Applications of immuno-magnetic bead and immunofluorescent flow cytometric techniques for the quantitative detection of HAB microalgae

Overthelastseveraldecades,harmfulalgalblooms(HABs)havebecomeaseriousenvironmental problem in many parts of the world. A rapid and accurate detection process for HAB algae has yet to be developed. Heterosigma akashiwo is one of the most important HABs species in China. The objective of this study was to develop an immunologic technique that can rapidly and sensitively count H. akashiwo cells. Five HABs species (Alexandrium catenella, Thalassiosira sp., Cryptomonas sp., Alexandrium tamarense and Symbiodinium sp.,) were used in this study to evaluate the analysis process we developed. A polyclonal antibody with high titers against H. akashiwo was obtained by injecting H. akashiwo cells into rabbits. Immuno-magnetic beads (IMB) were produced via conjugated polyclonal antibodies with magnetic beads and applied to isolate and count H. akashiwo cells from the culture. Results show that 66.7%-91.6% of the cells were captured from unialgal culture by IMBs, and only 5.3%-12.5% of the four other HAB microalgae species were captured, indicating that the constructed IMBs combined specifically with the H. akashiwo cells. At the same time, flow cytometry (FCM) sorting was exploited to screen H. akashiwo cells after labeling with FITC conjugated polyclonal antibodies. Using the FCM technique, 91.7% of the targeted cells were sorted out from mixed microalgae samples in just a few minutes. These results indicate that both antibody-involved IMB and antibody-based FCM techniques are highly effective at detecting and quantifying HAB species. These techniques, especially immuno-magnetic separation, have low associated cost, and are fast and simple processes compared with other techniques currently in use.     

Relationship between toxic and harmful microalgae and environmental factors in typical mariculture areas of East China Sea

Toxic and harmful algal blooms are usually more frequent in mariculture areas due to the abundant trophic conditions. To investigate the relationship between toxic and harmful microalgae and environmental factors, we set up 12 stations near three mariculture regions (Gouqi Island, Sandu Bay, and Dongshan Bay) in the East China Sea. We collected samples from all four seasons starting from May 2020 to March 2021. We identified 199 species belonging to 70 genera, of which 38 species were toxic and harmful, including 24 species of Dinophyceae, 13 species of Bacillariophyceae, and 1 species of Raphidophyceae. The species composition of toxic and harmful microalgae showed a predominance of diatoms in the summer (August), and dinoflagellates in the spring (May), autumn (November), and winter (March). The cell densities of toxic and harmful microalgae were higher in summer (with an average value of 15.34×10³ cells/L) than in other seasons, 3.53×10³ cells/L in spring, 1.82×10³ cells/L in winter, and 1.0×10³ cells/L in autumn. Pseudonitzschia pungens, Prorocentrum minimum, Paralia sulcata, and Prorocentrum micans were the dominant species and were available at all 12 stations in the three mariculture areas. We selected 10 toxic and harmful microalgal species with frequency >6 at the survey stations for the redundancy analysis (RDA), and the results show that NO ^?₃ , water temperature (WT), pH, DO, and NO ^?₂ were the main factors on distribution of toxic and harmful microalgae. We concluded that the rich nutrient conditions in the East China Sea mariculture areas increased the potential for the risk of toxic and harmful microalgal bloom outbreaks.

     

Daptonema parabreviseta sp. nov. (Xyalidae,Nematoda) from the Jiaozhou Bay of the Yellow Sea,China

A new species of free-living marine nematode of the genus Daptonema Cobb, 1920 is described from the sublittoral sediment of the Jiaozhou Bay, Qingdao. Daptonema parabreviseta sp. nov. is characterized by having narrowed anterior end; small funnel-shaped buccal cavity; short cephalic setae; spicules with bulbiform proximal end and pointed distal end; gubercunalum tubular shape with a blunt dorsal apophysis. The female genital system presents a spacious postvulval sac. The new species closely resembles Daptonema brevisetosum Thanh & Gagarin, 2009. However, it differs from Daptonema brevisetosum by having relatively narrowed head (10 μm vs 20 μm); spicules with bulbiform proximal end and pointed distal end, not bifurcated at distal end. Female genital system with a spacious postvulval sac (vs absent).

