Nitrogen availability improves the physiological resilience of coral endosymbiont Cladocopium goreaui to high temperature.

The physiological response of symbiotic Symbiodiniaceae to high temperature is believed to result in coral bleaching. However, the potential effect of nitrogen availability on heat acclimatization of symbiotic Symbiodiniaceae is still unclear. In this study, physiological responses of Symbiodiniaceae Cladocopium goreaui to temperature and nitrogen nutrient stress conditions were investigated. Nitrogen deficiency caused significant declines in cell concentration and chlorophyll content per cell, but significant increases in nitric oxide synthase activity, caspase3 activation level, and cellular carbon content of C. goreaui at normal temperature. Algal cells under high temperature and nitrogen deficiency showed significant rises in Fv/Fm, catalase activity, and caspase3 activation level, but no significant changes in cell yield, cell size, chlorophyll content, superoxide dismutase, nitric oxide synthase activity, and cellular contents of nitrogen and carbon, in comparison with those under normal temperature and nitrogen deficiency. Growth, chlorophyll, and nitrogen contents of algal cells under the high temperature and nitrogen-replete conditions were significantly higher than those under high temperature or nitrogen deficiency alone, whereas nitric oxide synthase activity, superoxide dismutase activity, catalase activity, carbon content, and caspase3 activation level exhibited opposite trends of variation. Transcriptomic and network analyses revealed ion transport and metabolic processes mainly involved in regulating these physiological activities under different temperature and nitrogen nutrient. The totality of results shows that high temperature activates stress responses, induces antioxidant capacity of apoptosis, and limits the growth rate of C. goreaui. Adequate nitrogen nutrient can improve the resilience of this Symbiodiniaceae against heat stress through repressed apoptosis, promoted ion transport, and optimized metabolism.

Oxidative stress, apoptosis activation and symbiosis disruption in giant clam Tridacna crocea under high temperature.

Giant clams are one of the most important animals in coral reef ecosystem, and its growth and reproduction are being threatened by heat stress due to global warming. In the present study, the symbiont density, the crucial enzyme activities and the transcriptome were investigated in the outer mantle of giant clam Tridacna crocea after the acute exposure of high temperature. The density of symbiotic zooxanthellae decreased significantly during 12-24 h, with the minimum level (7.75 × 105 cell cm-2, p < 0.05) at 12 h after heat stress. The activities of superoxide dismutase in the heat stress group was significantly lower than that in the control group at 24 h after heat stress, while no significant change in the activities of catalase was observed during the entire stress process. The activation level of caspase3 began to increase significantly at 12 h (1.22-fold, p < 0.05), and reached the highest level at 24 h (1.38-fold, p < 0.05) after heat stress. Six paired-end libraries were sequenced in two groups, including the heat stress and control group at 12 h after heat stress. Through the assembling of 187,116,632 paired-end reads with lengths of 2 × 150 bp, a total of 26,676 genes were obtained which derived from giant clam. Bioinformatics analysis revealed 47 significantly upregulated and 88 significantly downregulated genes at 12 h after the treatment. There were 12 overrepresented GO terms for significantly upregulated genes, mostly related to unfolded protein binding and ATP binding, whereas no GO term was overrepresented for significantly downregulated genes. These results collectively suggest high temperature could induce excessive oxidative stress through the repressed antioxidant ability, the apoptosis activated by the unfolded protein response, and further the collapse of the symbiosis between host and symbiont, which has been threatening the growth and reproduction of the giant clam T. crocea.

Effects of environmental factors on C-type lectin recognition to zooxanthellae in the stony coral Pocillopora damicornis.

C-type lectin is a superfamily of Ca2+-dependent carbohydrate-recognition proteins that play significant roles in nonself-recognition and pathogen clearance. In the present study, a C-type lectin (PdC-Lectin) was chosen from stony coral Pocillopora damicornis to understand its recognition characteristics to zooxanthellae. PdC-Lectin protein contained a signal peptide and a carbohydrate-recognition domain with EPN motif in Ca2+-binding site 2. The PdC-Lectin recombinant protein was expressed and purified in vitro. The binding of PdC-Lectin protein to zooxanthellae was determined with western blotting method, and the bound protein to 10-105 cell mL-1 zooxanthellae was detectable in a concentration-dependent manner. Less PdC-Lectin protein binding to zooxanthellae was observed for the incubation at 36 °C than that at 26 °C. Furthermore, the PAMP recognition spectrum of PdC-Lectin protein was tested through surface plasmon resonance method, and it bound to LPS and Lipid A, but not to LTA, ß-glucan, mannose or Poly (I:C). When PdC-Lectin protein was preincubated with LPS, there was less protein binding to zooxanthellae compared with that in non-preincubation group. These results collectively suggest that PdC-Lectin could recognize zooxanthellae, and the recognition could be repressed by high temperature and pathogenic bacteria, which would help to further understand the molecular mechanism of coral bleaching and the establishment of coral-zooxanthella symbiosis in the stony coral P. damicornis.

Suppression of NF-κB signal pathway by NLRC3-like protein in stony coral Acropora aculeus under heat stress.

Heat stress is the most common factor for coral bleaching, which has increased both in frequency and severity due to global warming. In the present study, the stony coral Acropora aculeus was subjected to acute heat stress and entire transcriptomes were sequenced via the next generation sequencing platform. Four paired-end libraries were constructed and sequenced in two groups, including a control and a heat stress group. A total of 120,319,751 paired-end reads with lengths of 2 × 100 bp were assembled and 55,021 coral-derived genes were obtained. After read mapping and abundance estimation, 9110 differentially expressed genes were obtained in the comparison between the control and heat stress group, including 4465 significantly upregulated and 4645 significantly downregulated genes. Twenty-three GO terms in the Biological Process category were overrepresented for significantly upregulated genes, and divided into six groups according to their relationship. These three groups were related to the NF-κB signal pathway, and the remaining three groups were relevant for pathogen response, immunocyte activation and protein ubiquitination. Forty-three common genes were found in four GO terms, which were directly related to the NF-κB signal pathway. These included 2 NACHT, LRR, PYD domains-containing protein, 5 nucleotide-binding oligomerization domain-containing protein, 29 NLRC3-like protein, 4 NLRC5-like protein, and 3 uncharacterized protein. For significantly downregulated genes, 27 overrepresented GO terms were found in the Biological Process category, which were relevant to protein ubiquitination and ATP metabolism. Our results indicate that heat stress suppressed the immune response level via the NLRC3-like protein, the fine-tuning of protein turnover activity, and ATP metabolism. This might disrupt the balance of coral-zooxanthellae symbiosis and result in the bleaching of the coral A. aculeus.

Ingested microplastics impair the metabolic relationship between the giant clam Tridacna crocea and its symbionts.

Microplastics are emerging as widespread pollutants in coral reef ecosystems worldwide; however, there is limited knowledge regarding their impact on giant clams, which are important reef builders. In the present study, the cytological, physiological, and molecular response of the giant clam Tridacna crocea to a 5 d exposure of microplastics was investigated. The concentration of microplastics in the intestine and outer mantle increased significantly and gradually after the exposure to microplastics. There were no significant changes in the density of symbiotic Symbiodiniaceae throughout the exposure period, but symbiont chlorophyll content increased significantly after 1 d of exposure. There was a significant increase in symbiont superoxide dismutase (SOD) activity, but a decrease in giant clam SOD activity and symbiont glutathione S-transferase (GST) activity. No significant changes in catalase (CAT) activity and caspase3 activation level were observed in the two symbiotic partners. Transcriptomic analysis of the giant clam revealed 138 significantly upregulated and 1390 significantly downregulated genes after 5 d of microplastic exposure. The top 20 GO terms overrepresented by these significantly downregulated genes were related to primary metabolic processes and cellular metabolic processes. No significantly upregulated genes were observed in symbionts, but 28 genes were significantly downregulated, including chloroplast oxygen-evolving enhancer, photosystem I reaction center subunit II, peptide/nitrate transporter, sodium-coupled neutral amino acid transporter, beta-glucosidase, and TPA: lipase. These results suggest that T. crocea ingests microplastics through the outer mantle and intestine, and these microplastics can suppress the photosynthesis, organic nutrient transportation, and detoxification ability of the symbionts, as well as the primary metabolism of the giant clam. This eventually could threaten their metabolic relationship and long-term survival.

Temperature regulates the recognition activities of a galectin to pathogen and symbiont in the scleractinian coral Pocillopora damicornis.

Lectins serve as essential pattern recognition receptors, and play important roles in the recognition of non-self and mediation of innate immune response in metazoans. Scleractinian corals are vulnerable to pathogen infection and endosymbiosis disruption under heat stress that can finally lead to coral bleaching. In this study, a cDNA sequence encoding one galectin was cloned in scleractinian coral Pocillopora damicornis (PdGLT-1). The deduced PdGLT-1 protein shared highest amino acid sequence similarity (99%) with galectin from Stylophora pistillata (XP_022806650.1), and was composed of one signal peptide, one Collagen domain and one Gal-Lectin domain. PdGLT-1 recombinant protein (rPdGLT-1) was expressed and purified in vitro. Binding activities of rPdGLT-1 to bacteria and symbiont were determined using western blotting method. Results showed that rPdGLT-1 was able to bind to gram-positive bacterium Streptococcus mutans, gram-negative bacteria Vibrio coralliilyticus and Escherichia coli, with the highest activity for V. coralliilyticus, and further agglutinated them. The bound rPdGLT-1 to Symbiodinium (10-104 cells mL-1) was detectable, and its binding ability was concentration-dependent. Furthermore, dual binding activities were determined under different temperatures (20, 25, 30 and 35 °C), and the optimal temperatures were found to be 25 and 30 °C for V. coralliilyticus and Symbiodinium, respectively. Results suggested that PdGLT-1 could recognize pathogenic bacteria and symbiotic dinoflagellates Symbiodinium. However, their recognition activities were repressed under high temperature (>30 °C). This study provided insights into the underlying mechanism of lectin modulation to heat bleaching through its pathogen and Symbiodinium recognition in the scleractinian coral P. damicornis.

Transcriptome, expression, and activity analyses reveal a vital heat shock protein 70 in the stress response of stony coral Pocillopora damicornis.

Coral bleaching occurs worldwide with increasing frequencies and intensities, which is caused by the stress response of stony coral to environmental change, especially increased sea surface temperature. In the present study, transcriptome, expression, and activity analyses were employed to illustrate the underlying molecular mechanisms of heat shock protein 70 (HSP70) in the stress response of coral to environmental changes. The domain analyses of assembled transcripts revealed 30 HSP70 gene contigs in stony coral Pocillopora damicornis. One crucial HSP70 (PdHSP70) was observed, whose expressions were induced by both elevated temperature and ammonium after expression difference analysis. The complete complementary DNA (cDNA) sequence of PdHSP70 was identified, which encoded a polypeptide of 650 amino acids with a molecular weight of 71.93 kDa. The deduced amino acid sequence of PdHSP70 contained a HSP70 domain (from Pro8 to Gly616), and it shared the highest similarity (95%) with HSP70 from Stylophora pistillata. The expression level of PdHSP70 gene increased significantly at 12 h, and returned to the initial level at 24 h after the stress of high temperature (32 °C). The cDNA fragment encoding the mature peptide of PdHSP70 was recombined and expressed in the prokaryotic expression system. The ATPase activity of recombinant PdHSP70 protein was determined, and it did not change significantly in a wide range of temperature from 25 to 40 °C. These results collectively suggested that PdHSP70 was a vital heat shock protein 70 in the stony coral P. damicornis, whose mRNA expression could be induced by diverse environmental stress and whose activity could remain stable under heat stress. PdHSP70 might be involved in the regulation of the bleaching owing to heat stress in the stony coral P. damicornis.

Systemic response of the stony coral Pocillopora damicornis against acute cadmium stress.

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 12-24h period, and reached lowest at 24h 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 12h 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.

Acute microplastic exposure raises stress response and suppresses detoxification and immune capacities in the scleractinian coral Pocillopora damicornis.

Microplastics are widespread emerging contaminants that have been found globally in the marine and freshwater ecosystem, but there is limited knowledge regarding its impact on coral reef ecosystem and underpinning mechanism. In the present study, using Pocillopora damicornis as a model, we investigated cytological, physiological, and molecular responses of a scleractinian coral to acute microplastic exposure. No significant changes were observed in the density of symbiotic zooxanthellae during the entire period of microplastic exposure, while its chlorophyll content increased significantly at 12 h of microplastic exposure. We observed significant increases in the activities of antioxidant enzymes such as superoxide dismutase and catalase, significant decrease in the detoxifying enzyme glutathione S-transferase and the immune enzyme alkaline phosphatase, but no change in the other immune enzyme phenoloxidase during the whole experiment period. Transcriptomic analysis revealed 134 significantly up-regulated coral genes at 12 h after the exposure, enriched in 11 GO terms mostly related to stress response, zymogen granule, and JNK signal pathway. Meanwhile, 215 coral genes were significantly down-regulated at 12 h after exposure, enriched in 25 GO terms involved in sterol transport and EGF-ERK1/2 signal pathway. In contrast, only 12 zooxanthella genes exhibited significant up-regulation and 95 genes down-regulation at 12 h after the microplastic exposure; genes regulating synthesis and export of glucose and amino acids were not impacted. These results suggest that acute exposure of microplastics can activate the stress response of the scleractinian coral P. damicornis, and repress its detoxification and immune system through the JNK and ERK signal pathways. These demonstrate that microplastic exposure can compromise the anti-stress capacity and immune system of the scleractinian coral P. damicornis, despite the minimal impact on the abundance and major photosynthate translocation transporters of the symbiont in the short term.