Amplicon-Based Illumina Sequencing and Quantitative PCR Reveals Nanoplankton Diversity and Biomass in Surface Water of Qinhuangdao Coastal Area, China

Aureococcus anophagefferens caused brown tides for three consecutive years from 2009 to 2011 in the coastal waters of Qinhuangdao, China, with numerous, widespread ecological and economic impact on ecosystems. To understand the population dy- namics of nanoplankton during the brown tides, sequences of the V9 region of the 18S rDNA gene, used as a marker, were analyzed by Illumina sequencing to assess nanoplankton biomass, and real-time fluorescence quantitative PCR was performed to analyze spa- tial variation in the 18S rDNA copy concentrations of nanoplankton off the Qinhuangdao coast in July, 2011. The results showed that A. anophagefferens and Minutocellus polymorphus were the dominant species in the local phytoplankton community during the brown tide in July 2011. The highest 18S rDNA copy concentrations of A. anophagefferens and M. polymorphus were detected at stations SHG and FN, respectively. The central area most strongly affected by the brown tide migrated southward from 2011 to 2013. Redundancy analysis (RDA) showed that the decreasing NOx concentration might provide suitable nutrient conditions for the A. anophagefferens outbreak. During the brown tide caused by A. anophagefferens, other phytoplankton, such as diatoms, cryptophytes, chlorophytes, dinoflagellates and other flagellates, could co-occur with it. For zooplankton, due to less selective feeding behavior, Amoebozoa was the most abundant zooplankton at station SHG, while Ciliophora was the most abundant zooplankton at other sta- tions for its more selective feeding.

Characterization of Pythium chondricola associated with red rot disease of Pyropia yezoensis (Ueda)(Bangiales,Rhodophyta) from Lianyungang,China

Pyropia yezoensis(formerly Porphyra yezoensis)is an economically important red alga that is cultured extensively in China.The red rot disease occurs commonly during Pyropia cultivation,causing serious economic losses.An incidence of red rot disease was found in a P.yezoensis farm from mid-November to mid-December 2015 at Lianyungang,Jiangsu Province,China.Histopathological examination revealed that the naturally infected thalli were infected apparently by a pathogen,leading to red rot symptoms.The causative agent was isolated and identified as the oomycete Pythium chondricola by morphological analysis and sequence analysis of the internal transcribed spacer and cytochrome oxidase subunit 1(cox 1).In artifi cial infection experiments on the P.yezoensis blades,the P.chondricola isolate was able to cause the same characteristic histopathology seen in natural infections.P.chondricola grew well at a wide range of temperatures in the range 8-31℃,salinities at 0-45 and pH 5-9.In an orthogonal test used to determine the effects of environmental factors(temperature,salinity,and zoospore concentration)on infection,the data revealed that temperature was the most important factor to affect red rot disease development,with the optimal conditions for disease expansion being 20℃,35 salinity,and a zoospore concentration of 10^6 zoospores/mL.The results obtained from the present study prompted us to set up a comprehensive epidemiological study on Pyropia,which will provide support to maintain the healthy development of the Pyropia industry in China.

Lyase Activities of Heterologous CpcS and CpcT for Phycocyanin Holo-β-subunit from Arthrospira platensis in Escherichia coli

Arthrospira platensis is an economically important cyanobacterium; and it has been used widely in food and pharmaceutical industries. The phycocyanin(PC) from A. platensis is extremely valuable in medicine and molecular biology due to its antioxidation and anti-tumoring activity and applicability as fluorescence protein tag. In present study, two recombinant plasmids, one contained the phycocyanobilin(PCB)-producing genes(hox1 and pcyA) while the other contained the phycobiliprotein gene(cpcB) and the lyase gene(either cpcS/U or cpcT), were constructed and synchronically transferred into E. coli in order to test the the activities of relevant lyases for catalysing PCB addition to CpcB during synthesizing fluorescent PC holo-β-subunit(β-PC) of A. platensis. As was evidenced by the fluorescence emitted at a peak specific for PC, CpcB was successfully synthesized in E. coli, to which co-expressed PCBs attached though at a relatively low efficiency. The results showed that the attachment of PCBs to CpcB were carried out mainly by co-expressed CpcS/U but CpcB also showed some autocatalytic activity. Currently, no CpcT activity was detected in this E. coli expression system. Further studies will be conducted to improve the efficiency of fluorescent PC synthesis in E. coli.

