CO2-driven ocean acidification weakens mussel shell defense capacity and induces global molecular compensatory responses

Chemosphere 243 (2020) 125415

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CO2-driven ocean acidification weakens mussel shell defense capacity and induces global molecular compensatory responses Xinguo Zhao a, b, c, Yu Han c, Bijuan Chen a, b, Bin Xia a, b, Keming Qu a, Guangxu Liu c, * a

Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, PR China Laboratory for Marine Ecology and Environment Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, PR China c College of Animal Sciences, Zhejiang University, Hangzhou, 310058, PR China b

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


OA will damage shell structure, and reduce shell strength and shell closure strength.
OA will lead to extracellular acidosis and Ca2þ deficiency.
OA will significantly alter gene expression profile in mantle tissue.
OA will weaken mussels’ shell defense capacity, and thus reduce their fitness.
The findings of this study have significant ecological and economic implications.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 September 2019 Received in revised form 6 November 2019 Accepted 18 November 2019 Available online 19 November 2019

Oceanic uptake of atmospheric CO2 is reducing seawater pH and shifting carbonate chemistry within, a process termed as ocean acidification (OA). Marine mussels are a family of ecologically and economically significant bivalves that are widely distributed along coastal areas worldwide. Studies have demonstrated that OA greatly disrupts mussels’ physiological functions. However, the underlying molecular responses (e.g., whether there were any molecular compensation mechanisms) and the extent to which OA affects mussel shell defense capacity remain largely unknown. In this study, the thick shell mussels Mytilus coruscus were exposed to the ambient pH (8.1) or one of two lowered pH levels (7.8 and 7.4) for 40 days. The results suggest that future OA will damage shell structure and weaken shell strength and shell closure strength, ultimately reducing mussel shell defense capacity. In addition, future OA will also disrupt haemolymph pH and Ca2þ homeostasis, leading to extracellular acidosis and Ca2þ deficiency. Mantle transcriptome analyses indicate that mussels will adopt a series of molecular compensatory responses to mitigate these adverse effects; nevertheless, weakened shell defense capacity will increase mussels’ susceptibility to predators, parasites and pathogens, and thereby reduce their fitness. Overall, the findings of this study have significant ecological and economic implications, and will enhance our understanding of the future of the mussel aquaculture industry and coastal ecosystems. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: Jim Lazorchak Keywords: Ocean acidification Mussel Calcification Acid-base status Defense capacity Mantle transcriptome sequencing

1. Introduction * Corresponding author. E-mail address: [email protected] (G. Liu). https://doi.org/10.1016/j.chemosphere.2019.125415 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

Anthropogenic activities (e.g., fossil fuel burning) emit large quantities of carbon dioxide (CO2) into the atmosphere.

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Approximately one third of the CO2 is eventually absorbed by the global ocean (Sabine et al., 2004), leading to surface seawater pH reduction and carbonate chemistry shifts, a phenomenon known as ocean acidification (OA) (Caldeira and Wickett, 2003). Since the Industrial Revolution, surface seawater pH has already decreased by 0.1 units, from approximately 8.21 to the current level of about 8.10 (Caldeira and Wickett, 2003). According to the Representative Concentration Pathway (RCP) 8.5 scenario of the Intergovernmental Panel on Climate Change (IPCC), the average surface seawater pH will further decrease by 0.3e0.4 and 0.7e0.8 units by the end of the 21st and 23rd centuries, respectively (IPCC et al., 2014). Previous studies have suggested that OA poses a threat to a wide variety of marine organisms, especially calcifying organisms (Hofmann et al., 2010; Kerr, 2010). However, the responses of marine organisms to OA have been found to be species-specific and to vary with life stage (Kroeker et al., 2010, 2013; Ries et al., 2009). For example, OA leads to decreases in metabolic rates in the blood clam Tegillarca granosa (Zhao et al., 2017b), wherease it leads to increases in metabolic rates in the blue mussel Mytilus edulis (Thomsen and Melzner, 2010). Similarly, the Ca2þ content of the red king crab Paralithodes camtschaticus is increased at the larvae stage, but remains unchanged at the juvenile stage upon OA exposure (Long et al., 2013a, 2013b). Regarding the species-specific and life stage variations, the current understanding of biological responses to OA cannot be simply applied to other species and/or life stages. Therefore, more research is needed to increase our understanding of the effects of OA on marine organisms and ecosystems. Marine mussels are a family of ecologically and economically significant bivalve species that are widely distributed along coastal areas worldwide. By aggregating into beds, marine mussels create habitats for other organisms, and are thus recognized as important marine ecosystem engineers (Borthagaray and Carranza, 2007). Many of them are important aquaculture species and have been human food for thousands of years (Ponce Oliva et al., 2019). According to a report by the Food and Agriculture Organization of the United Nations (FAO), the mussel aquaculture industry was worth approximately 4.0 billion USD in 2016 (FAO yearbook, 2018). Therefore, increasing attention has been paid to understanding how OA affects marine mussels. Current evidence demonstrates that OA will exert significant negative effects on various physiological processes in marine mussels, especially leading to the reduction in calcification and even to shell damage (Asplund et al., 2014; Fitzer et al., 2015; Li et al., 2015a; Melzner et al., 2011; Sadler et al., 2018; Thomsen et al., 2010, 2013). However, previous studies have mainly focused on the physiological effects of OA, leaving the underlying molecular responses (e.g., whether there were any molecular compensation mechanisms) largely overlooked. In addition, previous studies have mostly been performed with the blue mussel M. edulis. Considering the species-specific variations in responses to OA mentioned above, whether findings in other species also apply to mussel species such as M. coruscus needs to be investigated. Moreover, although shell defense capacity is crucial for the survival of individual mussels, the extent to which OA affects this capacity remains largely unknown. The present study was aimed to minimize these knowledge gaps. It was performed with the thick shell mussel M. coruscus, an ecologically and economically important species that is widely distributed along the coastal areas of China, Korea and Japan (Qi et al., 2019; Shang et al., 2019). Following 40-day exposure of M. coruscus to ambient condition (pH 8.1) or one of two acidified conditions (pH 7.8 and 7.4), the shell structure, shell strength and relative size of the posterior adductor muscle (a proxy for shell closure strength) were measured to investigate potential alterations in shell defense capacity upon OA exposure. In addition, the haemolymph pH and Ca2þ concentration were also determined to

show the physiological mechanisms underlying the observed alterations in shell defense capacity. Importantly, owing to mantle’s critical roles in shell formation and growth, gene expression profiles of mantle tissue were investigated through transcriptome sequencing to uncover the underlying molecular responses to OA.

