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Systemic response of the stony coral Pocillopora damicornis against acute cadmium stress
Aquatic Toxicology 194 (2018) 132–139
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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aqtox
Systemic response of the stony coral Pocillopora damicornis against acute cadmium stress Zhi Zhoua,b,c,
⁎,1
T
, Xiaopeng Yua,1, Jia Tanga, Yibo Wua, Lingui Wanga,b,c, Bo Huanga,c
a
Key Laboratory of Tropical Biological Resources of Ministry of Education, Hainan University, Haikou, Hainan, 570228, China State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, Hainan 570228, China c Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of Marine Science, Hainan University, Haikou, Hainan 570228, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Symbiosis Detoxification Antioxidation Apoptosis Coral
Heavy metals have become one of the main pollutants in the marine environment and a major threat to the growth and reproduction of stony corals. In the present study, the density of symbiotic zooxanthellae, levels of crucial physiological activities and the transcriptome were investigated in the stony coral Pocillopora damicornis after the acute exposure to elevated cadmium concentration. The density of symbiotic zooxanthellae decreased significantly during 12–24 h period, and reached lowest at 24 h after acute cadmium stress. No significant changes were observed in the activity of glutathione S-transferase during the entire stress exposure. The activities of superoxide dismutase and catalase, and the concentration of glutathione decreased significantly, but the activation level of caspase3 increased significantly after cadmium exposure. Furthermore, transcriptome sequencing and bioinformatics analysis revealed 3538 significantly upregulated genes and 8048 significantly downregulated genes at 12 h after the treatment. There were 12 overrepresented GO terms for significantly upregulated genes, mostly related to unfolded protein response, endoplasmic reticulum stress and apoptosis. In addition, a total of 32 GO terms were overrepresented for significantly downregulated genes, and mainly correlated with macromolecular metabolic processes. These results collectively suggest that acute cadmium stress could induce apoptosis by repressing the production of the antioxidants, elevating oxidative stress and activating the unfolded protein response. This cascade of reactions would result to the collapse of the coral-zooxanthella symbiosis and the expulsion of symbiotic zooxanthellae in the stony coral P. damicornis, ultimately leading to coral bleaching.
1. Introduction Stony corals are unique creatures living in shallow waters with relatively low nutrient levels and good illumination conditions (Zhang et al., 2015). They compose most of the great reef ecosystems in tropical seas, and heavily depend on symbiotic relationships with zooxanthellae, specifically with the dinoflagellate Symbiodinium (Tong et al., 2017). The zooxanthella lives within the intracellular vacuoles of the gastrodermal (endodermal) tissue, and provides the coral with nutrients through photosynthesis, a crucial energy source in an otherwise clear and nutrient-poor tropical waters (Ganot et al., 2011). In exchange, the coral provides the zooxanthellae with the carbon dioxide and ammonium needed for photosynthesis (Shinzato et al., 2011). However, owing to this distinct symbiosis, stony corals are more sensitive to changes in the marine environment, and exhibit systemic responses to environmental stress (Parkinson and Baums, 2014). Because of climate
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change and environmental pollution, stony corals have been subjected to frequent and severe diseases over the past decades (Vidal-Dupiol et al., 2011). The increasing mean annual sea surface temperature due to climate change and worsening marine pollution are both threats to the survival and reproduction of stony corals. During warming periods for example, stony corals undergo “bleaching” as a form of morphological and physiological response (Coles and Brown, 2003), a process where the host corals either lose their symbiotic zooxanthellae, or the zooxanthellae’s photosynthetic pigments degrade. The resulting collapse of the symbiosis between the coral and zooxanthellae causes decreased coral adaptation and increased mortality (Goreau and Macfarlane, 1990). Studies suggested that oxidative damage due to an excess of respiratory burst and reactive oxygen species (ROS) could induce apoptosis, cell death and tissue abscission of stony coral, and finally result to coral bleaching (Downs et al., 2002; Lesser, 1997).
Corresponding author at: 58 Renmin Road, Haikou 570228, China. E-mail address: [email protected] (Z. Zhou). These authors contributed equally to this work.
https://doi.org/10.1016/j.aquatox.2017.11.013 Received 8 September 2017; Received in revised form 21 November 2017; Accepted 22 November 2017 Available online 23 November 2017 0166-445X/ © 2017 Elsevier B.V. All rights reserved.
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tissue homogenates were prepared using Waterpik water jet to strip the tissue from the coral skeleton into approximately 10 mL filtered seawater (0.22 μm). The homogenates were then centrifuged at 1500g, 4 °C for 10 min, and the supernatants were used to determine enzyme activities and GSH concentration. The symbiotic zooxanthellae were resuspended in the filtered seawater, and counted using a Neubauer hemocytometer (QIUJING, China). The surface area of the nubbins used were determined by the aluminum foil method (Johannes et al., 1970). The density of symbiotic zooxanthellae was defined as the ratio of the number of symbiotic zooxanthellae to the surface area of coral nubbins (cell cm−2).
Heavy metals are one of the main marine pollutants mainly coming from industrial discharges, urban/agricultural run-off and anti-fouling paints. Its distribution and effects on coral reefs have recently received considerable attention (Hudspith et al., 2017; Morton and Blackmore, 2001). Heavy metal intoxications have been observed in the majority of coral reefs worldwide (Chen et al., 2010; Guzmán and Jiménez, 1992; Haynes and Johnson, 2000; Horwitz et al., 2014). Stony corals are able to absorb heavy metals from the environment, and accumulate them in their tissues and skeleton through mineralization (Barakat et al., 2015; Darvishnia et al., 2016; Esslemont et al., 2000; Harland et al., 2009). While some trace heavy metals such as strontium are essential to the growth and reproduction of stony corals (Meibom et al., 2003), high levels of other heavy metals such as copper, cadmium, lead, zinc and nickel have been reported to effect fertilization success, glutathione (GSH) levels, tissue integrity, bleaching and death of some stony coral species (Mitchelmore et al., 2003; Mitchelmore et al., 2007; ReicheltBrushett and Harrison, 1999, 2005; Sabdono, 2009). However, despite the high threat to heavy metal intoxication in most coastal environments, the molecular mechanisms underlying the toxic effects of heavy metals on stony coral remains little understood. Pocillopora damicornis is a species of stony coral in the family Pocilloporidae, and is native to tropical and subtropical parts of the Indian and Pacific Oceans. To understand the potential effects of heavy metal stress on coral symbiosis and physiology, the density of symbiotic zooxanthellae, the crucial factors to physiological activities, and the transcriptome of coral P. damicornis were investigated after the acute exposure to elevated cadmium. The present study would provide insights to further understand the molecular mechanisms underlying stress response and environmental adaption of stony corals.
