CO₂ induced seawater acidification impacts sea urchin larval development II: Gene expression patterns in pluteus larvae

Abstract

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 (2 days post-fertilization) and feeding (4 and 7 days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 8.1 and pCO₂ 41 Pa e.g. 399 μatm) and CO₂ acidified seawater with pH of 7.7 (pCO₂ 134 Pa e.g. 1318 μatm) 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.

Introduction

Due to anthropogenic CO₂ emissions, atmospheric pCO₂ is expected to reach values of 70–100 Pa (ca. 700–1000 μatm) by the end of this century (Caldeira and Wickett, 2005, Cao and Caldeira, 2008). This increase in pCO₂ will alter ocean surface water carbonate chemistry, resulting in a reduction of ocean carbonate concentrations and a decrease in pH by up to ca. 0.4 units. It has been suggested that this progressive ocean acidification will negatively impact marine heterotrophic organisms by slowing calcification rates or even causing dissolution of carbonate shells when saturation states of calcite (Ω_calc) or aragonite (Ω_arag) drop below unity (Orr et al., 2005, Hoegh-Guldberg et al., 2007). However, recent evidence indicates that elevated seawater pCO₂ impacts marine ectothermic organisms through multiple processes, such as disturbances in acid–base and ion homeostasis, metabolism, somatic growth and reproduction and thus not only impacts calcification. An altered energy partitioning between different physiological processes may be a key effect of seawater acidification on heterotrophic organisms (see Fabry, 2008, Pörtner and Farrell, 2008, Wood et al., 2008, Melzner et al., 2009, Dupont et al., 2010b for reviews).

In echinoderms, high mortalities and morphological abnormalities were observed in ophiuroid larvae exposed to a seawater pCO₂ of approximately 74 Pa (Dupont et al., 2008). Studies on other echinoderm species also reported growth delays (Kurihara and Shirayama, 2004, Kurihara, 2008, Clark et al., 2009, Brennand et al., 2010, O'Donnell et al., 2010), and reduced fertilization success (Havenhand et al., 2008), but in other cases could increase developmental success (Dupont et al., 2010a) or had no measurable effect on development or fertilization (Carr et al., 2006, Byrne et al., 2009, Byrne et al., 2010a, Byrne et al., 2010b, Ericson et al., 2010) all at seawater pCO₂ values expected for the next century. These conflicting findings indicate high species specificity of seawater pCO₂ impacts on echinoderms (for review see Dupont et al., 2010b).

Because of the direct impacts of rising pCO₂ on the carbonate chemistry of seawater, calcification has been the primary target of previous investigations. Responses of various invertebrate taxa are diverse, ranging from reductions in calcification rates (Langdon and Atkinson, 2005, Shirayama and Thornton, 2005, Clark et al., 2009, Thomsen et al., 2010) to increases in calcification rates (Gutowska et al., 2008, Wood et al., 2008, Gutowska et al., 2010). In sea urchin larvae, reduced growth has been observed in several studies. Although calcification rates appeared to be normal in relation to size in Paracentrotus lividus larvae (Martin et al., 2011), it is unclear whether hypercapnia effects on growth are due to reductions in calcification performance or whether calcification is affected in a secondary fashion due to changes in somatic growth rate (Kurihara and Shirayama, 2004, Clark et al., 2009, Brennand et al., 2010, O'Donnell et al., 2010). The few studies examining calcification under high pCO₂ by means of gene expression analysis mainly observed a down regulation of genes connected with calcification in early sea urchin larval stages (Todgham and Hofmann, 2009, O'Donnell et al., 2010). In contrast, Martin et al. (2011) demonstrated that expression of calcification genes in P. lividus was increased in response to elevated pCO₂. In lecitotrophic Crossaster papposus larvae, no impact on calcification and a higher growth rate was detected (Dupont et al., 2010a), implying that not only calcification, but other processes such as feeding, metabolism or ion regulation are affected by elevated pCO₂. In fact, metabolism has been found to be up-regulated with increasing seawater pCO₂ in the blue mussel Mytilus edulis (Thomsen et al., 2010, Thomsen and Melzner, 2010) and the ophiuroid Amphiura filiformis (Wood et al., 2008), potentially reflecting elevated energetic costs for calcification and/or cellular homeostasis in some marine invertebrates that cannot control the carbonate system speciation in their extracellular fluids. In other invertebrate species, metabolic depression, elicited by uncompensated extracellular pH, has been suggested to occur (see Pörtner, 2008). In early sea urchin larvae, no metabolic rate measurements under acidified conditions have been performed so far, but a down-regulation of several metabolic genes (e.g. Succinyl-CoA synthetase, citrate synthase, pyruvate dehydrogenase, ATP synthase F1 complex) has been reported (Todgham and Hofmann, 2009, O'Donnell et al., 2010). This could support the metabolic depression hypothesis and explain reduced rates of growth.

In this paper, we used gene expression profiling in an explorative way to investigate, whether transcripts of genes with a crucial importance in metabolism, calcification and ion homeostasis are affected by near-future (year 2100) levels of seawater acidification in pre-feeding (day 2) and feeding (days 4 and 7) Strongylocentrotus purpuratus pluteus larvae. In a companion study (Stumpp et al., 2011), we investigated the effects of pCO₂ on larval development success, morphology, feeding and metabolic rates. Larvae were raised at two seawater pCO₂ values: control (40 Pa, 399 μatm, pH 8.1) and elevated pCO₂ (134 Pa, 1318 μatm, pH 7.7).