2. Materials and methods 2.1. Animal collection and acclimation Thick shell mussels M. coruscus (shell length of 21.91 ± 2.34 mm) were collected from an intertidal site on Dongtou Island, Wenzhou, China (121.22 E, 27.75 N), where the natural seawater pH ranges from 8.0 to 8.2. The mussels were directly transported to the Qingjiang Station of Zhejiang Mariculture Research Institute, gently cleaned of epibionts without damaging the shells, and acclimated for two weeks in filtered and UV-irradiated natural seawater with continuous aeration prior to the experiments. The seawater pH was controlled at 8.10 ± 0.06, the temperature was controlled at 23 ± 0.4  C, and the salinity was controlled at 23 ± 0.5‰. The mussels were fed twice daily with the microalgae Platymonas subcordiformis at a rate of ~5% dry tissue weight, according to O’Donnell et al. (2013). Excess food and feces were removed daily through seawater changes, in which seawater was removed by siphoning, followed by refilling with seawater pre-equilibrated to the desired pH values.

2.2. Experimental design and seawater parameters Following acclimation, the mussels were randomly assigned to an ambient pH (8.1) group and two lowered pH (7.8 and 7.4) groups. The ambient seawater pH level (8.1) served as the control, while the lowered pH levels of 7.8 and 7.4 were set to mimic the oceanic surface pH conditions projected for the years 2100 and 2300, respectively (IPCC et al., 2014). The seawater pH levels were achieved and maintained by continuously bubbling with CO2 gas mixture, obtained by mixing CO2-free air and pure CO2 gas at controlled flow rates, according to Zhao et al. (2017a). All pH levels were conducted with 10 replicate chambers that contained 10 individuals each and filled with approximately 10 L of filtered and UV-irradiated natural seawater with the desired pH value (Table S1). The mussels were fed with the microalgae P. subcordiformis as described above. The seawater was maintained at 23  C using temperature regulators, and was changed daily. Seawater chemistry parameters, including the pH, salinity, total alkalinity (TA) and carbonate system parameters, were monitored daily to ensure that no substantial fluctuations occurred throughout the experiment period. Seawater pH levels were measured using a Sartorius PB-10 pH meter (Sartorius, Germany) calibrated with standard NBS buffers. Salinity was determined with a Multi 3410 conductivity meter (WTW, Germany). TA was assessed through potentiometric titration with an SM-Titrino 702 automatic titrator system (Metrohm, Switzerland). The carbonate system parameters, including CO2 partial pressure (pCO2), dissolved inorganic carbon (DIC), and aragonite saturation state (Uara) and calcite saturation state (Ucal), were calculated using the open-source program CO2SYS (Pierrot et al., 2006) with the pH, salinity, temperature, and TA values and the established constants (Dickson, 1990; Dickson and Millero, 1987; Mehrbach et al., 1973). The experiment lasted for 40 days. During the entire experimental period, no mussel mortality was observed. The seawater parameters of the experimental trials are summarized in Table S2.

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2.3. Shell integrity and structure analysis Following the 40-day exposure, the mussels were carefully dissected without damaging the shell surfaces. The shells were carefully cleaned and dried at room temperature. The internal and external shell surfaces were checked and photographed using a digital camera. To assess the severity of corrosion, the percentages of corroded areas for both the internal and external shell surfaces were quantified by image analysis using the open-access software ImageJ version 1.46r (http://imagej.nih.gov/ij/). The curvature of the shell was neglected in image analysis, according to Melzner et al. (2011). The severity of the external shell surface corrosion was further assessed by quantifying the occurrences of each type of corrosion, including periostracum discoloration, periostracum breakage and lifting, and prismatic layer dissolution. For microstructure analysis, target shell regions (Fig. S1) were fragmented along defined trajectories. Sections were mounted on pedestal stubs, coated with gold-palladium, and observed by a scanning electron microscopy (SEM; SU8010, Hitachi, Japan). 2.4. Shell strength measurement The left and right valves of five mussels from each replicate chamber (i.e., fifty mussels per pH level) were randomly chosen, and then shell strength measurement was performed following Burnett and Belk (2018). Briefly, a universal material-testing machine (AGS-J, Shimadzu, Japan) was used to determine shell strength. Each shell valve was placed between the horizontal jaws of the testing machine, as shown in Fig. S2. The valve was compressed at a constant loading rate of 10 mm/min until failure occurred, and the applied force was continuously recorded by a computer. Shell strength was measured as the force required to break the valve and is expressed in Newtons (N). In other words, shell strength is the maximum force that the shell valve could endure. For each mussel, the mean of the strengths of the left and right valves was recorded as the shell strength. 2.5. Adductor muscle size analysis Owing to the significant positive linear correlation between the relative size of the posterior adductor muscle and the shell closure strength (Christensen et al., 2012; Thomas, 1976). The relative size of the posterior adductor muscle was used a proxy for the shell closure strength of the mussel, and was determined following Christensen et al. (2012). Briefly, the posterior adductor muscle was severed with a scalpel along the plane of the shell edge, and its diameter was measured using a digital Vernier calliper with a precision of 0.01 mm. The relative size of the posterior adductor muscle was calculated as the ratio of the posterior adductor muscle diameter to the shell length. 2.6. Determination of haemolymph pH and Ca2þ concentration Mussel haemolymph samples were collected by pericardial puncture with gas-tight disposable syringes, and directly transferred to 1.5 mL sterile tubes. Haemolymph samples of five mussels from each replicate chamber were pooled in equal proportions and used as a biological replicate (i.e., ten biological replicates were tested per pH level). The haemolymph samples were centrifuged at 5000g for 5 min. For haemolymph sampling and handling, the temperature was controlled at 23  C (i.e., the seawater temperature during mussel incubation period). The supernatants (cell-free haemolymph) were used to measure the pH values and Ca2þ concentrations following Asplund et al. (2014). The pH values were determined with a pH microelectrode (WTW, Germany). The Ca2þ

