Analysis of Pacific oyster larval proteome and its response to high-CO 2 R. Dineshram a , Kelvin K.W. Wong a , Shu Xiao b , Ziniu Yu b , Pei Yuan Qian c , Vengatesen Thiyagarajan a,⇑ a The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong Kong, Hong Kong b South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China c Division of Life Science, The Hong Kong University of Science and Technology, The University of Hong Kong, Hong Kong article info Keywords: Environmental proteomics High-CO 2 Larval proteome Ocean acidification Pacific oyster Veliger abstract Most calcifying organisms show depressed metabolic, growth and calcification rates as symptoms to high-CO 2 due to ocean acidification (OA) process. Analysis of the global expression pattern of proteins (proteome analysis) represents a powerful tool to examine these physiological symptoms at molecular level, but its applications are inadequate. To address this knowledge gap, 2-DE coupled with mass spec- trophotometer was used to compare the global protein expression pattern of oyster larvae exposed to ambient and to high-CO 2 . Exposure to OA resulted in marked reduction of global protein expression with a decrease or loss of 71 proteins (18% of the expressed proteins in control), indicating a wide-spread depression of metabolic genes expression in larvae reared under OA. This is, to our knowledge, the first proteome analysis that provides insights into the link between physiological suppression and protein down-regulation under OA in oyster larvae. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The absorbed one-third of anthropogenic CO 2 by the oceans has started altering seawater carbonate chemistry equilibrium through the process known as ‘‘ocean acidification’’ (OA) (Doney et al., 2009). More data on OA impacts on calcium carbonate (CaCO 3 ) shell forming organisms (called ‘‘calcifiers’’) are urgently needed because OA could exert deleterious effects not only on organism’s ability to make their shells but also on their metabolism and phys- iology (Fabry et al., 2008). Global mean ocean pH has already de- creased 0.1 units because of OA, and is predicted to drop by 0.7 units before 2300 under the IPCC’s worst case scenario for CO 2 emissions (Zeebe et al., 2008). This excess H [+] combines with carbonate ions to form bicarbonates. The carbonate ions that are in depletion this way concurrently reduces the saturation state of all forms of CaCO 3 minerals, which makes marine organisms harder to form their shells and/or even trigger their shells to dissolve (Feely et al., 2009). Due to OA, southern oceans are already corrosive to shells of many invertebrates, making them harder to form their shells or even have their shell dissolved (Fabry et al., 2009). This OA effect is gradually spreading into tropical seas (Kleypas et al., 1999). The majority of calcifying shellfishes (e.g. edible oysters) have complex life cycles, during which the externally fertilized eggs pro- duce the pelagic larval stage, called ‘‘D-shaped’’ veliger. This newly hatched larva feeds on micro-algae, develops into advanced larval stage, called pediveliger, and finally enters into benthic life by attaching on hard substrates (Collet et al., 1999). Although this pe- lagic life aids them to disperse and colonize diverse habitats, it is achieved only at an extremely high cost (Thiyagarajan, 2010). Gen- erally, >90% of larvae dies before they reach attachment stage due to predation and environmental stress (Jessopp, 2007). Thus early larval life stages are not only highly susceptible to stressors; their physiological fitness would also determine the success of pre- and post-larval life (Pechenik, 1999). When analyzing the effects of OA on shellfishes, it is thus critical to study their effects not only on adult stage but also on larval stages (Dupont et al., 2008; Gazeau et al., 2010; Kurihara et al., 2007; Talmage and Gobler, 2010). The larvae of oysters are particularly at risk because they use ara- gonite (MgCO 3 ) in their shell, which is 30 times more sensitive to OA than normal calcite (CaCO 3 ) based adult shells (Medakovié et al., 1989). Reduced shell calcification rate (and thus growth rate), and metabolic depression are common symptoms of OA in early life stages (Dupont and Thorndyke, 2009; Talmage and Gobler, 2010). These symptoms could most probably be due to the down-regula- tion of genes responsible for calcification, and energy metabolism (Todgham and Hofmann, 2009). Expression of gene (s) does not al- ways correlate with their product (protein) (s) (Görg et al., 2004). Therefore, knowledge of protein expression pattern is necessary to understand the direct link between OA stress and larval physio- logical response (Hofmann et al., 2008). However, differential expression of proteins (proteome plasticity), especially in early lar- val life stages, in response to OA has not yet been well explored (Wong et al., 2011). Recently, two-dimensional electrophoresis 0025-326X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2012.07.043 ⇑ Corresponding author. E-mail address: rajan@hku.hk (V. Thiyagarajan). Marine Pollution Bulletin 64 (2012) 2160–2167 Contents lists available at SciVerse ScienceDirect Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul