Effects of Copper Exposure on Oxidative Stress, Apoptosis, Endoplasmic Reticulum Stress, Autophagy and Immune Response in Different Tissues of Chinese Mitten Crab (Eriocheir sinensis)

High concentrations of copper (Cu2+) pose a great threat to aquatic animals. However, the mechanisms underlying the response of crustaceans to Cu2+ exposure have not been well studied. Therefore, we investigated the alterations of physiological and molecular parameters in Chinese mitten crab (Eriocheir sinensis) after Cu2+ exposure. The crabs were exposed to 0 (control), 0.04, 0.18, and 0.70 mg/L of Cu2+ for 5 days, and the hemolymph, hepatopancreas, gills, and muscle were sampled. The results showed that Cu2+ exposure decreased the antioxidative capacity and promoted lipid peroxidation in different tissues. Apoptosis was induced by Cu2+ exposure, and this activation was associated with the mitochondrial and ERK pathways in the hepatopancreas. ER stress-related genes were upregulated in the hepatopancreas but downregulated in the gills at higher doses of Cu2+. Autophagy was considerably influenced by Cu2+ exposure, as evidenced by the upregulation of autophagy-related genes in the hepatopancreas and gills. Cu2+ exposure also caused an immune response in different tissues, especially the hepatopancreas, where the TLR2-MyD88-NF-κB pathway was initiated to mediate the inflammatory response. Overall, our results suggest that Cu2+ exposure induces oxidative stress, ER stress, apoptosis, autophagy, and immune response in E. sinensis, and the toxicity may be implicated following the activation of the ERK, AMPK, and TLR2-MyD88-NF-κB pathways.


Introduction
Copper (Cu) is an essential metal element for all living organisms and mainly exists in Cu 2+ and Cu + states. It is involved in a variety of physiological functions, such as electron transport, mitochondrial function, and free radical scavenging [1]. When present in excess, however, Cu becomes toxic and causes damage to cellular components [2]. It is also classified as a priority environmental pollutant [3]. Cu can accumulate in aquatic systems from both natural (e.g., erosion of rocks and soils, geological deposition) and anthropogenic sources (e.g., industrial, mining and agricultural activities, sewage discharge) [3,4]. In aquaculture, copper sulfate (CuSO 4 ) has been extensively used as a therapeutic agent to control skin lesions and gill diseases caused by parasites and pathogenic bacteria [5]. It is further used globally as an algicide to control harmful cyanobacterial blooms in freshwater [6]. The extensive use of Cu 2+ may lead to its short-term and/or repeated accumulation in aquatic environments. High concentrations of Cu 2+ (up to 100 mg/L) have further been detected in various aquatic ecosystems [7].
After 5 days of exposure, eight crabs from each tank were sampled randomly, and the hemolymph, hepatopancreas, gills, and muscle were immediately collected after anesthetization with an ice bath. Tissues from four crabs were mixed into one sample (six samples in total). The hemolymph was centrifuged (4000× g for 10 min at 4 • C) to obtain the supernatant. All samples were stored at −80 • C for gene expression and biochemistry analyses. The use of the crabs in the experiment was approved by the Freshwater Fisheries Research Center, and all experimental procedures were performed according to the Animal Care Guidelines.

Quantitative Real-Time PCR Analysis
Total RNA from the hepatopancreas, gills, and muscle was isolated using the RNAiso Plus reagent (TaKaRa, Beijing, China) according to the manufacturer's instructions. The quality and quantity of total RNA were evaluated using OD 260 , the ratio of OD 260 /OD 280 , and agarose gel electrophoresis. The isolated RNA was used to synthesize cDNA via reverse transcription PCR using the PrimeScript™ RT reagent (TaKaRa, No. RR047). In brief, the RNA (1 µg) was mixed with gDNA Eraser at 42 • C for 2 min to remove the genomic DNA. The mixture was then reacted with PrimeScript RT Enzyme Mix I (1 µL), RT Primer Mix (1 µL), 5× PrimeScript Buffer 2 (4 µL), and RNase-Free dH 2 O (4 µL) for 15 min at 37 • C and 5 s at 85 • C.
The mRNA levels of the target genes were measured by quantitative real-time PCR (qPCR) on a CFX96 Real-Time PCR instrument (Bio-Rad, Hercules, CA, USA). During the qPCR amplification process, cDNA (2 µL), TB Green Premix Ex Taq II (TaKaRa; 12.5 µL), forward and reverse specific primers (1 µL), and RNase-free water (8.5 µL) were mixed. The mixture was incubated for 30 s at 95 • C and subjected to 40 cycles at 95 • C for 5 s and 59-61 • C for 1 min. The expression of the target genes was analyzed using the 2 −∆∆Cq method [26]. The primers used are listed in Table S1. The ubiquitin-conjugating enzyme E2b (UBE) and β-actin genes were used as internal references to normalize the quantification cycle (Cq) values [27].

Integrated Biomarker Response Analysis
The integrated biomarker response (IBR) analysis for the oxidative stress parameters of different tissues was conducted using the method described by Sanchez, et al. [28]. The control group (without Cu 2+ ) was used as the reference condition. The IBR v2 value per concentration is the sum of the absolute values of the biomarker deviation index (A). The reference deviation of each biomarker is represented by the A value. In the star plot, the values above and below zero reflect the induction and reduction of the biomarker, respectively.