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concentrations were measured using a WFX-130A flame atomic spectrophotometer (Beijing Rayleigh Analytical Instruments Co, Ltd, China) according to Shi et al. (2016). 2.7. Mantle transcriptome sequencing, analysis and validation One mussel from each replicate chamber was randomly selected and dissected (for a total of ten mussels per pH level). The whole mantle tissue of the mussel was isolated and immediately frozen in liquid nitrogen. Total RNA was extracted using TRIzol Reagent (Invitrogen, 15,596,018) following the manufacturer’s protocol. The RNA sample was further treated with DNase I (Invitrogen, 18047019) to remove DNA contamination. The quality of the RNA sample was checked by 1.0% formaldehyde-denatured agarose gel electrophoresis. The concentration of the RNA sample was quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific). For each pH level, the ten mantle RNA samples were pooled in equal proportions to obtain a mixture. The standard Illumina protocol was followed for cDNA synthesis and library construction. Sequencing was performed by Shanghai OE Biotech Co, Ltd. (Shanghai, China) on an Illumina HiSeq2000 platform. The raw sequence reads were deposited in the Sequence Read Archive (SRA) at the National Center for Biotechnology Information (NCBI) with accession number PRJNA543748. The raw sequence reads were trimmed by removing adapters and low quality sequences. The quality of sequence reads was verified using the software FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). De novo transcriptome assembly was carried out using the software Trinity (Grabherr et al., 2011). The longest transcript of each locus was defined as an unigene and was used for downstream analyses. The unigenes were annotated by alignment to the Non-redundant (Nr), Swiss-Prot, Clusters of Orthologous Groups of proteins/ euKaryotic Orthologous Groups (KOG/COG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases using BlastX (E value < 105). The expression level of each unigene was calculated using the fragments per kilo bases per million mapped reads (FPKM) method (Trapnell et al., 2010). Differentially expressed unigenes (DEGs) between the control (pH 8.1) and acidified (pH 7.4) conditions were identified using the DESeq R package (Anders and Huber, 2010). Only unigenes with an absolute fold change ˃ 2 and a p value < 0.05 were considered true DEGs. Finally, the DEGs were subjected to GO enrichment and KEGG pathway analyses as described by Altermann and Klaenhammer (2005) and Ashburner et al. (2000) to determine the overall impacts of the experimental manipulation. The p values were adjusted by the Benjamini-Hochberg false discovery rate (FDR). An FDRadjusted p value < 0.01 was selected as the threshold to identify the most representative enriched KEGG and GO terms. In addition to GO and KEGG terms/pathways, the DEGs were also classified into three specific categories, including the ion and acid-base regulation, calcification, and adductor muscle categories, to reveal the specific responses of M. coruscus to OA, according to previous studies (Li et al., 2016a; Liao et al., 2015; Moya et al., 2016; Nagasawa, 2013; Zhang et al., 2012). To validate the transcriptome expression data, the expression levels of fifteen representative DEGs were determined through real-time quantitative PCR (RT-qPCR) using the same RNA samples as those used for transcriptome sequencing. The degree of agreement between the transcriptome expression data and the RT-qPCR data was estimated through linear regression, according to Li et al. (2016a). RT-qPCR was performed on a CFX96™ Real-Time System (Bio-Rad). The relative expression levels of the fifteen representative DEGs were calculated using the 2DDCt method (Livak and Schmittgen, 2001) with the 18S rRNA gene as the internal reference (Hüning et al., 2013; Zhao et al., 2017a). Detailed information

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of the fifteen representative DEGs and the gene-specific primers is listed in Table S3.

Person’s r ¼ 0.79, R2 ¼ 0.61, p < 0.0001) (Fig. 2c), indicating that internal and external shell surface corrosion occurred concurrently.

2.8. Statistical analysis

3.2. Shell strength and the relative size of the adductor muscle

One-way ANOVA was performed to show the effects of OA on the percentage of corroded shell surface area, shell strength, relative size of the posterior adductor muscle, haemolymph pH and haemolymph Ca2þ concentration. Post-hoc Tukey’s multiple tests were conducted to compare differences among groups. The normality assumption and homogeneity of variance were verified prior to analysis using Shapiro-Wilk’s test and Bartlett’s test, respectively. A Chi-square test was performed to compare the occurrences of the different types of external shell surface corrosion at the three pH levels. A p value < 0.05 was considered to indicate statistically significance. The Chi-square test was conducted using the software GraphPad Prism 5, while the other statistical analyses were performed with OriginPro 8.0.

OA significantly weakened shell strength (one-way ANOVA, df ¼ 2, p < 0.01). Shell strength was significantly decreased from 86 N in the control group to 56 N and 50 N in the pH 7.8 and pH 7.4 groups, respectively (Fig. 3a). OA also significantly decreased the relative size of the posterior adductor muscle (one-way ANOVA, df ¼ 2, p < 0.01). The relative size was significantly decreased from 0.154 at pH 8.1 to 0.147 and 0.133 at pH 7.8 and 7.4, respectively (Fig. 3b).

3. Results 3.1. Shell integrity and structure For mussels under ambient condition (pH 8.1), the internal shell surface was intact with a typical glossy appearance, and the external shell surface showed only periostracum discoloration (Fig. 1a). In contrast, for mussels under acidified conditions (pH 7.8 and 7.4), the typically glossy internal shell surface had become dull and white (Fig. 1a). In addition, the periostracum was broken, lifted or even absent extending from the umbo region to the shell margin on the external surface (Fig. 1a). SEM showed that the aragonite tablets of the nacreous layer and the calcite crystals of the prismatic layer on the normal internal shell surface had uniform structural orientations, while those of mussels under acidified conditions appeared to be disorientated or dissolved (Fig. 1b). The intact periostracum was purple-black to black, while the discolored periostracum had become beige (Fig. 1a). SEM illustrated that the intact region of periostracum had a smooth appearance, while the discolored region had become rough, suggesting slight damage to the periostracum surface microstructure (Fig. 1b). Additionally, SEM revealed that periostracum breakage and lifting ultimately surfaced the prismatic layer, and led to dissolution of calcite crystals on external surfaces (Fig. 1b). OA significantly increased the corroded area of the internal shell surface (one-way ANOVA, df ¼ 2, p < 0.01). The corroded area of inner shell surface (%) was significantly increased from 0% in the control group to 14% and 43% in the pH 7.8 and pH 7.4 groups, respectively (Fig. 2a). Similarly, the corroded area of the external shell surface was significantly affected by OA (one-way ANOVA, df ¼ 2, p < 0.01); the corroded area (%) was approximately 26% at pH 8.1, while it had increased to 31% and 47% at pH 7.8 and pH 7.4, respectively (Fig. 2b). Additionally, the severity of external shell surface damage (measured as the occurrence of each type of external shell surface corrosion) was also siginifcantly increased by OA (c2 ¼ 189.9, df ¼ 4, p < 0.0001). As shown in Fig. 2d, most (58%) of the analyzed mussels at pH 7.8 showed prismatic layer dissolution (the most severe type of external shell surface damage), and the others (42%) showed periostracum breakage and lifting; furthermore, 100% of the analyzed mussels at pH 7.4 showed prismatic layer dissolution. In contrast, the mussels at pH 8.1 showed only periostracum discoloration (the least severe type of external shell surface damage). These results suggest that the severity of shell damage increased with increasing OA. Moreover, there was a signifcant positive linear correlation between inner and external shell surface corrosion (linear fitting, y ¼ 0.46x þ 25.92,

