Mitigation effects of CO2-driven ocean acidification on Cd toxicity to the marine diatom Skeletonema costatum

Environmental Pollution 259 (2020) 113850

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Mitigation effects of CO2-driven ocean acidification on Cd toxicity to the marine diatom Skeletonema costatum* Fang Dong a, b, Pu Wang b, Wei Qian b, Xing Tang a, Xiaoshan Zhu b, c, *, Zhenyu Wang d, Zhonghua Cai b, Jiangxin Wang a a

College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, PR China Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China Southern Laboratory of Ocean Science and Engineering (Guangdong, Zhuhai), Zhuhai 519000, PR China d Institute of Environmental Processes and Pollution Control, School of Environmental and Civil Engineering, Jiangnan University, Wuxi 2141122, PR China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 July 2019 Received in revised form 28 November 2019 Accepted 16 December 2019 Available online 19 December 2019

Ocean acidification (OA) is a global problem to marine ecosystems. Cadmium (Cd) is a typical metal pollutant, which is non-essential but extremely toxic to marine organisms. The combined effects of marine pollution and climate-driven ocean changes should be considered for the effective marine ecosystem management of coastal areas. Previous reports have separately investigated the influences of OA and Cd pollution on marine organisms. However, little is known of the potential combined effects of OA and Cd pollution on marine diatoms. We investigated the sole and combined influences of OA (1500 ppm CO2) and Cd exposure (0.4 and 1.2 mg/L) on the coastal diatom Skeletonema costatum. Our results clearly showed that OA significantly alleviated the toxicity of Cd to S. costatum growth and mitigated the oxidant stress, although the intercellular Cd accumulation still increased. OA partially rescued S. costatum from the inhibition of photosynthesis and pyruvate metabolism caused by Cd exposure. It also upregulated genes involved in gluconeogenesis, glycolysis, the citrate cycle (TCA), Ribonucleic acid (RNA) metabolism, and especially the biosynthesis of non-protein thiol compounds. These changes might contribute to algal growth and Cd resistance. Overall, this study demonstrates that OA can alleviate Cd toxicity to S. costatum and explores the potential underlying mechanisms at both the cellular and molecular levels. These results will ultimately help us understand the impacts of combined stresses of climate change and metal pollution on marine organisms and expand the knowledge of the ecological risks of OA. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Climate change Ocean acidification Coastal pollution Cd Toxicity Skeletonema costatum

1. Introduction Ocean acidification (OA) represents an increased hydrogen ion (Hþ) concentration and decreased pH in surface ocean water. During the past century, the average pH of ocean surface water has fallen by 0.1 unit due to increased carbon dioxide emissions (Doney et al., 2009). At present, the rate of OA is the highest during the past 300 million years (Doney et al., 2009). By 2100, the atmosphere CO2 level is projected to reach 1000 ppm, and thus the average surface seawater pH may decline 0.3e0.4 units (Orr et al., 2005).

* This paper has been recommended for acceptance by Dr. Sarah Harmon. * Corresponding author. Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China. E-mail address: [email protected] (X. Zhu).

https://doi.org/10.1016/j.envpol.2019.113850 0269-7491/© 2019 Elsevier Ltd. All rights reserved.

Increased seawater acidity changes the chemical balance of the marine carbonate system, subsequently affecting the physiological processes (e.g., calcification, metabolism, immunity, and survival), growth, and reproduction of marine species (Iglesias-Rodriguez et al., 2008; Leite Figueiredo et al., 2016). Additionally, OA can influence the ecotoxicological effects of environmental pollutants. For example, OA alters the binding behavior of metals to sediments and increases the concentration of toxic free copper ions (Cu2þ) in coastal water (Ndungu, 2012; Roberts et al., 2013). Thus, investigations of marine pollution must consider the influences of climate-driven ocean change, which is a critical step for effective marine ecosystem management (Waldbusser and Salisbury, 2014). Cd is non-essential for marine organisms and reveals significant impacts on the marine environments (ANZECC, 1999). Its concentration in open surface seawater is generally low (generally at the level of mg/L or below, Apte et al., 1998), but may be greatly high in

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some coastal regions due to anthropogenic pollution (Simpson, 1981; Villanueva and Alfonoso, 1992). For example, Cd concentrations in coastal sediments outside of industrial plants in Northern China even reached 488 mg/kg in dry weight, and a major portion of Cd was strongly associated with exchangeable fractions (Pan and Wang, 2012). Moreover, Cd is easily absorbed and accumulated by algae, fishes, mussels, shrimps, and marine mammals (Odẑak et al., 1994; Shi and Wang, 2004; Smail et al., 2012). OA can modulate the accumulation and effects of Cd on fitness, survival, and physiological activities in marine organisms, subsequently affecting the tro€ tze et al., 2014; Liu phic transfer of Cd along marine food chains (Go et al., 2016; Nardi et al., 2017, 2018). However, most studies in relation to OA and Cd pollution were focusing on marine bivalves and copepods. Reports on marine algae are still deficient. As the major primary producers, diatoms are important to marine ecosystems (Rabosky and Sorhannus, 2009). Heavy metal pollution and OA both influence marine diatoms (Rijstenbil and Gerringa, 2002; Wei et al., 2003; Wang and Wang, 2008; Wu et al., 2010; Gao et al., 2012; Herzi et al., 2013). The combined effects of heavy metal pollution and OA on marine diatoms should be considered. Nevertheless, little is known of the potential combined effects of OA and Cd pollution on marine diatoms. To investigate the combined impacts of OA and Cd pollution on marine diatoms, we exposed S. costatum (an ecologically important and widely distributed coastal species) to different levels of CO2 and/or Cd, after which we measured changes in growth indices and Cd accumulation. To further explore the underlying mechanisms, antioxidant indices and transcriptome profiles were compared between treatments and the control. Our results provide a scientific basis to better understand the impacts of global climate change combined with Cd pollution on marine environments. 2. Materials and methods 2.1. Chemicals CdCl2$2.5H2O (analytical grade) was purchased from the Aladdin Industrial Corporation (Shanghai, China). The stock solution of Cd2þ (1.0 g/L) was prepared in ultrapure water. Working solutions of Cd (0.8 and 2.4 mg/L) were obtained by diluting the Cd2þ stock solution with natural seawater (pH 8.08, salinity 35.0 psm) which was collected from the coast of Xichong Village, Shenzhen City, China, and filtered through 0.22 mm Poretic membranes. The background concertation of Cd in the collected seawater was approximately 0.07 mg/L. 2.2. Algae culture S. costatum was purchased from the Guangyu Biology Company (Wenzhou, China). Cells were maintained in F/2-Si medium (Guillard and Ryther, 1962) at 25 ± 1  C with a photoperiod of 12 h: 12 h (light: dark) and a light intensity of approximately 170 mmoL/ m2$s. The culture was manually shaken three times per day. Cells were counted daily under a microscope using a hemocytometer (Primo Vert, Zeiss, Germany). 2.3. Experimental design The six treatments included OA (1500 ppm CO2; pH 7.55), 0.4 mg/L Cd (labeled as mCd), 1.2 mg/L Cd (hCd), 1500 ppm CO2 OA þ 0.4 mg/L Cd (OAmCd), and 1500 ppm CO2 OA þ 1.2 mg/L Cd (OAhCd). Treatment with 400 ppm CO2 was also included as the control. The levels of CO2 and Cd in each treatment are listed in Table 1. The CO2 level of 1500 ppm was chosen as the OA scenario, because the dynamic range of many coastal carbonate system

Table 1 Cd and CO2 levels in different treatments.

