CO 2 induced seawater acidi! cation impacts sea urchin larval development II: Gene expression patterns in pluteus larvae

Extensive use of fossil fuels is leading to increasing CO₂ concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO₂. As a result, the oceans' surface pH is expected to decrease by ca. 0.4 units by the year 2100, a major change with potentially negative consequences for some marine species. Because of their carbonate skeleton, sea urchins and their larval stages are regarded as likely to be one of the more sensitive taxa. In order to investigate sensitivity of pre-feeding (2days post-fertilization) and feeding (4 and 7days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 8.1 and pCO₂ 41Pa e.g. 399μatm) and CO₂ acidified seawater with pH of 7.7 (pCO₂ 134Pa e.g. 1318μatm) and investigated growth, calcification and survival. At three time points (day 2, day 4 and day 7 post-fertilization), we measured the expression of 26 representative genes important for metabolism, calcification and ion regulation using RT-qPCR. After one week of development, we observed a significant difference in growth. Maximum differences in size were detected at day 4 (ca. 10% reduction in body length). A comparison of gene expression patterns using PCA and ANOSIM clearly distinguished between the different age groups (two-way ANOSIM: Global R=1) while acidification effects were less pronounced (Global R=0.518). Significant differences in gene expression patterns (ANOSIM R=0.938, SIMPER: 4.3% difference) were also detected at day 4 leading to the hypothesis that differences between CO₂ treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO₂ effect. We found an up regulation of metabolic genes (between 10%and 20% in ATP-synthase, citrate synthase, pyruvate kinase and thiolase at day 4) and down regulation of calcification related genes (between 23% and 36% in msp130, SM30B, and SM50 at day 4). Ion regulation was mainly impacted by up regulation of Na⁺/K⁺-ATPase at day 4 (15%) and down regulation of NHE3 at day 4 (45%). We conclude that in studies in which a stressor induces an alteration in the speed of development, it is crucial to employ experimental designs with a high time resolution in order to correct for developmental artifacts. This helps prevent misinterpretation of stressor effects on organism physiology.