3.3. Haemolymph pH and Ca2þ concentration The haemolymph pH level was significantly decreased by OA (one-way ANOVA, df ¼ 2, p < 0.01). It was significantly reduced from 7.52 in the control group to nearly 7.38 and 7.24 in the pH 7.8 and pH 7.4 groups, respectively (Fig. 4a). Similarly, the haemolymph Ca2þ concentration was significantly reduced by OA (one-way ANOVA, df ¼ 2, p < 0.01). In control mussels, the haemolymph Ca2þ concentration was maintained at 297 mg/L, while in mussels exposed to pH 8.1 and 7.8, the concentration was reduced to 283 mg/L and 278 mg/L, respectively (Fig. 4b). 3.4. Mantle transcriptomic responses to OA Transcriptome sequencing of mantle tissues yielded 36, 539, 606 clean reads with a Q30 of 90.12% and 47, 196, 236 clean reads with a Q30 of 90.29% for the control (pH 8.1) and acidified (pH 7.4) groups, respectively (Table S4). De novo assembly generated 205,936 transcripts with an average length of 1078 bp and an N50 of 2058 bp, which represented 109,823 unigenes with an average length of 700 bp and an N50 of 1237 bp (Table S5). These results suggested that the results of transcriptome sequencing and de novo assembly for the mantle tissues of M. coruscus were of high quality and were reliable. The mantle gene expression profiles of M. coruscus were significantly altered by OA. A total of 2448 unigenes were found to be differentially expressed in acidified (pH 7.4) condition relative to control condition (pH 8.1), including 1624 upregulated DEGs and 824 down-regulated DEGs (Fig. S3). Linear fitting indicated that there was a strong positive linear correlation between the expression data generated by transcriptome sequencing analysis and that generated by RT-qPCR (R2 ¼ 0.9233, p < 0.01), validating the reliability and accuracy of the transcriptome sequencing data (Fig. S4). GO enrichment analyses revealed that among the up-regulated DEGs, a total of 15 GO terms were significantly enriched, including three terms in the “Cellular Component” category, seven terms in the “Molecular Function” category, and five terms in the “Biological Process” category (Table 1). In contrast, only eight GO terms were significantly enriched among the down-regulated DEGs, including two terms in the “Cellular Component” category, two terms in the “Molecular Function” category, and four terms in the “Biological Process” category (Table 1). KEGG enrichment analyses suggested that seven pathways were significantly enriched among the upregulated DEGs, including “focal adhesion”, “apoptosis”, “tight junction”, “ECM-receptor interaction”, “NF-kappa b signaling pathway”, “cardiac muscle contraction”, and “toll-like receptor signaling pathway” (Table 2). However, no pathway was significantly enriched among the down-regulated DEGs (Table 2). Among the DEGs, 26 were potentially involved in ion and acid-base regulation, including 23 up-regulated DEGs and three down-regulated DEGs (Table 3 and Table S6). Additionally, a total of 35 DEGs

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Fig. 1. Inner and external shell surfaces images of M. coruscus after 40 days treatment. (a) Representative stereomicroscopic images; (b) Representative SEM images. Capital letters indicate where the SEM images were taken. A: the normal nacreous layer on the internal shell surface; B: the corroded nacreous layer on the internal shell surface; C: the normal prismatic layer on the internal shell surface; D: the corroded prismatic layer on the internal shell surface; E: the normal region of periostracum on the external shell surface; F: the discoloration region of periostracum on the external shell surface; G: the breakage and lifting region of periostracum on the external shell surface; H: the periostracum loss and prismatic layer dissolution region on the external shell surface.

putatively participating in calcification were identified; 24 and 11 of them were up- and down-regulated in acidified condition (pH 7.4), respectively (Table 3 and Table S7). Finally, a total of 28 DEGs potentially involved in adductor muscle function were identified; all of them (28) were up-regulated (Table 3 and Table S8).

4. Discussion 4.1. OA impaired shell formation and maintenance capacity, and eventually damaged shell structure The results revealed that OA simultaneously corroded the internal and external shell surfaces of M. coruscus, and that the severity increased with increasing seawater acidity. The internal shell surfaces lost their typical glossy appearances and became white, while the external shell surfaces displayed obvious breakage and lifting of the periostracum and even prismatic layer dissolution extending from the umbo region to the shell margin. SEM analysis

suggested that microstructures of the periostracum, prismatic layer and nacreous layer were damaged by OA. Notably, the external shell surfaces of M. coruscus were also slightly damaged at the umbo region under ambient condition, displaying periostracum discolouration (a change from purple-black/black to beige), which commonly occurs among field mussels. A previous study revealed that periostracum discolouration is caused by the mutual friction of individuals in a wave-swept environment (Thomsen et al., 2010). The mutual friction of mussels in the field therefore hampers the protective function of the periostracum and acts as an accelerator for external shell surface damage. Nevertheless, this study suggests that OA markedly impairs the shell formation and maintenance capacity of M. coruscus and ultimately damages shell structure. These findings are consistent with those reported in other marine bivalve species, such as the blood clam T. granosa (Zhao et al., 2017b), the striped venus clam Chamelea gallina (Bressan et al., 2014), the Mediterranean mussel M. galloprovincialis (Gazeau et al., 2014) and the blue mussel M. edulis (Gazeau et al., 2007;

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Fig. 2. Effects of ocean acidification on shell intergity of M. corucus. (a) Corroded area of inner shell surface (%); (b) Corroded area of external shell surface (%); (c) Linear positive correlation between the corroded area of inner shell surface (%) and corroded area of external shell surface (%) of mussels at pH 7.8 and 7.4; (d) Occurrence (%) of each type of external shell surface corrosions (50 samples per pH level, c2 ¼ 189.9, df ¼ 4, p < 0.0001). Means not sharing the same superscript are significantly different (n ¼ 10, Tukey’s HSD, p < 0.05). Error bar represents SD.

Fig. 3. Effects of ocean acidification on (a) shell strength and (b) the relative size of the posterior adductor muscle of M. coruscus. Means not sharing the same superscript are significantly different (n ¼ 10, Tukey’s HSD, p < 0.05). Error bar represents SD.