Statistical Analysis
All statistical analyses were performed using SPSS 24.0 (SPSS, Chicago, IL, USA). The results are expressed as the mean ± standard error of the mean (SEM). The normal distribution and heterogeneity of variance were evaluated using the Shapiro-Wilk and Bartlett tests, respectively. For comparisons among different groups, a one-way analysis of variance (ANOVA) was performed, followed by an LSD post hoc test in cases of equal variance or the Kruskal-Wallis test for unequal variance. Differences were considered statistically significant at p < 0.05 among the different groups.

Alterations in the Redox State
There was a linear decrease in the levels of T-AOC, SOD, and GST and an increase in MDA after treatment with different concentrations of Cu 2+ in the hemolymph ( Figure 1A-E). Compared to the control group, the decreases in T-AOC, SOD, and GST were statistically significant in the 0.70 mg/L Cu 2+ -exposed group (p < 0.05; Figure 1A-C), while the increase in MDA was statistically significant in the 0.18 and 0.70 mg/L Cu 2+ -exposed groups (p < 0.05; Figure 1D). GSH was not influenced by Cu 2+ exposure in the hemolymph (p > 0.05; Figure 1E). In the hepatopancreas, the level of T-AOC decreased with increasing Cu 2+ concentrations, and the lowest value was observed after exposure to 0.70 mg/L of Cu 2+ (p < 0.05; Figure 1G). Similarly, the level of GSH underwent a dose-dependent decrease, which was statistically significant in the 0.70 mg/L Cu 2+ -exposed group (p < 0.05; Figure 1H). SOD activity and MDA content did not exhibit significant alterations in the hepatopancreas among the different Cu 2+ -exposed groups (p > 0.05; Figure 1F,I).
In the gills, SOD activity showed a downward trend after Cu 2+ exposure and was strongly decreased in crabs exposed to 0.70 mg/L of Cu 2+ (p < 0.05; Figure 1J). Conversely, the MDA content exhibited a rising tendency and was enhanced in crabs exposed to 0.70 mg/L of Cu 2+ (p < 0.05; Figure 1M). The levels of T-AOC and GSH showed a slight but non-significant alteration in the gills among the different groups (p > 0.05; Figure 1K,L).
In the muscle, exposure to 0.70 mg/L of Cu 2+ markedly decreased SOD activity and enhanced MDA formation (p < 0.05; Figure 1N,R) but did not influence other parameters (p > 0.05; Figure 1O,P).
To compare the differences among the tissues and groups exposed to different concentrations of Cu 2+ , four biomarkers related to the redox state were standardized and depicted in a star plot ( Figure 2). The IBRv2 index increased with increasing Cu 2+ concentrations and exhibited dose-dependent toxicity. Among the different tissues, the following order of average IBR v2 values was observed: hemolymph (5.64) > hepatopancreas (4.87) > gills (4.77) > muscle (4.53). In addition, after exposure to 0.70 mg/L of Cu 2+ , the highest IBR v2 value was observed in the hepatopancreas (9.26).

Alterations in the Expression of Apoptosis-Related Genes
To evaluate whether Cu 2+ exposure could induce apoptosis, we measured the mRNA levels of apoptosis-related genes, including caspase-3, caspase-8, B-cell lymphoma 2 (Bcl-2), Bcl2 X protein (Bax), p53, and cytochrome c (cytc1) in the hepatopancreas, gills, and muscle ( Figure 3). In the hepatopancreas, the mRNA levels of caspase-3, caspase-8, Bax, and p53 showed an increasing tendency, and the highest value of the expression levels of the genes was observed in the group treated with 0.70 mg/L of Cu 2+ (p < 0.05; Figure 3A).

Alterations in the Expression of MAPK Pathway-Related Genes
After Cu 2+ exposure, the genes associated with the MAPK signaling pathway showe various degrees of change ( Figure 4). In the hepatopancreas, the transcription of extrace lular signal-regulated protein kinase (erk) was elevated in the groups exposed to 0.18 an 0.70 mg/L of Cu 2+ , and jun (an AP-1 subunit) was elevated in the group exposed to 0.7 mg/L of Cu 2+ , both compared to that in the control group (p < 0.05; Figure 4A).
In the gills, erk expression was distinctly downregulated in the group exposed to 0.7 mg/L of Cu 2+ compared to that in the control group (p < 0.05; Figure 4B), while p38 expre sion was upregulated in the group exposed to 0.04 mg/L of Cu 2+ and gradually downreg ulated under exposure to 0.18 and 0.70 mg/L of Cu 2+ (p < 0.05; Figure 4B).  In the gills, the mRNA levels of caspase-3, Bax, p53, and cytc1 increased in treatments with 0.04 and/or 0.18 mg/L of Cu 2+ and then decreased to near normal values in treatment with 0.70 mg/L of Cu 2+ ( Figure 3B). The caspase-3 and cytc1 were markedly upregulated under exposure to 0.04 and 0.18 mg/L of Cu 2+ (p < 0.05; Figure 3B) and gradually decreased under exposure to 0.70 mg/L of Cu 2+ . Similarly, Bax and p53 were upregulated after exposure to 0.04 mg/L of copper (p < 0.05) and gradually decreased with increasing Cu 2+ concentrations ( Figure 3B).
In the muscle, caspase-3 and caspase-8 transcription were significantly upregulated compared to the control group after 5 days of Cu 2+ exposure (p < 0.05; Figure 3C). However, the mRNA levels of Bax, p53, and cytc1 were not significantly altered after copper exposure.

