Climate change has been associated with ecological variation, including alterations in biodiversity, abundance and distribution of species, and phenology (Parmesan and Yohe, 2003; Poloczanska et al., 2013). Within Europe, the Iberian Peninsula and Mediterranean area are regions with an elevated rate of warming, with a predicted increase between 4 and 7°C in air temperature by 2100 (Santos and Miranda, 2006; Christensen et al., 2007). Under such scenario, Portuguese coastal waters and the Mediterranean Sea will be 2–3°C and 3–4°C warmer, respectively (Miranda et al., 2002; Fischer and Schär, 2010). The impacts of such temperature changes, coupled with the effects of other anthropogenic activities may exert strong influences on ecosystem services, affecting economic activities such as fisheries and aquaculture. Climate change is predicted to affect such activities not only through effects of temperature on species physiology but also through changes on fish farming practices (Brander, 2007; Cochrane et al., 2009; Doubleday et al., 2013). Therefore, it is imperative to evaluate and understand biological responses of commercial fish species to the rise in temperature. This will allow researchers to identify the vulnerability of key species, and stakeholders to develop appropriate mitigation plans to reduce costs to society and environment (Poloczanska et al., 2013).

At a physiological level, temperature affects individual's metabolic rate, aerobic metabolism, performance, growth, reproduction, and survival with potential effects on population sustainability (Kordas et al., 2011; Dowd et al., 2015). Moreover, changes at the individual level are dependent upon the capacity to modulate gene expression under environmental variation (see Logan and Somero, 2011). Changes in cellular stress proteins (such as heat shock proteins and anti-oxidant enzymes) have been detected in marine organisms exposed to acute and chronic heat stress (e.g., Buckley and Hofmann, 2002; Tomanek, 2010; Madeira et al., 2013, 2014). Nevertheless, these studies have only focused on a few protein biomarkers. Additionally, research incorporating high throughput tools is highly skewed toward transcriptomics and therefore proteomics approaches are still scarce in marine biology (see Tomanek, 2014; Logan and Buckley, 2015 for reviews). Proteomics examines the ultimate product of gene expression, i.e., proteins, which are the functional units mediating stressor-biological system interactions and its subsequent responses (e.g., López, 2007). Thus, it can revolutionize our understanding of acclimation and adaptation processes and may be the basis of an integrative, systems biology approach (Karr, 2008), enabling a fast assessment of stress response networks affected by environmental cues. Currently, several pathways are known to be regulated by acute and chronic exposure to elevated temperature. When organisms are exposed to heat, hypoxaemia seems to set the first level of thermal limitation (Pörtner, 2010). Gradually, temperature will lead to other pervasive effects such as oxidative stress and protein damage. Accordingly, organisms usually modulate protein levels when temperature rises a few degrees above their acclimation temperature, in order to induce a stress response to protect cellular components from damage and/or meet potentially higher energetic demands caused by elevated metabolic rates. According to several “omics” studies, modulated proteins play roles in chaperoning activity, energetic metabolism, oxidative stress metabolism, immune response, cytoskeletal dynamics, transcriptional regulation, intracellular transport, protein synthesis and turnover, and signal transduction (see López et al., 2002; Gardeström et al., 2007; Stillman and Tagmount, 2009; Fields et al., 2012; Tomanek, 2014). By examining the full range of protein changes, the cells' phenotype can be assessed and directly related to the health and fitness of the organism and adaptation mechanisms (Dupont et al., 2007; Dalziel and Schulte, 2012; Diz et al., 2012). Consequently, predictions of cascading effects on populations can be improved, up-grading biogeographic forecasting, risk assessment, and prediction of potential costs to society.

Sparus aurata is an euryhaline and eurythermic species. Thus, it is widely distributed in the eastern Atlantic and is known from southern Ireland to Mauritania, including the Canary Islands, the Mediterranean Sea and the western and southern Black Sea (Froese and Pauly, 2006). In general, minimum and maximum survival temperatures are in the range of 5–34°C for adult sea breams. However, early life-stages (eggs, larvae and juveniles) have more limited temperature tolerance during ontogenesis. Moreover, temperature tolerance is highly dependent on acclimation. Accordingly, rapid and significant changes of temperature near to the thermal limits are likely to lead to poor welfare (European Food Safety Authority, 2008).

The aim of this study was to assess the vulnerability and acclimation capacity of the sea bream S. aurata (L., 1758), taken here as a proxy for some commercially important demersal fish species, to ocean warming and extreme events by (i) unraveling proteome changes in the muscle of juvenile fish exposed to warming, (ii) relating proteome plasticity to fitness measures (mortality and condition index) and (iii) inferring on the possible ecological consequences of warming for sea bream populations. Muscle tissue was chosen as target organ for several reasons, including (1) muscle proteome has been systematically characterized in S. aurata and is therefore well known in this species (Addis et al., 2010; Piovesana et al., 2016), (2) the commercial interest of this tissue, (3) muscle activity accounts for a great part of the organism's resting energy expenditure (Zurlo et al., 1990), and (4) muscular activity is crucial to dictate organismal functions such as locomotion and thus the capacity to escape unfavorable conditions and forage, determining the performance of the organism. We hypothesize that fitness outcomes depend on the capacity of fish to induce pro-survival pathways in extreme conditions (by up-regulating proteins with cytoprotective functions) and adjust energetic metabolism to meet the higher metabolic rates induced by warm water, determining the fate of sea bream populations in the face of climate change.

In this study, S. aurata juveniles modulated muscle proteins mostly when exposed to 30°C, while fish exposed to 24°C regulated fewer proteins, typically related to energy metabolism. These results coupled to a lack of a cellular stress response at 24°C suggest that this temperature is not very stressful to juvenile fish; alternatively, the cellular stress response could have been induced early on in the exposure period, but if this occurred, the fish were able to repair damage and acclimate as no changes related to the stress response/tissue damage were detected. Either way, S. aurata were able to cope with 24°C. This may be explained by the fact that juveniles inhabit low depth waters (i.e., estuaries and coastal lagoons) that can reach such temperatures quite often in the summer (Froese and Pauly, 2006; Madeira et al., 2012). However, 30°C was sufficiently warm to elicit the cellular stress response, indicated by the up-regulation of chaperones (heat shock proteins) and cytoskeletal re-arrangements, corroborating other acute and chronic experiments performed in aquatic animals (including S. aurata; Feidantsis et al., 2009; Madeira et al., 2014; Jayasundara et al., 2015; Tomalty et al., 2015).

Chaperones are cytoprotective proteins that stabilize denatured proteins and have a selective value (Hofmann, 2005) since they confer stress tolerance and delay thermal injury (Feder, 1996; Horowitz, 2001; Basu et al., 2002; Sorte and Hofmann, 2005; Bahrndorff et al., 2009). Moreover, several authors have reported a link between molecular chaperones and cytoskeletal components (Liang and MacRae, 1997; Mounier and Arrigo, 2002; Tomanek and Zuzow, 2010), suggesting that cytoskeletal proteins are very sensitive to temperature and chaperones may therefore play a decisive role on their modulation, function, and protection. In fact, cytoskeleton re-modeling may be essential to stabilize cytoskeletal components and maintain homeostatic balance upon challenging conditions (Buckley et al., 2006; Garlick and Robertson, 2007). This may be crucial not only to preserve cellular activities related to motility, signaling, and organelle movement but also to adjust ATP turn-over rates (see Garland et al., 2015) and maintain muscle contraction.

Besides up-regulating the cellular stress response, fish exposed to 30°C had significantly higher mortality rates. Increased mortality induced by temperature has also been observed in larval stages of S. aurata (Madeira et al., 2016) and throughout the life cycle of other fish species (e.g., Houde, 1989; Hinch and Martins, 2011). However, thermotolerance, survival and the potential for acclimation of each individual may depend on several factors such as thermal history, sex, nutritional condition, diseases, age and reproductive status (e.g., Becker and Genoway, 1979; Lutterschmidt and Hutchison, 1997; Underwood et al., 2012; Bruneaux et al., 2014). In accordance with higher mortality levels, fish exposed to 30°C presented signs of an inflammatory process or changed immune response, indicated by the up-regulation of sterile alpha motif domain-containing protein 9-like (from 24 to 30°C). Thermal stress is known to cause damage to cellular components and tissues in aquatic organisms, including S. aurata (e.g., Madeira et al., 2014, 2015; Raina et al., 2015). As such, proteins or transcripts involved in the inflammatory process and immune response are commonly regulated upon exposure to thermal stress (e.g., Thorne et al., 2010; Windisch et al., 2014; Basu et al., 2015; Garland et al., 2015; Tomalty et al., 2015).

Interestingly, no changes were detected in Fulton's K condition index. Stressful environments likely lead to higher energy demands, thus fish subjected to heat stress could have lower condition due to weight loss. However, juvenile fish did not seem to experience significant weight loss, despite the detected changes in energetic metabolism. Alternatively, we can also consider that the exposure time was not enough to observe changes in Fulton's K condition index. Nonetheless, proteins related to energetic pathways were both regulated at 24 and 30°C. Overall, most of these proteins were up-regulated upon warming. This indeed suggests a higher energy demand at higher temperatures, which is in accordance with other studies that show that metabolic rates increase in fish chronically exposed to high temperatures (e.g., Rosa et al., 2014; Vinagre et al., 2014). This is especially relevant in muscle, which has high energetic demands and has to provide energy during long-periods or increase the rate of energy production in response to explosive contractions (Westerblad et al., 2010), in order to maintain locomotion. In fact, adjustments in energy metabolism may be essential for fish to be able to move to thermal refugia or keep their activities of foraging.

Overall, changes in energetic metabolism suggest an enhanced glycolytic potential and increased carbohydrate metabolism mainly at 30°C but also, to some extent, at 24°C. This finding is supported by the up-regulation of alpha-enolase, triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase. Moreover, the up-regulation of creatine kinase isoforms and adenylate kinase indicates the regulation of ATP and AMP levels by producing ATP from phosphocreatine and ADP, and converting 2 ADP ⇆ ATP + AMP, respectively. These reactions contribute to the maintenance of ATP levels and regulate glycolytic enzymes (Berg et al., 2002; Fischer, 2013), being especially relevant in tissues with high energy requirements such as skeletal muscle. Its functional capacity depends on large energy budgets (Kenyon and Reed, 1983; Wallimann et al., 1992), which could hardly be sustained by the low pools of cellular ATP. Alternatively, phosphagens are accumulated in higher concentrations and can thus be used to produce energy and replenish ATP levels maintaining energy homeostasis (Wallimann et al., 1992). Although, most isoforms of creatine and adenylate kinases were up-regulated with temperature, there was also two down-regulated isoforms. The functional explanation for this opposed expression pattern is not clear but it may depend on the isoenzyme-specific subcellular localization and bounding properties, leading to slightly different physiological outcomes related to energetic metabolism and cellular structural properties (e.g., see Hornemann et al., 2000). However, as it was not possible to determine which specific isoenzymes were regulated, we cannot take further conclusions.

Changes in glycolytic enzymes have also been detected in other studies of acute (Garland et al., 2015) and chronic exposure to temperature (Jayasundara et al., 2015), usually increasing at higher temperatures. However, Garland et al. (2015) also detected that such enzymes could undergo antagonist changes, with enzymes at the preparatory phase decreasing and enzymes involved in the pay-off phase increasing. Moreover, there seemed to be a decrease in glycogenolysis at 24°C after 14 days of exposure indicated by the down-regulation of glycogen phosphorylase. This suggests that fish can maintain some glycogen reserves if the temperature increase is mild. After 21 days of exposure, glucose-6-phosphate isomerase was also down-regulated at 24°C suggesting a decrease in the inter-conversion of glucose-6-phosphate to fructose-6-phosphate (glycolysis/gluconeogenesis). This indicates that after 21 days of exposure to 24°C there is no longer the need for an enhanced glycolytic potential which points out that fish may be acclimating to this temperature. However, after 21 days of exposure to 30°C there seemed to be a decline in the tricarboxylic acid cycle (or related anaplerotic reactions), possibly suggesting a decrease in aerobic metabolism, since this cycle is dependent on the oxidation of acetyl-CoA and produces NADH to fuel the oxidative phosphorylation pathway. The potential decline in this pathway is supported by the down-regulation of glutamate dehydrogenase, which produces intermediate substrates for the tricarboxylic acid cycle (alpha ketoglutarate). Some studies suggest that exposure to high temperature induces a metabolic shift toward carbohydrate metabolism and the formation of glycogen stores (Brodte et al., 2006; Windisch et al., 2011, 2014), because these metabolic mechanisms are able to function under hypoxaemia, which is generally induced by hyperthermia (oxygen limited thermal tolerance, Pörtner and Farrell, 2008). Entering a state of hypoxaemia means a shift from aerobic to anaerobic metabolism at some point and this threshold for anaerobiosis is dependent on factors such as species and acclimation history (e.g., see Burleson and Silva, 2011). However, in this case, it is not entirely clear if pyruvate obtained from increased glycolysis is being used to maintain aerobic metabolism or is being converted to lactate in anaerobic conditions. Still, the down-regulation of glutamate dehydrogenase suggests that long-exposure to extreme temperatures decreases aerobic metabolism, potentially eliciting a transition to anaerobic metabolism.

Physiological adjustments to stress were influenced not only by temperature but also by the duration of exposure. While some mechanisms activated were common to both time-points (e.g., chaperoning), there seemed to be a transition in the predominant processes of response from T14 to T21. At T14, transcriptional/translational regulation, amino acid homeostasis and acid-base balance seemed more relevant while at T21, the shifting of the energetic metabolism was predominant and related to gluconeogenesis and cellular carbohydrate biosynthetic processes, which may be crucial to sustain metabolic performance under chronic stress.

Indeed, at T14, exposure to extreme temperature seems to have elicited the activation of endocrine processes and these may be responsible for the activation of molecular adaptive cascades, in which vesicular trafficking and translational regulation could play an important role. This finding is supported by the up-regulation of neuroendocrine convertase 1, eukaryotic initiation factor 4A-II and Rab GDP dissociation inhibitor beta in the muscle of juvenile fish exposed to 30°C. Several studies have shown that a number of hormones dictate responses to stress in fish and other organisms (e.g., Moalic et al., 1989; Barton and Iwama, 1991; Iwama et al., 1999; Barton, 2002; Pepels et al., 2004) and that modulation of translation and transcription is crucial to deal with acute and chronic thermal stress in fish (e.g., Long et al., 2012; Windisch et al., 2014; Tomalty et al., 2015). Moreover, the regulation of acid-base status may be an important mechanism for homeostatic balance in fish as suggested by the up-regulation of carbonic anhydrase 1 at T14 (30°C). However, the influence of hyperthermia in acid-base balance in fish is not well-known, as opposed to the effect of hypothermia (Pörtner et al., 1998) and other environmental factors such as salinity and pollutants (e.g., de Polo et al., 2014).

Molecular information, coupled with performance measurements, reflects the health status of fish and their ability to successfully acclimate to new environmental conditions. In this study, sea bream juveniles mildly responded to 24°C but showed a stronger response to 30°C. While exposure to 24°C only elicited energetic adjustments (higher glycolytic potential and regulation of ATP levels, maintenance of glycogen reserves), 30°C seems to induce much more molecular changes. At this temperature, the maturation of peptide hormones probably led to the activation of molecular cascades which potentially triggered energetic adjustments (enhanced glycolytic potential and regulation of ATP levels) necessary to induce the cellular stress response (increased chaperoning, cytoskeletal re-arrangements, and regulation of acid-base balance). Such changes are known to contribute to cellular homeostasis and muscle functioning. Nevertheless, the data indicate that aerobic metabolism may be compromised (down-regulation of the tricarboxylic acid cycle) and an inflammatory process could be ongoing in animals subjected to 30°C, which probably contributed to higher levels of mortality (Figure 6—summary figure). Overall, the molecular information coupled with mortality rates suggests that juvenile fish are able to acclimate to 24°C, but may be vulnerable to temperatures above 24°C and may not be able to fully acclimate to 30°C. S. aurata is an eurythermal subtropical species (with an upper thermal limit of 35.5°C—Madeira et al., 2014) that inhabits shallow habitats, in which a temperature of 24°C is fairly common during summer. However, 30°C (predicted future heat wave condition in Portuguese estuaries) seems somewhat deleterious to these sea breams suggesting that their health may be hindered if heat waves increase in frequency and intensity, as predicted by IPCC (2013). Therefore, recruitment and population levels of S. aurata may be affected by ocean warming, highlighting the need for improvement of management plans for fish stocks (e.g., limiting catch quotas as needed). Nevertheless, the ecological consequences of a warming ocean are dependent on several factors, including phenotypic plasticity throughout ontogeny, local adaptation (and thus larval dispersal), transgerational plasticity (e.g., Munday, 2014), and trophic-web cascading effects (e.g., Nagelkerken and Connell, 2015), warranting further research. Moreover, regardless of the enormous advances in proteomics and the significant findings of the present study, this approach still presents some limitations. Thus, for future studies a systems biology approach can be used to provide more information.

Accession numbers: GDIB_CANFA; NEC1_HUMAN; ENOA_XENLA; KCRM_BOVIN; TPISB_DANRE; KCRM_HUMAN; KCRM_PIG; HSP71_ORYLA; HSP7C_CRIGR; KAD1_CYPCA; KCRM_CANFA; ENOA_PONAB; ACT2_XENTR; PYGB_SHEEP; IF4A2_MACFA; KCRT_ONCMY; CAH1_CHIHA; HSP7C_ICTPU; TPIS_MESAU; G3P_DANRE; G3P_MELGA; SAM9L_HUMAN; HSP70_ONCTS; G6PI_BOVIN; DHE3_CHAAC. See supplemental materials for additional information concerning experimental setup, general categories of gene ontology, detailed functional categorization, masses and sequences of peptides obtained for each spot, protein expression levels and Tukey's post-hocs.

MD and CV designed the study; DM and MD carried out the experiments; DM and JA carried out sample preparation and 2-D gels, gel image analyses and protein digestion; RV performed mass spectrometry and protein identification; DM, RV, and PC carried out bioinformatics analyses; JC contributed with reagents and analytical advice; DM wrote the manuscript with relevant inputs from all authors.