A rapid and multi-endpoint ecotoxicological test using Mychonastes afer for efficient screening of metals and herbicides.

Microalgal growth-based tests are international standards for ecotoxicity assessment; however, their long exposure times, large sample volumes, and reliance on a single growth-endpoint make them inadequate for rapid toxicity screening. Here, we aimed to develop a rapid and simple ecotoxicological test using the fast-growing green alga Mychonastes afer, with multiple endpoints-growth, lipid content, and photosynthesis. We exposed M. afer to two metals-silver and copper-and two herbicides-atrazine and diuron-for 24 h and identified the most sensitive and reliable endpoints for each toxicant: the maximum electron transport rate (ETRmax) for Ag, Cu and atrazine, and the lipid content for diuron. Lipid content was found to be both a sensitive and reliable biomarker, meeting the effluent limit guidelines in both the Republic of Korea and the USA. The sensitivity of M. afer to Ag and atrazine also closely matched the HC5 values derived from the species sensitivity distribution approach, confirming its reliability for setting regulatory concentrations of these contaminants. Our calculated predicted no-effect concentration (PNEC) values were similar to established European Union PNECs for Ag, Cu, atrazine, and diuron, underlining the utility of these biological endpoints for ecological risk assessment and regulatory decision making. This method required lower sample volume (2 mL vs 100 mL) and exposure time (24 h vs 72-120 h) than conventional green algal tests, and eliminated the need for labour-intensive cell counting, expensive equipment, and chlorophyll fluorescence measurement expertise. Overall, this M. afer test can be a valuable tool for the rapid screening of wastewater for metals and herbicides, contributing to environmental protection and management practices.

New recorded diatoms in Holocene sediment cores from the Gonggeom-ji Wetland in Korea.

The Gonggeom-ji reservoir is an agricultural one built for rice farming during the Proto-Three Kingdoms period and was designated as Gyeongsangbuk-do monument No. 121 because of its high historical value. The Nakdonggang National Institute of Biological Resources has been conducting paleontological and paleoenvironmental studies on major wetlands from Korea since 2016, as well as diatom, geological, and depth distribution analyses on the sedimentary soil of Gonggeom-ji. This study summarized the description and ecological characteristics of six newly recorded diatoms (Gomphonema lacusrankala, Pinnularia diandae, P. gibba var. hyaline, P. lacunarum, Sellaphora labda var. nipponica, Stauroneis angustilancea) found in samples collected through drilling in Gonggeom-ji in 2019.

Proteomic profiling of cryopreserved Trichormus variabilis using various cryoprotectants.

Algae, which may be unicellular or multicellular, can carry out photosynthesis just like plants as they effectively utilize light energy. They contain various physiologically active substances and are, therefore, widely used commercially to produce healthy food and feed additives, cosmetics, and energy supplements. For useful applications, the cryopreservation technique has been used in various fields. Recently, to develop suitable cryopreservation methods for algal applications, various studies have been performed. However, adequate investigations have not been conducted to understand the mechanism underlying algal cryopreservation at the molecular level. Therefore, this study examined the profile alteration of the proteome using cryopreservation with various cryoprotectants (CPAs). Trichormus variabilis was cultured and then cryopreserved with 10% dimethyl sulfoxide, methanol, and glycerol, after which, proteome profiling was done. Finally, signaling pathway search was performed, and a new signaling pathway was established based on differentially expressed proteins. As a result, the expression levels of 17 proteins were observed. Additionally, it was confirmed that the differentially expressed proteins were related to 16 signaling pathways and that they were capable of interacting with each other. The findings suggest that the differentially expressed proteins may be applied as biomarkers for algal cryopreservation and to understand the mechanism underlying T. variabilis cryopreservation. Moreover, it is anticipated that the results from this study would be useful in selecting suitable CPAs and in upgrading the cryopreservation techniques.

Effect of cryopreservation on the bacterial community structure of filamentous cyanobacteria, Trichormus variabilis (Nostocales, Cyanobacteria).