2.4. Activity assay of antioxidases and detoxification enzyme The activities of superoxide dismutase (SOD, JIANCHENG, A001), catalase (CAT, JIANCHENG, A007) and glutathione S-transferase (GST, Solarbio, BC0350) in the supernatants were measured using commercial kits, following the manufacturer’s recommendations. Total SOD activity was determined by the hydroxylamine method (Kono, 1978), where 1 SOD activity unit was defined as the enzyme amount causing 50% inhibition in 1 mL reaction solution. Total CAT activity was determined using spectrophotometry to measure the yellowish complex compound generated after the reaction between hydrogen peroxide and ammonium molybdate. Here, 1 CAT enzyme activity unit referred to the amount of enzyme needed to degrade 1 mmol hydrogen peroxide per second. Spectrophotometry was also used to determine total GST activity, by measuring the compound produced from the conjugating reaction between 1-Chloro-2,4-dinitrobenzene (CDNB) and GSH. For this, 1 GST enzyme activity unit represented the amount of enzyme catalyzing the conjugation between 1 × 10−12 mol CDNB and GSH per min. The concentration of total protein in the supernatant was quantified by BCA method (Dodd and Drickamer, 2001). SOD, CAT and GST activities in the supernatant fraction of the samples were the ratio of the total enzyme activity unit to the total protein, and the results were expressed as U mg−1 protein.
2. Materials and methods 2.1. Coral Colonies of the stony coral P. damicornis were collected from a coral reef in Wenchang, Hainan Province, China, and transferred and cultured in flow-through aquaria (ca. 100 L) filled with filtered seawater (From the same sea area, 26 °C) in a facility located at Hainan University. Cultures were illuminated with two white and two blue cool fluorescent bulbs (Philips T5HO Activiva Active 54 W) in a 12 h/12 h light-dark cycle for one month to acclimatize in laboratory conditions.
2.5. Activity assay of caspase3 The caspase3 activity in the supernatant was measured by Caspase-3 Colorimetric Assay Kit (KeyGEN BioTECH) according to the manufacturer’s protocol. Briefly, 50 μL reaction buffer and 5 μL substrate were added in 50 μL supernatant, followed by incubation in the dark at 37 °C for 4 h. Then, the color change was measured spectrophotometrically at the wavelength of 405 nm. The activity of caspase3 was defined as the absorbance of the reaction solution at 405 nm (ABS405), and its activation level of caspase3 in coral nubbins was defined as the ratio of ABS405 in samples to that of the blank group.
2.2. Cadmium stress exposure A total of 50 coral nubbins were used for the cadmium stress experiment. Cadmium chloride (CdCl2) was added into the filtered seawater to prepare an elevated cadmium seawater with a final concentration of 110 nmol L−1 (20 μg L−1). Then, 20 of the coral nubbins were transferred into the seawater with elevated cadmium, hereafter referred to as the cadmium group, while another 20 were used as the control group which were incubated only in the filtered seawater, and the remaining 10 were the blank group. Coral nubbins were randomly sampled in the cadmium and control groups after 6, 12 and 24 h incubation, while the blank group was randomly sampled at 0 h. The 6 nubbins each from the cadmium and control groups after 12 h exposure were immediately stored in liquid nitrogen for subsequent RNA extraction and transcriptome sequencing. Meanwhile, 5 nubbins were prepared from each group at each time point and used for evaluating the density of symbiotic zooxanthellae, activity of crucial enzymes and the concentration of GSH.
2.6. Determination of GSH concentration The concentration of GSH in the supernatant was measured by the total glutathione assay kit (JIANCHENG, A061) as suggested in the product instructions. The total-GSH level (μmol L−1) was determined by the dithionitrobenzene or DTNB recycling reaction assay using the plate reader (TECAN, infinite F50) (Li et al., 2015; Ye et al., 2009). The GSH concentration in supernatant was defined as the ratio of total-GSH level to the protein concentration, and the results were expressed as μmol mg−1 protein. 2.7. Deep sequencing and reconstruction of P. damicornis transcriptome
2.3. The density of symbiotic zooxanthellae
Total RNA was isolated from each coral sample following the Trizol reagent protocol. The extracted total RNA was quantified by Nanodrop 2000 (Thermo Scientific) at 260/280 nm (ratio ≫ > 2.0) and its integrity was checked with Angilent 2100 Bioanalyzer (Agilent Technologies). The single-end fragment library (50 bp) was constructed
Variation in the density of symbiotic zooxanthellae after cadmium stress was determined by the method described by Higuchi et al. with few modifications (Higuchi et al., 2008; Higuchi et al., 2015). Briefly, 133
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and sequenced on the BGISEQ-500 platform according to the manufacturer’s instructions (BGI, Shenzhen, China). The generated raw sequencing reads were deposited at the NCBI Short Read Archive (SRA) under Accession No. SRP116721. 2.8. Reads mapping and identification of differentially expressed genes Raw reads were first processed using the Fastx-toolkit pipeline (http://hannonlab.cshl.edu/fastx_toolkit/index.html) to summarize data production, evaluate sequencing quality, and remove low quality reads and adaptor sequences. Quality filtration removed sequences in which 80% of the base pairs had a Phred score of less than 20, while adaptor sequences were removed using the trimmer package. A transcriptome of P. damicornis had been assembled in our previous study, where the coral-specific transcripts were further identified using the BLAST software (Yuan et al., 2017). A total of 77,199 transcripts from 42,399 coral genes were obtained, which then served as the reference sequences to map back the raw reads. The alignment of single-end reads was performed using the HISAT2 software (Pertea et al., 2016). StringTie and DESeq2 software were used to estimate transcript abundances and identify differentially expressed genes between the cadmium and control groups (Love et al., 2014).
Fig. 1. Density variation of the symbiotic zooxanthellae in the stony coral Pocillopora damicornis after acute cadmium stress. Vertical bars represent the mean ± S.D. (N = 5), and bars with different letters are significantly different (P < 0.05).
level (7.0 U mg−1, P < 0.05) observed at 6 h after cadmium stress (Fig. 2B). Furthermore, there was no significant difference in the activities of SOD and CAT between the control and blank groups for the entire duration of the experiment.
2.9. GO overrepresentation of differentially expressed genes Functional annotation and Gene Ontology (GO) term assignment of coral genes were completed using Trinotate 3.0.1 software (http:// trinotate.github.io/). After identifying of differentially expressed genes, the lists of significantly upregulated and downregulated genes were generated. GO overrepresentation analysis was implemented via the hypergeometric test with filter value of 0.05. The differentially expressed genes were selected as test set, while all genes were used as the reference set. The BiNGO tool was employed to calculate the overrepresented GO terms in the network and displayed them as a network of significant GO terms (Maere et al., 2005).
3.3. Effects of cadmium stress on the GST activity and GSH concentration GST activities in the cadmium group did not change significantly during the experiment, compared to those in the blank and control groups (Fig. 3). However, The GSH concentration in the cadmium group began to decrease significantly, and reached the lowest level (0.043 μmol mg−1, P < 0.05) at 12 h after exposure to elevated cadmium. The trend of the significant decrease continued into 24 h after cadmium stress (Fig. 4).
2.10. Statistical analysis
3.4. Effects of cadmium stress on the caspase3 activation level
All data was presented as means ± standard deviation (SD). All of the data was subjected to one-way analysis of variance (one-way ANOVA) followed by multiple comparison (S-N-K) using SPSS v22.0 (SPSS Inc., Chicago, Illinois) to determine significant differences among the treatments and controls. Differences were considered significant at P < 0.05.