Comparison of the In Vitro and In Vivo Toxic Effects of Three Sizes of Zinc Oxide(ZnO)Particles Using Flounder Gill(FG)Cells and Zebrafish Embryos

Nano-sized zinc oxide(n ZnO) particles are one kind of the most commonly used metal oxide nanoparticles(NPs). This study compared the cytotoxic and embryotoxic effects of three increasing sized ZnO particles(φ30 nm, 80-150 nm and 2 μm) in the flounder gill(FG) cells and zebrafish embryos, and analyzed the contribution of size, agglomeration and released Zn^(2+) to the toxic effects. All the tested ZnO particles were found to be highly toxic to both FG cells and zebrafish embryos. They induced growth inhibition, LDH release, morphological changes and apoptosis in FG cells in a concentration-, size-and time-dependent manner. Moreover, the release of LDH from the exposed FG cells into the medium occurred before the observable morphological changes happened. The ultrasonication treatment and addition of serum favored the dispersion of ZnO particles and alleviated the agglomeration, thus significantly increased the corresponding cytotoxicity. The released Zn^(2+) ions from ZnO particles into the extracellular medium only partially contributed to the cytotoxicity. All the three sizes of ZnO particles tested induced developmental malformations, decrease of hatching rates and lethality in zebrafish embryos, but size-and concentration-dependent toxic effects were not so obvious as in FG cells possibly due to the easy aggregation of ZnO particles in freshwater. In conclusion, both FG cells and zebrafish embryos are sensitive bioassay systems for safety assessment of ZnO particles and the environmental release of ZnO particles should be closely monitored as far as the safety of aquatic organisms is concerned.

Changes in the Photosynthetic Pigment Contents and Transcription Levels of Phycoerythrin-Related Genes in Three Gracilariopsis lemaneiformis Strains Under Different Light Intensities

Three Gracilariopsis lemaneiformis strains,including wild type and high-temperature-resistant cultivars 981 and 2007,were studied with the changes in their photosynthetic pigment contents and related gene transcription levels under different light intensities(10,60,100,and 200μmolm^(−2)s^(−1)).The three G.lemaneiformis strains had the following photosynthetic pigments with high-to-low contents:phycoerythrin(PE),phycocyanin(PC),allophycocyanin(APC),and chlorophyll a(Chl a).Among the three strains,cultivar 981 had the highest PE content,followed by cultivar 2007.The PC and APC contents were similar among the three strains,but they were higher in cultivars 981 and 2007 than in the wild type.The Chl a contents in the three G.lemaneiformis strains were equal.A low light intensity(10μmolm^(−2)s^(−1))promoted photosynthetic pigment accumulation in G.lemaneiformis and improved the relative PE gene transcription(peA and peB)in a short period(≤6 d).A high light intensity decreased the PE content.PebA and PebB,which catalyzed phycoerythrobilin synthesis,showed no compensatory upregulation at a low light intensity among the strains except for the wild type.At a high light intensity,transcription levels of pebA and pebB in the three strains were upregulated.This study provided an experimental basis for elucidating the photosynthesis of G.lemaneiformis.As key genes of algal growth,photo-synthesis-related genes served as useful gene markers for screening elite varieties with good traits in breeding.Cultivar 2007 was superior to cultivar 981 in terms of maintaining high pigment levels in a wide range of light intensities,which is the most suitable for aquaculture.

Distribution, Function and Polymorphism Characteristics of Microsatellites in Pyropia yezoensis Transcriptome

The distribution, putative function and polymorphism characteristics of simple sequence repeats(SSRs) in P. yezoensis transcriptome were analyzed in this study. In total, 3076 SSRs were detected among 2681 unigenes. Trinucleotide types were dominant, constituting 91.87% of all the microsatellites. The most abundant SSR was CCG(71.03%) and the second abundant one was AGC(234, 7.61%). A total of 111(3.61%) dinucleotide types were found and the most abundant one was AC(51, 1.66%) which was followed by CG(34, 1.11%). SSRs identified showed a GC bases preference;GC bases constituted 89.73% of all the SSR bases. SSRs occurrence number decreased as repetitions increased. Annotation results exhibited that the majority of SSRs-containing unigenes have the functions of ‘metabolic process’,‘binding’ and ‘catalytic activity’ and existed as the forms of ‘cell’,‘cell component’ and ‘organelle’. The dominant amino acids that SSRs coded were Ala(28.59%), Arg(26.02%), Gly(14.35%) and Pro(13.65%). Amplification results showed that 3 out 23 SSRs from transcriptome(13.04%) and 8 out 77 SSRs from genome(10.39%) were polymorphic.

