Physiological responses to cold stress in the gills of ...

Science of the Total Environment 640–641 (2018) 1372–1381

Contents lists available at ScienceDirect

Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Physiological responses to cold stress in the gills of discus fish (Symphysodon aequifasciatus) revealed by conventional biochemical assays and GC-TOF-MS metabolomics Bin Wen 1, Shi-Rong Jin 1, Zai-Zhong Chen ⁎, Jian-Zhong Gao ⁎ Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, China Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai Ocean University, Shanghai 201306, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Cold stress induced protective responses in the antioxidant system of discus fish. • Starch and sucrose metabolism and pentose phosphate pathway were activated. • Low temperature modified glycerolipid metabolism and sphingolipid metabolism. • The activation of glutathione metabolism agreed with increased glutathione level.

a r t i c l e

i n f o

Article history: Received 28 April 2018 Received in revised form 31 May 2018 Accepted 31 May 2018 Available online xxxx Editor: D. Barcelo Keywords: Cold stress Discus fish Metabolomics Antioxidant defence Gill

a b s t r a c t Discus fish (Symphysodon aequifasciatus) is a cichlid that is among the most popular fish for warm-water aquaria and also frequently used as the model animal for environmental science. However, little is known about the responses of S. aequifasciatus to low temperatures caused by environmental variation. Here, by using conventional biochemical assays and gas chromatography time-of-flight mass spectrometry metabolomics, we investigated the physiological responses of S. aequifasciatus gills exposed for 30 days to two temperature regimes: 28 °C and 20 °C. Low temperature resulted in elevated production of reactive oxygen species but not increased malondialdehyde. This might be partially related to protective responses in the antioxidant system, revealed by increased activities of superoxide dismutase and glutathione peroxidase, and level of reduced glutathione (GSH), compensating for the depletion of catalase activity. A total of 35 metabolites were identified as potential biomarkers of cold stress, showing the most influenced pathways including starch and sucrose metabolism, pentose phosphate pathway, glycerolipid metabolism, sphingolipid metabolism, glutathione metabolism, and arginine and proline metabolism. Moreover, the activation of glutathione metabolism agreed with the increased GSH level detected by biochemical assays. Overall, the results of this study suggest that low temperature can activate a protective antioxidant defence response and modify the metabolic pathways in gills of S. aequifasciatus, providing insights into the physiological regulation in response to cold stress in this tropical fish. © 2018 Elsevier B.V. All rights reserved.

⁎ Corresponding authors at: Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, China. E-mail addresses: [email protected] (Z.-Z. Chen), [email protected] (J.-Z. Gao). 1 These authors contributed equally to this work.

https://doi.org/10.1016/j.scitotenv.2018.05.401 0048-9697/© 2018 Elsevier B.V. All rights reserved.

B. Wen et al. / Science of the Total Environment 640–641 (2018) 1372–1381

1. Introduction Global climate change imposes increasing pressures on natural populations. All physiological processes in ectotherms, such as fish, are affected by temperature, and they are thus vulnerable to environmental variations. Changes in temperature have profound effects on the habitats of living things, causing population to face challenging environments (Przeslawski et al., 2008). Besides thermal stress from global warming (Ficke et al., 2007; Isaak et al., 2012), fishes can also experience cold events at a variety of time scales, including acute stress caused by daily temperature fluctuation and chronic exposure that may last from weeks to months (e.g., seasonal and regional temperature changes) (Tomanek, 2014; Stevens et al., 2016). To maintain physiological homeostasis despite unfavourable temperatures, fishes have developed adaptive mechanisms, both behavioural and physiological, to cope with temperature regimes outside their optimal range (Bonga, 1997; Barton, 2002). Discus fish (Symphysodon aequifasciatus) are an endemic species of the Amazon and among the most popular fish for warm-water aquaria. This species may be particularly susceptible to environmental stress due to an unusual reproductive strategy where newly hatched larvae were fed with epidermal mucus of the parental fish (Maunder et al., 2011). Thus it is frequently used as the model animal for environmental science (e.g., Lemgruber et al., 2013; Maunder et al., 2013; Wen et al., 2018). The temperature in native habitats of S. aequifasciatus has been reported to vary between 24 °C and 33 °C, depending on season and location (Bleher et al., 2007); however, relatively little research has addressed their responses to low temperatures from environmental change. A mechanistic understanding of the responses of S. aequifasciatus to low temperatures and of their ability to adjust in thermal sensitivity is helpful to predict the effects of temperature change on fish populations. Fish facing elevated temperature generally enhance their oxygen consumption and may, therefore, increase the production of reactive oxygen species (ROS) as by-products of intensified metabolism, resulting in oxidative stress (Lushchak, 2011). To prevent ROSinduced oxidative damage, e.g., lipid peroxidation (LPO), fish usually increase two classes of antioxidant defence: antioxidant enzymes (e.g., superoxide dismutase and catalase) and low-molecular-mass antioxidants (e.g., reduced glutathione) (Lushchak, 2011; Grim et al., 2013). Given the intensification of oxidative metabolism at elevated temperatures, one might expect a decrease in the risk of oxidative stress induction at decreased temperatures (Lushchak, 2011). Under some circumstances, however, low temperature can also cause the formation of ROS through increased mitochondrial density (Abele and Puntarulo, 2004; Kammer et al., 2011; Cheng et al., 2018) and thereby induce oxidative stress (e.g., Malek et al., 2004; Ibarz et al., 2010; Min et al., 2014). However, it is not clear why, in some cases, cold stress results in enhanced oxidative capacity and lipid remodelling without influencing antioxidant defence or susceptibility to LPO (Grim et al., 2010, 2013). Metabolomics, which provides a ‘snapshot’ profile of the metabolites in a biological system, has been suggested to be an effective tool for detecting fluctuations in metabolites in response to environmental stressors (e.g., Brandao et al., 2015; Lu et al., 2016; Cappello et al., 2016; Melis et al., 2017). Gas chromatography-mass spectrometry (GC–MS), liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) are the three most commonly used analytical technologies for metabolomic analysis. A few studies have used NMR-based metabolomics to examine the response of fish to thermal stress (Kullgren et al., 2013; Melis et al., 2017). However, NMR is generally considered less sensitive than gas chromatography-time of flightmass spectrometry (GC-TOF-MS), and it is difficult to assign the output signal to specific compounds as a result of resonance overlap (Barding et al., 2012). Additionally, responses to stress at higher levels of biological organization correspond to molecular events at lower levels (Brandao et al., 2015; Cappello et al., 2016). Therefore, given the

1373

elevated expression of ROS-associated processes and the molecular foundations of the stress response, the combination of biochemical assays and GC-TOF-MS metabolomics is a promising approach to understand the responsive mechanism towards cold stress. Among the functional organs in fish, the gills play a central role in environmental adaptation due to their involvement in respiration, iono- and osmoregulation, acid-base balance and waste nitrogen excretion, contributing critically to physiological homeostasis under stress (Evans et al., 2005). Therefore, responses to thermal stress may be particularly critical in this organ (Hu et al., 2015, 2016). Fish gills also show great morphological plasticity in response to temperature changes (Sollid et al., 2005; Bowden et al., 2014). Gills are believed to play a prominent role in physiological responses due to their direct and continuous interaction with the surrounding environment (Cappello et al., 2016). Therefore, we selected fish gills as the target organ for investigating cold stress response. Here, we hypothesized that low temperature would decrease the risk of oxidative stress induction, and result in changes in the metabolic profile and the modification of the related metabolic pathways in S. aequifasciatus. To test this hypothesis, we acclimated juvenile S. aequifasciatus to either 20 °C or 28 °C for a period of 30 days, because juvenile specimens are particularly susceptible to thermal stress (Madeira et al., 2014; He et al., 2015). Moreover, gender-specific responses may induced by thermal stress in fishes (Smith et al., 2013), however, it is usually difficult to identify the sex of S. aequifasciatus (Lin et al., 2017). In this case, the juveniles can be used to minimize the interference of variables such as gender (Cappello et al., 2016). By using a battery of biochemical indicators coupled with GC-TOF-MS metabolomics, we investigated the modulation of antioxidant system and the subsequent inhibition of oxidative damage, and the changes in metabolites and related metabolic pathways linked to cold stress in gills of S. aequifasciatus. We also clarified the interconnecting mechanisms between these two levels of response to gain deep insight into the physiological regulation underlying cold acclimation in S. aequifasciatus. 2. Materials and methods 2.1. Fish collection and laboratory acclimation Juvenile discus fish S. aequifasciatus were collected from a population located in Lake Manacapuru (part of the Manacapuru River) in the central Amazon basin of Brazil (03°17′S; 60°37′W) in August 2014. The obtained fish were transported live to the Ornamental Fish Breeding Laboratory, Shanghai Ocean University (Shanghai, China). Pirhonen et al. (2014) suggested that a temperature of 28–29 °C would be optimal for juvenile discus Symphysodon spp. As such, the fish were acclimated at a temperature of 28 °C for a period of 30 days before the temperature trials were initiated (Smith et al., 2013). During acclimation and the subsequent trials, the fish was fed a diet consisting of beef heart, duck heart and shrimp, called ‘beef heart hamburger’, to apparent satiation twice daily (0800 h and 1700 h). All animal care was conducted in accordance with the Administrative Measures for Experimental Animals in Shanghai, and the experimental protocols were approved by the Animal Ethics Committee of Shanghai Ocean University. 2.2. Experimental design After the acclimation period, a total of 80 juvenile S. aequifasciatus with similar size (body weight ~10 g and body length ~5 cm) were randomly distributed among eight 150-L experimental tanks, with ten individuals per tank. The eight tanks were further divided into two groups, with four tanks, i.e., 40 individuals for each group. The water temperature in the control group was maintained at 28 °C which was close to the average environmental temperature of S. aequifasciatus. The temperature in the cold group was decreased by 1 °C per day until it reached

1374


20 °C. Our preliminary experiments indicated that the critical low temperature of S. aequifasciatus was estimated to be 13 °C, and the low temperature of 20 °C was selected based on 13 °C and 24 °C. Moreover, S. aequifasciatus reared at 20 °C were feeding and showing no external signs of abnormal behaviour (Wen et al., 2017). The acclimation period commenced once the final temperature had been sustained for 24 h. During the exposure, water quality (e.g., ammonia, nitrate, nitrite and pH) was monitored, and all tanks were continuously aerated to ensure comparable O2 saturation levels. The animals were maintained on a 12 h:12 h light/dark cycle throughout the acclimation period. After 30 days of acclimation, a period suggested to be sufficient to allow the necessary physiological adjustments involved in thermal acclimation (Vergauwen et al., 2010), four individuals were randomly sampled from each tank, with 16 individuals per treatment. The rest of the individuals were not dedicated to the subsequent analysis and maintained for further study. The collected fish were sacrificed by anesthesia with MS-222 and dissected on ice. The sampled gills were frozen in liquid nitrogen and stored at −80 °C. A total of 16 gills were collected from each group, with eight gills from each treatment used for biochemical parameter determination and another eight gills used for further GC-TOF-MS analysis. 2.3. Biochemical parameter determination Samples of gill tissue were homogenized (1:4, w/v) in ice-cold phosphate buffer (0.1 M, pH 7.4). Afterwards, the obtained homogenates were centrifuged at 3000g at 4 °C for 10 min. The supernatants were then collected for measurements of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) activities; and reduced glutathione (GSH), reactive oxygen species (ROS) and malondialdehyde (MDA) levels. MDA was estimated by measurement of thiobarbituric acid reactive substances (TBARS) content. SOD activity was measured at 550 nm by using the xanthine oxidase method (Fridovich, 1969). CAT activity was determined by monitoring residual H2O2 absorbance at 405 nm (Goth, 1991). GPx activity, estimated by the rate of GSH oxidation, was measured at 412 nm (Hafeman et al., 1974). GR activity was determined by measuring nicotinamide adenine dinucleotide phosphate (NADPH) oxidation at 340 nm (Carlberg and Mannervik, 1975). The level of reduced GSH was measured at 412 nm by using 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) reagent, following the method of Tietze (1969). DTNB was reduced by the free sulfhydryl groups of GSH to form the yellow compound 5-thio-2-nitrobenzoic acid (TNB). ROS content was measured by using 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma) and a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA) with excitation at 500 nm and emission at 525 nm (Delaporte et al., 2003). MDA concentration was determined by a TBARS assay at 530 nm, following the methodology described by Satoh (1978). The protein concentrations in the samples were estimated according to the Bradford protein assay (Bradford, 1976), by using bovine serum albumin as a standard. The absorbance was measured using a Synergy H4 Hybrid Multi-Mode microplate reader (BioTek Instruments, Winooski, VT, USA). 2.4. Sample preparation and GC-TOF-MS analysis A subsample of 100 mg from gill was transferred into 2 mL Eppendorf tubes and mixed with 0.5 mL of extracting solution (methanol and chloroform, 3:1, v/v). The tubes were vortexed for 30 s. Afterwards, the sample was homogenized in a ball mill for 4 min at 45 Hz and treated via ultrasound for 5 min while being incubated in ice water. After centrifugation at 13,000g for 15 min at 4 °C, an aliquot of 400 μL of supernatant was transferred into a fresh 2 mL GC-TOF-MS glass vial and dried in a vacuum concentrator. The residue was derivatized by addition of 50 μL of methoxyamine hydrochloride (20 mg/mL in pyridine) incubated for 30 min at 80 °C, followed by the

addition of 60 μL of bis-(trimethylsilyl)-trifluoroacetamide reagent (1% trimethylchlorosilane, TMCS, v/v) and incubated for 90 min at 70 °C. According to Begley et al. (2009), trimethylsilyl derivatives can be stable for 30 h after derivatization. As such, 1 μL of solution was immediately injected into an Agilent 7890 gas chromatograph system (Agilent Corporation, Santa Clara, CA, USA) coupled with a Pegasus HT time-of-flight mass spectrometer (GC-TOF-MS) in splitless mode. The system was equipped with a DB-5MS capillary column coated with 5% diphenyl cross-linked with 95% dimethylpolysiloxane (30 m × 0.25 mm inner diameter, 0.25 μm film thickness; J&W Scientific, Folsom, CA, USA). Helium was used as the carrier gas, with a constant flow rate of 1 mL min−1. The temperature program was set up as follows: the initial temperature was kept at 50 °C for 1 min, then elevated to 310 °C at a rate of 10 °C min−1 and sustained for 6 min at 310 °C. The temperatures of the injector, transfer line and ion source were set to 280, 270 and 220 °C, respectively. A mass range of 50–500 m/z in full-scan mode for electron impact ionization (70 eV) was applied. The solvent delay time was set to 6.04 min. 2.5. Data processing and metabolite identification The acquired GC-TOF-MS data were analysed as previously described by Kind et al. (2009). The Chroma TOF4.3X software of LECO Corporation and LECO-Fiehn Rtx5 database were used for raw peak extracting, data baselines filtering and calibration of the baseline, peak alignment, deconvolution analysis, peak identification and integration of the peak area. The missing values of the raw data were filled up by half of the minimum value, and the detection of peaks was through the interquartile range denoising method. The peak area was normalized to the total peak area. The RI (retention time index) method was used in the peak identification, and the RI tolerance was 5,000. The internal standard normalization method was also employed for the GCTOF-MS analysis. The identification of gill metabolites was achieved similarly to a previous report (Chang et al., 2015). In brief, metabolites were annotated using commercial mass spectral libraries (Mainlib, NIST, Wiley, and Fiehn) and a home-made metabolite library. The discriminating compounds were further verified by matching the RI with data from the LECO-Fiehn Metabolomics Library, which gave a similarity value (SV) for evaluating the accuracy of the compound identification. An SV N 700 indicated that metabolite identification was reliable, whereas an SV b 200 meant that the identified metabolite was unreliable. A compound with a SV between 200 and 700 was considered a putative annotation. 2.6. Statistical analysis and pathway analysis The biochemical indicators were shown as means ± SD (n = 8). The differences in these parameters between treatments were compared by using Student's t-test at a significance level of 0.05 (p b 0.05). Prior to statistical analysis, the raw data were examined for normality of distribution and homogeneity of variance with Kolmogorov-Smirnov test and Levene's test, respectively. The data were statistically analysed with the software SPSS for Windows (release 20.0). The resulting GC-TOF-MS data, including the peak numbers, sample names, and normalized peak areas, were inputted to the SIMCA 14.1 software package (MKS Data Analytics Solutions, Umea, Sweden) for principal component analysis (PCA) and orthogonal projections to latent structure–discriminate analysis (OPLS-DA). PCA showed the distribution of origin data. Supervised OPLS-DA was applied to obtain a high level of group separation and a good understanding of variables responsible for classification. Afterwards, a 7-fold cross-validation was conducted to estimate the robustness and the predictive ability of the model derived by OPLS-DA. The intercepts of R2 and Q2 were obtained after permutation tests with 200 iterations. On the basis of variable importance in projection (VIP) values N1.0 obtained from the OPLS-DA model and p-values b0.05 from Student's t-test, differential metabolites


A

B

B 12

A

10 100

SOD activity -1 U mg protein

ROS production % of control

120

14

B

140

80 60

8

A

6

40

4

20

2

0

0

Control

Cold

C

Control

D

B

Cold

1.0

1200

B .8

GSH content -1 nmol mg protein

1000

GPx activity -1 U mg protein

1375

A 800

600

400

.6

A .4

.2

200

0

0.0

Control

E

Cold

Control

F

500

25 A

B

A

20

400 A

GR activity -1 U mg protein

CAT activity -1 U mg protein

Cold

300

200

15

10

5

100

0

0

Control

Cold

G

Control

2.5

Cold

A

2.0

MDA content -1 nmol mg protein

A

1.5

1.0

.5

0.0

Control

Cold

Fig. 1. Oxidative stress responses in the gills of S. aequifasciatus exposed to control (black) and low (grey) temperatures. ROS (A), SOD (B), GPx (C), GSH (D) CAT (E), GR (F), and MDA (G). Different upper-case letters indicate significant differences between treatments (p b 0.05). The same upper-case letters indicate no differences between treatments (p N 0.05).

1376


were identified. The Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) was used to search for the pathways of the metabolites. A free web-based tool, MetaboAnalyst 3.0, which uses the high-quality KEGG metabolic pathway database as the backend knowledgebase, was employed for pathway analysis and visualization (http://www.metaboanalyst.ca).

inorganic compound (Table 1). To intuitively inspect the tendencies in the variation of metabolite concentrations between the control and cold treatments, we produced a heat map of differential metabolites according to the relative quantities of each marker. In comparison with the control, as shown in Fig. 4, a total of 15 metabolites were upregulated (red) and a total of 20 metabolites were down-regulated (blue) in the cold group, as listed in Table 1.

3. Results 3.4. Metabolic pathway analysis 3.1. Biochemical indicators After 30 days of cold acclimation, cold-exposed fish showed increased ROS production (Student's t-test, p = 0.023; Fig. 1A), SOD activity (Student's t-test, p = 0.001; Fig. 1B), GPx activity (Student's ttest, p = 0.001; Fig. 1C), and GSH level (Student's t-test, p = 0.001; Fig. 1D) compared with the control fish. In contrast, cold-acclimated fish expressed decreased CAT activity compared to the control fish (Student's t-test, p = 0.008; Fig. 1E). GR activity (Student's t-test, p = 0.250; Fig. 1F) and MDA content (Student's t-test, p = 0.061; Fig. 1G), however, did not show significant differences between the control and cold treatments.

To explore the potential metabolic pathways that were affected by cold acclimation, we imported the differential metabolites (Table 1) into MetaboAnalyst 3.0. The pathway analysis results provided a detailed account of the metabolic pathway changes associated with cold acclimation (Fig. 5 and Table S1). On the basis of both −lnp-values N 1.0 and pathway impact scores N 0.01, the most relevant metabolic pathways were identified as starch and sucrose metabolism, the pentose phosphate pathway (PPP), glycerolipid metabolism, sphingolipid metabolism, glutathione metabolism and arginine and proline metabolism. 4. Discussion

3.2. Overall changes in metabolites in response to cold stress A total of 767 metabolite peaks were extracted by GC-TOF-MS. Unsupervised PCA showed an obvious separation between the cold and control treatments, although there appeared to be a tank-dependent separation between the controls (Fig. 2), indicating that the coldacclimated fish exhibited changes in their metabolic profiles relative to the control fish. To maximize the discrimination between the two classes, we employed OPLS-DA to identify differences in metabolite levels between the cold and control treatments. The score scatter plots for the GC-TOF-MS data in the training set showed clear discrimination between the cold and control groups (Fig. 3A). The OPLS-DA score plot had the cumulative values of R2Y and Q2 being 97.6% and 62.8%, respectively, suggesting that the model derived by OPLS-DA had good fit and high predictability (Fig. 3B) and could be exploited in the subsequent analyses. 3.3. Cold-responsive differential metabolites On the basis of the OPLS-DA results, a total of 35 differential metabolites were identified, including twelve carbohydrates and carbohydrate conjugates, six lipids and lipid-like molecules, nine organic acids and derivatives, and seven other organic compounds, as well as one

Fig. 2. PCA for the metabolite profiles of gills of S. aequifasciatus, showing separation between the control (green) and cold (blue) treatments. Each dot represents one gill sample from each treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

A battery of biochemical indicators were employed to characterize the antioxidant status of the gills of S. aequifasciatus during cold exposure (Fig. 6). The SOD-CAT system is frequently considered the first line of defence against ROS formation under stress (Pandey et al., 2003). SOD converts the superoxide radical into H2O2, which is then metabolized by CAT. In this study, cold stress led to enhanced gill SOD activity but CAT inhibition. Similarly, Malek et al. (2004) found upregulated gene expression of SOD but not CAT in the muscles of zebrafish after 1-year cold acclimation. Joy et al. (2017), however, reported increases in both SOD and CAT activities in Etroplus suratensis exposed to acute low temperature. In addition to CAT, GPx can also prevent the production of ROS by neutralizing H2O2. Enhanced gill GPx activity would help clear accumulated H2O2 during cold acclimation. Min et al. (2014) also observed the activation of GPx activity in the gills of black rockfish Sebastes schlegelii in response to decreased temperature. The response to CAT depletion involves compensatory changes in the activity levels of enzymes, e.g., GPx, involved in glutathione metabolism (Bagnyukova et al., 2005). In this way, CAT inhibition could be partially compensated for by an increase in GPx activity. Non-enzymatic small organic molecules such as GSH also play important roles in the maintenance of cellular redox status. An increase in GSH was observed in the gills of cold-acclimated fish, corresponding to a protective response to cold exposure. He et al. (2015) also found the activation of GSH in the liver of tilapia Oreochromis niloticus in response to cold stress. Such increase in GSH might suggest the activation of a glutathione-dependent system of antioxidant defence in the gills of S. aequifasciatus exposed to low temperatures. As a protective response, organisms can augment the levels of GSH through biosynthesis or by increasing its regeneration by GR (Peña-Llopis et al., 2003). The unaltered GR activity indicated inability of the gills to regenerate GSH efficiently. Min et al. (2014) also observed similar GR response to thermal stress in the gills of the black rockfish S. schlegelii. Consequently, the increase in the amount of GSH may be associated with de novo synthesis. Cold acclimation typically cause an increase in intracellular lipids and a remodelling of lipids in biological membranes (Abele and Puntarulo, 2004; Grim et al., 2013), which can impact the susceptibility of biological membranes to LPO. The risk of LPO can be magnified because fishes acclimated to cold generally have enhanced oxidative capacity (Kammer et al., 2011; Grim et al., 2013). In this study, despite elevated ROS production under cold exposure, no significant increases in MDA levels were observed, probably partially due to the protective responses in the antioxidant system of S. aequifasciatus gills. Similarly, Malek et al. (2004) reported that zebrafish maintained at 18 °C for


1377

Fig. 3. OPLS-DA for discriminating the metabolite profiles of gills of the control (green) and cold (blue) treatments, with each dot representing one gill sample from each treatment (A); corresponding permutation test obtained from GC-TOF-MS (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

1 year had MDA levels similar to fish maintained at 28 °C. Joy et al. (2017), however, found increased MDA levels in E. suratensis during 72 h of cold exposure. These distinct results suggest that cold-induced oxidative stress is related to the duration of exposure. The alterations in biochemical parameters indicated the ability of S. aequifasciatus gills to increase the activities of SOD and GPx, and the level of GSH, in the process of cold exposure. The metabolomic analysis provided an integrated description of the cold-induced metabolic changes in gills, allowing the identification of differential metabolites during cold acclimation. Changes in metabolites (i.e., glucose, D-fructose, 6-phospho-D-gluconate, 3-phosphoglycerate and phosphate) related to carbohydrate and energy metabolism (Fig. 6), suggesting that cold acclimation appears to strongly influence energy pathways. The reduced glucose and D-fructose stores in the gills of cold-acclimated fish indicated increased energy expenditure or turnover at decreased temperatures. A decrease in glucose has also been detected in the liver of the gilthead sea bream Sparus aurata after cold exposure (Vargas-Chacoff et al., 2009). The increases in 3phosphoglycerate might suggest the metabolization of glucose via glycolysis. Such result is in accord with increased lactate dehydrogenase activity of S. aequifasciatus under cold stress (Wen et al., 2017). Phosphate (Pi) signalling can result in balanced modulation of oxidative

phosphorylation by influencing ATP production, contributing to the homeostasis of energy metabolism (Bose et al., 2003). The great increase in Pi abundance might indicate the hydrolysis of high-energy phosphate bonds to liberate Pi to combat ROS production (Dalvi et al., 2017). Such result can be supported by enhanced alkaline phosphatase (ALP) activity of S. aequifasciatus exposed to cold stress (Wen et al., 2017), because ALP plays an important role in the metabolism of phosphorus. The PPP is an alternative route of glucose catabolism that functions in the formation of NADPH for biosynthetic reactions. PPP activity is reported to increase with decreasing temperatures (e.g., Hochachka and Hayes, 1962; Johnston and Dunn, 1987). The increase in 6-phospho-Dgluconate with cold treatment indicated the activation of PPP, resulting in large amounts of NADPH which can then be employed for biosynthesis of unsaturated fatty acids. To maintain membrane fluidity, coldacclimated fishes possess elevated amounts of unsaturated fatty acids within phospholipids (Tiku et al., 1996; Barnes et al., 2014). This may be supported by the increases in cis-gondoic acid and behenic acid (Fig. 6). Additionally, GR can use NADPH to convert oxidized glutathione (GSSG) into GSH, and increased production of NADPH promotes the regeneration of GSH (Lewis et al., 2014; Fan et al., 2014). Given the similar GR activity between the cold and control groups as revealed

1378


Table 1 The relative concentrations of differential metabolites in response to cold stress in the gills of S. aequifasciatus. Metabolites

SV

Control

Cold

VIPa

p-Valueb

Dihydroxyacetone Glucose

931 929 910

0.002629 0.015235 0.001995

0.001115 0.008685 0.001066

2.35 2.08 2.21

0.002 0.009 0.003

906 877 853 834 828 824 798 769 758 721 687

7.82E−08 0.001241 0.000654 0.000721 0.000684 0.000231 0.000381 6.07E−05 7.82E−08 0.002789 0.000284

0.001017 0.000537 0.002252 0.002162 0.000255 0.00046 0.000234 0.000133 0.000326 0.000533 0.000132

2.54 2.42 1.54 1.49 2.35 1.72 2.06 1.38 2.80 2.56 1.07

0.002 0.002 0.004 0.039 0.004 0.037 0.014 0.008 0.000 0.003 0.024

686 645 627 563

0.000496 6.22E−06 0.000114 3.71E−05

0.002461 2.27E−05 0.000369 9.8E−05

1.46 1.27 1.68 1.46

0.044 0.027 0.042 0.002

D-Glucoheptose

561 536 525 505 491 486 428 387 381 378

6.17E−05 0.000166 0.000145 0.000112 0.000139 0.000165 0.000164 0.000506 2.09E−06 8.59E−05

0.000225 6.51E−08 4.98E−06 6.51E−08 7.68E−05 6.16E−05 6.51E−08 0.000794 3.3E−05 6.51E−08

1.88 1.68 1.14 1.63 1.13 2.29 2.07 1.77 1.60 1.62

0.006 0.037 0.042 0.045 0.040 0.004 0.013 0.024 0.026 0.046

Tartaric acid N-(2-hydroxyethyl)-iminodiacetic acid 9-Fluorenone 10-Hydroxydecanoic acid 4-Acetylbutyric acid 4-Aminobenzoic acid Isoxanthopterin

360 347 330 311 295 275 263

3.71E−05 0.00015 5.05E−05 0.000183 3.52E−05 7.79E−06 0.002738

0.000115 1.12E−05 1.39E−05 7.69E−05 2.27E−06 6.51E−08 0.000698

1.03 1.73 1.66 2.17 1.74 1.81 1.88

0.026 0.015 0.010 0.020 0.034 0.033 0.029

D-Talose

Phosphate Sorbose O-Phosphorylethanolamine 3-Aminoisobutyric acid Glyceric acid Behenic acid Levoglucosan 3-Phosphoglycerate cis-Gondoic acid Sedoheptulose D-Fructose

6-Phospho-D-gluconate 11-epi-Prostaglandin F2alpha Spermidine L-Ornithine

Maltitol Carnitine Prostaglandin A2 Cetadiol 3-Hydroxypropionic acid 4-Hydroxybutyrate Nicotianamine S-Carboxymethylcysteine Adrenosterone

Trendc

Similarity value (SV) was used to evaluate the accuracy of the discriminating metabolite. a Variable importance in the projection (VIP) was obtained from OPLS-DA with a threshold of 1.0. b p-Value was calculated from Student's t-test. c The red and blue arrows indicate up and down trends, respectively in response to cold stress.

Fig. 4. Heat-map visualization of the differential metabolites in response to cold stress. Colour denotes the abundance of metabolites, from the highest (red) to the lowest (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)


1379

Fig. 5. Pathway mapping based on differential metabolites, as visualized by bubble plots. Bubble size is proportional to the impact of each pathway, and bubble colour denotes the degree of significance, from the highest (red) to the lowest (white). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

by biochemical assays, enhanced PPP may instead account for the increased gill GSH. In addition to the biosynthesis of unsaturated fatty acids, some of the differential metabolites (i.e., glyceric acid, dihydroxyacetone, Ophosphorylethanolamine and carnitine) appeared to be linked to perturbation of other lipid metabolic pathways (Fig. 6). Glyceric acid is a branch point between glycerolipid and carbohydrate metabolism (Kirkwood et al., 2012). The depletions of glyceric acid and dihydroxyacetone suggested the mobilization of glycerolipids as an energy source via β-oxidation (Kirkwood et al., 2012). This possibility is supported by the changes in carnitine (a lipid-transport-related metabolite) of the cold-acclimated fish. These results suggest that S. aequifasciatus exposed

to prolonged cold stress rely on at least two different energy sources (glycolysis and β-oxidation) to generate enough energy for maintenance. The up-regulation of O-phosphorylethanolamine reflected the occurrence of sphingolipid metabolism. During temperature acclimation, organisms can mobilize sphingolipid signalling to control stress response (Hrobuchak et al., 2017). Phosphatidylethanolamines (PEs) are rich in readily oxidizable amino acids (AAs) and docosahexaenoic acid, and PE-derived unsaturated fatty acids usually serve as the substrate of sphingolipid peroxidation under stress (González-Domínguez et al., 2014). An increase of O-phosphorylethanolamine, a precursor of PE, can also reflect an enhanced antioxidant defence mechanism (Duarte et al., 2012).

Fig. 6. The hypothetical framework based on the changed metabolites and biochemical indicators in the gills of cold-exposed fish compared with control fish. The metabolites and biochemical parameters are coloured according to the type of change (black, no change; red, up-regulation; blue, down-regulation). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

1380


Changes in metabolites (i.e., ornithine, spermidine and 3hydroxypropionic acid) related to protein metabolism were also observed in gills in response to cold exposure (Fig. 6). Spermidine and ornithine were concomitantly up-regulated, indicating the activations of glutathione metabolism and of arginine and proline metabolism (Melis et al., 2017). Moreover, the activation of glutathione metabolism agreed with the increased GSH level found among the biochemical indicators, demonstrating that the increases in spermidine and ornithine are potentially indicative of a glutathione dependent system of antioxidant defence in the gills (Eisenberg et al., 2009). Thermal stress, especially long-term cold acclimation, can also induce a switch in metabolic fuels from carbohydrates and lipids to protein oxidation (e.g., Tseng and Hwang, 2008; Vergauwen et al., 2010). The modification of beta-alanine metabolism revealed by the changes in spermidine and 3-hydroxypropionic acid indicated a temperature compensation and an increased demand for or turnover of specific AAs. This may be supported by enhanced activities of alanine aminotransferase and aspartate aminotransferase of S. aequifasciatus under low temperature (Wen et al., 2017). 5. Conclusion This study suggests that low temperature resulted in marked changes in both the antioxidant status and the metabolic profile of S. aequifasciatus gills. As shown in Fig. 6, a battery of biochemical parameters coupled with GC-TOF-MS metabolomics allowed a good understanding of the physiological responses involved in cold acclimation, namely, the mobilizations of starch and sucrose metabolism and glycerolipid metabolism for meeting energy demand; the activation of PPP for biosynthesis of unsaturated fatty acids; the inductions of SOD and GPx activities, and GSH level as revealed by the activation of GSH metabolism for antioxidant defence. Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.05.401. Acknowledgements The study presented in the manuscript was funded by the China Postdoctoral Science Foundation (2017M621433); and the Doctoral Scientific Research Foundation of Shanghai Ocean University (A2-020317-100306). The authors are grateful to Biotree Bio-Technique Co., Ltd. (Shanghai, China) for technical assistance with the metabolomics analysis. References Abele, D., Puntarulo, S., 2004. Formation of reactive species and induction of antioxidant defence systems in polar and temperate marine invertebrates and fish. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 138, 405–415. Bagnyukova, T.V., Storey, K.B., Lushchak, V.I., 2005. Adaptive response of antioxidant enzymes to catalase inhibition by aminotriazole in goldfish liver and kidney. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 142, 335–341. Barding, G.A., Béni, S., Fukao, T., Bailey-Serres, J., Larive, C.K., 2012. Comparison of GC-MS and NMR for metabolite profiling of rice subjected to submergence stress. J. Proteome Res. 12, 898–909. Barnes, K.R., Cozzi, R.R., Robertson, G., Marshall, W.S., 2014. Cold acclimation of NaCl secretion in a eurythermic teleost: mitochondrial function and gill remodeling. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 168, 50–62. Barton, B.A., 2002. Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids. Integr. Comp. Biol. 42, 517–525. Begley, P., Francis-Mcintyre, S., Dunn, W.B., Broadhurst, D.I., Halsall, A., Tseng, A., Knowles, J., Consortium, H., Goodacre, R., Kell, D.B., 2009. Development and performance of a gas chromatography-time-of-flight mass spectrometry analysis for large-scale nontargeted metabolomic studies of human serum. Anal. Chem. 81, 7038–7046. Bleher, H., Stölting, K.N., Salzburger, W., Meyer, A., 2007. Revision of the genus Symphysodon Heckel, 1840 (Teleostei: Perciformes: Cichlidae) based on molecular and morphological characters. Aqua 12, 133–174. Bonga, S.W., 1997. The stress response in fish. Physiol. Rev. 77, 591–625. Bose, S., French, S., Evans, F.J., Joubert, F., Balaban, R.S., 2003. Metabolic network control of oxidative phosphorylation multiple roles of inorganic phosphate. J. Biol. Chem. 278, 39155–39165.