The activation level of caspase3 increased significantly in the stony coral P. damicornis after cadmium stress. It reached the highest level at 24 h (1.5-fold, P < 0.05), which was significantly higher than those in the blank and control groups (Fig. 5), while no significant difference was observed at the other time points. Furthermore, there was no significant difference in the caspase3 activation level between the control and blank groups.
3. Result 3.5. Gene expression 3.1. Temporal variation in the density of symbiotic zooxanthellae after cadmium stress
After filtering of low-quality and adaptor sequences, there was a total of 182,683,827 single-end reads obtained from all six transcriptome libraries. The number of reads in each library is summarized in Table 1. The high-quality reads were processed in HISAT2 software for alignment using the reference sequences built from the previously assembled coral transcripts. The mapped rates ranged from 23.97 to 41.03% for the 6 libraries (Table. 1). Gene expression abundance was analyzed using StringTie software, and the expression level of each gene was calculated following the Fragments Per Kilobase of exon per Million fragments mapped (FPKM) method. The counts of mapped reads in each gene were further submitted to the DESeq2 software to identify differentially expressed genes (Table S1).
The density of symbiotic zooxanthellae began to decrease significantly (3.9 × 105 cell cm−2, P < 0.05) at 12 h, and reached the minimum level (3.3 × 105 cell cm−2, P < 0.05) at 24 h after cadmium exposure (Fig. 1). No significant differences were detected in the density of symbiotic zooxanthellae between the control group and blank group during the whole experiment. 3.2. Effects of cadmium stress on the SOD and CAT activities The activities of both SOD and CAT decreased significantly in the stony coral P. damicornis after cadmium stress. Specifically, the SOD activities in the cadmium group were significantly lower (106.2 U mg−1, P < 0.05) after 12 h of stress exposure, in comparison with those in the blank and control groups (Fig. 2A). In contrast, CAT levels in the cadmium group became significantly lower than those in the blank and control groups during the 6–12 h period, with the lowest
3.6. Identification of differentially expressed genes The differences in expression of coral genes were analyzed with the DESeq2 software. After library calibration, the expression levels of coral genes were compared between the cadmium (treatment) and control 134
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Fig. 2. Temporal activities of the redox-related enzymes in the stony coral Pocillopora damicornis after acute cadmium stress. (A) Superoxide dismutase (SOD). (B) Catalase (CAT). Vertical bars represent the mean ± S.D. (N = 5), and bars with different letters are significantly different (P < 0.05).
Fig. 3. Temporal activities of glutathione S-transferase (GST) in the stony coral Pocillopora damicornis after acute cadmium stress. Vertical bars represent the mean ± S.D. (N = 5), and no significant difference was observed between groups.
Fig. 5. Temporal activities of caspase3 in the stony coral Pocillopora damicornis after acute cadmium stress. Vertical bars represent the mean ± S.D. (N = 5), and bars with different letters are significantly different (P < 0.05).
Table 1 Transcriptome mapping statics. Library
Total reads
Mapped reads
Mapped rate
Control_1 Control_2 Control_3 Cd_1 Cd_2 Cd_3
30,846,679 31,226,383 29,244,058 30,777,208 31,344,353 29,245,146
8,604,985 7,485,209 8,471,962 12,263,934 12,586,678 11,999,165
27.90% 23.97% 28.97% 39.85% 40.16% 41.03%
3.7. Functional annotation of the differentially expressed genes after cadmium stress The expression levels of 3538 genes in the cadmium group were higher than those in the control group, while the expression levels of 8048 genes were significantly lower (FDR < 0.05). The GO overrepresentation of these differentially expressed genes in the control/ cadmium comparison was further analyzed at multiple GO levels in the Biological Process category. For the 3538 significantly upregulated genes, there were 12 major overrepresented GO terms (Table S3, Fig. 6). These overrepresented GO terms were mainly related to protein modification, unfolded protein response, endoplasmic reticulum stress and apoptosis. Furthermore, a total of 32 overrepresented GO terms were detected for 8048 significantly downregulated genes (Table S3, Fig. 7), which were mainly
Fig. 4. Changes in glutathione (GSH) concentration in the stony coral Pocillopora damicornis after acute cadmium stress. Vertical bars represent the mean ± S.D. (N = 5), and bars with different letters are significantly different (P < 0.05).
groups, which revealed 11,586 differentially expressed genes (Table S2). Two lists of these differentially expressed genes were obtained, namely for significantly upregulated and downregulated genes containing 3538 and 8048 differentially expressed genes, respectively.
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Fig. 6. Overrepresented GO terms of the 3538 significantly upregulated genes in the cadmium group at 12 h after acute cadmium stress. A total of 12 GO terms were overrepresented for these significantly upregulated genes. “Ref” referred to the proportion of genes annotated to a GO term in all reference gene of the stony coral Pocillopora damicornis, while “test” referred to the proportion of genes annotated to that GO term in all significantly upregulated genes.
cadmium stress could lead to coral bleaching, and even death, which could be the same thing happening in the stony coral P. damicornis. To systematically understand the response of stony corals towards acute cadmium stress and the underlying mechanisms behind symbiosis collapse, crucial physiological parameters representing the detoxification, redox and apoptosis systems were monitored in the stony coral P. damicornis. The temporal variation in GST activity was determined upon exposure to cadmium, which was the phase II metabolic enzymes in the detoxification process of all eukaryotes (Nicosia et al., 2014). We observed that the GST activity did not change significantly before and after exposure to the heavy metal, demonstrating that acute cadmium stress (20 μg L−1) could not trigger the detoxification system in the stony coral P. damicornis. Several studies reported that cnidarians were able to accumulate heavy metal in tissues and skeletons, especially cadmium (Metian et al., 2015; Mohammed and Dar, 2010). The ability to bioaccumulate allows the cnidarians to tolerate high levels of cadmium. For example, the 96 h LC50 and 6 h EC50 reached as high as 946 and 355 μg L−1 for cadmium in the anemone Aiptasia pulchella, a usual cnidarian model for ecotoxicological studies in tropical marine environments (Howe et al., 2014). Similarly, tolerance against heavy metals could have accounted for the insignificant change of GST activity in the detoxification system of the stony coral P. damicornis. This then suggests that although cadmium did not directly affect the physiology of the coral, its accumulation however decreased the symbiotic zooxanthellae, which could result to bleaching and then to death in the stony coral P. damicornis. Changes in the activity of SOD and CAT were also explored after acute cadmium stress, which were the main antioxidases in the redox system. Results showed that both SOD and CAT activities decreased significantly compared to those in the blank and control groups. This suggests that the high cadmium level (20 μg L−1) could suppress the antioxidant abilities of the stony coral P. damicornis. However, it was different from the previous observations where the increased ROS level induced the expressions and activities of antioxidases under high
correlated with the metabolic processes and macromolecule localization. 4. Discussion Coral reef ecosystems are increasingly being impacted by a wide variety of anthropogenic pollution, including heavy metal accumulation, which could be contributing to coral reef stress leading to bleaching episodes (Brock and Bielmyer, 2013; Mitchelmore et al., 2007). Coral bleaching is characterized by the collapse of coral-zooxanthella symbiosis and the decrease in symbiotic zooxanthellae (Ganot et al., 2011). In the present study, the systemic responses of stony corals towards acute cadmium stress, focusing on P. damicornis, were investigated to reveal its adaptation mechanisms. The change in the density of symbiotic zooxanthellae was determined first to understand the balance of coral-zooxanthella symbiosis after cadmium stress. Results showed that it decreased significantly during 12–24 h period after exposure to elevated cadmium, and reached the lowest level after 24 h. This demonstrates that an acute stress from cadmium (20 μg L−1) could induce a significant decrease of symbiotic zooxanthellae in the stony coral P. damicornis. This however was inconsistent with previous observations, which reported that the density of symbiotic zooxanthellae did not change significantly after cadmium stress (Mitchelmore et al., 2007). The disparity in the observations could have resulted from the differences in cadmium concentrations used and the sampling time, since the insignificant change was observed between 4 and 14 days after exposure to cadmium (5 μg L−1). The present results demonstrated that acute cadmium stress could lead to the collapse of coralzooxanthella symbiosis and the expulsion of symbiotic zooxanthellae. The reason for the collapse after cadmium stress might be similar to the response after heat stress, attributed to the changes in the internal environment of the stony coral, such as the increase of ROS level (Lesser, 1997), the activation of apoptosis (Tchernov et al., 2011), and so on. The continued decrease of symbiotic zooxanthellae after acute 136
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Fig. 7. Overrepresented GO terms of the 8048 significantly downregulated genes in the cadmium group at 12 h after acute cadmium stress. A total of 32 GO terms were overrepresented for these significantly downregulated genes. “Ref” referred to the proportion of genes annotated to a GO term in all reference gene of the stony coral Pocillopora damicornis, while “test” referred to the proportion of genes annotated to that GO term in all significantly downregulated genes.
caspase3 increased significantly after 24 h exposure to cadmium stress, demonstrating that cadmium also induced apoptosis in the stony coral P. damicornis. Apoptosis was also observed when the same stony coral P. damicornis suffered from heat and ammonium stresses (Tchernov et al., 2011; Yuan et al., 2017). Specifically, heat stress could induce apoptosis through TNF signal pathway and caspase3 in the stony coral (Quistad et al., 2014; Zhou et al., 2017). In the present study, apoptosis was induced after cadmium stress because of the upregulated oxidative stressors in the internal environment through the repression of antioxidant production. An example of this is the accumulation of nitric oxide, which then induced apoptosis through caspase3 also in the stony coral P. damicornis (Hawkins et al., 2014). Apoptosis would ultimately result to the collapse of the symbiotic balance between coral and zooxanthellae, and lead to the expulsion of symbiotic zooxanthellae causing bleaching. To have a deeper insight into the molecular mechanisms underlying the response of coral to elevated cadmium, we sequenced the transcriptome of the stony coral P. damicornis at 12 h after acute cadmium stress exposure. A total of 3538 significantly upregulated genes and 8048 significantly downregulated genes were obtained. Further, inspection of the overrepresented GO terms revealed that significantly upregulated genes were mainly related to protein modification,
temperature, air exposure or other stressful conditions (Levy et al., 2006; Teixeira et al., 2013; Yakovleva et al., 2004). The significant decreases of SOD and CAT activities in the present study could have been a result of the change in their expression or structure under cadmium stress. Previous studies reported that cadmium could change the conformation of SOD protein to decrease its enzyme activity, and cause oxidative stress-induced neural cell apoptosis (Huang et al., 2006). It was also reported that cadmium stress induced the expression level of CAT mRNA, but suppressed its activity and decreased its protein content in Pisum sativum (Romero-Puertas et al., 2007). Furthermore, the significant decline of GSH concentration supports the hypothesis that the antioxidant ability was suppressed by acute cadmium stress in the stony coral P. damicornis. Environmental stressors generally induce ROS levels in the internal environment of the organism: therefore, the decline of antioxidant production implied that the oxidative stress would become stronger under cadmium stress. This could even trigger apoptosis, collapse in the symbiosis, and bleaching of the stony coral (Baird et al., 2009; Lesser, 1997). Caspase3 is the key executory enzyme and final effector for apoptosis to occur (Hengartner, 2000). Consequently, we surveyed the activation level of caspase3 to understand the apoptosis status of the stony coral P. damicornis after acute cadmium stress. The activation level of 137
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unfolded protein response, endoplasmic reticulum stress and apoptosis. The unfolded protein response was also observed in two species of Acropora after heat stress (Ruiz-Jones and Palumbi, 2017; TraylorKnowles et al., 2017). In this study, the said response could account for the upward oxidative stress in the body of the organisms by repressing production of antioxidants, since ROS is known to induce the emergence of unfolded or misfolded proteins (Runkel et al., 2013). These unfolded or misfolded proteins would accumulate in the lumen of the endoplasmic reticulum and activate the unfolded protein response. If these proteins are not repaired within a certain time span or the disruption is prolonged, the unfolded protein response would lead to apoptosis (Erguler et al., 2013). These studies corroborate our observation on the physiological activities during stressful conditions in the stony coral P. damicornis. On the other hand, the 32 overrepresented GO terms for significantly downregulated genes correlated well with the metabolic processes involving macromolecules, especially for nucleic acid. This further demonstrated that gene expressions could be regulated positively during acute cadmium stress, and mRNA transcription in the stony coral P. damicornis would be remodeled under elevated cadmium to maintain homeostasis and improve adaptability
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Acknowledgements The authors were grateful to all of the laboratory members for their continuous technical advice and helpful discussions. This research was supported by a grant (No. 31772460) from National Science Foundation of China, Natural Science Foundation (No. 20164158) from Hainan Province, and the Scientific Research Foundation (kyqd1554) from Hainan University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox.2017.11.013. References Baird, A.H., Bhagooli, R., Ralph, P.J., Takahashi, S., 2009. Coral bleaching: the role of the host. Trends Ecol. Evol. 24, 16–20. Barakat, S.A., Al-Rousan, S., Al-Trabeen, M.S., 2015. Use of scleractinian corals to indicate marine pollution in the northern Gulf of Aqaba Jordan. Environ. Monit. Assess. 187. Brock, J.R., Bielmyer, G.K., 2013. Metal accumulation and sublethal effects in the sea anemone Aiptasia pallida, after waterborne exposure to metal mixtures. Comp. Biochem. Physiol. Toxicol. Pharmacol. CBP 158, 150–158. Chen, T.R., Yu, K.F., Li, S., Price, G.J., Shi, Q., Wei, G.J., 2010. Heavy metal pollution recorded in porites corals from daya bay, northern south China sea. Mar. Environ. Res. 70, 318–326. Coles, S.L., Brown, B.E., 2003. Coral bleaching–capacity for acclimatization and adaptation. Adv. Mar. Biol. 46, 183–223. Darvishnia, Z., Bakhtiari, A.R., Kamrani, E., Sajjadi, M.M., 2016. Bioaccumulation of Fe, Zn and Pb in faviidae and poritidae corals from Qeshm Island, Persian Gulf, Iran. Indian J. Geo. Mar. Sci. 45, 1727–1732. Dodd, R.B., Drickamer, K., 2001. Lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity. Glycobiology 11, 71R–79R. Downs, C.A., Fauth, J.E., Halas, J.C., Dustan, P., Bemiss, J., Woodley, C.M., 2002. Oxidative stress and seasonal coral bleaching. Free Radic. Biol. Med. 33, 533–543. Erguler, K., Pieri, M., Deltas, C., 2013. A mathematical model of the unfolded protein stress response reveals the decision mechanism for recovery, adaptation and apoptosis. BMC Syst. Biol. 7, 16. Esslemont, G., Harriott, V.J., McConchie, D.M., 2000. Variability of trace-metal concentrations within and between colonies of Pocillopora damicornis. Mar. Pollut. Bull. 40, 637–642. Ganot, P., Moya, A., Magnone, V., Allemand, D., Furla, P., Sabourault, C., 2011. Adaptations to endosymbiosis in a cnidarian-dinoflagellate association: differential gene expression and specific gene duplications. PLoS Genet. 7, e1002187. Goreau, T.J., Macfarlane, A.H., 1990. Reduced growth rate of Montastrea annularis following the 1987–1988 coral-bleaching event. Coral Reefs 8, 211–215. Guzmán, H.M., Jiménez, C.E., 1992. Contamination of coral reefs by heavy metals along the Caribbean coast of Central America (Costa Rica and Panama). Mar. Pollut. Bull. 24, 554–561. Harland, A.D., Bryan, G.W., Brown, B.E., 2009. Zinc and cadmium absorption in the symbiotic anemone Anemonia viridis and the non-symbiotic anemone Actinia equina. J. Mar. Biol. Assoc. U. K. 70, 789.
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Systemic response of the stony coral Pocillopora damicornis against acute cadmium stress Zhi Zhoua,b,c,
⁎,1
T
, Xiaopeng Yua,1, Jia Tanga, Yibo Wua, Lingui Wanga,b,c, Bo Huanga,c
a
Key Laboratory of Tropical Biological Resources of Ministry of Education, Hainan University, Haikou, Hainan, 570228, China State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, Hainan 570228, China c Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of Marine Science, Hainan University, Haikou, Hainan 570228, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Symbiosis Detoxification Antioxidation Apoptosis Coral
Heavy metals have become one of the main pollutants in the marine environment and a major threat to the growth and reproduction of stony corals. In the present study, the density of symbiotic zooxanthellae, levels of crucial physiological activities and the transcriptome were investigated in the stony coral Pocillopora damicornis after the acute exposure to elevated cadmium concentration. The density of symbiotic zooxanthellae decreased significantly during 12–24 h period, and reached lowest at 24 h after acute cadmium stress. No significant changes were observed in the activity of glutathione S-transferase during the entire stress exposure. The activities of superoxide dismutase and catalase, and the concentration of glutathione decreased significantly, but the activation level of caspase3 increased significantly after cadmium exposure. Furthermore, transcriptome sequencing and bioinformatics analysis revealed 3538 significantly upregulated genes and 8048 significantly downregulated genes at 12 h after the treatment. There were 12 overrepresented GO terms for significantly upregulated genes, mostly related to unfolded protein response, endoplasmic reticulum stress and apoptosis. In addition, a total of 32 GO terms were overrepresented for significantly downregulated genes, and mainly correlated with macromolecular metabolic processes. These results collectively suggest that acute cadmium stress could induce apoptosis by repressing the production of the antioxidants, elevating oxidative stress and activating the unfolded protein response. This cascade of reactions would result to the collapse of the coral-zooxanthella symbiosis and the expulsion of symbiotic zooxanthellae in the stony coral P. damicornis, ultimately leading to coral bleaching.
1. Introduction Stony corals are unique creatures living in shallow waters with relatively low nutrient levels and good illumination conditions (Zhang et al., 2015). They compose most of the great reef ecosystems in tropical seas, and heavily depend on symbiotic relationships with zooxanthellae, specifically with the dinoflagellate Symbiodinium (Tong et al., 2017). The zooxanthella lives within the intracellular vacuoles of the gastrodermal (endodermal) tissue, and provides the coral with nutrients through photosynthesis, a crucial energy source in an otherwise clear and nutrient-poor tropical waters (Ganot et al., 2011). In exchange, the coral provides the zooxanthellae with the carbon dioxide and ammonium needed for photosynthesis (Shinzato et al., 2011). However, owing to this distinct symbiosis, stony corals are more sensitive to changes in the marine environment, and exhibit systemic responses to environmental stress (Parkinson and Baums, 2014). Because of climate
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1
change and environmental pollution, stony corals have been subjected to frequent and severe diseases over the past decades (Vidal-Dupiol et al., 2011). The increasing mean annual sea surface temperature due to climate change and worsening marine pollution are both threats to the survival and reproduction of stony corals. During warming periods for example, stony corals undergo “bleaching” as a form of morphological and physiological response (Coles and Brown, 2003), a process where the host corals either lose their symbiotic zooxanthellae, or the zooxanthellae’s photosynthetic pigments degrade. The resulting collapse of the symbiosis between the coral and zooxanthellae causes decreased coral adaptation and increased mortality (Goreau and Macfarlane, 1990). Studies suggested that oxidative damage due to an excess of respiratory burst and reactive oxygen species (ROS) could induce apoptosis, cell death and tissue abscission of stony coral, and finally result to coral bleaching (Downs et al., 2002; Lesser, 1997).
Corresponding author at: 58 Renmin Road, Haikou 570228, China. E-mail address: [email protected] (Z. Zhou). These authors contributed equally to this work.
https://doi.org/10.1016/j.aquatox.2017.11.013 Received 8 September 2017; Received in revised form 21 November 2017; Accepted 22 November 2017 Available online 23 November 2017 0166-445X/ © 2017 Elsevier B.V. All rights reserved.
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tissue homogenates were prepared using Waterpik water jet to strip the tissue from the coral skeleton into approximately 10 mL filtered seawater (0.22 μm). The homogenates were then centrifuged at 1500g, 4 °C for 10 min, and the supernatants were used to determine enzyme activities and GSH concentration. The symbiotic zooxanthellae were resuspended in the filtered seawater, and counted using a Neubauer hemocytometer (QIUJING, China). The surface area of the nubbins used were determined by the aluminum foil method (Johannes et al., 1970). The density of symbiotic zooxanthellae was defined as the ratio of the number of symbiotic zooxanthellae to the surface area of coral nubbins (cell cm−2).