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Fig. 4. Effects of ocean acidification on (a) haemolymph pH and (b) haemolymph Ca2þ concentration of M. coruscus. Means not sharing the same superscript are significantly different (n ¼ 10, Tukey’s HSD, p < 0.05). Error bar represents SD.

Table 1 GO enrichment table (pH 7.4 vs pH 8.1). CC: Cellular Component. MF: Molecular Function. BP: Biological Process. Up: terms enriched in the set of up-regulated DEGs in pH 7.4. Down: terms enriched in the set of down-regulated DEGs in pH 7.4. regulation

category

GO ID

GO term

No. of DEGs

adjusted p-value

Up

CC

GO:0016021 GO:0005576 GO:0005938 GO:0005509 GO:0005516 GO:0004713 GO:0004197 GO:0002020 GO:0008061 GO:0051015 GO:0007155 GO:0007275 GO:0043123 GO:0051592 GO:0048085 GO:0005576 GO:0005578 GO:0003714 GO:0008134 GO:0006030 GO:0051260 GO:0045892 GO:0006310

Integral component of membrane Extracellular region Cell cortex Calcium ion binding Calmodulin binding Protein tyrosine kinase activity Cysteine-type endopeptidase activity Protease binding Chitin binding Actin filament binding Cell adhesion Multicellular organismal development Positive regulation of I-kappa b kinase/NF-kappa b signaling Response to calcium ion Adult chitin-containing cuticle pigmentation Extracellular region Proteinaceous extracellular matrix Transcription corepressor activity Transcription factor binding Chitin metabolic process Protein homooligomerization Negative regulation of transcription, DNA-templated DNA recombination

126 42 12 98 15 12 12 10 10 7 20 15 12 8 2 19 7 4 4 6 5 5 5

8.06E-03 3.91E-06 1.03E-03 6.45E-16 1.18E-04 1.11E-04 5.99E-03 3.14E-04 1.30E-02 6.55E-03 9.48E-03 6.94E-03 1.91E-03 5.79E-05 3.40E-03 1.36E-07 1.84E-05 1.55E-03 9.02E-03 1.63E-04 2.22E-03 3.25E-03 4.42E-03

MF

BP

Down

CC MF BP

Table 2 KEGG enrichment table (pH 7.4 vs pH 8.1). Up: pathways enriched in the set of up-regulated DEGs in pH 7.4. Down: pathways enriched in the set of down-regulated DEGs in pH 7.4. regulation

KEGG ID

KEGG term

No. of DEGs

adjusted p-value

Up

ko04510 ko04210 ko04530 ko04512 ko04064 ko04260 ko04620

Focal adhesion Apoptosis Tight junction ECM-receptor interaction NF-kappa b signaling pathway Cardiac muscle contraction Toll-like receptor signaling pathway

29 16 13 12 11 9 9

1.52E-05 1.20E-03 2.93E-03 1.34E-02 4.69E-03 6.52E-03 9.90E-03

Thomsen et al., 2010). It is highly likely that shell formation and maintenance capacity impairment and therefore shell structural damage are widespread responses of marine bivalves to OA.

4.2. OA disrupted haemolymph pH and Ca2þ homeostasis In the present study, the haemolymph pH levels of M. coruscus were markedly reduced by OA, indicating disruption of

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Table 3 Summary of the genes responsive to acidified condition (pH 7.4). category

gene family

No. of unigenes

regulation

Ion- and acid-base regulation

Ca2þ/Mg2þ-permeable cation channels Ca2þ-modulated nonselective cation channel L-type voltage-dependent Ca2þ channel Calmodulin Calcium-binding protein Carbonic anhydrase Sulfate/bicarbonate/oxalate exchanger SAT-1 Electroneutral sodium bicarbonate exchanger 1 Naþ/Kþ ATPase, beta subunit Potassium channel subfamily K member 18 Sodium/glucose cotransporter Sodium/myo-inositol cotransporter Solute carrier organic anion transporter family member 2B1 Asparagine-rich protein Calponin-like protein Caltractin Cartilage matrix protein Chitin synthase Calmodulin Calcium-binding protein Carbonic anhydrase Lectin Mucin Nacre protein Perlucin Perlwapin Pif Tyrosinase Shell matrix protein Dynein Filamin-A Filamin-C Myosin Paramyosin Titin Transgelin

5 1 3 5 3 1 1 1 1 1 2 1 1 1 1 1 1 2 5 3 1 9 3 1 2 1 1 2 1 1 1 2 9 1 13 1

up up up up up down down down up up up up up down up up up up up up down 6 up, 3 down 2 up, 1 down down down down down up down up up up up up up up

Calcification

Adductor muscle

extracellular acid-base homeostasis and therefore induction of extracellular acidosis upon OA exposure. Similar effects have also been detected in other marine bivalve species, such as the Pacific oyster Crassostrea gigas (Lannig et al., 2010), the blood clam T. granosa (Zhao et al., 2017b), the blue mussel M. edulis (Mangan et al., 2017; Ramesh et al., 2017; Thomsen et al., 2013) and the Mediterranean mussel M. galloprovincialis (Michaelidis et al., 2005). Notably, a linear relationship between haemolymph pH and seawater pH has even been found in the blue mussels M. edulis (Thomsen et al., 2013). It seems that extracellular acidosis is also a widespread response of marine bivalves to OA. Previous studies have reported that extracellular acidosis subsequently dissolves CaCO3 crystals, leading to the observed corrosion on inner shell surfaces (Lindinger et al., 1984; Michaelidis et al., 2005). In addition to HCO- 3, dissolution of shell CaCO3 crystals also releases free Ca2þ, which is predicted to increase the Ca2þ level in haemolymph (Michaelidis et al., 2005). However, the haemolymph Ca2þ concentrations of M. coruscus were found to be significantly reduced in this study with OA. Similar effects have been observed in the blood clam T. granosa (Zhao et al., 2017b). A paradox between theoretical speculation and empirical observation has also been revealed in studies on the blue mussel M. edulis (Asplund et al., 2014; Thomsen et al., 2010) and the Pacific oyster C. gigas (Lannig et al., 2010), in which OA caused obvious dissolution of shell CaCO3 crystals without significantly changing haemolymph Ca2þ concentrations. This paradox may be attributable to reduced Ca2þ uptake from food and seawater, because it has been demonstrated that the filter-feeding behavior of M. coruscus (Sui et al., 2016; Wang et al., 2015), T. granosa (Zhao et al., 2017b), M. edulis (Stapp

et al., 2017; Sun et al., 2016) and C. gigas (Ginger et al., 2013) is significantly suppressed by OA. In addition, such inference is supported by studies on the pearl oyster Pinctada fucata, in which OA conversely elevates filter-feeding behavior (Liu and He, 2012) while increasing haemolymph Ca2þ concentration (Li et al., 2015b) and shell CaCO3 crystal dissolution (Li et al., 2016a; Liu et al., 2017). We therefore suggest that OA inhibits Ca2þ uptake and thereby decreases the haemolymph Ca2þ concentration in M. coruscus.

