Identification of circRNAs and circRNA-mRNA network of Epinephelus coioides during Singapore grouper iridovirus infection.

Circular RNA (circRNA), one of the important non-coding RNA molecules with a closed-loop structure, plays a key regulatory role in cell processing. In this study, circRNAs of Epinephelus coioides, an important marine cultured fish in China, were isolated and characterized, and the network of circRNAs and mRNA was explored during Singapore grouper iridovirus (SGIV) infection, one of the most important double stranded DNA virus pathogens of marine fish. 10 g of raw data was obtained by high-throughput sequencing, and 2599 circRNAs were classified. During SGIV infection, 123 and 37 circRNAs occurred differential expression in spleen and spleen cells, indicating that circRNAs would be involved in the viral infection. GO annotation and KEGG demonstrated that circRNAs could target E. coioides genes to regulate cell activity and the activation of immune factors. The results provide some insights into the circRNAs mediated immune regulatory network during bony fish virus infection.

Molecular characterization, expression and function analysis of Epinephelus coioides PKC-ɑ response to Singapore grouper iridovirus (SGIV) infection.

Protein kinase C (PKC) constitutes the main signal transduction pathway, and participates in the signal pathway of cell proliferation and movement in mammals. In this study, PKC-ɑ was obtained from Epinephelus coioides, an important marine fish cultivated in the coastal areas of southern China and Southeast Asia. The full length cDNA of PKC-ɑ was 3362 bp in length containing a 23 bp 5'UTR, a 1719 bp 3'UTR, and a 1620 bp open reading frame encoding 539 amino acids. It contains three conservative domains including protein kinase C conserved region 2 (C2), Serine/Threonine protein kinases, catalytic domain (S_TKc) and ser/thr-type protein kinases (S_TK_X). Its mRNA can be detected in all 11 tissues examined of E. coioides, and the expression was significantly upregulated response to Singapore grouper iridovirus (SGIV) infection, one of the important pathogens of marine fish. Upregulated E. coioides PKC-ɑ significantly inhibited the activation of nuclear factor kappa-B (NF-κB) and activator protein-1 (AP-1), and SGIV-induced cell apoptosis. The results indicated that the PKC-ɑ may play an important role in pathogenic stimulation.

Antifungal peptides from the marine gorgonian-associated fungus Aspergillus sp. SCSIO41501.

Three undescribed cyclic lipopeptides maribasins C-E and four undescribed linear peptides aspergillipeptides H-K together with three known analogous maribasins A-B and marihysin A were isolated from the marine gorgonian-associated fungus Aspergillus sp. SCSIO 41501 (Trichocomaceae). Their structures were determined by spectroscopic analysis, and their absolute configurations were further confirmed by Marfey's methods. Maribasins C-E and maribasins A-B showed significant antifungal activity against five phytopathogenic fungal strains with MIC values of 3.12-50 µg/disc. Structure-bioactivity relationship exhibited that the ß-amino fatty acid chain could significantly affect the antifungal activity of this type of cyclic lipopeptides.

Molecular cloning, inducible expression with SGIV and Vibrio alginolyticus challenge, and function analysis of Epinephelus coioides PDCD4.

Programmed cell death 4 (PDCD4) in mammals, a gene closely associated with apoptosis, is involved in many biological processes, such as cell aging, differentiation, regulation of cell cycle, and inflammatory response. In this study, grouper Epinephelus coioides PDCD4, EcPDCD4-1 and EcPDCD4-2, were obtained. The open reading frame (ORF) of EcPDCD4-1 is 1413 bp encoding 470 amino acids with a molecular mass of 52.39 kDa and a theoretical pI of 5.33. The ORF of EcPDCD4-2 is 1410 bp encoding 469 amino acids with a molecular mass of 52.29 kDa and a theoretical pI of 5.29. Both EcPDCD4-1 and EcPDCD4-2 proteins contain two conserved MA3 domains, and their mRNA were detected in all eight tissues of E. coioides by quantitative real-time PCR (qRT-PCR) with the highest expression in liver. The expressions of two EcPDCD4s were significantly up-regulated after Singapore grouper iridovirus (SGIV) or Vibrio alginolyticus infection. In addition, over-expression of EcPDCD4-1 or EcPDCD4-2 can inhibit the activity of the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), and regulate SGIV-induced apoptosis. The results demonstrated that EcPDCD4s might play important roles in E. coioides tissues during pathogen-caused inflammation.

