Dataset: lipid

Lipid content
To test for changes in total lipid content in the larvae following incubations under ambient and high pCO2, the initial lipid content of larvae was determined in three samples of 50 larvae, freshly released from the maternal colonies at 06:00 hrs and pooled among them. Each batch of larvae was placed in 1.5-mL vials for processing. The final lipid content of the larvae (i.e., 24 h after the start of incubations) was determined by processing batches of larvae in the same way following 24 h in ambient or high pCO2 in either shallow (July) or deep (August) locations. Tubes incubated on the reef were returned to the lab 24 h after the start of each experiment (i.e., 08:00 hrs the following day), and all living larvae were collected by rinsing the contents of each tube into a collection container. Retrieved larvae were counted, and a single batch of 50 larvae from each tube at both depths was placed in a 1.5 mL vial for measurement of lipid content.

After transferring the batches of larvae to 1.5-mL vials, excess seawater was removed from the vials using a Pasteur pipette, and the larvae were frozen at -80°C for lipid analysis within 2–4 weeks. Following the protocol of Harii et al. (2010) with slight modifications, lipids were extracted with 5 mL dichloromethane-methanol (6:4) in an ultrasonic bath at room temperature for 15 minutes. Each batch of 50 larvae was extracted three times, with the solvent and eluted lipid pooled among extracts. Extracted lipids were concentrated in a rotary evaporator for 15–20 min. Following complete evaporation, the residue was dissolved in dichloromethane 6 times, and centrifuged to separate lipids from water. The solvent containing dissolved lipids was transferred to a new 50 mL flask after each separation. After washing 6 times, the samples were partially evaporated once again for ~ 5 min, and passed through a ~ 1 cm column of Na2SO4 crystals and plastic wool to remove water. Samples drained by gravity from the column into 4-mL pre-combusted glass vials (400°C for 4 hrs), in which they were then dried under nitrogen gas and weighed using a microbalance (± 1 µg, MT5, Mettler-Toledo, USA). Larval lipid content was expressed as µg larva-1. 

Statistical analysis
Statistical analyses were conducted using SYSTAT Version 11 software (Systat Software, San Jose, CA). A two-factor RM ANOVA was used to compare the effects of depth and pCO2 on larval position in the tubes, with time of day as the RM factor and arcsine-transformed values of the percentage of larvae found in the top of each tube as the dependent variable. Differences in lipid content of larvae were evaluated using a two-sample t-test to compare the effect of pCO2 on total lipid between depths, and a Kruskal-Wallis non-parametric test (due to violations of normality in the data) to compare lipid content after pCO2 incubations regardless of month. Assumptions of normality and homogeneity of variance for the RM-ANOVA were assessed through graphical analyses of the residuals.