4.3. OA induced potential mantle tissue injury, but also triggered compensatory responses Genes involved in the “apoptosis” KEGG pathway were significantly up-regulated, suggesting that OA induced mantle cell death in M. coruscus. This finding was supported by GO enrichment analyses, in which the “cysteine-type endopeptidase activity” and “protease binding” terms in the “Molecular Function” category were significantly enriched for the up-regulated DEGs, and the “protein homooligomerization” and “DNA recombination” terms in the “Biological Process” category were significantly enriched for the down-regulated DEGs. Endopeptidase and protease are critical enzymes catalyzing protein degradation, which is an important process of cell death (Lecker et al., 2006). Therefore, the upregulation of genes involved in “cysteine-type endopeptidase activity” and “protease binding” indicates that OA triggered protein degradation and thereby cell death. Additionally, the downregulation of genes involved in “protein homooligomerization” suggests that OA inhibited the formation of protein quaternary structures, leading to protein dysfucntion and ultimately cell death.

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Furthermore, the down-regulation of genes related to “DNA recombination” indicates that OA hindered homologous recombination repair of DNA damage, which in turn increased the accumulation of DNA damage and eventually resulted in cell death (Zada et al., 2019). These data suggest that OA induced mantle tissue injury in M. coruscus. Notably, GO enrichment analyses revealed the up-regulation of genes associated with the “integral component of membrane”, “extracellular region” and “cell cortex” terms in the “Cellular Component” category, and with the “cell adhesion” term in the “Biological Process” category under OA conditions. These findings are in agreement with the results of KEGG analyses, which revealed that the “focal adhesion”, “tight junction” and “ECM-receptor interaction” pathways were significantly enriched for the upregulated genes. Up-regulation of genes related to cell adhesion has also been detected in the pearl oyster P. fucata (Li et al., 2016a). These results indicate the enhancement of cell-matrix adhesion, which plays essential roles in important biological processes including cell motility, cell proliferation, cell differentiation, cell survival and cell communication. Additionally, GO enrichment analyses also revealed the up-regulation of genes involved in “multicellular organismal development” term in the “Biological Process” category, suggesting positive effects of OA on cell proliferation and differentiation. Furthermore, the GO terms “transcription corepressor activity” and “transcription factor binding” in the “Molecular Function” category, and “negative regulation of transcription, DNA-templated” in the “Biological Process” category were significantly enriched for the down-regulated DEGs, indicating the promotion of DNA transcription and protein synthesis by OA, with positive effects on cell proliferation. Therefore, these data suggest that there were compensatory responses against mantle tissue injury. Together, the findings of this study reveal that OA induced mantle tissue injury and compensatory responses. In addition to calcification, the mantle also plays essential roles in reproduction and sensory processes (Gosling, 2015). Structural damage in mantle tissue could lead to global physiological and behavioral alterations. We therefore suggest that future work should endeavor to determine the extent to which OA affects mantle tissue. 4.4. The molecular mechanism and compensatory responses underlying shell damage Genes encoding Ca2þ channels, including Ca2þ/Mg2þ-permeable cation channel, Ca2þ-modulated nonselective cation channel and Ltype voltage-dependent Ca2þ channel, were significantly upregulated, suggesting that extracellular Ca2þ influx was evoked under OA conditions. Additionally, genes involved in Ca2þ regulation, including calmodulin and calcium-binding protein, were also up-regulated, indicating that much of the internalized Ca2þ was temporarily stored and released upon use. This conclusion is supported by the results of GO enrichment analyses, in which the terms “calcium ion binding” and “calmodulin binding” in the “Molecular Function” category, and “response to calcium ion” in the “Biological Process” category were significantly enriched for the up-regulated DEGs. For bivalves, intracellular Ca2þ not only participates in intracellular signal transduction but also drives the intracellular formation of amorphous CaCO3, which is the precursor material for shell formation (Li et al., 2016b; Mount et al., 2004; Xiang et al., 2014). Therefore, Ca2þ influx might promote the formation of amorphous CaCO3. This possibility is consistent with the findings of a previous study (DeCarlo et al., 2018), showing that the coral Pocillopora damicornis increases the transport of Ca2þ into calcification sites and thereby increases CaCO3 formation to resist OA. Interestingly, the expression levels of genes regulating CaCO3