The first complete organellar genomes of an Antarctic red alga,Pyropia endiviifolia:insights into its genome architecture and phylogenetic position within genus Pyropia(Bangiales,Rhodophyta)

Pyropia species grow in the intertidal zone and are cold-water adapted. To date, most of the information about the whole plastid and mitochondrial genomes(ptDNA and mtDNA) of this genus is limited to Northern Hemisphere species. Here, we report the sequencing of the ptDNA and mtDNA of the Antarctic red alga Pyropia endiviifolia using the Illumina platform. The plastid genome(195 784 bp, 33.28% GC content) contains 210 protein-coding genes, 37 tRNA genes and 6 rRNA genes. The mitochondrial genome(34 603 bp, 30.5% GC content) contains 26 protein-coding genes, 25 tRNA genes and 2 rRNA genes. Our results suggest that the organellar genomes of Py. endiviifolia have a compact organization. Although the collinearity of these genomes is conserved compared with other Pyropia species, the genome sizes show significant differences, mainly because of the different copy numbers of rDNA operons in the pt DNA and group II introns in the mtDNA. The other Pyropia species have 2–3 distinct intronic ORFs in their cox 1 genes, but Py. endiviifolia has no introns in its cox 1 gene. This has led to a smaller mtDNA than in other Pyropia species. The phylogenetic relationships within Pyropia were examined using concatenated gene sets from most of the available organellar genomes with both the maximum likelihood and Bayesian methods. The analysis revealed a sister taxa affiliation between the Antarctic species Py. endiviifolia and the North American species Py. kanakaensis.

Divergence time, historical biogeography and evolutionary rate estimation of the order Bangiales (Rhodophyta) inferred from multilocus data

Bangiales is the only order of the Bangiophyceae and has been suggested to be monophyletic. This order contains approximately 190 species and is distributed worldwide. Previous molecular studies have produced robust phylogenies among the red algae, but the divergence times, historical biogeography and evolutionary rates of Bangiales have rarely been studied. Phylogenetic relationships within the Bangiales were examined using the concatenated gene sets from all available organellar genomes. This analysis has revealed the topology((( Bangia, Porphyra) Pyropia) Wildemania). Molecular dating indicates that Bangiales diversifi ed approximately 246.40 million years ago(95% highest posterior density(HPD)= 194.78–318.24 Ma, posterior probability(PP)=0.99) in the Late Permian and Early Triassic, and that the ancestral species most likely originated from eastern Gondwanaland(currently New Zealand and Australia) and subsequently began to spread and evolve worldwide. Based on pairwise comparisons, we found a slower rate of nucleotide substitutions and lower rates of diversifi cation in Bangiales relative to Florideophyceae. Compared with Viridiplantae(green algae and land plants), the evolutionary rates of Bangiales and other Rhodophyte groups were found to be dramatically faster, by more than 3-fold for plastid genome(ptDNA) and 15-fold for mitochondrial genome(mtDNA). In addition, an average 2.5-fold lower dN/dS was found for the algae than for the land plants, which indicates purifying selection of the algae.

Culture observation and molecular phylogenetic analysis on the blooming green alga Chaetomorpha valida (Cladophorales, Chlorophyta) from China

The marine green alga Chaetomorpha valida fouls aquaculture ponds along the coastal cities of Dalian and Rongcheng, China. Unialgal cultures were observed under a microscope to determine the developmental morphological characters of C. valida. Results reveal that gametophytic filaments often produce lateral branches under laboratory culture conditions, suggesting an atypical heteromorphic life cycle of C. valida between unbranched sporophytes and branched gametophytes, which differs from typical isomorphic alternation of Chaetomorpha species. The shape of the basal attachment cell, an important taxonomic character within the genus, was found variable depending on environmental conditions. The 18S rDNA and 28S rDNA regions were used to explore the phylogenetic affinity of the taxa. Inferred trees from 18S rDNA sequences revealed a close relationship between C. valida and Chaetomorpha moniligera. These results would enrich information in general biology and morphological plasticity of C. valida and provided a basis for future identification of green tide forming algae.