Alterations in the Expression of MAPK Pathway-Related Genes
After Cu 2+ exposure, the genes associated with the MAPK signaling pathway showed various degrees of change ( Figure 4). In the hepatopancreas, the transcription of extracellular signal-regulated protein kinase (erk) was elevated in the groups exposed to 0.18 and 0.70 mg/L of Cu 2+ , and jun (an AP-1 subunit) was elevated in the group exposed to 0.70 mg/L of Cu 2+ , both compared to that in the control group (p < 0.05; Figure 4A).

Alterations in the Expression of ER Stress-Related Genes
The mRNA levels of the ER stress-related genes showed irregular variations after Cu 2+ exposure in the hepatopancreas, gills, and muscle ( Figure 5). In the hepatopancreas, the mRNA levels of activating transcription factor 6 (atf6) and atf4 exhibited a linear rising trend with increasing Cu 2+ concentrations and were upregulated in the group treated with 0.70 mg/L of Cu 2+ (p < 0.05; Figure 5A). Compared to those in the control group, exposure to 0. 18   In the gills, erk expression was distinctly downregulated in the group exposed to 0.70 mg/L of Cu 2+ compared to that in the control group (p < 0.05; Figure 4B), while p38 expression was upregulated in the group exposed to 0.04 mg/L of Cu 2+ and gradually downregulated under exposure to 0.18 and 0.70 mg/L of Cu 2+ (p < 0.05; Figure 4B).
In the muscle, c-Jun N-terminal kinase (jnk) mRNA was downregulated in the groups exposed to 0.18 and 0.70 mg/L of Cu 2+ , and jun mRNA was downregulated in the groups exposed to 0.04, 0.18, and 0.70 mg/L of Cu 2+ , compared to those in the control group (p < 0.05; Figure 4C).

Alterations in the Expression of ER Stress-Related Genes
The mRNA levels of the ER stress-related genes showed irregular variations after Cu 2+ exposure in the hepatopancreas, gills, and muscle ( Figure 5). In the hepatopancreas, the mRNA levels of activating transcription factor 6 (atf6) and atf4 exhibited a linear rising trend with increasing Cu 2+ concentrations and were upregulated in the group treated with 0.70 mg/L of Cu 2+ (p < 0.05; Figure 5A). Compared to those in the control group, exposure to 0.18 and 0.70 mg/L of Cu 2+ upregulated the transcription of eukaryotic translation initiation factor 2 α (eif2α), and 0.70 mg/L of Cu 2+ upregulated inositol-requiring enzyme 1 (ire1) transcription (p < 0.05; Figure 5A).
Antioxidants 2022, 11, x FOR PEER REVIEW 9 of 22 0.70 mg/L of Cu 2+ relative to that in the control group (p < 0.05; Figure 5B). In addition, exposure to 0.70 mg/L of Cu 2+ decreased grp78 transcription (p < 0.05; Figure 5B). In the muscle, the mRNA level of atf6 was significantly enhanced in the 0.18 and 0.70 mg/L Cu 2+ -exposed groups compared to that in the control group (p < 0.05), but other genes were not significantly changed ( Figure 5B).

Alterations in the Expression of Autophagy-Related Genes
Eight autophagy-related genes, including 5-AMP-activated protein kinase β (ampkβ), beclin, p62, microtubule-associated proteins 1A/1B light chain 3a (lc3a), lc3c, autophagyrelated gene 7 (atg7), transcription factor EB (tfeb), and lysosome-associated membrane protein 1 (lamp1), were used to evaluate the autophagic response to Cu 2+ exposure in the In the gills, the mRNA levels of atf6 exhibited an initial upregulation followed by a decreasing tendency, and a peak value was observed in the crabs exposed to 0.04 mg/L of Cu 2+ (p < 0.05; Figure 5B). The expression of atf6 was downregulated under exposure to 0.70 mg/L of Cu 2+ relative to that in the control group (p < 0.05; Figure 5B). In addition, exposure to 0.70 mg/L of Cu 2+ decreased grp78 transcription (p < 0.05; Figure 5B).
In the muscle, the mRNA level of atf6 was significantly enhanced in the 0.18 and 0.70 mg/L Cu 2+ -exposed groups compared to that in the control group (p < 0.05), but other genes were not significantly changed ( Figure 5B).