Cryopreservation is an efficient method used to preserve microorganisms for long periods of time, such as up to 30 years, without changes in genetic and physiological characteristics. As cyanobacteria and microalgae are usually maintained as both axenic and xenic cultures, knowledge of co-cultured bacteria and changes in their community structure is important for the successful maintenance of microbial culture collections. In this study, research on the changes in co-cultured bacterial community structure during cyanobacterial cryopreservation were investigated using three different experimental groups by next generation sequencing (NGS): 1) cultured Trichormus variabilis without cryopreservation (control group), 2) cultured T. variabilis after cryopreservation in 10% dimethyl sulfoxide (Me2SO) for 14 days (cryo-cell group), and 3) cultured T. variabilis after cryopreservation in 10% Me2SO for 14 days within alginate beads (cryo-bead group). The results showed that the abundance of Sphingomonas and Hydrogenophaga (belonging to phylum Proteobacteria) was significantly increased in the cryo-bead group (Sphingomonas, control: 0.25%, cryo-cell: 1.32%, cryo-bead: 41.70%; Hydrogenophaga, control: 5.47%, cryo-cell: 5.24%, cryo-bead: 12.32%). However, the abundance of the phylum Bacteroidetes was significantly decreased in the cryo-bead group compared to that in the other groups (control: 26.29%, cryo-cell: 38.84%, cryo-bead: 11.43%). Bacterial diversity was generally reduced after cryopreservation in the cryo-bead group, where the overgrowth of a few unique bacteria was observed in the co-cultured bacterial community. These results imply that changes in the co-cultured bacterial community during preservation should be considered as an important factor for the development of methods for cyanobacterial cryopreservation.

Seasonal Dynamics and Metagenomic Characterization of Marine Viruses in Goseong Bay, Korea.

Viruses are the most abundant biological entities in the oceans, and account for a significant amount of the genetic diversity of marine ecosystems. However, there is little detailed information about the biodiversity of viruses in marine environments. Rapid advances in metagenomics have enabled the identification of previously unknown marine viruses. We performed metagenomic profiling of seawater samples collected at 6 sites in Goseong Bay (South Sea, Korea) during the spring, summer, autumn, and winter of 2014. The results indicated the presence of highly diverse virus communities. The DNA libraries from samples collected during four seasons were sequenced using Illumina HiSeq 2000. The number of viral reads was 136,850 during March, 70,651 during June, 66,165 during September, and 111,778 during December. Species identification indicated that Pelagibacter phage HTVC010P, Ostreococcus lucimarinus OIV5 and OIV1, and Roseobacter phage SIO1 were the most common species in all samples. For viruses with at least 10 reads, there were 204 species during March, 189 during June, 170 during September, and 173 during December. Analysis of virus families indicated that the Myoviridae was the most common during all four seasons, and viruses in the Polyomaviridae were only present during March. Viruses in the Iridoviridae were only present during three seasons. Additionally, viruses in the Iridoviridae, Herpesviridae, and Poxviridae, which may affect fish and marine animals, appeared during different seasons. These results suggest that seasonal changes in temperature contribute to the dynamic structure of the viral community in the study area. The information presented here will be useful for comparative analyses with other marine viral communities.

Differential gene expression patterns during embryonic development of sea urchin exposed to triclosan.

Triclosan (TCS; 2,4,4'-trichloro-2'-hydroxydiphenyl ether) is a broad-spectrum antibacterial agent used in common industrial, personal care and household products which are eventually rinsed down the drain and discharged with wastewater effluent. It is therefore commonly found in the aquatic environment, leading to the continual exposure of aquatic organisms to TCS and the accumulation of the antimicrobial and its harmful degradation products in their bodies. Toxic effects of TCS on reproductive and developmental progression of some aquatic organisms have been suggested but the underlying molecular mechanisms have not been defined. We investigated the expression patterns of genes involved in the early development of TCS-treated sea urchin Strongylocentrotus nudus using cDNA microarrays. We observed that the predominant consequence of TCS treatment in this model system was the widespread repression of TCS-modulated genes. In particular, empty spiracles homeobox 1 (EMX-1), bone morphogenic protein, and chromosomal binding protein genes showed a significant decrease in expression in response to TCS. These results suggest that TCS can induce abnormal development of sea urchin embryos through the concomitant suppression of a number of genes that are necessary for embryonic differentiation in the blastula stage. Our data provide new insight into the crucial role of genes associated with embryonic development in response to TCS. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 426-433, 2017.

Detection of coat protein gene of nervous necrosis virus using loop-mediated isothermal amplification.

OBJECTIVE: To establish a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the identification of nervous necrosis virus (NNV) infection. METHODS: A set of synthesized primers was used to match the sequences of a specific region of the nnv gene from the National Center for Biotechnology Information database, not originating from NNV-infected fish, the efficiency and specificity of LAMP were measured dependent on the concentration of DNA polymerase and the reaction temperature and time. In addition, to determine species-specific LAMP primers, cross reactivity testing was applied to the reaction between NVV and other virus families including viral hemorrhagic septicemia virus and marine birnavirus. RESULTS: The optimized LAMP reaction carried out at 64 °C for 60 min, and above 4 U Bst DNA polymerase. The sensitivity of LAMP for the detection of nnv was thus about 10 times greater than the sensitivity of polymerase chain reaction. The LAMP assay primers were specific for the detection NNV infection in Epinephelus septemfasciatus. CONCLUSIONS: The development of LAMP primers based on genetic information from a public database, not virus-infected samples, may provide a very simple and convenient method to identify viral infection in aquatic organisms.

Detection of the dinoflagellate, Cochlodinium polykrikoides, that forms algal blooms using sandwich hybridization integrated with nuclease protection assay.

OBJECTIVES: To detect Cochlodinum polykrikoides in long-term monitoring and high-throughput sampling projects using an integrated sandwich hybridization and nuclease protection assay (NPA-SH). RESULTS: The specificity of the probes was verified with individual and mixed cultures as well as field collection, and the quantity of C. polykrikoides determined by NPA-SH analysis showed a good correlation with that determined by cell-counting with a light microscope. In addition a standard curve for C. polykrikoides was established to represent the correlation between optical absorbance in the NPA-SH assay and cell density. CONCLUSIONS: This approach provides an efficient alternative to traditional, morphology-based methods for the rapid identification and quantification of harmful algal species and could be used to monitor phytoplankton in field surveys.

Seasonal Dynamics of Marine Microbial Community in the South Sea of Korea.

High-resolution 16S rRNA tag pyrosequencing was used to obtain seasonal snapshots of the bacterial diversity and community structure at two locations in Gosung Bay (South Sea, Korea) over a one year period. Seasonal sampling from the water column at each site revealed highly diverse bacterial communities containing up to 900 estimated Operational Taxonomic Units (OTUs). The Alphaproteobacteria and Gammaproteobacteria were the most abundant groups, and the most frequently recorded OTUs were members of Pelagibacter and Glaciecola. In particular, it was observed that Arcobacter, a genus of the Epsilonproteobacteria, dominated during summer. In addition, Psedoalteromonadaceae, Vibrionaceae and SAR11-1 were predominant members of the OTUs found in all sampling seasons. Environmental factors significantly influenced the bacterial community structure among season, with the phosphate and nitrate concentrations contributing strongly to the spatial distribution of the Alphaproteobacteria; the Gammaproteobacteria, Flavobacteria, and Actinobacteria all showed marked negative correlations with all measured nutrients, particularly silicon dioxide and chlorophyll-a. The results suggest that seasonal changes in environmental variables contribute to the dynamic structure of the bacterial community in the study area.

Fatty acid methyl ester profiles and nutritive values of 20 marine microalgae in Korea.

OBJECTIVE: To screen the fatty acid (FA) composition of 20 marine microalgae species, including seven Diophyceae, six Bacillariophyceae, four Chlorophyceae, two Haptophyceae and one Raphidophyceae species. METHODS: Microalgal cells cultured at the Korea Institute of Ocean Science & Technology were harvested during the late exponential growth phase and the FA composition analyzed. RESULTS: The FA composition of microalgae was species-specific. For example, seven different species of Dinophyceae were composed primarily of C14:0, C16:0, C18:0, C20:4n-6, C20:5n-3 and C22:6n-3, while C14:0, C16:0, C16:1, C18:0, C20:5n-3 and C22:6n-3 were abundant FAs in six species of Bacillariophyceae. In addition, four Chlorophyceae, two Haptophyceae and one Raphidophyceae species all contained a high degree of C16:1n-7 [(9.28-34.91)% and (34.48-35.04)%], C14:0 [(13.34-25.96)%] and [(26.69-28.24)%], and C16:0 [(5.89-29.15)%] and [(5.70-16.81)%]. Several factors contribute to the nutritional value of microalgae, including the polyunsaturated FA content and n-3 to n-6 FA ratio, which could be used to assess the nutritional quality of microalgae. CONCLUSIONS: This study is the first comprehensive assessment of the FA composition and nutritional value of microalgae species in South Korea, and identifies the potential utility of FAs as species-specific biomarkers.

Anti-inflammation activities of mycosporine-like amino acids (MAAs) in response to UV radiation suggest potential anti-skin aging activity.

Certain photosynthetic marine organisms have evolved mechanisms to counteract UV-radiation by synthesizing UV-absorbing compounds, such as mycosporine-like amino acids (MAAs). In this study, MAAs were separated from the extracts of marine green alga Chlamydomonas hedleyi using HPLC and were identified as porphyra-334, shinorine, and mycosporine-glycine (mycosporine-Gly), based on their retention times and maximum absorption wavelengths. Furthermore, their structures were confirmed by triple quadrupole MS/MS. Their roles as UV-absorbing compounds were investigated in the human fibroblast cell line HaCaT by analyzing the expression levels of genes associated with antioxidant activity, inflammation, and skin aging in response to UV irradiation. The mycosporine-Gly extract, but not the other MAAs, had strong antioxidant activity in the 2,2-diphenyl-1-picryhydrazyl (DPPH) assay. Furthermore, treatment with mycosporine-Gly resulted in a significant decrease in COX-2 mRNA levels, which are typically increased in response to inflammation in the skin, in a concentration-dependent manner. Additionally, in the presence of MAAs, the UV-suppressed genes, procollagen C proteinase enhancer (PCOLCE) and elastin, which are related to skin aging, had increased expression levels equal to those in UV-mock treated cells. Interestingly, the increased expression of involucrin after UV exposure was suppressed by treatment with the MAAs mycosporine-Gly and shinorine, but not porphyra-334. This is the first report investigating the biological activities of microalgae-derived MAAs in human cells.

Distinct patterns of marine bacterial communities in the South and North Pacific Oceans.

The study of oceanic microbial communities is crucial for our understanding of the role of microbes in terms of biomass, diversity and ecosystem function. In this study, 16S rRNA gene tag pyrosequencing was used to investigate change in bacterial community structure between summer and winter water masses from Gosung Bay in the South Sea of Korea and Chuuk in Micronesia, located in the North and South Pacific Oceans, respectively. Summer and winter sampling from each water mass revealed highly diverse bacterial communities, containing ~900 Operational Taxonomic Units (OTUs). The microbial distribution and highly heterogeneous composition observed at both sampling sites were different from those of most macroorganisms. The bacterial communities in the seawater at both sites were most abundant in Proteobacteria during the summer in Gosung and in Bacterioidetes during the winter. The proportion of Cyanobacteria was higher in summer than in winter in Chuuk and similar in Gosung. Additionally, the microbial community during summer in Gosung was significantly different from other communities observed based on the unweighted UniFrac distance. These data suggest that in both oceanic areas sampled, the bacterial communities had distinct distribution patterns with spatially- and temporally-heterogeneous distributions.

Hypoxia-modulated gene expression profiling in sea urchin (Strongylocentrotus nudus) immune cells.

Hypoxia is an issue that affects ocean coastal waters worldwide. It has severe consequences for marine organisms, including death and rapid adaptive changes in metabolic organization. Although some aquatic animals are routinely exposed and resistant to severe environmental hypoxia, others such as sea urchins (Strongylocentrotus nudus) have a limited capacity to withstand this stress. In this study, hypoxia induced a significant increase in the number of red spherule cells among coelomocytes, which function as immune cells. This suggests that sea urchin immune cells could be used as a biological indicator of hypoxic stress. In the current study, we used cDNA microarrays to investigate the differential expression patterns of hypoxia-regulated genes to better understand the molecular mechanisms underlying the response of immune cells to hypoxia. Surprisingly, the predominant major effect of hypoxia was the widespread suppression of gene expression. In particular, the expression of RNA helicase and GATA-4/5/6 was decreased significantly in response to hypoxia, even in field conditions, suggesting that they could be utilized as sensitive bioindicators of hypoxic stress in the sea urchin.

Phenol content, antioxidant and tyrosinase inhibitory activity of mangrove plants in Micronesia.

OBJECTIVE: To find out and compare the in vitro antioxidant and tyrosinase inhibitory activities of two species of mangrove plants. METHODS: Mangrove samples were harvested at the shoreline on the island of Weno, Chuuk State in Micronesia. The phenol content, antioxidant activity (based on DPPH-free radical scavenging) and tyrosinase inhibitory activity in different tissues (leaves, barks and roots) of Rhizophora stylosa (R. stylosa) and Sonneratia alba (S. alba), collected from the island of Weno. RESULTS: Total phenol content ranged from 4.87 to 11.96 mg per g of freeze dried samples. The highest antioxidant activity was observed in R. stylosa bark (85.5%). The highest tyrosinase inhibitory activity was found in S. alba bark. Also, total phenol content and antioxidant activity were higher in methanol extracts than in aqueous extracts. CONCLUSIONS: Taken together, the results of this study proved that mangroves can be excellent sources of antioxidant compounds.