Characterization of SOX3 Gene in an Ovoviviparous Teleost, Black Rockfish(Sebastes schlegeli)

Evolutionary evidence suggests that Sox3, a member of the high-mobility-group(HMG) family of transcription factors, is an ancestral precursor of Sry and is involved in sex determination similar to Sry. However, there is limited information regarding the SOX3 gene of the black rockfish(Sebastes schlegeli). In this study, we first isolated SOX3 gene from the gonads of S. schlegeli by homology cloning. The full-length of S. schlegeli SOX3(SsSOX3) c DNA was 1386 bp, comprising a 906-bp open reading frame, which encodes a peptide showing 93.6% and 93.9% homology with the Sox3 proteins of Epinephelus coioides and Oryzias latipe, respectively. Comparison of the cDNA sequence of the Ss SOX3 gene with the corresponding genomic DNA fragment revealed that the SsSOX3 gene consists of a single exon. Phylogenetic analysis demonstrated the evolutionary relationship of Ss SOX3 with other known SOXB1 genes in fish and tetrapods. The promoter region contains binding sites of several transcriptional factors that might participate in the regulation of Ss SOX3 expression. Quantitative real-time PCR analysis indicated that SsSOX3 was expressed in all the investigated larval developmental stages from 1 to 35 days after birth and the level of expression gradually decreased as the development proceeded. SsSOX3 exhibited sexually dimorphic expression in adult gonads, with high expression in the ovary but low expression in the testis. In situ hybridization revealed that SsSOX3 was strongly expressed in oocytes and follicular cells of ovaries but slightly expressed in germ cells of testicular tissues. Therefore, this study suggests that Ss SOX3 plays an important role in oogenesis and ovary differentiation in S. schlegeli.

Development of a PCR method for detection of Pseudoalteromonas marina associated with green spot disease in Pyropia yezoensis

Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease(GSD)in Pyropia yezoensis.To prevent GSD from development and spread,an effective method to detect the pathogen at early GSD infection stages need to be established.In this research,PCR methods were established targeting the dnaA gene(encoding chromosome replication initiator protein)and the dnaN gene(encodingβsliding clamp of DNA polymeraseⅢprotein)to detect P.marina with three primer pairs pws-dnaA2(Forward,5'-ACCGCATTAACGAACTACTCGTG-3';Reverse,5'-TGCCATTACCTACAGCATGG-3'),pcs-dnaN2(Forward,5'-CTTACAACGTTATCAGCGGC-3';Reverse,5'-GTTGAGTATTAAGTGATTGAGTAAGC-3')or pws-dnaN3(Forward,5'-ACTTACAA-CGTTATCAGCGGC-3';Reverse,5'-ACTGCTGTTTGAGTCTGCTAAC-3').Three PCR methods corresponding to the three primer pairs sufficiently distinguished P.marina from 22 bacterial species,thus resulting in detection limits of 4 to 4×10^2 CFU cells or 2.37×10^1 to 2.37×10^3 fg of P.marina DNA per PCR reaction.In an artificial infection experiment ofP.yezoensis infected with P.marina,all established PCRs successfully detected P.marina at early GSD infection stages.The results show that the established PCRs are specific and sensitive,and are potential for applications in early diagnosis of GSD in Pyropia.