Heavy metals are one of the main marine pollutants mainly coming from industrial discharges, urban/agricultural run-off and anti-fouling paints. Its distribution and effects on coral reefs have recently received considerable attention (Hudspith et al., 2017; Morton and Blackmore, 2001). Heavy metal intoxications have been observed in the majority of coral reefs worldwide (Chen et al., 2010; Guzmán and Jiménez, 1992; Haynes and Johnson, 2000; Horwitz et al., 2014). Stony corals are able to absorb heavy metals from the environment, and accumulate them in their tissues and skeleton through mineralization (Barakat et al., 2015; Darvishnia et al., 2016; Esslemont et al., 2000; Harland et al., 2009). While some trace heavy metals such as strontium are essential to the growth and reproduction of stony corals (Meibom et al., 2003), high levels of other heavy metals such as copper, cadmium, lead, zinc and nickel have been reported to effect fertilization success, glutathione (GSH) levels, tissue integrity, bleaching and death of some stony coral species (Mitchelmore et al., 2003; Mitchelmore et al., 2007; ReicheltBrushett and Harrison, 1999, 2005; Sabdono, 2009). However, despite the high threat to heavy metal intoxication in most coastal environments, the molecular mechanisms underlying the toxic effects of heavy metals on stony coral remains little understood. Pocillopora damicornis is a species of stony coral in the family Pocilloporidae, and is native to tropical and subtropical parts of the Indian and Pacific Oceans. To understand the potential effects of heavy metal stress on coral symbiosis and physiology, the density of symbiotic zooxanthellae, the crucial factors to physiological activities, and the transcriptome of coral P. damicornis were investigated after the acute exposure to elevated cadmium. The present study would provide insights to further understand the molecular mechanisms underlying stress response and environmental adaption of stony corals.
2.4. Activity assay of antioxidases and detoxification enzyme The activities of superoxide dismutase (SOD, JIANCHENG, A001), catalase (CAT, JIANCHENG, A007) and glutathione S-transferase (GST, Solarbio, BC0350) in the supernatants were measured using commercial kits, following the manufacturer’s recommendations. Total SOD activity was determined by the hydroxylamine method (Kono, 1978), where 1 SOD activity unit was defined as the enzyme amount causing 50% inhibition in 1 mL reaction solution. Total CAT activity was determined using spectrophotometry to measure the yellowish complex compound generated after the reaction between hydrogen peroxide and ammonium molybdate. Here, 1 CAT enzyme activity unit referred to the amount of enzyme needed to degrade 1 mmol hydrogen peroxide per second. Spectrophotometry was also used to determine total GST activity, by measuring the compound produced from the conjugating reaction between 1-Chloro-2,4-dinitrobenzene (CDNB) and GSH. For this, 1 GST enzyme activity unit represented the amount of enzyme catalyzing the conjugation between 1 × 10−12 mol CDNB and GSH per min. The concentration of total protein in the supernatant was quantified by BCA method (Dodd and Drickamer, 2001). SOD, CAT and GST activities in the supernatant fraction of the samples were the ratio of the total enzyme activity unit to the total protein, and the results were expressed as U mg−1 protein.
2. Materials and methods 2.1. Coral Colonies of the stony coral P. damicornis were collected from a coral reef in Wenchang, Hainan Province, China, and transferred and cultured in flow-through aquaria (ca. 100 L) filled with filtered seawater (From the same sea area, 26 °C) in a facility located at Hainan University. Cultures were illuminated with two white and two blue cool fluorescent bulbs (Philips T5HO Activiva Active 54 W) in a 12 h/12 h light-dark cycle for one month to acclimatize in laboratory conditions.
2.5. Activity assay of caspase3 The caspase3 activity in the supernatant was measured by Caspase-3 Colorimetric Assay Kit (KeyGEN BioTECH) according to the manufacturer’s protocol. Briefly, 50 μL reaction buffer and 5 μL substrate were added in 50 μL supernatant, followed by incubation in the dark at 37 °C for 4 h. Then, the color change was measured spectrophotometrically at the wavelength of 405 nm. The activity of caspase3 was defined as the absorbance of the reaction solution at 405 nm (ABS405), and its activation level of caspase3 in coral nubbins was defined as the ratio of ABS405 in samples to that of the blank group.
2.2. Cadmium stress exposure A total of 50 coral nubbins were used for the cadmium stress experiment. Cadmium chloride (CdCl2) was added into the filtered seawater to prepare an elevated cadmium seawater with a final concentration of 110 nmol L−1 (20 μg L−1). Then, 20 of the coral nubbins were transferred into the seawater with elevated cadmium, hereafter referred to as the cadmium group, while another 20 were used as the control group which were incubated only in the filtered seawater, and the remaining 10 were the blank group. Coral nubbins were randomly sampled in the cadmium and control groups after 6, 12 and 24 h incubation, while the blank group was randomly sampled at 0 h. The 6 nubbins each from the cadmium and control groups after 12 h exposure were immediately stored in liquid nitrogen for subsequent RNA extraction and transcriptome sequencing. Meanwhile, 5 nubbins were prepared from each group at each time point and used for evaluating the density of symbiotic zooxanthellae, activity of crucial enzymes and the concentration of GSH.
2.6. Determination of GSH concentration The concentration of GSH in the supernatant was measured by the total glutathione assay kit (JIANCHENG, A061) as suggested in the product instructions. The total-GSH level (μmol L−1) was determined by the dithionitrobenzene or DTNB recycling reaction assay using the plate reader (TECAN, infinite F50) (Li et al., 2015; Ye et al., 2009). The GSH concentration in supernatant was defined as the ratio of total-GSH level to the protein concentration, and the results were expressed as μmol mg−1 protein. 2.7. Deep sequencing and reconstruction of P. damicornis transcriptome
2.3. The density of symbiotic zooxanthellae
Total RNA was isolated from each coral sample following the Trizol reagent protocol. The extracted total RNA was quantified by Nanodrop 2000 (Thermo Scientific) at 260/280 nm (ratio ≫ > 2.0) and its integrity was checked with Angilent 2100 Bioanalyzer (Agilent Technologies). The single-end fragment library (50 bp) was constructed
Variation in the density of symbiotic zooxanthellae after cadmium stress was determined by the method described by Higuchi et al. with few modifications (Higuchi et al., 2008; Higuchi et al., 2015). Briefly, 133
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and sequenced on the BGISEQ-500 platform according to the manufacturer’s instructions (BGI, Shenzhen, China). The generated raw sequencing reads were deposited at the NCBI Short Read Archive (SRA) under Accession No. SRP116721. 2.8. Reads mapping and identification of differentially expressed genes Raw reads were first processed using the Fastx-toolkit pipeline (http://hannonlab.cshl.edu/fastx_toolkit/index.html) to summarize data production, evaluate sequencing quality, and remove low quality reads and adaptor sequences. Quality filtration removed sequences in which 80% of the base pairs had a Phred score of less than 20, while adaptor sequences were removed using the trimmer package. A transcriptome of P. damicornis had been assembled in our previous study, where the coral-specific transcripts were further identified using the BLAST software (Yuan et al., 2017). A total of 77,199 transcripts from 42,399 coral genes were obtained, which then served as the reference sequences to map back the raw reads. The alignment of single-end reads was performed using the HISAT2 software (Pertea et al., 2016). StringTie and DESeq2 software were used to estimate transcript abundances and identify differentially expressed genes between the cadmium and control groups (Love et al., 2014).
Fig. 1. Density variation of the symbiotic zooxanthellae in the stony coral Pocillopora damicornis after acute cadmium stress. Vertical bars represent the mean ± S.D. (N = 5), and bars with different letters are significantly different (P < 0.05).
level (7.0 U mg−1, P < 0.05) observed at 6 h after cadmium stress (Fig. 2B). Furthermore, there was no significant difference in the activities of SOD and CAT between the control and blank groups for the entire duration of the experiment.