     

Diversity and community structure of eukaryotic microalgae in surface sediments in the central Bohai Sea,China, based on a metabarcoding approach

Sediment samples were collected at 17 stations in the central Bohai Sea, China, and the diversity and community structure of eukaryotic microalgae were assessed by metabarcoding the V4 region of 18S rDNA. A total of 930 operational taxonomic units (OTUs) were detected for microeukaryotes, including 98 algal OTUs. The algal communities comprised 42 genera belonging to 19 classes of six phyla, and they were dominated by chrysophytes and dinoflagellates. Dinoflagellates were also the most diverse microalgal group. The nano-sized dinoflagellates Biecheleria halophila and Azadinium trinitatum occurred abundantly in the study area; however, they have not been reported previously, as they may be overlooked or misidentified in light microscopy. Many pico-sized chlorophytes were detected in the sediment samples. Sixteen of the detected OTUs were assigned to potentially harmful and/or bloom-forming microalgae, suggesting some potential risks of harmful algal blooms in the central Bohai Sea. The capacity of metabarcoding to detect morphologically cryptic and small species makes this method a sufficiently sensitive means of detection for assessing eukaryotic microalgae in sediments.

     

Preliminary identification of three new isolates in genus Alexandrium (Dinophyceae) from China sea area

The 5.8S ribosomal DNA sequences (5.8S rDNA) and their flanking regions, internal transcribed spacer 1 and spacer 2 (ITS1 and ITS2) of three new isolates in genus Alexandrium (Alexandrium sp. qd1, Alexandrium sp. qd2, Alexandrium sp. gz) from China were amplified, sequenced, and subjected to phylogenetic analysis. Alexandrium sp. gz and Alexandrium sp. qd1 were grouped with high bootstrap values with four strains/species, i.e., A. catenella South Korea strain, A. catenella Japan strain, and two from China, Alexandrium sp. AC03 and Alexandrium sp. AN01 being proposed to be A. catenalla in a previous study. Then Alexandrium sp. gz and Alexandrium sp. qd1 were identified as Alexandrium catenella. As A. catenella was isolated from Qingdao and Guangzhou sea areas, it supposedly distributed at least in these two areas and was genetically different. Alexandrium sp. qd2 differed greatly from species in Alexandrium. It clustered with Symbiodinium californium, Symbiodinium sp. G15 and Gymnodinium sp. Zhao 01 with 100% bootstrap value; so Alexandrium sp. qd2 affiliates to genus Symbiodinium, and is probably a free-living Symbiodinium species.     

Species identification and phylogenetic analysis of genus Nassarius (Nassariidae) based on mitochondrial genes

Genus Nassarius contains many subgenera, such as Zeuxis, Telasco, Niotha, Varicinassa, Plicarcularia, Nassarius s. str. and Reticunassa. On the basis of morphological characteristics of the shell and radula and sequences of mitochondrial cytochrome oxidase subunit I (COI) and 16S rRNA genes, Nassarius specimens collected from the South China Sea were identified and phylogenetically analyzed. Although Nassarius sp. and Nassarius (Varicinassa) variciferus were morphologically different in their shells, few variations were found among their radular teeth and sequences of mtCOI and mt16S RNA genes. Therefore, Nassarius sp. should be classified as N. (Varicinassa) variciferus. Nassarius (Zeuxis) sp. has only a subtle difference from Nassarius (Zeuxis) algidus on the shell, but it shows obvious differences in radular teeth and DNA sequence, indicating that they are two distinct species. Sequence divergence of mtCOI and mt16S RNA genes within Nassarius species was much lower than that between species, suggesting that these two genes are suitable for Nassarius species identification. Phylogenetic analysis (neighbor-joining and maximum parsimony) based on mtCOI and mt16S rRNA genes revealed the presence of two groups in genus Nassarius and a closest relationship between subgenera Zeuxis and Telasco. Species of subgenus Plicarcularia did not form a single clade. The molecular phylogeny was not congruent with the previous morphological phylogeny. The subgeneric divisions of genus Nassarius appear to be uncertain and unreliable.     

Extracellular release of β-lactamase by osmotic shock inSynechococcus transformant

A unicellular cyanobacteriumSynechococcus sp. strain PCC 7002 was transformed with plasmid pQL1, on which β-lactamase gene (bla) and β-galactosidase gene (lacZ) were encoded. The transformant cells released β-lactamase into medium by an abrupt drop of osmotic pressure. This result indicates that this cyanobacterium recognizes and processes the signal sequence of β-lactamase, and accumulates the enzyme in periplasm. Repeated release of β-lactamase was possible by repeated osmotic shocks wihout, impairing cell viability. On the other hand, most of the β-galactosidase remained in cytoplasm under the osmotic shock.