Bowden, A.J., Gardiner, N.M., Couturier, C.S., Stecyk, J.A.W., Nilsson, G.E., Munday, P.L., Rummer, J.L., 2014. Alterations in gill structure in tropical reef fishes as a result of elevated temperatures. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 175, 64–71. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brandao, F., Cappello, T., Raimundo, J., Santos, M.A., Maisano, M., Mauceri, A., Pacheco, M., Pereira, P., 2015. Unravelling the mechanisms of mercury hepatotoxicity in wild fish (Liza aurata) through a triad approach: bioaccumulation, metabolomic profiles and oxidative stress. Metallomics 7, 1352–1363. Cappello, T., Brandão, F., Guilherme, S., Santos, M.A., Maisano, M., Mauceri, A., Canário, J., Pacheco, M., Pereira, P., 2016. Insights into the mechanisms underlying mercuryinduced oxidative stress in gills of wild fish (Liza aurata) combining 1H NMR metabolomics and conventional biochemical assays. Sci. Total Environ. 548, 13–24. Carlberg, I.N.C.E.R., Mannervik, B.E.N.G.T., 1975. Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J. Biol. Chem. 250, 5475–5480. Chang, C., Guo, Z.G., He, B., Yao, W.Z., 2015. Metabolic alterations in the sera of Chinese patients with mild persistent asthma: a GC-MS-based metabolomics analysis. Acta Pharmacol. Sin. 36, 1356–1366. Cheng, C.H., Guo, Z.X., Wang, A.L., 2018. The protective effects of taurine on oxidative stress, cytoplasmic free-Ca2+ and apoptosis of pufferfish (Takifugu obscurus) under low temperature stress. Fish Shellfish Immunol. 77, 457–464. Dalvi, R.S., Das, T., Debnath, D., Yengkokpam, S., Baruah, K., Tiwari, L.R., Pal, A.K., 2017. Metabolic and cellular stress responses of catfish, Horabagrus brachysoma (Günther) acclimated to increasing temperatures. J. Therm. Biol. 65, 32–40. Delaporte, M., Soudant, P., Moal, J., Lambert, C., Quéré, C., Miner, P., Choquet, G., Paillard, C., Samain, J.F., 2003. Effect of a mono-specific algal diet on immune functions in two bivalve species-Crassostrea gigas and Ruditapes philippinarum. J. Exp. Biol. 206, 3053–3064. Duarte, J.M., Lei, H., Mlynárik, V., Gruetter, R., 2012. The neurochemical profile quantified by in vivo 1H NMR spectroscopy. NeuroImage 61, 342–362. Eisenberg, T., Knauer, H., Schauer, A., Büttner, S., Ruckenstuhl, C., Carmona-Gutierrez, D., Fussi, H., 2009. Induction of autophagy by spermidine promotes longevity. Nat. Cell Biol. 11 (1305-Ue102). Evans, D.H., Piermarini, P.M., Choe, K.P., 2005. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol. Rev. 85, 97–177. Fan, J., Ye, J., Kamphorst, J.J., Shlomi, T., Thompson, C.B., Rabinowitz, J.D., 2014. Quantitative flux analysis reveals folate-dependent NADPH production. Nature 510, 298. Ficke, A.D., Myrick, C.A., Hansen, L.J., 2007. Potential impacts of global climate change on freshwater fisheries. Rev. Fish Biol. Fish. 17, 581–613. Fridovich, I., 1969. Superoxide dismutase and enzymatic function for erythrocuprein (Hemocuprein). J. Biol. Chem. 244, 6049–6055. González-Domínguez, R., García-Barrera, T., Gómez-Ariza, J.L., 2014. Combination of metabolomic and phospholipid-profiling approaches for the study of Alzheimer's disease. J. Proteome 104, 37–47. Goth, L., 1991. A simple method for determination of serum catalase activity and revision of reference range. Clin. Chim. Acta 196, 143–151. Grim, J.M., Miles, D.R.B., Crockett, E.L., 2010. Temperature acclimation alters oxidative capacities and composition of membrane lipids without influencing activities of enzymatic antioxidants or susceptibility to lipid peroxidation in fish muscle. J. Exp. Biol. 213, 445–452. Grim, J.M., Simonik, E.A., Semones, M.C., Kuhn, D.E., Crockett, E.L., 2013. The glutathionedependent system of antioxidant defense is not modulated by temperature acclimation in muscle tissues from striped bass, Morone saxatilis. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 164, 383–390. Hafeman, D.G., Sunde, R.A., Hoekstra, W.G., 1974. Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat. J. Nutr. 104, 580–587. He, J., Qiang, J., Yang, H., Xu, P., Zhu, Z.X., Yang, R.Q., 2015. Changes in the fatty acid composition and regulation of antioxidant enzymes and physiology of juvenile genetically improved farmed tilapia Oreochromis niloticus (L.), subjected to short-term low temperature stress. J. Therm. Biol. 53, 90–97. Hochachka, P.W., Hayes, F.R., 1962. The effect of temperature acclimation on pathways of glucose metabolism in the trout. Can. J. Zool. 40, 261–270. Hrobuchak, H.P., Ardasheva, A.P., Servello, D.P., Nolan, A.P., Chan, J.P., 2017. The role of the sphingolipid metabolism pathway in healthy aging. FASEB J. 31, 935.4. Hu, P., Liu, M., Zhang, D., Wang, J., Niu, H., Liu, Y., Chen, L., 2015. Global identification of the genetic networks and cis-regulatory elements of the cold response in zebrafish. Nucleic Acids Res. 43, 9198–9213. Hu, P., Liu, M., Liu, Y., Wang, J., Zhang, D., Niu, H., Jiang, S., Wang, J., Zhang, D., Han, B., Xu, Q., Chen, L., 2016. Transcriptome comparison reveals a genetic network regulating the lower temperature limit in fish. Sci. Rep. 6, 28952. Ibarz, A., Martín-Pérez, M., Blasco, J., Bellido, D., de Oliveira, E., Fernández-Borràs, J., 2010. Gilthead sea bream liver proteome altered at low temperatures by oxidative stress. Proteomics 10, 963–975. Isaak, D.J., Wollrab, S., Horan, D., Chandler, G., 2012. Climate change effects on stream and river temperatures across the northwest US from 1980–2009 and implications for salmonid fishes. Clim. Chang. 113, 499–524. Johnston, I.A., Dunn, J.E.F.F., 1987. Temperature acclimation and metabolism in ectotherms with particular reference to teleost fish. Symp. Soc. Exp. Biol. 41, 67–93. Joy, S., Alikunju, A.P., Jose, J., Sudha, H.S.H., Parambath, P.M., Puthiyedathu, S.T., Philip, B., 2017. Oxidative stress and antioxidant defense responses of Etroplus suratensis to acute temperature fluctuations. J. Therm. Biol. 70, 20–26. Kammer, A.R., Orczewska, J.I., O'Brien, K.M., 2011. Oxidative stress is transient and tissue specific during cold acclimation of threespine stickleback. J. Exp. Biol. 214, 1248–1256.

