco, Inc). Two FAs, 12:0 and 15:1, were below detection limit or invariant across egg batches and were therefore excluded from subsequent analyses.