Control Ocean acidification Cd OA þ Cd

Treatment

Cd concentration (mg/L)

CO2 level (ppm)

C OA mCd hCd OAmCd OAhCd

0 0 0.4 1.2 0.4 1.2

400 1500 400 400 1500 1500

parameters often reaches or even exceeds projections of end-ofcentury change in oceanic carbonate chemistry due to complex ecological environments, global OA and eutrophication (Zhai et al., 2012; Waldbusser and Salisbury, 2014; Gu et al., 2017). As predicted, the pH value in coastal areas might decrease to 7.4e7.7 at the end of the 21st century (Gu et al., 2017). In the present study, the pH of the seawater was 7.55 at the CO2 level of 1500 ppm, which is suitable for mimicking the near-future OA scenario. For Cd, concentrations of 0.4 and 1.2 mg/L were chosen in the experiments, which are the levels of Cd in highly polluted areas inducing significant toxicity to diatoms (Simpson, 1981; Villanueva and Alfonoso, 1992; Wang and Wang, 2011). At these concentrations, clearer responses of diatoms to Cd stress can be observed, including changes of transcripts participating in cell signaling and detoxification process (Brembu et al., 2011). The diatoms were exposed to OA and Cd treatments for 7 days. A special system was manufactured to control the CO2 level (Supplemental Fig. S1). The variance of CO2 concentration was less than 5%. 2-[4-(2-Hydroxyethyl)-1piperazinyl] ethanesulfonic acid (HEPES, Sigma), that has no toxic effects on cells and can stabilize the pH for long time, was used as the biological buffer (Riebesell et al., 2010; Gu et al., 2017). S. costatum cells at the exponential growth phase were harvested by centrifugation at 3500 g for 10 min at 4  C, washed twice with filtered (0.25 mm membrane) seawater, and finally suspended in F/ 2-Si medium without ethylene diamine tetraacetie acid (EDTA) (to avoid interaction between EDTA and Cd). The initial concentration of algae was adjusted to 0.8  105 cells/ml and then subjected to corresponding treatments in beakers. Each treatment was independently repeated three times. The beakers were shaken three times every day, and their positions were randomly exchanged to reduce differences in illumination. The detailed methods of eliminating external heavy metal pollution and metal adsorption interference by glass walls are presented in Supplemental Annotation 1.

2.4. Determination of algal density and chlorophyll a content Algal density and chlorophyll a (chl-a) content were monitored daily. All experimental samples were taken at the same time (13:00 p.m.) every day for density counting and chl-a determination. Alga counting was performed using a hemocytometer under a microscope (Primo Vert, Zeiss, Germany). The chl-a contents were determined using a Chlorophyll Fluorescence System (Phyto-PAM, Walz company, Germany). The Phyto-PAM was calibrated using chla extracted from S. costatum using the acetone extraction method. Each sample was counted and measured for at least three times as technical repeats.

2.5. Determination of Cd content The extracellular and intracellular Cd contents (mg/kg) were measured using an atomic absorption spectrometry (AAS; Analytik Jena AG, ZEEnit 700, Germany). The detailed methods are presented in Supplementary Fig. S2.

F. Dong et al. / Environmental Pollution 259 (2020) 113850

2.6. Determination of antioxidant indices After exposure, activity of superoxide dismutase (SOD) and contents of malondialdehyde (MDA), glutathione (GSH), and phytochelatins (PCs) were determined. In brief, 40 ml of algal solution was centrifuged at 5000 g for 10 min at 4  C. The pellet was rinsed twice with double distilled water and re-suspended in 5 mL of 0.1 M phosphate buffer (PBS, pH 7.8). After sonication on ice for 5 min, the mixture was centrifuged at 8000g for 10 min at 4  C. The supernatant was collected to determine activity of SOD and contents of MDA, GSH, and PC as described in Supplemental Annotation 2. 2.7. Comparative transcriptome analyses S. costatum cells at the exponential growth phase were collected during the exposure for transcriptome sequencing. Three biological replicates were sequenced for each treatment. The raw data were deposited in GenBank with the accession number of PRJNA555443. Methods for RNA isolation, Illumina sequencing, De novo assembly, gene function annotation, analysis of differentially expressed genes (DEGs), and enrichment of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways follow Zhang et al. (2016). 2.8. Real-time quantitative polymerase chain reaction (PCR) validation The relative expression levels of 20 randomly selected unigenes were validated using real-time quantitative PCR (RT-qPCR). Total RNA samples were the same for Illumina sequencing. Complementary deoxyribonucleic acid (cDNA) was synthesized using a BioRT cDNA first strand synthesis kit (Bioer, Hangzhou, China) with oligo(dT) primer. RT-qPCR was carried out using a BioEasy master mix kit (Bioer, Hangzhou, China) on a Line Gene9600 Plus qPCR machine (Bioer, Hangzhou, China). Each reaction was repeated three times as technical replicates. All primers used are listed in Supplemental Table S1. NADP-dependent glyceraldehyde-3phosphate dehydrogenase (NADP) was used as the internal control. The relative expression levels of each gene were calculated using the typical 2-△△Ct method (Livak and Schmittgen, 2001). Three biological replicates were performed for each treatment. 2.9. Statistical analyses Data are expressed as mean ± standard deviation (SD, n ¼ 3). Statistical analysis was performed using one-way ANOVA by SPSS 18.0. P < 0.05 was considered significantly different. 3. Results and discussion 3.1. Growth of S. costatum under exposure to OA and Cd During the exposure, algal densities and chl-a contents continuously increased with time in all treatments (Fig. 1). Compared with the control, treatment with OA significantly increased algal density and chl-a content. At the same level of CO2, addition of Cd (0.4 and 1.2 mg/L) significantly decreased algal density and chl-a content. However, at the same concentration of Cd after treatment for three days, algal density and chl-a content were significantly higher under OA conditions than under normal CO2 condition (Fig. 1, P < 0.05). These results indicate that OA promotes S. costatum growth and mitigates Cd toxicity to S. costatum. 3.2. Effects of OA on Cd accumulation in S. costatum Cd accumulation influences toxic behaviors of Cd to algae (Miao