Molecular characterization, expression and function analysis of Epinephelus coioides MKK4 response to SGIV and Vibrio alginolyticus infection.

Mitogen-activated protein kinase 4 (MKK4), a member of the MAP kinase family, play important roles in response to many environmental and cellular stresses in mammals. In this study, three MKK4 subtypes, EcMKK4-1, EcMKK4-2 and EcMKK4-3, were obtained from grouper Epinephelus coioides. The open reading frame (ORF) of EcMKK4s are obtained and the EcMKK4s proteins contain highly conserved domains: a S_TKc domain, a canonical diphosphorylation group and two conserved MKKK ATP binding motifs, Asp-Phe-Gly (DFG) and Ala-Pro-Glu (APE). EcMKK4s could be found both in the cytoplasmic and nuclear. The EcMKK4s mRNA were detected in all E. coioides tissues examined with the different expression levels, and the expression were up-regulated during SGIV (Singapore grouper iridescent virus) or Vibrio alginolyticus infection. EcMKK4 could significantly reduce the activation of AP-1 reporter gene. The results suggested that EcMKK4s might play important roles in pathogen-caused inflammation.

New pyrone and cyclopentenone derivatives from marine-derived fungus Aspergillus sydowii SCSIO 00305.

Two new 2-pyrone derivatives sydowiones A-B (1, 2), one new cyclopentenone derivative sydowione C (3), and one new mycotoxin 6-methoxyl austocystin A (4) along with two known analogues paecilpyrone A (5) and austocystin A (6), were isolated from the marine-derived fungus Aspergillus sydowii SCSIO 00305. The structures of 1-4 were elucidated by extensive spectroscopic analysis. The absolute configuration of C-8 in 1 was established by Mosher method, and further confirmed by calculation of the electronic circular dichroism (ECD) spectra. The absolute configuration of C-11 in 3 was also determined by calculation of ECD spectra. The absolute configuration of 6 was determined by a single-crystal X-ray diffraction experiment for the first time. Compounds 1-4 showed moderate toxicity towards brine shrine naupalii with LC50 values of 19.5, 14.3, 8.3 and 2.9 µM, respectively. And 1 and 2 also showed antioxidant activity against 2,2-diphenyl-picrylhydrazyl (DPPH) radicals with IC50 values of 46.0 and 46.6 µM, respectively.[Formula: see text].

Analysis of nifH DNA and RNA reveals a disproportionate contribution to nitrogenase activities by rare plankton-associated diazotrophs.

BACKGROUND: Holobionts comprising nitrogen-fixing diazotrophs and phytoplankton or zooplankton are ubiquitous in the pelagic sea. However, neither the community structure of plankton-associated diazotrophs (PADs) nor their nitrogenase transcriptional activity are well-understood. In this study, we used nifH gene Illumina sequencing and quantitative PCR to characterize the community composition and nifH expression profile of PADs with > 100 µm size fraction in the euphotic zone of the northern South China Sea. RESULTS: The results of DNA- and RNA-derived nifH gene revealed a higher alpha-diversity in the active than in the total community. Moreover, the compositional resemblance among different sites was less for active than for total communities of PADs. We characterized the 20 most abundant OTUs by ranking the sum of sequence reads across 9 sampling stations for individual OTUs in both nifH DNA and RNA libraries, and then assessed their phylogenetic relatedness. Eight of the 20 abundant OTUs were phylogenetically affiliated with Trichodesmium and occurred in approximately equal proportion in both the DNA and RNA libraries. The analysis of nifH gene expression level showed uneven attribute of the abundance and nitrogenase activities by the remaining 12 OTUs. Taxa belonging to cluster III and Betaproteobacteria were present at moderate abundance but exhibited negligible nitrogenase transcription activity. Whereas, the abundances of Richelia, Deltaproteobacteria and Gammaproteobacteria were low but the contribution of these groups to nitrogenase transcription was disproportionately high. CONCLUSIONS: The substantial variation in community structure among active dizatrophic fractions compared to the total communities suggests that the former are better indicators of biological response to environmental changes. Altogether, our study highlights the importance of rare PADs groups in nitrogen fixation in plankton holobionts, evidenced by their high level of nitrogenase transcription.

Evolution of the sink and source of dissolved inorganic nitrogen with salinity as a tracer during summer in the Pearl River Estuary.

In this study, we evaluated the sink and source of the surface water along the PRE using a mixing model method with salinity as tracer. The observational data showed that the decreasing of dissolved inorganic nitrogen (DIN) did not closely follow the physical mixing lines of freshwater and modified seawater. In the western part, DIN consumption by phytoplankton and bacteria uptake (ΔDIN)varied from 15.81 µmol L-1 to 88.53 µmol L-1. On the contrary, in the eastern part, ΔDIN varied from -63.66 µmol L-1 to -10.45 µmol L-1. DIN source in the eastern part may be mainly caused by organic matter decomposition, while DIN remove is strongly associated with phytoplankton growth and bacteria consumption. These differential behaviors of the estuary with respect to DIN are largely due to varying degrees of hydrodynamics due to different topography in the two areas. Sensitivity analysis indicated reduction strategies of DIN inputs to coastal waters may improve environment quality in the PRE, due to DIN changes in the freshwater end-member having a determined influence on biological activities (R). Our results indicate that the model may be a valuable way to address the sources and sink of DIN in the river-dominated estuaries.

Seasonal and spatial variations of water quality and trophic status in Daya Bay, South China Sea.

Coastal water quality and trophic status are subject to intensive environmental stress induced by human activities and climate change. Quarterly cruises were conducted to identify environmental characteristics in Daya Bay in 2013. Water quality is spatially and temporally dynamic in the bay. Cluster analysis (CA) groups 12 monitoring stations into two clusters. Cluster I consists of stations (S1, S2, S4-S7, S9, and S12) located in the central, eastern, and southern parts of the bay, representing less polluted regions. Cluster II includes stations (S3, S8, S10, and S11) located in the western and northern parts of the bay, indicating the highly polluted regions receiving a high amount of wastewater and freshwater discharge. Principal component analysis (PCA) identified that water quality experience seasonal change (summer, winter, and spring-autumn seasons) because of two monsoons in the study area. Eutrophication in the bay is graded as high by Assessment of Estuarine Trophic Status (ASSETS).

Cultivation-dependent analysis of the microbial diversity associated with the seagrass meadows in Xincun Bay, South China Sea.

Microbial communities have largely existed in the seagrass meadows. A total of 496 strains of the bacteria in the seagrass meadows, which belonged to 50 genera, were obtained by the plate cultivation method from three sites of Xincun Bay, South China Sea. The results showed that Bacillales and Vibrionales accounted for the highest proportions of organisms in all communities. The diversity of the bacteria in the sediment was higher than that associated with seagrass. Thalassia hemperichii possessed the highest abundance of bacteria, followed by Enhalus acoroides and Cymodocea rotundata. Robust seasonal dynamics in microbial community composition were also observed. It was found that microbial activities were closely tied to the growth stage of the seagrass. The microbial distribution was the lowest in site 3. The abundance of the bacteria was linked to the interactions between bacteria and plants, the condition of plant and even the coastal water quality and the nutrition level in the sediment.

Responses of bacterial communities in seagrass sediments to polycyclic aromatic hydrocarbon-induced stress.