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formation, including calponin-like protein, caltractin, cartilage matrix protein and calmodulin, were significantly up-regulated, indicating increased intracellular amorphous CaCO3 formation. We thus conclude that M. coruscus enhanced intracellular amorphous CaCO3 formation to alleviate the negative effects on shell formation. Generally, the formed amorphous CaCO3 is transported onto the growing shell and eventually transforms into CaCO3 crystals (i.e., crystalline aragonite and calcite) (Radha et al., 2010; Weiss et al., 2002). However, the gene expression levels of shell matrix proteins regulating CaCO3 crystal formation, growth and orientation (Nagasawa, 2013; Suzuki et al., 2009), including asparagine-rich protein, nacre protein, perlucin, perlwapin, pif and shell matrix protein, were significantly down-regulated, suggesting that the transformation of amorphous CaCO3 and subsequent orientation of CaCO3 crystals were hampered by OA. This conclusion is in accordance with our SEM data and with the findings of previous studies on the blue mussels M. edulis (Fitzer et al., 2014a, 2014b, 2016) and the pearl oyster P. fucata (Li et al., 2016a), showing disorientated aragonite tablets and calcite crystals on internal shell surfaces. Therefore, the down-regulation of these shell matrix proteins might be a possible explanation for the observed corrosion on the internal shell surface. This study revealed significant up-regulation of tyrosinase in M. coruscus under OA conditions. Similar effects have also been observed in the blue mussel M. edulis (Hüning et al., 2013) and the Mediterranean pteropod Heliconoides inflatus (Moya et al., 2016). Previous studies have also demonstrated the distinctive roles of tyrosinase in forming the shell periostracum (Nagasawa, 2013). Thus, the up-regulation of tyrosinase could be a compensatory response to attenuate periostracum damage. We also observed significant up-regulation of chitin synthase, which catalyzes chitin synthesis, consistent with the results of previous studies on the blue mussel M. edulis (Hüning et al., 2013) and the Chilean scallop Argopecten purpuratus (Ramajo et al., 2016). GO enrichment analyses showed that “chitin binding” in the “Molecular Function” category and “adult chitin-containing cuticle pigmentation” in the “Biological Process” category were significantly enriched among the up-regulated DEGs, while “chitin metabolic process” in the “Biological Process” category was significantly enriched among the down-regulated DEGs. These results suggest the promotion of chitin synthesis and the inhibition of chitin degradation in OAexposed M. coruscus. Chitin plays critical roles in creating the organic framework of mollusk shells (Schonitzer and Weiss, 2007). Therefore, alterations in these processes and functions might be adaptive responses of M. coruscus to cope with OA. It should be noted that among the shell matrix proteins identified in this study, the respective transcripts encoding lectin and mucin (two families of shell matrix proteins) (Moya et al., 2016; Nagasawa, 2013), were differentially regulated. Some were downregulated, while others were up-regulated by OA. In addition to palying important roles in the calcification process, these two protein families also participate in cell-cell adhesion, immune responses and signal transduction (Drickamer, 1999; Strous and Dekker, 1992). It is therefore likely that different isoforms of the two protein families played different roles in the responses of M. coruscus to OA and thus were differentially regulated. 4.5. Energy-consuming compensatory responses were mounted to maintain haemolymph ion and acid-base homeostasis The gene expression levels of Naþ/Kþ-ATPase, Kþ channel, Naþrelated cotransporters and solute carrier organic anion transporter were significantly up-regulated, which might have facilitated the establishment of a transmembrane electrochemical potential

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gradient and in turn promoted the transmembrane transport of a variety of ions and small molecules. This result indicates a potential compensatory response of M. coruscus to maintain ion and acidbase homeostasis. This conclusion is in agreement with previous studies on the pearl oyster P. fucata (Li et al., 2016a) and the yesso scallop Patinopecten yessoensis (Liao et al., 2019) reporting a similar compensation mechanism. However, the gene expression level of carbonic anhydrase was significantly down-regulated by OA. Down-regulation of carbonic anhydrase has also been observed in the blue mussel M. edulis under OA conditions at 22  C and 25  C (Li et al., 2015a). Carbonic anhydrase is an enzyme that catalyzes the inter conversion between CO2 and HCO- 3 and thus plays important roles in acid-base regulation (Wang et al., 2017). Down-regulation of carbonic anhydrase should therefore lead to decreased interconversion between CO2 and HCO- 3, thereby weakening the acidbase regulation capacity. This possibility is consistent with the down-regulation of the two HCO- 3 exchangers (i.e., sulfate/bicarbonate/oxalate exchanger sat-1 and electroneutral sodium bicarbonate exchanger 1). We therefore conclude that M. coruscus unsuccessfully compensated for OA-induced impairment of acidbase regulation capacity and that ineffective compensation eventually resulted in extracellular acidosis. Notably, Naþ/Kþ-ATPase must consume ATP energy to drive active transport of Naþ and Kþ, implying that the compensatory response of M. coruscus is an energy-consuming process. Although it was not investigated in this study, M. coruscus metabolism has been found to be suppressed by OA (Shang et al., 2018; Sui et al., 2016; Wang et al., 2015). It is widely accepted that temporal metabolic depression is a main adaptive strategy of marine invertebrates to survive abiotic environmental stress (Guppy, 2004). We therefore propose that M. coruscus adopted metabolic depression to decrease overall energy demand and to provide sufficient energy only for essential biological processes to enable survival under OA conditions. However, the up-regulation of Naþ/Kþ-ATPase indicates that additional energy was required for ion and acid-base regulation, which are essential biological processes. Under these circumstances, relatively less energy was available for other energy-consuming biological processes. This scenario suggests that there were trade-offs among energy-consuming biological processes in M. coruscus, such as ion/acid-base status regulation and calcification. Therefore, the ability to differentially reallocate energy among essential biological processes may determine the sensitivity of marine mussel species to OA (Pan et al., 2015; €rtner, 2013). Wittmann and Po 4.6. OA weakened shell defense capacity by damaging shell structure, and decreasing shell strength and closure strength

snail Nucella lapillus (Sherker et al., 2017). Generally, bivalves close their shells tightly upon encountering predators and/or other threats. Therefore, shell closure strength (i.e., the force required to open the shell) has been adopted as another indicator of bivalves’ shell defense capacity (Aoki et al., 2010). In this study, shell closure strength was estimated using the relative size of the posterior adductor muscle as a proxy, because there is a significant positive linear correlation between these two parameters (Christensen et al., 2012; Thomas, 1976). For mussels exposed to pH 7.8 and 7.4, the relative size of the posterior adductor muscle was significantly decreased to approximately 95% and 86% of that in control mussels, respectively. We therefore deduced that the shell closure strength of M. coruscus was significantly decreased under OA conditions. This finding is consistent with that of a previous study on the same mussel species M. coruscus (Sui et al., 2017). The weak shell closure strength should greatly decrease mussels’ resistance to parasites, such as the pea crab Pinnotheres sinensis (Sun et al., 2006); and to shell-entering predators, such as the starfish Asterias rubens (Reimer and Tedengren, 1996). These results suggest that along with damaging the shell structure, OA also weakened the shell strength and shell closure strength of M. coruscus. In other words, the projected OA will dramatically decrease mussels’ shell defense capacity and thereby elevate their vulnerability to predators, parasites and pathogens. Fortunately, the transcriptome analyses revealed that the GO terms “protein tyrosine kinase activity” in the “Molecular Function” category, “positive regulation of I-kappa b kinase/NF-kappa b signaling” in the “Biological Process” category, and the KEGG pathways “NF-kappa b signaling pathway” and “Toll-like receptor signaling pathway” were significantly enriched among the upregulated DEGs. Given the essential roles of these functions, processes and pathways in immunity (Liu et al., 2016), these findings indicate the elevation of immune responses against pathogens. Additionally, the GO term “actin filament binding” in the “Molecular Function” category, and the KEGG pathway “cardiac muscle contraction” were significantly enriched among the up-regulated DEGs, suggesting positive effects of OA on adductor muscle function and shell closure strength. These results are consistent with the up-regulation of adductor muscle-related genes, including dynein, filamin-A, filamin-C, myosin, paramyosin, titin and transgelin. As discussed above, there were also compensatory responses related to shell formation and maintenance. The mounting of these compensatory responses indicated that the negative effects of OA on shell defense capacity had been partially mitigated; otherwise, they would have been more severe. 5. Conclusion