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and Wang, 2006; García-Ríos et al., 2007; Wang and Wang, 2008). Therefore, after exposure, the intracellular and extracellular Cd contents in S. costatum were determined. The intracellular and extracellular accumulation of Cd was positively correlated with Cd concentration in culture media (p < 0.05, Fig. 2). In the same treatment, the extracellular Cd contents were 18e27 times higher than intracellular contents, which was consistent with the previous finding that accumulation of Cd was mainly localized to cell wall of algae (Chojnacka et al., 2005). Charged groups (such as peptidoglycan, uronic acid, polysaccharide) on cell walls could adsorb and retain Cd, forming precipitates and establishing an efficient barrier to prevent intracellular accumulation of Cd (Chojnacka et al., 2005). However, in comparison to treatment with Cd alone, the OA þ Cd treatment significantly increased intracellular and extracellular accumulation of Cd. For example, the intracellular Cd contents in the OAhCd and OAmCd treatments increased by 1.70 and 1.46 times, respectively; the extracellular Cd accumulation in OAhCd and OAmCd treatments increased by 1.34 and 1.40 times, respectively. These results display an enhanced accumulation of pollutants in algae under OA, which was supposed to increase the toxicity of Cd to S. costatum. However, our results conflicted with this hypothesis (Fig. 1). We therefore considered that OA may somehow induce the detoxification of Cd pollution. As reported, exposure to Cd always induces oxidative damage in algae (Herzi et al., 2013). To repair the oxidative damages, algae have evolved various enzymatic and non-enzymatic antioxidant mechanisms (Kawakami et al., 2006a, 2006b). In order to further investigate the changes of Cd detoxification mechanisms in S. costatum under OA conditions, typical enzymatic and non-enzymatic antioxidant strategies were compared between treatments and the control. We measured (1) SOD activity and MDA content, which reflect intracellular oxidative stress, damage, and the capacity of enzymatic antioxidant system, and (2) GSH and PCs, which are the main constituents of the non-enzymatic antioxidant system in algae and play dominant roles in alleviation of metal-induced oxidative stress. 3.3. Variations of two antioxidant indices under exposure to OA and Cd 3.3.1. Variations of SOD activity and MDA content SOD is an enzyme associated with cellular oxygen metabolism in living organisms and acts as a dominant biomarker of oxidative stress. MDA is an important product of lipid peroxidation and is regarded as a reliable indicator of oxidative injuries in cells (Demiral and Türkan, 2005). Therefore, SOD activity and MDA content reflect intracellular oxidative stress, damage and the capacity of the enzymatic antioxidant system in response to heavy metals. In the present study, compared with the control, treatments with mCd and hCd significantly increased SOD activity in S. costatum (Fig. 3, p < 0.05), and treatment with hCd also significantly elevated MDA content (p < 0.01). These results indicate that Cd induces significant oxidative stress and damage to S. costatum, which might explain the inhibition of Cd on S. costatum growth observed in the present study. Compared with single Cd treatment, exposure to OA þ Cd significantly decreased SOD activity (in treatments with mCd and hCd, p < 0.05) and MDA content (in treatment with hCd, p < 0.05). These results indicate alleviative effects of OA on Cd-induced oxidative toxicity to S. costatum, consistent with the changes of growth indices. 3.3.2. Variations of GSH and PCs contents GSH and PCs are important intracellular non-protein thiols that directly resist Cd pollution in algae, since they bind metals through thiolate coordination and form stable metal complexes (Lee et al., 1996; Pistocchi et al., 2000; Wei et al., 2003; Kawakami et al.,

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Fig. 1. Cell densities (cells/mL) and chl-a contents (mg/L) of Skeletonema costatum during 7-day exposure to different Cd concentrations (panels A and B, respectively). Data are expressed as mean ± SD of three biological replicates. Those with different letters above error bars are significantly different (p < 0.05).

Fig. 2. The intracellular (A) and extracellular Cd accumulation (B) in Skeletonema costatum after exposure for 7 days (mg/kg, dry weight). Data are expressed as mean ± SD of three biological replicates. Those with different letters above error bars are significantly different (p < 0.05).

2006a, 2006b). Contents of GSH and PCs serve as alternative indices to assess the extent of metal stress in phytoplankton and to investigate the underlying toxic mechanisms (Tang et al., 2000; Kawakami et al., 2006a, 2006b). Compared with the control, exposure to Cd alone significantly promoted GSH and PCs synthesis in S. costatum (Fig. 3, p < 0.05). The PCs content also increased significantly with the increasing Cd concentration, indicating that Cd stress induces a large amount of PCs synthesis. However, compared to the mCd group, GSH content decreased but PCs synthesis significantly increased in the hCd group. GSH is the primary precursor of PCs and metal transporter proteins (Ahner et al., 2002). Studies on metal-stressed plants and algae have reported

significant fluctuations in GSH content upon the onset of PCs (Rüegsegger et al., 1990; Rauser et al., 1991; Rijstenbil and Wijnholds, 1996; Kawakami et al., 2006a, 2006b), which supports our findings and indicates that the decreased intracellular GSH levels is caused by substantial PCs synthesis. Compared with the control, exposure to OA significantly elevated GSH content (Fig. 3, p < 0.05). However, treatment with OA þ Cd significantly decreased PCs contents in comparison to the treatment with Cd alone (Fig. 3, p < 0.05). Due to the high affinity between SH-groups and metals, both GSH and PCs are efficient intracellular metal complexing ligands. However, GSH shows more complex responses to metal levels in both laboratory and field

F. Dong et al. / Environmental Pollution 259 (2020) 113850

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Fig. 3. SOD activities (U/mg.prot) and MDA (nmol/mg.prot), GSH (mmol/g.prot), and PC (mmol/g.prot) contents of Skeletonema costatum after exposure to different Cd concentrations for 7 days (panels A, B, C, and D, respectively). Data are expressed as mean ± SD of three biological replicates. Those with different letters above error bars are significantly different (p < 0.05).