The seagrass meadows represent one of the highest productive marine ecosystems, and have the great ecological and economic values. Bacteria play important roles in energy flow, nutrient biogeochemical cycle and organic matter turnover in marine ecosystems. The seagrass meadows are experiencing a world-wide decline, and the pollution is one of the main reasons. Polycyclic aromatic hydrocarbons (PAHs) are thought be the most common. Bacterial communities in the seagrass Enhalus acoroides sediments were analyzed for their responses to PAHs induced stress. Dynamics of the composition and abundance of bacterial communities during the incubation period were explored by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR assay, respectively. Both the incubation time and the PAHs concentration played significant roles in determining the microbial diversity, as reflected by the detected DGGE bands. Analysis of sequencing results showed that the Gammaproteobacteria were dominant in the seagrass sediments, accounting for 61.29 % of all sequenced bands. As PAHs could be used as carbon source for microbes, the species and diversity of the PAH-added groups (group 1 and 2) presented higher Shannon Wiener index than the group CK, with the group 1 showing the highest values almost through the same incubation stage. Patterns of changes in abundance of the three groups over the experiment time were quite different. The bacterial abundance of the group CK and group 2 decreased sharply from 4.15 × 10(11) and 6.37 × 10(11) to 1.17 × 10(10) and 1.07 × 10(10) copies/g from day 2 to 35, respectively while bacterial abundance of group 1 increased significantly from 1.59 × 10(11) copies/g at day 2 to 8.80 × 10(11) copies/g at day 7, and then dropped from day 14 till the end of the incubation. Statistical analysis (UMPGA and PCA) results suggested that the bacterial community were more likely to be affected by the incubation time than the concentration of the PAHs. This study provided the important information about dynamics of bacterial community under the PAHs stress and revealed the high bacterial diversity in sediments of E. acoroides. Investigation results also indicated that microbial community structure in the seagrass sediment were sensible to the PAHs induced stress, and may be used as potential indicators for the PAHs contamination.

Illumina-based analysis the microbial diversity associated with Thalassia hemprichii in Xincun Bay, South China Sea.

In order to increase our understanding of the microbial diversity associated with seagrass Thalassia hemprichii in Xincun Bay, South China Sea, 16S rRNA gene was identified by highthrough sequencing method. Bacteria associated with seagrass T. hemprichii belonged to 37 phyla, 99 classes. The diversity of bacteria associated with seagrass was similar among the geographically linked coastal locations of Xincun Bay. Proteobacteria was the dominant bacteria and the α-proteobacteria had adapted to the seagrass ecological niche. As well, α-proteobacteria and Pseudomonadales were associated microflora in seagrass meadows, but the interaction between the bacteria and plant is needed to further research. Burkholderiales and Verrucomicrobiae indicated the influence of the bay from anthropogenic activities. Further, Cyanobacteria could imply the difference of the nutrient conditions in the sites. γ-proteobacteria, Desulfobacterales and Pirellulales played a role in the cycle of sulfur, organic mineralization and meadow ecosystem, respectively. In addition, the less abundance bacteria species have key functions in the seagrass meadows, but there is lack knowledge of the interaction of the seagrass and less abundance bacteria species. Microbial communities can response to surroundings and play key functions in the biochemical cycle.

Spatial variations of bacterial community and its relationship with water chemistry in Sanya Bay, South China Sea as determined by DGGE fingerprinting and multivariate analysis.

Bacteria play important roles in the structure and function of marine food webs by utilizing nutrients and degrading the pollutants, and their distribution are determined by surrounding water chemistry to a certain extent. It is vital to investigate the bacterial community's structure and identifying the significant factors by controlling the bacterial distribution in the paper. Flow cytometry showed that the total bacterial abundance ranged from 5.27 × 10(5) to 3.77 × 10(6) cells/mL. Molecular fingerprinting technique, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing has been employed to investigate the bacterial community composition. The results were then interpreted through multivariate statistical analysis and tended to explain its relationship to the environmental factors. A total of 270 bands at 83 different positions were detected in DGGE profiles and 29 distinct DGGE bands were sequenced. The predominant bacteria were related to Phyla Protebacteria species (31 %, nine sequences), Cyanobacteria (37.9 %, eleven sequences) and Actinobacteria (17.2 %, five sequences). Other phylogenetic groups identified including Firmicutes (6.9 %, two sequences), Bacteroidetes (3.5 %, one sequences) and Verrucomicrobia (3.5 %, one sequences). Conical correspondence analysis was used to elucidate the relationships between the bacterial community compositions and environmental factors. The results showed that the spatial variations in the bacterial community composition was significantly related to phosphate (P = 0.002, P < 0.01), dissolved organic carbon (P = 0.004, P < 0.01), chemical oxygen demand (P = 0.010, P < 0.05) and nitrite (P = 0.016, P < 0.05). This study revealed the spatial variations of bacterial community and significant environmental factors driving the bacterial composition shift. These results may be valuable for further investigation on the functional microbial structure and expression quantitatively under the polluted environments in the world.