B. Wen et al. / Science of the Total Environment 640–641 (2018) 1372–1381 Kind, T., Wohlgemuth, G., Lee, D.Y., Lu, Y., Palazoglu, M., Shahbaz, S., Fiehn, O., 2009. FiehnLib: mass spectral and retention index libraries for metabolomics based on quadrupole and time-of-flight gas chromatography/mass spectrometry. Anal. Chem. 81, 10038–10048. Kirkwood, J.S., Lebold, K.M., Miranda, C.L., Wright, C.L., Miller, G.W., Tanguay, R.L., Barton, C.L., Traber, M.G., Stevens, J.F., 2012. Vitamin C deficiency activates the purine nucleotide cycle in zebrafish. J. Biol. Chem. 287, 3833–3841. Kullgren, A., Jutfelt, F., Fontanillas, R., Sundell, K., Samuelsson, L., Wiklander, K., Kling, P., Koppe, W., Larsson, D.G.J., Björnsson, B.T., Jönsson, E., 2013. The impact of temperature on the metabolome and endocrine metabolic signals in Atlantic salmon (Salmo salar). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 164, 44–53. Lemgruber, R.D.S.P., dos Anjos Marshall, N.A., Ghelfi, A., Fagundes, D.B., Val, A.L., 2013. Functional categorization of transcriptome in the species Symphysodon aequifasciatus Pellegrin 1904 (Perciformes: Cichlidae) exposed to benzo [a] pyrene and phenanthrene. PLoS One 8, e81083. Lewis, C.A., Parker, S.J., Fiske, B.P., McCloskey, D., Gui, D.Y., Green, C.R., Vokes, N.I., Feist, A.M., Vander Heiden, M.G., Metallo, C.M., 2014. Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells. Mol. Cell 55, 253–263. Lin, R., Wang, L., Zhao, Y., Gao, J., Chen, Z., 2017. Gonad transcriptome of discus fish (Symphysodon haraldi) and discovery of sex-related genes. Aquac. Res. 48, 5993–6000. Lu, J., Shi, Y., Wang, S., Chen, H., Cai, S., Feng, J., 2016. NMR-based metabolomic analysis of Haliotis diversicolor exposed to thermal and hypoxic stresses. Sci. Total Environ. 545, 280–288. Lushchak, V.I., 2011. Environmentally induced oxidative stress in aquatic animals. Aquat. Toxicol. 101, 13–30. Madeira, D., Vinagre, C., Costa, P.M., Diniz, M.S., 2014. Histopathological alterations, physiological limits, and molecular changes of juvenile Sparus aurata in response to thermal stress. Mar. Ecol. Prog. Ser. 505, 253–266. Malek, R.L., Sajadi, H., Abraham, J., Grundy, M.A., Gerhard, G.S., 2004. The effects of temperature reduction on gene expression and oxidative stress in skeletal muscle from adult zebrafish. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 138, 363–373. Maunder, R.J., Buckley, J., Val, A.L., Sloman, K.A., 2011. Accumulation of dietary and aqueous cadmium into the epidermal mucus of the discus fish Symphysodon sp. Aquat. Toxicol. 103, 205–212. Maunder, R.J., Buckley, J., Val, A.L., Sloman, K.A., 2013. A toxic diet: transfer of contaminants to offspring through a parental care mechanism. J. Exp. Biol. 216, 3587–3590. Melis, R., Sanna, R., Braca, A., Bonaglini, E., Cappuccinelli, R., Slawski, H., Roggio, T., Uzzau, S., Anedda, R., 2017. Molecular details on gilthead sea bream (Sparus aurata) sensitivity to low water temperatures from 1H NMR metabolomics. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 204, 129–136. Min, E.Y., Baeck, S.K., Kang, J.C., 2014. Combined effects of copper and temperature on antioxidant enzymes in the black rockfish Sebastes schlegeli. J. Fish. Aquat. Sci. 17, 345–353. Pandey, S., Parvez, S., Sayeed, I., Haque, R., Bin-Hafeez, B., Raisuddin, S., 2003. Biomarkers of oxidative stress: a comparative study of river Yamuna fish Wallago attu (Bl. & Schn.). Sci. Total Environ. 309, 105–115.

1381

Peña-Llopis, S., Ferrando, M.D., Peña, J.B., 2003. Fish tolerance to organophosphateinduced oxidative stress is dependent on the glutathione metabolism and enhanced by N-acetylcysteine. Aquat. Toxicol. 65, 337–360. Pirhonen, J., Aaltonen, S., Järvenpää, H., 2014. Growth of domesticated discus Symphysodon sp. at constant temperatures. Aquac. Res. 45, 940–943. Przeslawski, R., Ahyong, S., Byrne, M., Woerheide, G., Hutchings, P.A.T., 2008. Beyond corals and fish: the effects of climate change on noncoral benthic invertebrates of tropical reefs. Glob. Chang. Biol. 14, 2773–2795. Satoh, K., 1978. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin. Chim. Acta 90, 37–43. Smith, S., Bernatchez, L., Beheregaray, L.B., 2013. RNA-seq analysis reveals extensive transcriptional plasticity to temperature stress in a freshwater fish species. BMC Genomics 14, 375. Sollid, J., Weber, R.E., Nilsson, G.E., 2005. Temperature alters the respiratory surface area of crucian carp Carassius carassius and goldfish Carassius auratus. J. Exp. Biol. 208, 1109–1116. Stevens, P.W., Blewett, D.A., Boucek, R.E., Rehage, J.S., Winner, B.L., Young, J.M., Paperno, R., 2016. Resilience of a tropical sport fish population to a severe cold event varies across five estuaries in southern Florida. Ecosphere 7, e01400. Tietze, F., 1969. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27, 502–522. Tiku, P.E., Gracey, A.Y., Macarteny, A.I., Beynon, R.J., Cossins, A.R., 1996. Cold-induced expression of delta9-desaturase in carp by transcriptional and posttranslational mechanisms. Science 271, 815–818. Tomanek, L., 2014. Proteomics to study adaptations in marine organisms to environmental stress. J. Proteome 105, 92–106. Tseng, Y.C., Hwang, P.P., 2008. Some insights into energy metabolism for osmoregulation in fish. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 148, 419–429. Vargas-Chacoff, L., Arjona, F.J., Polakof, S., del Río, M.P.M., Soengas, J.L., Mancera, J.M., 2009. Interactive effects of environmental salinity and temperature on metabolic responses of gilthead sea bream Sparus aurata. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 154, 417–424. Vergauwen, L., Benoot, D., Blust, R., Knapen, D., 2010. Long-term warm or cold acclimation elicits a specific transcriptional response and affects energy metabolism in zebrafish. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 157, 149–157. Wen, B., Jin, S.R., Chen, Z.Z., Gao, J.Z., Wang, L., Liu, Y., Liu, H.P., 2017. Plasticity of energy reserves and metabolic performance of discus fish (Symphysodon aequifasciatus) exposed to low-temperature stress. Aquaculture 481, 169–176. Wen, B., Zhang, N., Jin, S.R., Chen, Z.Z., Gao, J.Z., Liu, Y., Liu, H.P., Xu, Z., 2018. Microplastics have a more profound impact than elevated temperatures on the predatory performance, digestion and energy metabolism of an Amazonian cichlid. Aquat. Toxicol. 195, 67–76.