experiments due to its multifunctionality (Kawakami et al., 2006a, 2006b). PCs production was related to the aqueous free metal ion concentration (Ahner et al., 1994; Ahner and Morel, 1995; Morelli and Scarano, 2001) and can serve as a bioindicator of metal exposure in plants (Grill et al., 1988; Robinson, 1989). Therefore, in the present study, PCs synthesis at the cellular level objectively reflects the bioavailability and toxicity of free Cd ions. Briefly, compared with sole Cd treatment, exposure to OA þ Cd decreased the activities of SOD, contents of MDA and PCs. These changes explicitly indicate alleviative effects of OA on the Cdinduced oxidative toxicity to S. costatum, which are consistent with the changes of growth indices among treatments. To explore the molecular mechanisms underlying the OA-alleviated Cd toxicity to S. costatum, transcriptomes of S. costatum were sequenced and bioinformatics analyses were conducted to compare molecular differences between treatments and the control. 3.4. Indications from transcriptome analysis 3.4.1. Transcriptome sequencing, qPCR validation and functional annotation Transcriptome sequencing resulted in 1.03 Me2.90 M of clean reads with Q20 values higher than 97.73% for all samples (Supplemental Table S2). Transcriptome assembly revealed that 23283 unigenes were expressed in all treatments and the control (Fig. 4). Compared with the control, 1489, 3251, 4381 unigenes were significantly upregulated, and 2990, 3022, 4402 unigenes were significantly downregulated in treatment with OA, hCd and OAhCd, respectively. Compared with hCd, 1274 unigenes were significantly upregulated and 2267 unigenes were significantly downregulated in treatment with OAhCd. RT-qPCR validation on 20 selected unigenes showed similar changing trends to the results calculated by FPKM (fragments per kilobase of exon per million reads mapped) values (Fig. S3), suggesting the reliability of the transcriptome sequencing results. DEGs between OA and the control enriched five KEGG pathways (Table 2). Compared with the control, treatment with OA significantly upregulated genes in the “ribosome biogenesis”, “RNA transport”, “steroid biosynthesis”, and “glycolysis” categories, but

Fig. 4. The Venn diagram indicates the number of unigenes identified in different treatments and the control.

downregulated “ribosome” pathways. Compared with the control, five KEGG pathways were significantly enriched in the hCd treatment (Table 2). In response to hCd treatment, “homologous recombination (HR)” and “amino sugar and nucleotide sugar metabolism” were significantly downregulated. In addition, many genes involved in “pyruvate metabolism” and “carbon fixation” were dramatically downregulated, while genes encoding photosynthetic antenna proteins were upregulated. Compared with the control, five KEGG pathways were enriched under OA þ hCd conditions. Besides “steroid biosynthesis”, two KEGG pathways were related to “ribosome metabolism” and two

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F. Dong et al. / Environmental Pollution 259 (2020) 113850

Table 2 KEGG enriched pathways under different treatments. C: control; OA: treatment with 1500 ppm CO2; hCd: treatment with 1.2 mg/L Cd; OA þ hCd: treatment with 1500 ppm CO2 and 1.2 mg/L Cd. Comparisons

KEGG

OA vs C

Ribosome Ribosome biogenesis in eukaryotes RNA transport Steroid biosynthesis Glycolysis/Gluconeogenesis Homologous recombination Pyruvate metabolism Photosynthesis - antenna proteins Amino sugar and nucleotide sugar metabolism Carbon fixation in photosynthetic organisms Ribosome biogenesis in eukaryotes Ribosome RNA transport Steroid biosynthesis RNA degradation Ribosome

hCd vs C

OA þ hCd vs C

OA þ hCd vs hCd

were related to “RNA metabolism” (Table 2). In comparison to treatment with hCd, exposure to OA þ hCd only significantly enriched the “ribosome” pathway. 3.4.2. Effects of OA and Cd on DNA, RNA, and protein metabolism HR is required for accurate chromosome segregation during meiotic division and constitutes a key repair and tolerance pathway for complex DNA damage (Heyer et al., 2010). Oxidative stress causes recombinogenic lesions on chromosomes, which may then activate the HR mechanism leading to chromosome rearrangement. This response contributes to the survival of cells afflicted by oxidative DNA damages (Wang et al., 2016). In response to treatment with hCd, genes involved in HR were significantly downregulated, suggesting the possible genotoxicity of Cd to S. costatum, which deserves further investigations. RNA transport from the nucleus to the cytoplasm is fundamental for gene expression (Rodriguez et al., 2004). The ribosome is a large RNAeprotein machine that synthesizes all cellular proteins. Ribosome biogenesis is the major consumer of cellular energy among all life processes and is sensitive to many stresses, including DNA damage, oncogenic stress, low oxygen, oxidative stress, and nutritional stress (Orsolic et al., 2016). In the present study, treatment with hCd reduced gene expression levels of RNA transport and ribosome biogenesis, suggesting that exposure to Cd inhibits RNA transcription and protein biosynthesis. However, under OA conditions, RNA transport and ribosome biogenesis were both upregulated, indicating increased gene expression. Furthermore, amide biosynthesis and peptide metabolism were significantly upregulated under OA conditions. These changes might produce more mRNAs and proteins, which might alleviate the toxic effects of Cd on algal growth. 3.4.3. Transcriptional changes in photosynthesis and energy metabolism The photosynthesis absorbs energy of sunlight and converts CO2 into useful carbohydrates (Collini et al., 2010). This process contributes materials and energy to plant growth and metabolism. In the present study, key genes in photosynthesis including psbM, psbU, psaA, psaB, and psaC were significantly downregulated in the hCd treatment compared with the control, displaying the harmful effects of Cd on photosynthesis in S. costatum. The similar inhibition of Cd on photosynthesis was reported in various alga species (Takamura et al., 1989). However, in the OAhCd treatment, psbA and psbC were still downregulated, but the expression levels of psbM,

psbU, and psaB showed no significant differences or even greater values in comparison to the control or the hCd treatment (Table 3). These results suggest that OA partially rescues S. costatum from photosynthetic inhibition by Cd, which might explain the increased blooming growth. The gluconeogenesis process converts a variety of non-sugar substances into glucose or glycogen, which acts as a bridge in the energy cycle between photosynthesis and tricarboxylic acid (TCA) cycle (Hers and Hue, 2003). Photosynthesis contributes 3C intermediates (glyceraldehyde-3-phosphate) to the biosynthesis of glucose through the gluconeogenesis pathway (Buchanan, 1992; Oosten and Besford, 1996), which is then consumed by the TCA cycle to generate adenosine-triphosphate (ATP). In the present study, hCd treatment showed lower levels of SHDA/SDH1, SHDB/ SDH2/ACADM/acd, MDH1, GAPDH/gapA, and pgm/PGM2 than those in the control, suggesting the inhibition of gluconeogenesis and TCA cycle, which might result from the reduced carbohydrates caused by the downregulated photosynthesis in hCd treatment. In comparison to the control, treatment with OA upregulated genes involved in the gluconeogenesis process (glms/GFPT, TPI/tpiA, GAPDH, gapA, pgm/PGM2, and LDH) and SDHA/SDH1 in the TCA cycle. These results suggest that S. costatum accelerates the accumulation and metabolism of energy species under OA conditions, which not only promotes cell growth and division, but also supplies more energy to resist the harmful effects of Cd. Upregulation of genes in gluconeogenesis and the TCA cycle in comparison to the control support this viewpoint. Pyruvate, the end-product of glycolysis, is derived from additional sources in the cellular cytoplasm, and is ultimately transported into mitochondria as the material for the TCA cycle. In the present study, hCd treatment reduced pyruvate metabolism, probably inhibiting the TCA cycle. Compared with the control, treatment with hCd þ OA showed no significant changes in pyruvate metabolism, demonstrating that OA might alleviate Cd toxicity to pyruvate metabolism. 3.4.4. Changes in antioxidant genes in response to OA and Cd treatments Photosynthesis-antenna proteins are responsible for the capture and conversion of light into chemical energy, which is the first step of photosynthesis (Ben-Shem et al., 2003). In comparison to the control, the increased expression levels of photosynthetic antenna proteins in hCd treatment might absorb more light energy. The extra light energy possibly increases production of reactive oxygen species (ROS) and induces oxidative stress in S. costatum. In addition to the increased SOD activity and elevated GSH and PCs contents, five unigenes involved in the formation and proliferation of the peroxisome, were upregulated in the hCd treatment compared with the control (Table 3). As an important part of the antioxidant system in organisms, the peroxisome contains a large number of oxidases (De Duve and Baudhuin, 1966; Schrader and Fahimi, 2006). The significant upregulation of PEX activated the peroxisomes to resist the Cd toxicity. Interestingly, there were no significant differences in the expression levels of the five PEX unigenes between treatments with OAhCd and hCd, probably because OA has little influence on peroxisome expression in S. costatum. cysK is an important gene regulating the synthesis of cysteine (Nicholson et al., 1995; Tanous et al., 2008), which is a precursor of GSH. In the present study, cysK expression was significantly upregulated in S. costatum under OA conditions compared with the control, which is consistent with the GSH content results, suggesting that OA might activate cysK expression and then directly stimulate GSH production. Glutathione peroxidase catalyzes the conversion of GSH to oxidized glutathione (GSSG), and is therefore an important