Alterations in the Expression of Autophagy-Related Genes
Eight autophagy-related genes, including 5-AMP-activated protein kinase β (ampkβ), beclin, p62, microtubule-associated proteins 1A/1B light chain 3a (lc3a), lc3c, autophagyrelated gene 7 (atg7), transcription factor EB (tfeb), and lysosome-associated membrane protein 1 (lamp1), were used to evaluate the autophagic response to Cu 2+ exposure in the hepatopancreas, gills, and muscle ( Figure 6). In the hepatopancreas, the mRNA levels of atg7, tfeb, ampkβ, beclin, p62, and lc3a increased with Cu 2+ concentrations in a linear or non-linear manner, and they were significantly upregulated in the 0.70 mg/L Cu 2+ -exposed group compared to the control group (p < 0.05; Figure 6A). A significant upregulation was also observed in atg7 under exposure to 0.18 mg/L of Cu 2+ and in tfeb and p62 under exposure to 0.04 and 0.18 mg/L Cu 2+ (p < 0.05; Figure 6A).  Figure 6A). In the gills, Cu 2+ exposure caused a significant increase in the atg7 mRNA level in the 0.04 mg/L Cu 2+ -exposed group, tfeb in the 0.18 and 0.70 mg/L Cu 2+ -exposed groups, and p62 in the 0.04 and 0.18 mg/L Cu 2+ -exposed groups compared with those in the control group (p < 0.05; Figure 6B). In contrast, Cu 2+ exposure caused a significant decrease in lc3c mRNA in the 0.70 mg/L Cu 2+ -exposed group (p < 0.05; Figure 6B).
In the muscle, only the transcription of tfeb and lc3a was significantly changed by Cu 2+ exposure ( Figure 6C). The transcription of tfeb was lower in the 0.18 and 0.70 mg/L Cu 2+exposed groups than in the 0 mg/L Cu 2+ -exposed group (p < 0.05; Figure 6C). Furthermore, the transcription of lc3a was lower in the 0.70 mg/L Cu 2+ -exposed group than in the 0 mg/L Cu 2+ -exposed group (p < 0.05; Figure 6C).  In the gills, Cu 2+ exposure caused a significant increase in the atg7 mRNA level in the 0.04 mg/L Cu 2+ -exposed group, tfeb in the 0.18 and 0.70 mg/L Cu 2+ -exposed groups, and p62 in the 0.04 and 0.18 mg/L Cu 2+ -exposed groups compared with those in the control group (p < 0.05; Figure 6B). In contrast, Cu 2+ exposure caused a significant decrease in lc3c mRNA in the 0.70 mg/L Cu 2+ -exposed group (p < 0.05; Figure 6B).
In the muscle, only the transcription of tfeb and lc3a was significantly changed by Cu 2+ exposure ( Figure 6C). The transcription of tfeb was lower in the 0.18 and 0.70 mg/L Cu 2+exposed groups than in the 0 mg/L Cu 2+ -exposed group (p < 0.05; Figure 6C). Furthermore, the transcription of lc3a was lower in the 0.70 mg/L Cu 2+ -exposed group than in the 0 mg/L Cu 2+ -exposed group (p < 0.05; Figure 6C).

Alterations in the Expression of Immune Response-Related Genes
The immune response to Cu 2+ exposure was assessed by determining the immune response-related genes in the hepatopancreas, gills, and muscle (Figure 7). In the hepatopancreas, the mRNA levels of Toll-like receptor 2 (tlr2), myeloid differentiation protein-88 (myd88), relish, interleukin-16 (il-16), lipopolysaccharide-induced TNF-α factor (litaf ), and pelle were higher in the 0.70 mg/L Cu 2+ -exposed group than in the control group (p < 0.05; Figure 7A). Higher mRNA levels of tlr2 and myd88 were also observed in the 0.18 mg/L Cu 2+ -exposed group (p < 0.05; Figure 7A).

Alterations in the Expression of Immune Response-Related Genes
The immune response to Cu 2+ exposure was assessed by determining the immune response-related genes in the hepatopancreas, gills, and muscle (Figure 7). In the hepatopancreas, the mRNA levels of Toll-like receptor 2 (tlr2), myeloid differentiation protein-88 (myd88), relish, interleukin-16 (il-16), lipopolysaccharide-induced TNF-α factor (litaf), and pelle were higher in the 0.70 mg/L Cu 2+ -exposed group than in the control group (p < 0.05; Figure 7A). Higher mRNA levels of tlr2 and myd88 were also observed in the 0.18 mg/L Cu 2+ -exposed group (p < 0.05; Figure 7A).
In the gills, the mRNA level of tlr2 was strongly upregulated in the 0.70 mg/L Cu 2+treated group compared to that in the control group (p < 0.05; Figure 7B). The litaf in the three Cu 2+ -treated groups and lysozyme (lzm) in the 0.04 and 0.18 mg/L Cu 2+ -treated groups were highly expressed (p < 0.05; Figure 7B).
In the muscle, the transcription of tlr2 was upregulated in the three Cu 2+ -treated groups compared to that in the control group (p < 0.05; Figure 7C). Likewise, the expression of myd88 and lzm was upregulated in the 0. In the gills, the mRNA level of tlr2 was strongly upregulated in the 0.70 mg/L Cu 2+treated group compared to that in the control group (p < 0.05; Figure 7B). The litaf in the three Cu 2+ -treated groups and lysozyme (lzm) in the 0.04 and 0.18 mg/L Cu 2+ -treated groups were highly expressed (p < 0.05; Figure 7B).
In the muscle, the transcription of tlr2 was upregulated in the three Cu 2+ -treated groups compared to that in the control group (p < 0.05; Figure 7C). Likewise, the expression of myd88 and lzm was upregulated in the 0.70 mg/L Cu 2+ -treated group (p < 0.05; Figure 7C).

Alterations in the Expression of Stress-and Detoxification-Related Genes
In the hepatopancreas, the mRNA levels of shock protein 90 (hsp90), cytochrome P450 (cyp) 2b, and cyp4 exhibited a linear rising trend with increasing Cu 2+ concentrations, and upregulation was observed in the 0.70 mg/L Cu 2+ -treated group (p < 0.05; Figure 8A). The mRNA levels of hsp70 and metallothioneins (mt) were first upregulated and then downregulated with increasing Cu 2+ concentrations, as evidenced by higher hsp70 expression under exposure to 0.04 mg/L of Cu 2+ and higher mt and cyp2a expression under exposure to 0.04 and 0.18 mg/L of Cu 2+ (p < 0.05; Figure 8A).

Alterations in the Expression of Stress-and Detoxification-Related Genes
In the hepatopancreas, the mRNA levels of shock protein 90 (hsp90), cytochrome P450 (cyp) 2b, and cyp4 exhibited a linear rising trend with increasing Cu 2+ concentrations, and upregulation was observed in the 0.70 mg/L Cu 2+ -treated group (p < 0.05; Figure 8A). The mRNA levels of hsp70 and metallothioneins (mt) were first upregulated and then downregulated with increasing Cu 2+ concentrations, as evidenced by higher hsp70 expression under exposure to 0.04 mg/L of Cu 2+ and higher mt and cyp2a expression under exposure to 0.04 and 0.18 mg/L of Cu 2+ (p < 0.05; Figure 8A) In the gills, the mRNA levels of hsp60 and hsp70 were significantly upregulated under 0.04 mg/L Cu 2+ exposure (p < 0.05; Figure 8B) but gradually reduced to the same level as that in the control group. Similarly, hsp90 expression was upregulated in the group exposed to 0.18 mg/L of Cu 2+ but downregulated in the group exposed to 0.70 mg/L of Cu 2+ (p < 0.05; Figure 8B). Other genes were not markedly affected by Cu 2+ exposure.
In the muscle, only hsp90 expression was significantly reduced in the 0.70 mg/L Cu 2+exposed group compared to that in the control group (p < 0.05; Figure 8C).

Discussion
Excess copper has been widely confirmed to be toxic to crustaceans, and the toxic effect is linked not only to concentration but also to exposure time. The median lethal concentration (24-96 h LC50) of Cu 2+ decreased with the extension of exposure time in In the gills, the mRNA levels of hsp60 and hsp70 were significantly upregulated under 0.04 mg/L Cu 2+ exposure (p < 0.05; Figure 8B) but gradually reduced to the same level as that in the control group. Similarly, hsp90 expression was upregulated in the group exposed to 0.18 mg/L of Cu 2+ but downregulated in the group exposed to 0.70 mg/L of Cu 2+ (p < 0.05; Figure 8B). Other genes were not markedly affected by Cu 2+ exposure.
In the muscle, only hsp90 expression was significantly reduced in the 0.70 mg/L Cu 2+ -exposed group compared to that in the control group (p < 0.05; Figure 8C).

Discussion
Excess copper has been widely confirmed to be toxic to crustaceans, and the toxic effect is linked not only to concentration but also to exposure time. The median lethal concentration (24-96 h LC 50 ) of Cu 2+ decreased with the extension of exposure time in crustaceans [12,13]. Exposure to 0.75 mg/L Cu 2+ for 7 days resulted in abnormal gill tip structure of M. rosenbergii [29]. The stress biomarkers showed an increased tendency in a time-dependent manner (1-7 days) in Macrobrachium scabriculum exposed to Cu 2+ at doses of 0.032-0.352 mg/L [30]. A study of 3-48 h of exposure showed that Cu 2+ treatments (5-20 mg/L) began to negatively influence the immune ability of L. vannamei after 12 h [31]. Similar to previous studies, our data also exhibited that exposure to Cu 2+ (0.04-0.70 mg/L) for 5 days had adverse effects on antioxidative status, apoptosis, ER stress, and immune response in E. sinensis. It is worth noting that the Cu 2+ toxicity showed tissue-specificity, and hepatopancreas was more sensitive to Cu 2+ exposure in E. sinensis. In invertebrates, metals, including copper, are commonly taken in via gills and accumulate in the hepatopancreas [32]. Yang et al. reported that the accumulation of copper in the hepatopancreas was higher than in other tissues in E. sinensis after Cu 2+ exposure [33]. Meanwhile, the hepatopancreas is considered a primary organ of excretion and detoxification for metals in crustaceans [34]. Thus, it may be more susceptible to copper exposure.

Effects of Copper Exposure on Antioxidative Status
Oxidative stress is a physiological imbalance state in which the production of reactive oxygen species (ROS) overwhelms the cellular antioxidant defense capacity, eventually resulting in damage to cellular macromolecules, such as DNA, proteins, and lipids. Copper is known to participate in the formation of ROS, and its overload may result from repetitive radical formation via redox cycling [35,36]. Excessive ROS can induce oxidative stress and impair the antioxidant defense system. Indeed, strong evidence exists that acute or chronic Cu 2+ exposure induces oxidative stress in different aquatic animals. For example, Cu 2+ exposure enhances the activities of antioxidative enzymes such as SOD and glutathione peroxidase (Gpx) in hepatopancreas of L. vannamei [37] and Callinectes sapidus [38], and gills of O. niloticus [39], reflecting an occurrence of oxidative stress. In contrast, exposure to high levels of waterborne Cu 2+ decreases enzymatic and non-enzymatic antioxidants and induces oxidative damage in the gills of P. clarkia [40], the hepatopancreas of Minuca rapax [41], and the brain of Cyprinus carpio [42]. Our study further showed that the antioxidant capacity in different tissues of E. sinensis decreased following exposure to 0.70 mg/L of Cu 2+ , indicating that a higher level of Cu 2+ exposure induces oxidative damage. In addition, our data showed a variable intensity of oxidative stress in different tissues after Cu 2+ exposure, which was supported by a previous study in Carassius auratus [43], indicating the tissue specificity of Cu 2+ toxicity.
Peroxidative damage to membrane lipids is another common consequence of excess Cu 2+ . Lipid peroxy radicals formed during lipid peroxidation may change the fluidity and permeability of the cell membrane in injured cells [44]. MDA, a lipid peroxidation product, is a typical indicator used to evaluate lipid peroxidation. It is increased in multiple fish tissues after Cu 2+ exposure [45][46][47]. Similarly, Cu 2+ -overloaded Procambarus clarkii has a significantly increased MDA concentration in the hemolymph, hepatopancreas, and gills [40,48,49]. Our data also exhibited enhanced MDA content in the hemolymph, gills, and muscle of E. sinensis after exposure to 0.70 mg/L of Cu 2+ , indicating that high levels of Cu 2+ exposure induce lipid peroxidation and augment oxidative damage.
It has been reported that the toxic effect of copper on redox state was related to cultured conditions, such as salinity, temperature, and pH, in aquatic animals. Moderate salinity levels increased GST activity to alleviate the lethal toxicity of Cu 2+ , but high salinity levels worsen the Cu 2+ -induced oxidative damage in Danio rerio embryos [50]. In M. rapax, the higher temperature (35 • C) significantly increased Cu 2+ -induced oxidative stress [41]. Carvalho et al. (2015) suggested that the effect of Cu 2+ on the response of antioxidant defense systems was determined by water pH in Prochilodus lineatus [51]. The evidence revealed that copper combined with other factors causes more significant toxicity in aquatic animals than copper alone. Thus, interactive effects between copper exposure and cultured conditions will be examined in future research.

Effects of Copper Exposure on Apoptosis
Apoptosis is considered a sensitive parameter for assessing the toxicity of environmental pollutants [52]. It has been reported that Cu, a common environmental pollutant, can induce apoptosis in aquatic animals. High concentrations of Cu 2+ increase the incidence of TUNEL-positive cells (apoptosis) in the gills of D. rerio and C. auratus [53,54]. Acute exposure to Cu 2+ increases the apoptotic hemocyte ratio and caspase-3 gene expression in L. vannamei [55]. In our study, apoptosis-related genes such as caspase-3, caspase-8, Bax, p53, and cytc were upregulated in the hepatopancreas, gills, and/or muscle of E. sinensis, indicating that mitochondria-mediated apoptosis was activated by Cu 2+ exposure. Cu 2+induced apoptosis is likely elicited by the induction of ROS [56]. Our data support this view, given the strong oxidative stress that was found after Cu 2+ exposure. Additionally, in D. rerio, the central nervous system and liver show higher sensitivity to apoptosis induced by Cu 2+ exposure [57]. Similarly, Cu 2+ -exposed E. sinensis exhibits stronger apoptosis in the hepatopancreas and gills. In the hepatopancreas, the activation of apoptosis was mainly observed under exposure to 0.7 mg/L of Cu 2+ , while in the gills, it was mainly observed under exposure to 0.04 and 0.18 mg/L of Cu 2+ . Thus, Cu-triggered apoptosis may occur in a tissue-specific manner.
MAPK signaling pathways, including ERK, JNK, and p38, play critical roles in apoptosis [58]. The ERK-AP-1 and JNK-AP-1 pathways have been reported to regulate oxidative stress-induced apoptosis [59]. A previous study reported that Cu 2+ exposure causes apoptosis via the activation of ERK and p38 in the hepatocytes of O. mykiss [60]. Mitochondrial apoptosis induced by copper nanoparticles has been associated with the activation of the ERK signaling pathway in female mice [61]. In our study, the mRNA levels of erk and jun (a AP-1 subunit) were upregulated in the hepatopancreas after Cu 2+ exposure and significantly associated with apoptosis, indicating that the ERK-AP-1 pathway may be involved in Cu 2+ -induced apoptosis. In the gills, p38 gene expression was upregulated in the 0.04 mg/L copper-exposed group, which implies that the p38 pathway may be activated to regulate apoptosis after exposure to lower dose of Cu 2+ . In the muscle, however, the mRNA levels of jnk and jun were downregulated after Cu 2+ exposure, although the underlying mechanisms remain unclear. We hypothesize that the downregulation may be related to tissue damage caused by Cu 2+ exposure.

Effects of Copper Exposure on ER Stress
The ER is a pivotal organelle that is responsible for protein assembly, folding, and transportation. Protein misfolding and ER stress trigger a complex signaling process, known as the unfolded protein response (UPR), to restore ER homeostasis [62]. A triggered UPR is a protective mechanism to reinstate ER homeostasis, but persistent or severe ER stress can initiate cell death via mitochondrial pathways [63]. Environmental pollutants, such as Cu 2+ , activate ER stress and impair mitochondrial function in aquatic animals [64]. Cu 2+ exposure for 30 days leads to upregulated ER stress-related genes, such as grp78, perk, eif2a, ire-1α, and atf6 in the liver of Synechogobius hasta and Pelteobagrus fulvidraco [65,66].
We also observed a marked upregulation of eif2a, atf4, atf6, and ire1 in the hepatopancreas of E. sinensis, indicating that exposure to 0.7 mg/L of Cu 2+ induced ER stress. In the gills, exposure to 0.04 mg/L of Cu 2+ upregulated atf6, while 0.7 mg/L of Cu 2+ downregulated atf6 and grp78. We hypothesize that the downregulation of these genes was related to ER damage under exposure to higher concentrations of Cu 2+ . Similar data have also been found in the liver of S. hasta exposed to a higher level (0.055 mg/L) of Cu 2+ for 60 days [65]. In addition, increased ROS production under Cu 2+ exposure can induce ER stress and activate the ATF6 and IRE1 signaling pathways, leading to apoptosis [67].

Effects of Copper Exposure on Autophagy
Autophagy is a crucial cell-clearing process that regulates the degradation of damaged organelles and unfolded proteins by fusion with lysosomes in cells. LC3 and p62 are widely used as markers of autophagy. In the later stages of autophagy, TFEB coordinates lysosomal activation and autophagosome-lysosome fusion [68]. A recent study reported that excess dietary copper induces oxidative stress and autophagy, as evidenced by the upregulated expression of beclin1, lc3B, and p62 in P. fulvidraco, which then protected against copperinduced lipid accumulation [69]. Activated autophagy has also been reported in GC-1 cells [70], pig testes [71], and the hypothalamus of broilers [72] following Cu 2+ exposure due to oxidative stress. In contrast, Cu 2+ exposure has been found to downregulate the mRNA levels of lc3 in D. rerio gills, indicating the impairment of macroautophagy [73]. Furthermore, the AMPK signaling pathway has been shown to regulate Cu 2+ -induced autophagy [74,75]. In our study, the mRNA levels of autophagy-related genes, including ampkβ, beclin, lc3a, tfeb, p62, and atg7, were upregulated in the hepatopancreas, suggesting that Cu 2+ exposure may activate autophagy via the AMPK-Beclin pathway. Unlike those in the hepatopancreas, the mRNA levels of atg7 and p62 in the gills were upregulated following exposure to lower doses of Cu 2+ (0.04 and/or 0.18 mg/L) but returned to similar levels as those in the control group after being exposed to a higher dose of Cu 2+ (0.7 mg/L). The expression of lc3c was even downregulated in the 0.7 mg/L Cu 2+ -exposed gills. The findings suggest that a low dose of Cu 2+ may initiate autophagy, but a high dose can impair the autophagic process in the gills. The detailed mechanisms require further study. The activation of autophagy may be linked to oxidative stress and ER stress induced by Cu 2+ exposure [76].

Effects of Copper Exposure on the Immune Response
The immune response is a key mechanism following pollutant toxicity in aquatic organisms. TLRs, widely existing pattern-recognition receptors, are considered major regulators of the immune response [77]. Numerous studies have suggested that environmental pollutants, including heavy metals, can activate the TLRs to regulate immune response in animals. For example, Cr(VI) exposure upregulated tlr2 and myd88 expression in Geloina erosa gills [78], and microbiota-dependent TLR2 signaling reduced silver nanoparticle toxicity to D. rerio larvae [79]. Relish, an NF-κB transcription factor, also plays a key role in the innate immunity of crustaceans [80]. In crustaceans, the TLR2-MyD88 pathway regulates the immune response to pathogenic bacterial infections [81,82]. A transcriptomic analysis revealed that Cu 2+ exposure significantly affects the TLR pathway in Mizuhopecten yessoensis [83]. In L. vannamei, the gene expression of TLRs was significantly increased in the 0.05 mg/L Cu 2+ -treated group, but returned to the control level following treatments with higher doses of Cu 2+ [37]. Aksakal and Ciltas [84] also reported that a low expression of immune-related genes such as tlr4 and tlr22 resulted in immunosuppression in D. rerio after exposure to copper oxide nanoparticles. In our study, the immune response to Cu 2+ exposure was tissue-specific. In the hepatopancreas, a significant inflammatory response occurred via the TLR2-MyD88-NF-κB pathway after Cu 2+ exposure. Despite the upregulation of tlr2 and/or myd88 in the gills and muscle, relish and il-16 (an important pro-inflammatory cytokine in crabs) were not altered, which may indicate no obvious inflammatory response in the two tissues, especially the muscle. In addition, the ERK pathway may also be involved in Cu 2+ -induced inflammation in the hepatopancreas [85].
In addition to NF-κB, LITAF is a pivotal transcription factor in the inflammatory response and regulates the transcription of TNF-α and other cytokines [86]. Tang et al. [87] suggested that LITAF is a mediator from the NF-κB pathway in the lipopolysaccharideinduced inflammatory response. It has been reported that litaf is upregulated and involved in the immune response in E. sinensis after Edwardsiella tarda and Vibrio anguillarum infections [86]. Similarly, our data show that Cu 2+ exposure upregulated the expression of litaf in the hepatopancreas, suggesting that the TLR2-MyD88-LITAF pathway may be triggered in response to Cu 2+ toxicity.

Effects of Copper Exposure on the Stress Response and Detoxification
HSPs are molecular chaperones that play well-established roles in protein folding and transport. HSP60, HSP70, and HSP90 are well-studied HSPs that are abundantly induced under a variety of chemical exposures [88], which is a protective response to stressors [89]. A previous study reported that Cu 2+ exposure upregulated the mRNA levels of hsp60, hsp70, and hsp90 in the liver of C. carpio [90]. A study on freshwater prawns (Macrobrachium malcolmsonii) further showed that the synthesis of HSP70 appeared from the 1st to 24th hour in the gills under Cu 2+ exposure but was not recorded after the 24-h mark [91]. We also observed that the mRNA levels of hsp60, hsp70, and hsp90 exhibited an initial upregulation followed by a decreasing tendency in the gills. We therefore conjectured that Cu 2+ exposure at lower doses triggered an HSP-mediated protective mechanism. In addition, the downregulation of hsp90 may be interpreted as a result of the strong oxidative stress induced by higher doses of Cu 2+ [92].
MT, a metal-binding protein with a high affinity for metals, is involved in the regulation of essential metal ion homeostasis and the detoxification of non-essential metal ions [93]. After 4 days of Cu 2+ exposure, the MT level was found to increase in Gasterosteus aculeatus [94]. An increased MT level has also been reported after Cu 2+ exposure in Pacifastacus leniusculus [95] and constitutes a protective response to Cu 2+ accumulation. Similarly, our data show upregulated mt expression in the hepatopancreas after exposure to 0.04 and 0.18 mg/L of Cu 2+ , indicating that a lower concentration of Cu 2+ induces a positive response, but a higher concentration inhibits the response.
CYP enzymes have been implicated in the detoxification and metabolism of environmental pollutants, including heavy metals, in aquatic animals. The CYP 1-4 families are considered reliable biomarkers for monitoring environmental toxicants [96]. The induction of CYP enzymes may be an adaptive response to metal exposure, whereas their decrease inhibits detoxification [97]. In Cu 2+ -exposed Diaphanosoma celebensis, CYP-related genes, including cyp2 and cyp4, were found to be upregulated at an earlier exposure time (6 h) but downregulated at a later exposure time (24h) [98]. In C. auratus, Cu 2+ exposure upregulated the expression of cyp1a and cyp3a, but combined treatment with Cu 2+ and diclofenac decreased their expression [99]. In our study, the mRNA levels of cyp2A, cyp2B, and cyp4 were markedly increased in the hepatopancreas, implying that CYP enzymes may be involved in the phase I detoxification of Cu 2+ toxicity. In addition, our data revealed that detoxification predominantly occurred in the hepatopancreas but not in the gills or muscle.
Apart from acute toxicity, long-term Cu 2+ exposure also causes its accumulation in different tissues of crustaceans. In E. sinensis, the copper accumulation was positively related to its level in water, and the hepatopancreas was the primary target organ [33]. The accumulation presents potential for bio-magnification through the food chain [100], which may pose a health risk to humans, since humans are the primary consumers of E. sinensis and other aquatic animals. In order to maintain cellular homeostasis, many organisms possess a purification ability of toxic elements. Boada et al. [101] reported that Mugil curema eliminated enriched copper for14 days after Cu 2+ exposure. Similarly, the purification process in juvenile Petenia kraussii exposed to Cu 2+ was achieved after 14 days [102]. In P. clarkii, enrichment of copper in hepatopancreas was completely eliminated after 7 days [103]. However, the depuration time of E. sinensis for excessive copper has not been reported until now, and will be further evaluated in our future research. Furthermore, the potential health risks of copper accumulation from aquatic food consumption should be investigated.

Conclusions
In this study, we examined the adverse effects of Cu 2+ exposure on different tissues of E. sinensis. Cu 2+ exposure suppressed antioxidative parameters and promoted lipid peroxidation in different tissues, resulting in oxidative damage. After Cu 2+ exposure, apoptosis-related genes were upregulated, implying that apoptosis was activated, and the activation may be related to the upregulation of the MAPK pathway and ER stress. In the hepatopancreas and gills, the regulation of autophagy-related genes indicated that the autophagic response was involved in Cu 2+ toxicity. In addition, Cu 2+ exposure increased immune-related gene expression in different tissues, especially the hepatopancreas, where the TLR2-MyD88-NF-κB pathway may be initiated to mediate the inflammatory response. Furthermore, the upregulation of anti-stress and detoxification genes revealed that an adaptive mechanism was activated in different tissues following Cu 2+ exposure. Overall, the toxicity response of Cu 2+ in E. sinensis was associated with oxidative stress, apoptosis, ER stress, autophagy, and immune response. This study enriches our understanding of the potential toxicity response of Cu 2+ in crustaceans, which may provide more reference data for the environmental risk assessments of Cu 2+ .

Conflicts of Interest:
The authors declare no conflict of interest.