Identification of Surface Water Quality along the Coast of Sanya, South China Sea.

Principal component analysis (PCA) and cluster analysis (CA) are utilized to identify the effects caused by human activities on water quality along the coast of Sanya, South China Sea. PCA and CA identify the seasonality of water quality (dry and wet seasons) and polluted status (polluted area). The seasonality of water quality is related to climate change and Southeast monsoons. Spatial pattern is mainly related to anthropogenic activities (especially land input of pollutions). PCA reveals the characteristics underlying the generation of coastal water quality. The temporal and spatial variation of the trophic status along the coast of Sanya is governed by hydrodynamics and human activities. The results provide a novel typological understanding of seasonal trophic status in a shallow, tropical, open marine bay.

The diversity of coral associated bacteria and the environmental factors affect their community variation.

Coral associated bacterial community potentially has functions relating to coral health, nutrition and disease. Culture-free, 16S rRNA based techniques were used to compare the bacterial community of coral tissue, mucus and seawater around coral, and to investigate the relationship between the coral-associated bacterial communities and environmental variables. The diversity of coral associated bacterial communities was very high, and their composition different from seawater. Coral tissue and mucus had a coral associated bacterial community with higher abundances of Gammaproteobacteria. However, bacterial community in seawater had a higher abundance of Cyanobacteria. Different populations were also found in mucus and tissue from the same coral fragment, and the abundant bacterial species associated with coral tissue was very different from those found in coral mucus. The microbial diversity and OTUs of coral tissue were much higher than those of coral mucus. Bacterial communities of corals from more human activities site have higher diversity and evenness; and the structure of bacterial communities were significantly different from the corals collected from other sites. The composition of bacterial communities associated with same coral species varied with season's changes, geographic differences, and coastal pollution. Unique bacterial groups found in the coral samples from more human activities location were significant positively correlated to chemical oxygen demand. These coral specific bacteria lead to coral disease or adjust to form new function structure for the adaption of different surrounding needs further research.

Bacterial dynamics within the mucus, tissue and skeleton of the coral Porites lutea during different seasons.

Investigation of the response of coral microbial communities to seasonal ecological environment at the microscale will advance our understanding of the relationship between coral-associated bacteria community and coral health. In this study, we examined bacteria community composition from mucus, tissue and skeleton of Porites lutea and surrounding seawater every three months for 1 year on Luhuitou fringing reef. The bacterial communities were analyzed using pyrosequencing of the V1-V2 region of the 16S rRNA gene, which demonstrated diverse bacterial consortium profiles in corals. The bacterial communities in all three coral compartments studied were significantly different from the surrounding seawater. Moreover, they had a much more dynamic seasonal response compared to the seawater communities. The bacterial communities in all three coral compartments collected in each seasonal sample tended to cluster together. Analysis of the relationship between bacterial assemblages and the environmental parameters showed that the bacterial community correlated to dissolved oxygen and rainfall significantly at our study site. This study highlights a dynamic relationship between the high complexity of coral associated bacterial community and seasonally varying ecosystem parameters.

Paenibacillus abyssi sp. nov., isolated from an abyssal sediment sample from the Indian Ocean.

A Gram-positive, rod-shaped bacterium, designated strain SCSIO N0306(T), was isolated from an abyssal sediment sample collected from the Indian Ocean. The isolate was found to grow optimally at 0-2 % (w/v) NaCl, pH 7.0 and 30 °C. Comparative analysis of the 16S rRNA gene sequence showed that the isolate SCSIO N0306(T) belongs phylogenetically to the genus Paenibacillus, and to be most closely related to P. algorifonticola XJ259(T) (with 95.47 % sequence similarity), sharing less than 95.0 % sequence similarity with all other taxa of this genus. Chemotaxonomic analysis revealed MK-7 as the major isoprenoid quinone, the DNA G+C content was determined to be 45.5 mol%, and anteiso-C15:0, C16:0, and iso-C15:0 were identified as the major fatty acids. On the basis of this polyphasic taxonomic data, isolate SCSIO N0306(T) is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus abyssi sp. nov. is proposed. The type strain is SCSIO N0306(T) (= DSM 26238(T) = CGMCC 1.12987(T)).

