Dataset: larvae count

Response of larval behavior to high pCO2
To determine the position of P. damicornis larvae in the seawater in response to high pCO2, larvae were transferred from collection containers to UV-transparent acrylic tubes (UV-T, ACRYLITE Colorless 0070 GT, Evonik Industries, New Jersey, USA) and incubated in situ at two depths, which centered the tubes vertically at ~ 0.3 m (July 2016) or ~ 3.3 m (August 2016). The tubes had a wall thickness of 6 mm, were 68-cm long with an inner diameter of 4.5 cm, and were sealed at either end with UV-T acrylic caps. A 6-mm hole was drilled in the top cap so the tubes could be filled with seawater; the hole was later covered with vinyl electrical tape. While the objective was to completely fill the tubes with seawater, in most cases small bubbles were introduced during the filling process and combined when the tubes were positioned vertically to create an air gap of ≤ 1-cm height at the top of the tube. The acrylic in these tubes transmitted ~ 92% of ultraviolet radiation (UV-R) (280–400 nm), ensuring larvae were exposed to ecologically relevant light conditions for shallow seawater. Previous studies of the vertical movement of P. damicornis larvae in acrylic tubes, as well as preliminary observations of larval behavior used in the present study, show that larvae of P. damicornis exposed to ambient seawater generally aggregate either in the top or bottom 0–2 cm of tubes regardless of light conditions (i.e., light versus dark). Therefore, the vertically-oriented tubes were marked into two sections for the purpose of scoring the larvae by position: an upper section (21-cm length) and lower section (47-cm length), since we reasoned it would be unlikely that the larvae would accumulate in the center of the tubes. Of the larvae found in the lower section, 83–88% were located in the bottom 23 cm of the tube, which suggested that the greater size of the lower scoring section in the tubes did not upwardly bias estimates of larvae scored as moving downward.

Eight tubes were used for each experiment, during which four were filled with ambient (~ 400 µatm pCO2) seawater, and four with treatment (~ 1000 µatm pCO2) seawater that simulated the elevated pCO2 conditions predicted under a pessimistic scenario of human activity to occur by 2100 (RCP6.0). Once tubes were filled with seawater, 50 larvae were haphazardly selected from the larval stock obtained by pooling larvae released that morning from maternal colonies, and added using a Pasteur pipette through the 6-mm hole in the cap of the tube. The hole was sealed when the tubes were stocked with larvae. Each tube was carefully inverted to gently mix the 50 larvae, so each trial began with the larvae scattered along the length of the tube. Although the pelagic larval duration (PLD) of P. damicornis can be > 100 d, their larvae commonly settle ≤ 24 h following release, and therefore, the present incubations were designed to last 12 h starting at 08:00 hrs.

After the tubes were stocked with larvae on the shore, they were transported by a snorkeler to an adjacent fringing reef and suspended vertically, with the midpoint of the tubes at a median depth (MD) of either ~ 0.34 m (July) or 3.34 m (August) (hereafter referred to as 0.3 and 3.3-m MD) (Fig. 1). The median depth was recorded at the middle of the vertically oriented tubes, thus the depth range for the 0.3 m MD tubes extended from the surface to 0.68 m, and from 3 – 3.68 m for the 3.3 m MD tubes. To maintain this configuration, the tubes were attached to a weight at 5-m depth, and a float was used to keep them suspended vertically. While it is not possible to exclude an effect of time (July versus August) in confounding the contrast of depth, experimental conditions were kept virtually identical between months to reduce temporal bias. Tubes were stocked with larvae shortly after sunrise, and were installed on the reef within 30 minutes of filling at ~ 07:30 hrs. Thereafter, the vertical position of the larvae was scored every 4 hours starting at 08:00 hrs (initial) and finishing at ~19:30–20:00 hrs (sunset); the tubes were removed from the water at 08:00 hrs the following day and the larvae processed for lipid content (described below). At each census, the position of the larvae in the tubes was scored as the number of larvae in either the top or bottom sections of the tube. Tubes were not evaluated at night because of logistical constraints associated with nighttime snorkeling. The percentage of the total number of larvae present in the tubes at the time of each sampling was calculated for the top section of the tubes and used as the response variable to evaluate the effects of the treatments (depth and pCO2).

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.