Trace Metals Analysis Along the Fildes Peninsula Coastline Using Two Red Algae, Rhodymenia antarctica and Iridaea cordata, as Monitors

Two red algae, Rhodymenia antarctica and Iridaea cordata(Rhodophyta), were employed to investigate the pollution situation along Fildes Peninsula coastline, King George Island. Ten sites from east and west coastlines were investigated, and the concentrations of Cu, Pb, Zn, Cd, Cr, As and Hg were determined. The metal pollution index(MPI) was used to evaluate the overall pollution level represented by the investigated sites and for interregional comparisons. The two algae exhibited different preference to special trace metal. R. antarctica could accumulate more Cd(0.63 mg kg^(-1)) and Hg(0.026 mg kg^(-1)) than I. cordata(Cd 0.34 mgkg^(-1), Hg 0.019 mg kg^(-1)). I. cordata could accumulate more As(15.53 mg kg^(-1)) than R. antarctica(10.11 mg kg^(-1)). There was no significant difference in accumulating Cu, Pb, Zn and Cr between the two algae. R. antarctica could be used to monitor Cd and Hg. I. cordata would be more appropriate for monitoring As. MPI monitored by the two algae were from 1.02 to 2.26(R. antarctica), and 1.03 to 1.25(I. cordata), respectively. Pollution situation of Fildes Peninsula was becoming serious, especially of the east coastline. The objective of this research was to gather the baseline information for trace metals investigation in Antarctic.

Glutathione S-transferase genes in scallops and their diverse expression patterns after exposure to PST-producing dinoflagellates

The glutathione S-transferases(GSTs)are a superfamily of enzymes that function in cellular protection against toxic substances and oxidative stress.Bivalves could accumulate high concentration of paralytic shellfish toxins(PSTs)from harmful algae.To understand the possible involvement of GSTs in protecting bivalves during PST accumulation and metabolism,the GST genes were systemically analyzed in two cultured scallops,Azumapecten farreri and Mizuhopecten yessoensis,which were reported for PST deposition during harmful algae bloom.A total of 35 and 37 GSTs were identified in A.farreri(AfGSTs)and M.yessoensis(MyGSTs)genome,respectively,and the expansion of the sigma class from the cytosolic subfamily was observed.In both scallop species,sigma class GSTs showed higher expression than other members.The high GSTs expression was detected mainly during/after larvae stages and in the two most toxic organs,hepatopancreas and kidney.After ingesting PST-producing dinoflagellates,all the regulated AfGSTs in the hepatopancrcas were from the sigma class,but with opposite regulation pattern between Alexandrium catenella and A.minutum exposure.In scallop kidneys,where PSTs transformed into higher toxicity,more AfGSTs were regulated than in the hepatopancreas,and most of them were from the sigma class,with similar regulation pattern between A.catenella and A.minutum exposure.In M.yessoensis exposed to A.catenella,MyGST-σ2 was the only up-regulated MyGST in both hepatopancreas and kidney.Our results suggested the possible diverse function of scallop GSTs and the importance of sigma class in the defense against PSTs,which would contribute to the adaptive evolution of scallops in marine environments.

Evolutionary dynamics and conserved function of the Tudor domain-containing(TDRD)proteins in teleost fish

Tudor domain-containing(TDRD)proteins,the germline enriched protein family,play essential roles in the process of gametogenesis and genome stability through their interaction with the PIWI-interacting RNA(piRNA)pathway.Several studies have suggested the rapid evolution of the piRNA pathway in teleost lineages with striking reproductive diversity.However,there is still limited information about the function and evolution of Tdrd genes in teleost species.In this study,through genome wide screening,13 Tdrd family genes were identified in economically important aquaculture fish,including spotted sea bass(Lateolabrax maculatus),Asian sea bass(Lates calcarifer),and tongue sole(Cynoglossus semilaevis).With copy number,structure,phylogeny,and synteny analysis,duplication of Tdrd6 and Tdrd7,as well as loss of Stk31 and Tdrd10,were characterized in teleost lineages.Codon based molecular evolution analysis indicated faster evolution of teleost Tdrd genes than that in mammals,potentially associated with the accelerated evolution of the piRNA pathway in teleost lineages.The evolutionary diversity of Tdrd genes was also detected between different teleost lineages.RNA-seq analysis showed that most teleost Tdrd genes were dominantly expressed in gonads,particularly highly expressed in testis,such as Tdrd6,Tdrd7a,Tdrd9,Ecat8,and Tdrd15.The varied expression and evolutionary pattern between the duplicated Tdrd6 and Tdrd7 in teleosts may indicate their functional diversification.All these results suggest a conserved function of teleost Tdrd family in gametogenesis and the piRNA pathway,which could lay a foundation for the evolution of Tdrd genes and be helpful for further deciphering of Tdrd functions in teleosts.