Since shell structure was markedly damaged, it was reasonable to hypothesize that shell strength should be weakened by OA. To test this hypothesis and further elucidate the impacts of OA on shell defense capacity, shell strength was analyzed in this study. We found that for mussels exposed to pH 7.8 and 7.4, shell strength was significantly reduced to approximately 65% and 58% of that of control mussels, respectively. These results suggest that the shell strength of M. coruscus was indeed weakened by OA. Similar effects have also been observed in other mussel species, such as the California mussel M. californianus (Gaylord et al., 2011) and the blue mussel M. edulis (Li et al., 2015a; Mackenzie et al., 2014). Hence, a decrease in shell strength seems to be a common response of marine mussels to OA. A fragile shell structure and reduced mechanical strength should tremendously elevate mussels’ vulnerability to pathogens, such as the bacterium Vibrio tubiashii (Asplund et al., 2014); to shell-crushing predators, such as the crab Carcinus maenas (Edgell et al., 2008); and to shell-drilling predators, such as the

In conclusion, this study demonstrates that OA weakens mussel shell defense capacity. Although mussels will adopt a series of molecular compensatory responses to resist OA, weakened shell defense capacity will increase mussels’ susceptibility to predators, parasites and pathogens, and thereby reduce their fitness. Mussels are important aquaculture bivalves and marine ecosystem engineers. The findings of this study thus have significant ecological and economic implications. Overall, this study sheds light on the effects of OA on mussel defense and the underlying molecular responses. It will also improve our understanding of the future of the mussel aquaculture industry and coastal ecosystems. Author contribution X.Z. and G.L. conceived and designed this study. X.Z. and Y.H. performed the whole experiments and collected the data. All

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authors contributed to data analysis and interpretation. X.Z. wrote the manuscript, B.C., B.X., K.Q. and G.L. revised the manuscript. G.L. provided oversight of the project. All authors gave final approval for publication. Declaration of competing interest The authors declare no competing interests. Acknowledgements This work was funded by National Key R & D Program of China (No. 2018YFD0900603), National Natural Science Foundation of China (No. 31672634) and China Postdoctoral Science Foundation (No. 2017M622323). The authors greatly thank Yichen Wang and Xingguan Lin for their assistance with animal incubation. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125415. References Altermann, E., Klaenhammer, T.R., 2005. PathwayVoyager: pathway mapping using the Kyoto Encyclopedia of genes and Genomes (KEGG) database. BMC Genomics 6, 60. Anders, S., Huber, W., 2010. Differential expression analysis for sequence count data. Genome Biol. 11, R106. Aoki, H., Ishikawa, T., Fujiwara, T., Atsumi, T., Nishikawa, H., Okamoto, C., et al., 2010. Utility of shell-closing strength as the indicator of good health in breeding and culture management of Japanese pearl oyster Pinctada fucata. Aquaculture 308, S115eS118. Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., et al., 2000. Gene Ontology: tool for the unification of biology. Nat. Genet. 25, 25. Asplund, M.E., Baden, S.P., Russ, S., Ellis, R.P., Gong, N., Hernroth, B.E., 2014. Ocean acidification and host-pathogen interactions: blue mussels, Mytilus edulis, encountering Vibrio tubiashii. Environ. Microbiol. 16, 1029e1039. Borthagaray, A.I., Carranza, A., 2007. Mussels as ecosystem engineers: their contribution to species richness in a rocky littoral community. Acta Oecol. 31, 243e250. Bressan, M., Chinellato, A., Munari, M., Matozzo, V., Manci, A., Mar ceta, T., et al., 2014. Does seawater acidification affect survival, growth and shell integrity in bivalve juveniles? Mar. Environ. Res. 99, 136e148. Burnett, N.P., Belk, A., 2018. Compressive strength of Mytilus californianus shell is time-dependent and can influence the potential foraging strategies of predators. Mar. Biol. 165, 42. Caldeira, K., Wickett, M.E., 2003. Oceanography: anthropogenic carbon and ocean pH. Nature 425, 365-365. Christensen, H.T., Dolmer, P., Petersen, J., Tørring, D., 2012. Comparative study of predatory responses in blue mussels (Mytilus edulis L.) produced in suspended long line cultures or collected from natural bottom mussel beds. Helgol. Mar. Res. 66, 1e9. DeCarlo, T.M., Comeau, S., Cornwall, C.E., McCulloch, M.T., 2018. Coral resistance to ocean acidification linked to increased calcium at the site of calcification. Proc. R. Soc. Biol. Sci. 285, 20180564. Dickson, A.G., 1990. Thermodynamics of the dissociation of boric-acid in potassiumchloride solutions form 273.15 K to 318.15 K. J. Chem. Eng. Data 22, 113e127. Dickson, A.G., Millero, F.J., 1987. A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media. Deep-Sea Res. 34, 1733e1743. Drickamer, K., 1999. C-type lectin-like domains. Curr. Opin. Struct. Biol. 9, 585e590. Edgell, T.C., Brazeau, C., Grahame, J.W., Rochette, R., 2008. Simultaneous defense against shell entry and shell crushing in a snail faced with the predatory shorecrab Carcinus maenas. Mar. Ecol. Prog. Ser. 371, 191e198. FAO yearbook, F.A.O., 2018. Fishery and Aquaculture Statistics 2016. Fitzer, S.C., Chung, P., Maccherozzi, F., Dhesi, S.S., Kamenos, N.A., Phoenix, V.R., et al., 2016. Biomineral shell formation under ocean acidification: a shift from order to chaos. Sci. Rep. 6, 21076. Fitzer, S.C., Cusack, M., Phoenix, V.R., Kamenos, N.A., 2014a. Ocean acidification reduces the crystallographic control in juvenile mussel shells. J. Struct. Biol. 188, 39e45. Fitzer, S.C., Phoenix, V.R., Cusack, M., Kamenos, N.A., 2014b. Ocean acidification impacts mussel control on biomineralisation. Sci. Rep. 4, 6218. Fitzer, S.C., Zhu, W., Tanner, K.E., Phoenix, V.R., Kamenos, N.A., Cusack, M., 2015. Ocean acidification alters the material properties of Mytilus edulis shells. J. R. Soc. Interface 12, 20141227. Gaylord, B., Hill, T.M., Sanford, E., Lenz, E.A., Jacobs, L.A., Sato, K.N., et al., 2011.

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