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Gluconeogenesis

Citrate cycle (TCA)

Antioxidant process

psbM psbU psaB psbA psbC PEX1 PEX3 PEX5, PXR1 PEX6, PXAAA1 PEX10 gpx cysK SDHA, SDH1 E4.2.1.2A, fumA, fumB SDHB, SDH2 MDH1 glmS, GFPT TPI, tpiA GAPDH, gapA pgm;PGM2 LDH Photosynthesis

0.82 0.03 0.82 0.77 0.19 0.42 1.20 1.01 0.54 0.61 2.01 1.97 1.59 0.79 0.11 0.39 2.34 2.55 6.09 1.89 4.06

1.34E-01 9.59E-01 3.79E-01 1.27E-01 7.05E-01 6.56E-01 5.45E-02 1.98E-01 3.86E-01 3.49E-01 1.02E-03 3.63E-03 3.43E-02 3.42E-01 8.59E-01 5.49E-01 3.10E-03 2.25E-02 2.23E-09 1.45E-02 8.40E-05

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7.06E-04 4.84E-02 9.12E-03 4.68E-06 3.25E-06 1.24E-02 4.48E-02 1.04E-03 4.62E-03 4.56E-02 3.45E-01 6.11E-01 5.76E-01 2.98E-02 2.18E-02 3.86E-04 2.66E-01 5.70E-01 5.30E-04 3.09E-03 5.70E-01 1.81 1.03 2.61 2.55 2.54 1.89 1.19 2.02 1.49 1.16 0.41 0.33 0.35 1.64 1.32 1.80 1.71 0.50 3.31 1.73 0.50

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1.03 0.92 1.32 1.22 1.55 1.52 2.27 2.40 2.06 1.32 1.55 1.38 2.15 1.10 1.38 1.31 1.42 3.08 5.25 1.33 3.50

1.14E-01 6.67E-02 2.20E-01 3.72E-02 3.42E-03 7.12E-02 2.85E-03 4.51E-04 3.93E-04 4.35E-02 5.09E-03 7.84E-02 3.04E-03 1.53E-01 3.75E-02 1.73E-02 5.59E-01 7.25E-03 3.31E-07 4.25E-02 1.65E-03

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0.70 0.09 0.82 1.22 0.92 0.48 0.99 0.35 0.48 0.07 1.36 1.00 1.73 0.52 0.03 0.55 2.65 1.62 1.31 0.51 2.00

1.81E-01 8.32E-01 2.32E-01 1.46E-02 4.42E-02 4.23E-01 8.42E-02 3.96E-01 2.99E-01 8.88E-01 4.20E-02 5.75E-02 6.92E-04 4.39E-01 9.48E-01 1.80E-01 4.65E-01 3.11E-02 3.08E-02 2.64E-01 5.90E-03

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significant regulate Corrected p-value log2FC Corrected p-value log2FC Corrected p-value

significant regulate log2FC

significant regulate

log2FC

Corrected p-value

significant regulate

OA þ hCd vs hCd OA þ hCd vs C hCd vs C OA vs C gene Category

Table 3 Differentially expressed genes (DEGs) involved in significantly enriched pathways (KEGG). C: control; OA: treatment with 1500 ppm CO2; hCd: treatment with 1.2 mg/L Cd; OA þ hCd: treatment with 1500 ppm CO2 and 1.2 mg/L Cd.

F. Dong et al. / Environmental Pollution 259 (2020) 113850

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peroxide-degrading enzyme in many organisms (Toppo et al., 2008). It also contributes to the conversion of toxic peroxides to non-toxic hydroxy compounds. gpx is involved in the mediation of glutathione peroxidase, promotes the decomposition of hydrogen peroxide (H2O2) and protects the structure and function of cell membranes from damages caused by toxic substances (Jones, 2002). In the present study, gpx was significantly upregulated in OA treatments compared with those at the normal CO2 level, further supporting the implication that the GSH pathway contributes to the alleviative effects of OA on Cd toxicity. 3.5. Underlying mechanisms of Cd toxicity mitigation by OA Cd often forms chlorine complexes in oceans. Since chloride ion (Cl) is not sensitive to pH changes, it seems unlikely that differences in Cd speciation and accumulation at different CO2 levels are due to changes in pCO2 (atmospheric CO2 partial pressure) or pH (Millero et al., 2009; Stockdale et al., 2016), suggesting that the effects of OA on metal accumulation might be mediated by biological speciation. Previous studies have achieved similar conclusions when investing the effects of CO2 on metal uptake in cephalopod eggs and embryos (Lacoue-Labarthe et al., 2009), € tze et al., 2014). Crassostrea virginica and Mercenaria mercenaria (Go In the present study, we found that two genes closely related to non-protein thiol groups were significantly upregulated under OA, which would increase the formation of organic ligands-Cd species in algae. Overall, we infer that the influence of OA on Cd toxicity to S. costatum was mainly regulated by physiological process and the labile-Cd species associated non-protein thiol. In treatment with Cd alone, PCs synthesis might be a detoxification mechanism of Cd in S. costatum. Under OA, upregulated genes in gluconeogenesis, glycolysis, TCA cycle, ribosome biogenesis, RNA transport, and peptide metabolism promoted chl-a production, algal growth and resistance to Cd toxicity (Fig. 5). More importantly, the upregulated levels of cysK and gpx (which are closely related to GSH synthesis-metabolism and metal detoxification) enhance algal resistance to direct Cd exposure. Upregulated expression of cysK promotes the biosynthesis of cysteine and GSH, which could (1) directly bind to free Cd irons to produce GSH-metal complexes and/or (2) produce GSH-organic conjugates through the catalysis of GSH S-transferase, which would decrease the intracellular content of free Cd (Fig. 5), alleviate the intracellular oxidative stress and reduce PCs synthesis. In treatments with OA þ Cd, the accumulation of total Cd increased, but intracellular oxidative stress in S. costatum was alleviated, suggesting that level of free Cd ions might decrease, because the toxicity of Cd to algae is closely related to its highly toxic free ionic state (Herzi et al., 2013). It is still unknown whether the alleviative effects of OA on Cd toxicity are a general response in different alga species, which requires further study in future. 4. Conclusions and implications 4.1. Conclusions This study clearly shows the mitigated toxicity of Cd to S. costatum under CO2-driven OA conditions. Treatment with Cd caused significant oxidative stress in S. costatum, which damaged algal cells and inhibited growth. In response to OA condition, S. costatum showed upregulated levels of gluconeogenesis, glycolysis, and TCA, accumulating more organic materials and energy, and promoting RNA metabolism, protein biosynthesis, and the antioxidant process. OA also rescued S. costatum from the inhibition of Cd on photosynthesis and pyruvate metabolism. In particular, OA significantly upregulated cysK and gpx expression, thereby