2.9. GO overrepresentation of differentially expressed genes Functional annotation and Gene Ontology (GO) term assignment of coral genes were completed using Trinotate 3.0.1 software (http:// trinotate.github.io/). After identifying of differentially expressed genes, the lists of significantly upregulated and downregulated genes were generated. GO overrepresentation analysis was implemented via the hypergeometric test with filter value of 0.05. The differentially expressed genes were selected as test set, while all genes were used as the reference set. The BiNGO tool was employed to calculate the overrepresented GO terms in the network and displayed them as a network of significant GO terms (Maere et al., 2005).
3.3. Effects of cadmium stress on the GST activity and GSH concentration GST activities in the cadmium group did not change significantly during the experiment, compared to those in the blank and control groups (Fig. 3). However, The GSH concentration in the cadmium group began to decrease significantly, and reached the lowest level (0.043 μmol mg−1, P < 0.05) at 12 h after exposure to elevated cadmium. The trend of the significant decrease continued into 24 h after cadmium stress (Fig. 4).
2.10. Statistical analysis
3.4. Effects of cadmium stress on the caspase3 activation level
All data was presented as means ± standard deviation (SD). All of the data was subjected to one-way analysis of variance (one-way ANOVA) followed by multiple comparison (S-N-K) using SPSS v22.0 (SPSS Inc., Chicago, Illinois) to determine significant differences among the treatments and controls. Differences were considered significant at P < 0.05.
The activation level of caspase3 increased significantly in the stony coral P. damicornis after cadmium stress. It reached the highest level at 24 h (1.5-fold, P < 0.05), which was significantly higher than those in the blank and control groups (Fig. 5), while no significant difference was observed at the other time points. Furthermore, there was no significant difference in the caspase3 activation level between the control and blank groups.
3. Result 3.5. Gene expression 3.1. Temporal variation in the density of symbiotic zooxanthellae after cadmium stress
After filtering of low-quality and adaptor sequences, there was a total of 182,683,827 single-end reads obtained from all six transcriptome libraries. The number of reads in each library is summarized in Table 1. The high-quality reads were processed in HISAT2 software for alignment using the reference sequences built from the previously assembled coral transcripts. The mapped rates ranged from 23.97 to 41.03% for the 6 libraries (Table. 1). Gene expression abundance was analyzed using StringTie software, and the expression level of each gene was calculated following the Fragments Per Kilobase of exon per Million fragments mapped (FPKM) method. The counts of mapped reads in each gene were further submitted to the DESeq2 software to identify differentially expressed genes (Table S1).
The density of symbiotic zooxanthellae began to decrease significantly (3.9 × 105 cell cm−2, P < 0.05) at 12 h, and reached the minimum level (3.3 × 105 cell cm−2, P < 0.05) at 24 h after cadmium exposure (Fig. 1). No significant differences were detected in the density of symbiotic zooxanthellae between the control group and blank group during the whole experiment. 3.2. Effects of cadmium stress on the SOD and CAT activities The activities of both SOD and CAT decreased significantly in the stony coral P. damicornis after cadmium stress. Specifically, the SOD activities in the cadmium group were significantly lower (106.2 U mg−1, P < 0.05) after 12 h of stress exposure, in comparison with those in the blank and control groups (Fig. 2A). In contrast, CAT levels in the cadmium group became significantly lower than those in the blank and control groups during the 6–12 h period, with the lowest
3.6. Identification of differentially expressed genes The differences in expression of coral genes were analyzed with the DESeq2 software. After library calibration, the expression levels of coral genes were compared between the cadmium (treatment) and control 134
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Fig. 2. Temporal activities of the redox-related enzymes in the stony coral Pocillopora damicornis after acute cadmium stress. (A) Superoxide dismutase (SOD). (B) Catalase (CAT). Vertical bars represent the mean ± S.D. (N = 5), and bars with different letters are significantly different (P < 0.05).
Fig. 3. Temporal activities of glutathione S-transferase (GST) in the stony coral Pocillopora damicornis after acute cadmium stress. Vertical bars represent the mean ± S.D. (N = 5), and no significant difference was observed between groups.
Fig. 5. Temporal activities of caspase3 in the stony coral Pocillopora damicornis after acute cadmium stress. Vertical bars represent the mean ± S.D. (N = 5), and bars with different letters are significantly different (P < 0.05).
Table 1 Transcriptome mapping statics. Library
Total reads
Mapped reads
Mapped rate
Control_1 Control_2 Control_3 Cd_1 Cd_2 Cd_3
30,846,679 31,226,383 29,244,058 30,777,208 31,344,353 29,245,146
8,604,985 7,485,209 8,471,962 12,263,934 12,586,678 11,999,165
27.90% 23.97% 28.97% 39.85% 40.16% 41.03%
3.7. Functional annotation of the differentially expressed genes after cadmium stress The expression levels of 3538 genes in the cadmium group were higher than those in the control group, while the expression levels of 8048 genes were significantly lower (FDR < 0.05). The GO overrepresentation of these differentially expressed genes in the control/ cadmium comparison was further analyzed at multiple GO levels in the Biological Process category. For the 3538 significantly upregulated genes, there were 12 major overrepresented GO terms (Table S3, Fig. 6). These overrepresented GO terms were mainly related to protein modification, unfolded protein response, endoplasmic reticulum stress and apoptosis. Furthermore, a total of 32 overrepresented GO terms were detected for 8048 significantly downregulated genes (Table S3, Fig. 7), which were mainly
Fig. 4. Changes in glutathione (GSH) concentration in the stony coral Pocillopora damicornis after acute cadmium stress. Vertical bars represent the mean ± S.D. (N = 5), and bars with different letters are significantly different (P < 0.05).
groups, which revealed 11,586 differentially expressed genes (Table S2). Two lists of these differentially expressed genes were obtained, namely for significantly upregulated and downregulated genes containing 3538 and 8048 differentially expressed genes, respectively.
