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.

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.

Microplastic exposure represses the growth of endosymbiotic dinoflagellate Cladocopium goreaui in culture through affecting its apoptosis and metabolism.

Microplastics are widespread emerging marine pollutants that have been found in the coral reef ecosystem. In the present study, using Cladocopium goreaui as a symbiont representative, we investigated cytological, physiological, and molecular responses of a Symbiodiniaceae species to weeklong microplastic exposure (Polystyrene, diameter 1.0 µm, 9.0 × 109 particles L-1). The density and size of algal cells decreased significantly at 7 d and 6-7 d of microplastic exposure, respectively. Chlorophyll a content increased significantly at 7 d of exposure, whereas Fv/Fm did not change significantly during the entire exposure period. We observed significant increases in superoxide dismutase activity and caspase3 activation level, significant decrease in glutathione S-transferase activity, but no change in catalase activity during the whole exposure period. Transcriptomic analysis revealed 191 significantly upregulated and 71 significantly downregulated genes at 7 d after microplastic exposure. Fifteen GO terms were overrepresented for these significantly upregulated genes, which were grouped into four categories including transmembrane ion transport, substrate-specific transmembrane transporter activity, calcium ion binding, and calcium-dependent cysteine-type endopeptidase activity. Thirteen of the significantly upregulated genes encode metal ion transporter and ammonium transporter, and five light-harvesting protein genes were among the significantly downregulated genes. These results demonstrate that microplastics can act as an exogenous stressor, suppress detoxification activity, nutrient uptake, and photosynthesis, elevate oxidative stress, and raise the apoptosis level through upregulating ion transport and apoptotic enzymes to repress the growth of C. goreaui. These effects have implications in negative impacts of microplastics on coral-Symbiodiniaceae symbiosis that involves C. goreaui.

Possible roles of glutamine synthetase in responding to environmental changes in a scleractinian coral.

Glutamine synthetase is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. In this study, the activity and responses of glutamine synthetase towards environmental changes were investigated in the scleractinian coral Pocillopora damicornis. The identified glutamine synthetase (PdGS) was comprised of 362 amino acids and predicted to contain one Gln-synt_N and one Gln-synt_C domain. Expression of PdGS mRNA increased significantly after 12 h (1.28-fold, p < 0.05) of exposure to elevated ammonium, while glutamine synthetase activity increased significantly from 12 to 24 h, peaking at 12 h (54.80 U mg-1, p < 0.05). The recombinant protein of the mature PdGS (rPdGS) was expressed in E. coli BL21, and its activities were detected under different temperature, pH and glufosinate levels. The highest levels of rPdGS activity were observed at 25 °C and pH 8 respectively, but decreased significantly at lower temperature, and higher or lower pH. Furthermore, the level of rPdGS activities was negatively correlated with the concentration of glufosinate, specifically decreasing at 10-5 mol L-1 glufosinate to be less than 50% (p < 0.05) of that in the blank. These results collectively suggest that PdGS, as a homologue of glutamine synthetase, was involved in the nitrogen assimilation in the scleractinian coral. Further, its physiological functions could be suppressed by high temperature, ocean acidification and residual glufosinate, which might further regulate the coral-zooxanthella symbiosis via the nitrogen metabolism in the scleractinian coral P. damicornis.

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.

Pathways with PathWhiz.

PathWhiz (http://smpdb.ca/pathwhiz) is a web server designed to create colourful, visually pleasing and biologically accurate pathway diagrams that are both machine-readable and interactive. As a web server, PathWhiz is accessible from almost any place and compatible with essentially any operating system. It also houses a public library of pathways and pathway components that can be easily viewed and expanded upon by its users. PathWhiz allows users to readily generate biologically complex pathways by using a specially designed drawing palette to quickly render metabolites (including automated structure generation), proteins (including quaternary structures, covalent modifications and cofactors), nucleic acids, membranes, subcellular structures, cells, tissues and organs. Both small-molecule and protein/gene pathways can be constructed by combining multiple pathway processes such as reactions, interactions, binding events and transport activities. PathWhiz's pathway replication and propagation functions allow for existing pathways to be used to create new pathways or for existing pathways to be automatically propagated across species. PathWhiz pathways can be saved in BioPAX, SBGN-ML and SBML data exchange formats, as well as PNG, PWML, HTML image map or SVG images that can be viewed offline or explored using PathWhiz's interactive viewer. PathWhiz has been used to generate over 700 pathway diagrams for a number of popular databases including HMDB, DrugBank and SMPDB.

SMPDB 2.0: big improvements to the Small Molecule Pathway Database.

The Small Molecule Pathway Database (SMPDB, http://www.smpdb.ca) is a comprehensive, colorful, fully searchable and highly interactive database for visualizing human metabolic, drug action, drug metabolism, physiological activity and metabolic disease pathways. SMPDB contains >600 pathways with nearly 75% of its pathways not found in any other database. All SMPDB pathway diagrams are extensively hyperlinked and include detailed information on the relevant tissues, organs, organelles, subcellular compartments, protein cofactors, protein locations, metabolite locations, chemical structures and protein quaternary structures. Since its last release in 2010, SMPDB has undergone substantial upgrades and significant expansion. In particular, the total number of pathways in SMPDB has grown by >70%. Additionally, every previously entered pathway has been completely redrawn, standardized, corrected, updated and enhanced with additional molecular or cellular information. Many SMPDB pathways now include transporter proteins as well as much more physiological, tissue, target organ and reaction compartment data. Thanks to the development of a standardized pathway drawing tool (called PathWhiz) all SMPDB pathways are now much more easily drawn and far more rapidly updated. PathWhiz has also allowed all SMPDB pathways to be saved in a BioPAX format. Significant improvements to SMPDB's visualization interface now make the browsing, selection, recoloring and zooming of pathways far easier and far more intuitive. Because of its utility and breadth of coverage, SMPDB is now integrated into several other databases including HMDB and DrugBank.