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F. Dong et al. / Environmental Pollution 259 (2020) 113850

Fig. 5. Mechanisms underlying the mitigation effects of CO2-driven ocean acidification on Cd-induced growth inhibition and oxidative stress in Skeletonema costatum. Straight red arrows indicate significant increase (in the textboxes with grey background) or upregulation (in the textboxes with orange background), and straight blue arrows indicate significant decrease (in the textboxes with grey background) or downregulation (in the textboxes with orange background). Transverse lines indicate no significant variation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

promoting the binding of non-protein thiol compounds to Cd and reducing the toxic effects of oxidative stress (Fig. 5). 4.2. Implications Diatoms contribute up to 40% of marine primary productivity and provide one of the first signals of impacts on ecosystem due to their short response times (Nelson et al., 1995). To the best of our knowledge, this is the first study to demonstrate that OA alleviates Cd toxicity to S. costatum and to identify the underlying molecular mechanisms. However, whether this alleviation is specific to S. costatum or is common in diverse marine algal taxa remains unknown. If this is a common response in diatoms, the combined OA-Cd stress could increase the primary productivity in oceanic ecosystems but would also magnify the accumulation of Cd in the ocean food chain/web. Therefore, the risk assessment of heavy pollution on material transfer in the food web, seafood safety, and ocean carbon circulation should consider influences of climate change, especially when these assessments were conducted in coastal areas, because coastal areas showed more severe heavy metal pollution and higher acidification rate than the open ocean. Notes The authors declare no competing financial interest. CRediT authorship contribution statement Fang Dong: Investigation, Formal analysis, Data curation, Validation, Writing - original draft. Pu Wang: Formal analysis, Data curation, Software. Wei Qian: Validation. Xing Tang: Software,

Data curation. Xiaoshan Zhu: Funding acquisition, Project administration, Supervision, Conceptualization, Resources. Zhenyu Wang: Resources. Zhonghua Cai: Resources. Jiangxin Wang: Funding acquisition, Project administration, Supervision, Conceptualization. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (41573094, 41877352, and 31670116), the Guangdong Innovation Research Team Fund (2014ZT05S078), the Shenzhen Grant Plan for Science & Technology (JCYJ20160308095910917, and JCYJ20170818100339597), and the Guangdong MEPP fund (GDOE[2019]A06). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.113850. References Ahner, B.A., Morel, F.M.M., 1995. Phytochelatin production in marine algae. 2. Induction by various metals. Limnol. Oceanogr. 40, 658e665. Ahner, B.A., Wei, L.P., Oleson, J.R., Ogura, N., 2002. Glutathione and other low molecular weight thiols in marine phytoplankton under metal stress. Mar. Ecol. Prog. Ser. 232, 93e103. Ahner, B.A., Price, N.M., Morel, F.M.M., 1994. Phytochelatin production by marine phytoplankton at low free metal ion concentrations: laboratory studies and field data from Massachusetts Bay. Proc. Natl. Acad. Sci. U. S. A. 91, 8433e8436. ANZECC, 1999. Australian and New Zealand Guidelines for Fresh and Marine Water Quality. I. The Guidelines. Australian and New Zealand Environment and Conservation Council and Agriculture and Resource Management Council of Australia and New Zealand, Canberra.