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Fig. 6. Overrepresented GO terms of the 3538 significantly upregulated genes in the cadmium group at 12 h after acute cadmium stress. A total of 12 GO terms were overrepresented for these significantly upregulated genes. “Ref” referred to the proportion of genes annotated to a GO term in all reference gene of the stony coral Pocillopora damicornis, while “test” referred to the proportion of genes annotated to that GO term in all significantly upregulated genes.
cadmium stress could lead to coral bleaching, and even death, which could be the same thing happening in the stony coral P. damicornis. To systematically understand the response of stony corals towards acute cadmium stress and the underlying mechanisms behind symbiosis collapse, crucial physiological parameters representing the detoxification, redox and apoptosis systems were monitored in the stony coral P. damicornis. The temporal variation in GST activity was determined upon exposure to cadmium, which was the phase II metabolic enzymes in the detoxification process of all eukaryotes (Nicosia et al., 2014). We observed that the GST activity did not change significantly before and after exposure to the heavy metal, demonstrating that acute cadmium stress (20 μg L−1) could not trigger the detoxification system in the stony coral P. damicornis. Several studies reported that cnidarians were able to accumulate heavy metal in tissues and skeletons, especially cadmium (Metian et al., 2015; Mohammed and Dar, 2010). The ability to bioaccumulate allows the cnidarians to tolerate high levels of cadmium. For example, the 96 h LC50 and 6 h EC50 reached as high as 946 and 355 μg L−1 for cadmium in the anemone Aiptasia pulchella, a usual cnidarian model for ecotoxicological studies in tropical marine environments (Howe et al., 2014). Similarly, tolerance against heavy metals could have accounted for the insignificant change of GST activity in the detoxification system of the stony coral P. damicornis. This then suggests that although cadmium did not directly affect the physiology of the coral, its accumulation however decreased the symbiotic zooxanthellae, which could result to bleaching and then to death in the stony coral P. damicornis. Changes in the activity of SOD and CAT were also explored after acute cadmium stress, which were the main antioxidases in the redox system. Results showed that both SOD and CAT activities decreased significantly compared to those in the blank and control groups. This suggests that the high cadmium level (20 μg L−1) could suppress the antioxidant abilities of the stony coral P. damicornis. However, it was different from the previous observations where the increased ROS level induced the expressions and activities of antioxidases under high
correlated with the metabolic processes and macromolecule localization. 4. Discussion Coral reef ecosystems are increasingly being impacted by a wide variety of anthropogenic pollution, including heavy metal accumulation, which could be contributing to coral reef stress leading to bleaching episodes (Brock and Bielmyer, 2013; Mitchelmore et al., 2007). Coral bleaching is characterized by the collapse of coral-zooxanthella symbiosis and the decrease in symbiotic zooxanthellae (Ganot et al., 2011). In the present study, the systemic responses of stony corals towards acute cadmium stress, focusing on P. damicornis, were investigated to reveal its adaptation mechanisms. The change in the density of symbiotic zooxanthellae was determined first to understand the balance of coral-zooxanthella symbiosis after cadmium stress. Results showed that it decreased significantly during 12–24 h period after exposure to elevated cadmium, and reached the lowest level after 24 h. This demonstrates that an acute stress from cadmium (20 μg L−1) could induce a significant decrease of symbiotic zooxanthellae in the stony coral P. damicornis. This however was inconsistent with previous observations, which reported that the density of symbiotic zooxanthellae did not change significantly after cadmium stress (Mitchelmore et al., 2007). The disparity in the observations could have resulted from the differences in cadmium concentrations used and the sampling time, since the insignificant change was observed between 4 and 14 days after exposure to cadmium (5 μg L−1). The present results demonstrated that acute cadmium stress could lead to the collapse of coralzooxanthella symbiosis and the expulsion of symbiotic zooxanthellae. The reason for the collapse after cadmium stress might be similar to the response after heat stress, attributed to the changes in the internal environment of the stony coral, such as the increase of ROS level (Lesser, 1997), the activation of apoptosis (Tchernov et al., 2011), and so on. The continued decrease of symbiotic zooxanthellae after acute 136
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Fig. 7. Overrepresented GO terms of the 8048 significantly downregulated genes in the cadmium group at 12 h after acute cadmium stress. A total of 32 GO terms were overrepresented for these significantly downregulated genes. “Ref” referred to the proportion of genes annotated to a GO term in all reference gene of the stony coral Pocillopora damicornis, while “test” referred to the proportion of genes annotated to that GO term in all significantly downregulated genes.
caspase3 increased significantly after 24 h exposure to cadmium stress, demonstrating that cadmium also induced apoptosis in the stony coral P. damicornis. Apoptosis was also observed when the same stony coral P. damicornis suffered from heat and ammonium stresses (Tchernov et al., 2011; Yuan et al., 2017). Specifically, heat stress could induce apoptosis through TNF signal pathway and caspase3 in the stony coral (Quistad et al., 2014; Zhou et al., 2017). In the present study, apoptosis was induced after cadmium stress because of the upregulated oxidative stressors in the internal environment through the repression of antioxidant production. An example of this is the accumulation of nitric oxide, which then induced apoptosis through caspase3 also in the stony coral P. damicornis (Hawkins et al., 2014). Apoptosis would ultimately result to the collapse of the symbiotic balance between coral and zooxanthellae, and lead to the expulsion of symbiotic zooxanthellae causing bleaching. To have a deeper insight into the molecular mechanisms underlying the response of coral to elevated cadmium, we sequenced the transcriptome of the stony coral P. damicornis at 12 h after acute cadmium stress exposure. A total of 3538 significantly upregulated genes and 8048 significantly downregulated genes were obtained. Further, inspection of the overrepresented GO terms revealed that significantly upregulated genes were mainly related to protein modification,
temperature, air exposure or other stressful conditions (Levy et al., 2006; Teixeira et al., 2013; Yakovleva et al., 2004). The significant decreases of SOD and CAT activities in the present study could have been a result of the change in their expression or structure under cadmium stress. Previous studies reported that cadmium could change the conformation of SOD protein to decrease its enzyme activity, and cause oxidative stress-induced neural cell apoptosis (Huang et al., 2006). It was also reported that cadmium stress induced the expression level of CAT mRNA, but suppressed its activity and decreased its protein content in Pisum sativum (Romero-Puertas et al., 2007). Furthermore, the significant decline of GSH concentration supports the hypothesis that the antioxidant ability was suppressed by acute cadmium stress in the stony coral P. damicornis. Environmental stressors generally induce ROS levels in the internal environment of the organism: therefore, the decline of antioxidant production implied that the oxidative stress would become stronger under cadmium stress. This could even trigger apoptosis, collapse in the symbiosis, and bleaching of the stony coral (Baird et al., 2009; Lesser, 1997). Caspase3 is the key executory enzyme and final effector for apoptosis to occur (Hengartner, 2000). Consequently, we surveyed the activation level of caspase3 to understand the apoptosis status of the stony coral P. damicornis after acute cadmium stress. The activation level of 137
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unfolded protein response, endoplasmic reticulum stress and apoptosis. The unfolded protein response was also observed in two species of Acropora after heat stress (Ruiz-Jones and Palumbi, 2017; TraylorKnowles et al., 2017). In this study, the said response could account for the upward oxidative stress in the body of the organisms by repressing production of antioxidants, since ROS is known to induce the emergence of unfolded or misfolded proteins (Runkel et al., 2013). These unfolded or misfolded proteins would accumulate in the lumen of the endoplasmic reticulum and activate the unfolded protein response. If these proteins are not repaired within a certain time span or the disruption is prolonged, the unfolded protein response would lead to apoptosis (Erguler et al., 2013). These studies corroborate our observation on the physiological activities during stressful conditions in the stony coral P. damicornis. On the other hand, the 32 overrepresented GO terms for significantly downregulated genes correlated well with the metabolic processes involving macromolecules, especially for nucleic acid. This further demonstrated that gene expressions could be regulated positively during acute cadmium stress, and mRNA transcription in the stony coral P. damicornis would be remodeled under elevated cadmium to maintain homeostasis and improve adaptability
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