Detection of polyketide synthase and nonribosomal peptide synthetase biosynthetic genes from antimicrobial coral-associated actinomycetes.

The diversity and properties of actinobacteria, predominant residents in coral holobionts, have been rarely documented. In this study, we aimed to explore the species diversity, antimicrobial activities and biosynthetic potential of culturable actinomycetes within the tissues of the scleractinian corals Porites lutea, Galaxea fascicularis and Acropora millepora from the South China Sea. A total of 70 strains representing 13 families and 15 genera of actinobacteria were isolated. The antimicrobial activity and biosynthetic potential of fifteen representative filamentous actinomycetes were estimated. Crude fermentation extracts of 6 strains exhibited comparable or greater activities against Vibrio alginolyticus than ciprofloxacin. Seven of the 15 actinomycetes strains possess type I polyketide synthases (PKS-I) and/or nonribosomal peptide synthetases (NRPS) genes. Nine tested strains possess type II polyketide synthases (PKS-II). Phylogenetic analysis based on 16S rRNA gene sequences indicated that these PKS and NRPS gene screening positive strains belong to genera Nocardiopsis, Pseudonocardia, Streptomyces, Micromonospora, Amycolatopsis and Prauserella. One PKS-I and four NRPS fragments showed <70% similarity to their closest relatives, which suggested the novelty of these genes. This study helps uncover the genetic capacity of stony coral-associated actinomycetes to produce bioactive molecules.

Mangrovibacterium diazotrophicum gen. nov., sp. nov., a nitrogen-fixing bacterium isolated from a mangrove sediment, and proposal of Prolixibacteraceae fam. nov.

A nitrogen-fixing bacterium, designated strain SCSIO N0430(T), was isolated from a mangrove sediment sample. Analysis of the sequence of the nifH gene responsible for nitrogen fixation in this strain indicated a close relationship to an uncultured bacterium ZNZ-D11 (GenBank accession no. JF896696). 16S rRNA gene sequence analysis revealed that this isolate had less than 93 % similarity to its closest relative, Sunxiuqinia elliptica DQHS4(T). A phylogenetic tree reconstructed based on 16S rRNA gene sequences revealed that strain SCSIO N0430(T) was a member of the phylum Bacteroidetes. Chemotaxonomic and physiological characteristics, including phospholipids and major fatty acids, readily distinguished the isolate from established members of the phylum Bacteroidetes. It is concluded that strain SCSIO N0430(T) represents a novel genus and species, for which the name Mangrovibacterium diazotrophicum gen. nov., sp. nov. is proposed, with the type strain of the species SCSIO N0430(T) ( = KCTC 32129(T) = DSM 27148(T) = JCM 19152(T)). Based on phylogenetic characteristics and 16S rRNA gene signature nucleotide patterns, the three genera Sunxiuqinia, Prolixibacter and Mangrovibacterium are proposed to make up a novel family, Prolixibacteraceae fam. nov., in the order Bacteroidales.

Spatial variation of bacterial community structure of the Northern South China Sea in relation to water chemistry.

Spatial distribution, diversity and composition of bacterial communities of the northern South China Sea (SCS) surface water and the relationship with the in situ environmental chemistry were investigated. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) was used to investigate the bacterial community structure. The DGGE gel showed that each sample harbored a distinct bacterial community structure and spatial variations of bacterial community composition among all samples were obviously. A total of 17 intensive bands were excised and the sequence analysis of these DGGE bands revealed that Proteobacteria were the dominant bacterial group of surface water in the north part of SCS. Results of the taxonomic analysis showed that the communities consisted of Proteobacteria (α-subdivision, ß-subdivision, γ-subdivision), Actinobacteria, Cyanobacteria, Bacteroidetes and Firmicutes. Unweighted pair group method with arithmetic averages clustering of the sampling stations indicated that all stations were classified mainly based on geographical proximity. Canonical correspondence analysis (CCA) was employed to further investigate the relationships between DGGE band pattern and the environmental variables and the first two CCA ordination axes suggested that the structure of the bacterial community was significantly correlated with the variables of nitrate (F = 1.24, P < 0.05).