F. Dong et al. / Environmental Pollution 259 (2020) 113850 Apte, S.C., Bateley, G.E., Szymczak, R., Randall, P., Lee, R., Waite, T.D., 1998. Baseline trace metal concentrations in New South Wales coastal waters. Mar. Freshw. Res. 49 (3), 203e214. Ben-Shem, A., Frolow, F., Nelson, N., 2003. Crystal structure of plant photosystem I. Nature 426 (6976), 630e635. Brembu, T., Jørstad, M., Winge, P., Valle, K.C., Bones, A.M., 2011. Genome-wide profiling of responses to cadmium in the diatom Phaeodactylum tricornutum. Environ. Sci. Technol. 45 (18), 7640e7647. Buchanan, B.B., 1992. Carbon dioxide assimilation in oxygenic and anoxygenic photosynthesis. Phytosnth. Res. 33 (2), 147e162.  recka, H., 2005. Biosorption of Cr3þ, Cd2þ and Cu2þ Chojnacka, K., Chojnacki, A., Go ions by blue-green algae Spirulina sp.: kinetics, equilibrium and the mechanism of the process. Chemosphere 59 (1), 75e84. Collini, E., Wong, C.Y., Wilk, K.E., Curmi, P.M.G., Brumer, P., Scholes, G.D., 2010. Coherently wired light-harvesting in photosynthetic marine algae at ambient temperature. Nature 463 (7281), 644e647. De Duve, C., Baudhuin, P., 1966. Peroxisomes (microbodies and related particles). Physiol. Rev. 46 (2), 323e357. _ 2005. Comparative lipid peroxidation, antioxidant defense Demiral, T., Türkan, I., systems and proline content in roots of two rice cultivars differing in salt tolerance. Environ. Exp. Bot. 53, 247e257. Doney, S.C., Fabry, V.J., Feely, R.A., Kleypas, J.A., 2009. Ocean acidification: the other CO2 problem. Annu. Rev. Mar. Sci. 1 (1), 169e192. Gao, K.S., Xu, J.T., Gao, G., Li, Y.H., Hutchins, D.A., Huang, B.Q., Wang, L., Zheng, Y., Jin, P., Cai, X.N., et al., 2012. Rising CO2 and increased light exposure synergistically reduce marine primary productivity. Nat. Clim. Change 2, 519e523.  zatl, D., MorenoGarcía-Ríos, V., Freile-Pelegrín, Y., Robledo, D., Mendoza-Co S anchez, R., Gold-Bouchot, G., 2007. Cell wall composition affects Cd2þ accumulation and intracellular thiol peptides in marine red algae. Aquat. Toxicol. 81 (1), 65e72. € tze, S., Matoo, O.B., Beniash, E., Saborowski, R., Sokolova, I.M., 2014. Interactive Go effects of CO2 and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria. Aquat. Toxicol. 149 (8), 65e82. Grill, E., Winnacker, E.L., Zeng, M.H., 1988. Occurrence of heavy metal binding phytochelatins in plants growing in a mining refuse area. Experientia 44 (6), 539e540. Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms. 1. Cyclotella nana Hustedt and Detonula confervacea (cleve) gran. Can. J. Microbiol. 8, 229e239. Gu, X.Y., Li, K.Q., Pang, K., Ma, Y.P., Wang, X.L., 2017. Effects of pH on the growth and NH4-N uptake of Skeletonema costatum and Nitzschia closterium. Mar. Pollut. Bull. 124, 946e952. Herzi, F., Jean, N., Zhao, H.Y., Mounier, S., Mabrouk, H.H., Hlaili, A.S., 2013. Copper and cadmium effects on growth and extracellular exudation of the marine toxic dinoflagellate Alexandrium catenella: 3D-fluorescence spectroscopy approach. Chemosphere 93 (6), 1230e1239. Hers, H.G., Hue, L., 2003. Gluconeogenesis and related aspects of glycolysis. Annu. Rev. Biochem. 52 (1), 617e653. Heyer, W.D., Ehmsen, K.T., Liu, J., 2010. Regulation of homologous recombination in eukaryotes. Annu. Rev. Genet. 44 (1), 113e139. Iglesias-Rodriguez, M.D., Halloran, P.R., Rickaby, R.E.M., Hall, I.R., ColmeneroHidalgo, E., Gittins, J.R., Green, D.R.H., Tyrrell, T., Gibbs, S.J., von Dassow, P., et al., 2008. Phytoplankton calcification in a high-CO2 world. Science 320 (5874), 336e340. Jones, D.P., 2002. Redox potential of GSH/GSSG couple: assay and biological significance. Methods Enzymol. 348, 93e112. Kawakami, S.K., Gledhill, M., Achterberg, E.P., 2006a. Effects of metal combinations on the production of phytochelatins and glutathione by the marine diatom Phaeodactylum tricornutum. Biometals 19, 51e60. Kawakami, S.K., Gledhill, M., Achterberg, E.P., 2006b. Production of phytochelatins and glutathione by marine phytoplankton in response to metal stress. J. Phycol. 42, 975e989. €nsli, F., Teyssie , J.L., Markich, S., Jeffree, R., Lacoue-Labarthe, T., Martin, S., Obeha Bustamante, P., 2009. Effects of increased pCO2 and temperature on trace element (Ag, Cd and Zn) bioaccumulation in the eggs of the common cuttlefish, Sepia officinalis. Biogeosciences 6 (11), 2561e2573. Lee, J.G., Ahner, B.A., Morel, F.M.M., 1996. Export of cadmium and phytochelatin by the marine diatom Thalassiosira weissflogii. Environ. Sci. Technol. 30, 1814e1821. Leite Figueiredo, D.A., Branco, P.C., Dos Santos, D.A., Emerenciano, A.K., Iunes, R.S., Shimada Borges, J.C., Machado Cunha da Silva, J.R., 2016. Ocean acidification affects parameters of immune response and extracellular pH in tropical sea urchins Lytechinus variegatus and Echinometra luccunter. Aquat. Toxicol. 180, 84e94. Liu, F.J., Li, S.X., Zheng, F.Y., Huang, X.G., 2016. Risk assessment of excessive CO2 emission on diatom heavy metal consumption. Sci. Total Environ. 566e567, 1349e1354. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2DDCt method. Methods 25, 402e408. Miao, A.J., Wang, W.X., 2006. Cadmium toxicity to two marine phytoplankton under different nutrient conditions. Aquat. Toxicol. 78 (2), 114e126. Millero, F.J., Woosley, R., Ditrolio, B., Waters, J., 2009. Effects of ocean acidification on the speciation of metals in seawater. Oceanography 22, 72e85. Morelli, E., Scarano, G., 2001. Synthesis and stability of phytochelatins induced by cadmium and lead in the marine diatom Phaeodactylum tricornutum. Mar.

9

Environ. Res. 52, 383e395. Nardi, A., Mincarelli, L.F., Benedetti, M., Fattorini, D., d’Errico, G., Regoli, F., 2017. Indirect effects of climate changes on cadmium bioavailability and biological effects in the Mediterranean mussel Mytilus galloprovincialis. Chemosphere 169, 493e502. Nardi, A., Benedetti, M., Fattorini, D., Regoli, F., 2018. Oxidative and interactive challenge of cadmium and ocean acidification on the smooth scallop Flexopecten glaber. Aquat. Toxicol. 196, 53e60. Ndungu, K., 2012. Model predictions of copper speciation in coastal water compared to measurements by analytical voltammetry. Environ. Sci. Technol. 46 (14), 7644e7652. guer, P., Brzezinski, M.A., Leynaert, A., Que guiner, B., 1995. ProNelson, D.M., Tre duction and dissolution of biogenic silica in the ocean: revised global estimates, comparison with regional data and relationship to biogenic sedimentation. Glob. Biogeochem. Cycles 9 (3), 359e372. Nicholson, M.L., Gaasenbeek, M., Laudenbach, D.E., 1995. Two enzymes together capable of cysteine biosynthesis are encoded on a cyanobacterial plasmid. Mol. Gen. Genet. 247 (5), 623e632. , T., Branica, M., 1994. Bioaccumulation rate of Cd Odẑak, N., Martin ci c, D., Zvonarie and Pb in Mytilus galloprovincialis foot and gills. Mar. Chem. 46 (1e2), 119e131. Oosten, J.J., Besford, R.T., 1996. Acclimation of photosynthesis to elevated CO2 through feedback regulation of gene expression: climate of opinion. Phytosnth. Res. 48 (3), 353e365. Orr, J.C., Fabry, V.J., Aumont, O., Bopp, L., Doney, S.C., Feely, R.A., Ganadesikan, A., Gruber, N., Ishida, A., Joos, F., et al., 2005. Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature 437 (7059), 681e686. Orsoli c, I., Jurada, D., Pullen, N., Oren, M., Eliopoulos, A.G., Volarevic, S., 2016. The relationship between the nucleolus and cancer: current evidence and emerging paradigms. Semin. Cancer Biol. 37e38, 36e50. Pan, K., Wang, W.X., 2012. Trace metal contamination in estuarine and coastal environments in China. Sci. Total Environ. 421e422, 3e16. Pistocchi, R., Mormile, M.A., Guerrini, F., Isani, G., Boni, L., 2000. Increased production of extra- and intracellular metal-ligands in phytoplankton exposed to copper and cadmium. J. Appl. Phycol. 12, 469e477. Rabosky, D.L., Sorhannus, U., 2009. Diversity dynamics of marine planktonic diatoms across the Cenozoic. Nature 457 (7226), 183e186. Rauser, W.E., Schupp, R., Rennenberg, H., 1991. Cysteine, gamma-glutamylcysteine, and glutathione levels in maize seedlings: distribution and translocation in normal and cadmium-exposed plants. Plant Physiol. 97, 128e138. Riebesell, U., Fabry, V.J., Hansson, L., Gattuso, J.P., 2010. Guide to best practices for ocean acidification research and data reporting. Publ. Off. Eur. Union, Oceanogr. (Washington D. C.) 22 (4), 260. Rijstenbil, J.W., Gerringa, L.J.A., 2002. Interactions of algal ligands, metal complexation and availability, and cell responses of the diatom Ditylum brightwellii with a gradual increase in copper. Aquat. Toxicol. 56 (2), 115e131. Rijstenbil, J.W., Wijnholds, J.A., 1996. HPLC analysis of nonprotein thiols in planktonic diatoms: pool size, redox state and response to copper and cadmium exposure. Mar. Biol. 127, 45e54. Roberts, D.A., Birchenough, S.N., Lewis, C., Sanders, M.B., Bolam, T., Sheahan, D., 2013. Ocean acidification increases the toxicity of contaminated sediments. Glob. Change Biol. 19 (2), 340e351. Rodriguez, M.S., Dargemont, C., Stutz, F., 2004. Nuclear export of RNA. Biol. Cell (Paris) 96 (8), 639e655. Robinson, N.J., 1989. Algal metallothioneins: secondary metabolites and proteins. J. Appl. Phycol. 1 (1), 5e18. Rüegsegger, A., Schmutz, D., Brunold, C., 1990. Regulation of glutathione synthesis by cadmium in Pisum sativum L. Plant Physiol. 93 (4), 1579e1584. Schrader, M., Fahimi, H.D., 2006. Peroxisomes and oxidative stress. Biochim. Biophys. Acta 1763 (12), 1755e1766. Shi, D.L., Wang, W.X., 2004. Understanding the differences in Cd and Zn bioaccumulation and subcellular storage among different populations of marine clams. Environ. Sci. Technol. 38 (2), 449e456. Simpson, W.R., 1981. A critical review of cadmium in the marine environment. Prog. Oceanogr. 10 (1), 1e70. Stockdale, A., Tipping, E., Lofts, S., Mortimer, R.J.G., 2016. The effect of ocean acidification on organic and inorganic speciation of trace metals. Environ. Sci. Technol. 50 (4), 1906e1913. ~ udo-Wilhelmy, S.A., 2012. Smail, E.A., Webb, E.A., Franks, R.P., Bruland, K.W., San Status of metal contamination in surface waters of the coastal ocean off Los Angeles, California since the implementation of the clean water. Act. Environ. Sci. Technol. 46 (8), 4304e4311. Tang, D., Hung, C., Warnken, K.W., Santschi, P.H., 2000. The distribution of biogenic thiols in surface waters of Galveston Bay. Limnol. Oceanogr. 45 (6), 1289e1297. Tanous, C., Soutourina, O., Raynal, B., Hullo, M.F., Mervelet, P., Gilles, A.M., Noirot, P., Danchin, A., England, P., Martin-Verstraete, I., 2008. The CymR regulator in complex with the enzyme CysK controls cysteine metabolism in Bacillus subtilis. J. Biol. Chem. 283 (51), 35551e35560. Takamura, N., Kasai, F., Watanabe, M.M., 1989. Effects of Cu, Cd and Zn on photosynthesis of freshwater benthic algae. J. Appl. Phycol. 1 (1), 39e52. Toppo, S., Vanin, S., Bosello, V., Tosatto, S.C., 2008. Evolutionary and structural insights into the multifaceted glutathione peroxidase (Gpx) superfamily. Antioxidants Redox Signal. 10 (9), 1501e1514. Villanueva, S., Alfonoso, V., 1992. Heavy metals in the coastal zone from Gulf of Mexico and Mexican Caribbean: a review (Metales pesados en la zona costera

10

F. Dong et al. / Environmental Pollution 259 (2020) 113850

xico y Caribe Mexicano: una revisio n). Red Rev. Cient Ame r del Golfo de Me Latin Carib Esp Port 8, 47e61. Waldbusser, G.G., Salisbury, J.E., 2014. Ocean acidification in the coastal zone from an organism’s perspective: multiple system parameters, frequency domains, and habitats. Annu. Rev. Mar. Sci. 6 (6), 221e247. Wang, C., Yang, J.H., Lu, D.Z., Fan, Y.S., Zhao, M.R., Li, Z.Y., 2016. Oxidative stressrelated DNA damage and homologous recombination repairing induced by N,N-dimethylformamide. J. Appl. Phycol. 36 (7), 936e945. Wang, M.J., Wang, W.X., 2011. Cadmium sensitivity, uptake, subcellular distribution and thiol induction in a marine diatom: recovery from cadmium exposure. Aquat. Toxicol. 101 (2), 377e386. Wang, M.J., Wang, W.X., 2008. Cadmium toxicity in a marine diatom as predicted by the cellular metal sensitive fraction. Environ. Sci. Technol. 42 (3), 940e946. Wei, L.P., Donat, J.R., Fones, G., Ahner, B.A., 2003. Interactions between Cd, Cu, and

Zn influence particulate phytochelatin concentrations in marine phytoplankton: laboratory results and preliminary field data. Environ. Sci. Technol. 37 (16), 3609e3618. Wu, Y.P., Gao, K.S., Riebesell, U., 2010. CO2-induced seawater acidification affects physiological performance of the marine diatom Phaeodactylum tricornutum. Biogeosciences 7 (9), 2915e2923. Zhai, W.D., Zhao, H.D., Zheng, N., Xu, Y., 2012. Coastal acidification in summer bottom oxygen-depleted waters in northwestern-northern Bohai Sea from June to August in 2011. Chin. Sci. Bull. 57 (9), 1062e1068. Zhang, S.F., Yuan, C.J., Ying, C., Chen, X.H., Li, D.X., Liu, J.L., Lin, L., Wang, D.Z., 2016. Comparative transcriptomic analysis reveals novel insights into the adaptive response of Skeletonema costatumto changing ambient phosphorus. Front. Microbiol. 7 (6307), 1476.