     

Will harmful dinoflagellate Karenia mikimotoi grow phagotrophically?

We studied the phagotrophic ability of dinoflagellate strain Karenia mikimotoi KM-Lü (isolated from the South China Sea), using fluorescent microspheres, bacteria isolated from the culture of K. mikimotoi and a marine microalgae Isochrysis galbana. We found that K. mikimotoi cultured under conditions of high light intensity could ingest fluorescent microspheres (diameters 0.5 and 2.0 μm) and fluorescence-labeled bacteria and microalgae. Under a low light intensity, however, only fluorescent microspheres (diameter 0.5 μm) and fluorescence-labeled microalgae were ingested. K. mikimotoi showed better growth by ingesting living marine bacteria or microalgae I. galbana than the controls, either in nutrient-depleted or nutrient-replete conditions. In nutrient-depleted conditions, the growth of K. mikimotoi was more significant with I. galbana as the prey item. In conclusion, the harmful dinoflagellate K. mikimotoi from the South China Sea has apparent phagotrophic ability, and some marine bacteria and microalgae may promote the growth of K. mikimotoi.     

Laboratory study on the ecological impact of sophorolipid used for harmful algae elimination

We studied the role of sophorolipid in inhibiting harmful algae bloom (HAB). Different sophorolipid concentrations were tested on marine microalgae, zooplankton, fish, and bivalve (Mytilus edulis) in laboratory. The result shows that sophorolipid could inhibit the growth of algal species selectively. Among three algae species selected, Platymonas helgolandica var. tsingtaoensis was promoted with increasing sophorolipid concentration; Isochrysis galbana was inhibited seven days later in sophorolipid concentration below 40 mg/L; and Nitzschia closterium f. minutissima was inhibited obviously in only a high sophorolipid concentration over 20 mg/L. Therefore, sophorolipid in a low concentration at <20 mg/L could remove certain harmful algae species effectively without harming other non-harmful microalgae. For other animals, sophorolipid could inhibit the growth of ciliate Strombidium sp. by 50% at 20 mg/L sophorolipid concentration after 96 h. The concentration in 96-h LC₅₀ for Calanus sinicus, Neomysis awatschensis, Lateolabrax japonicus, and Paralichthys olivaceus was 15, 150, 60, and 110 mg/L, respectively. The 24 h LC₅₀ value for Artemia salina was 600 mg/L. The relative clearance rate of mussel Mytilus edulis decreased to 80%, 40%, and 20% of the control group after being exposed to 20, 50, and 100 mg/L sophorolipid for 24 h. Therefore, the toxicity for mitigation of harmful algae bloom at previously recommended concentration of 5–20 mg/L sophorolipid is low for most tested organisms in this reaserch.     

A method of batch-purifying microalgae with multiple antibiotics at extremely high concentrations

Axenic microalgal strains are highly valued in diverse microalgal studies and applications. Antibiotics, alone or in combination, are often used to avoid bacterial contamination during microalgal isolation and culture. In our preliminary trials, we found that many microalgae ceased growing in antibiotics at extremely high concentrations but could resume growth quickly when returned to an antibiotics-free liquid medium and formed colonies when spread on a solid medium. We developed a simple and highly efficient method of obtaining axenic microalgal cultures based on this observation. First, microalgal strains of different species or strains were treated with a mixture of ampicillin, gentamycin sulfate, kanamycin, neomycin and streptomycin (each at a concentration of 600 mg/L) for 3 days; they were then transferred to antibiotics-free medium for 5 days; and finally they were spread on solid f/2 media to allow algal colonies to form. With this method, five strains of Nannochloropsis sp. (Eustigmatophyceae), two strains of Cylindrotheca sp. (Bacillariophyceae), two strains of Tetraselmis sp. (Chlorodendrophyceae) and one strain of Amphikrikos sp. (Trebouxiophyceae) were purified successfully. The method shows promise for batch-purifying microalgal cultures.     

Screening for lipophilic marine toxins and their potential producers in coastal waters of Weihai in autumn, 2020

Lipophilic marine toxins (LMTs) produced by some microalgae in the sea could accumulate in shellfish and pose potential threats to the health of seafood consumers. Phytoplankton and shellfish samples were collected from coastal waters of Weihai in Shandong Peninsula, China in autumn, 2020, and screened for lipophilic marine toxins and their potential producers using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and high throughput sequencing of partial DNA (V4 region of the 18S rRNA gene) extracted from phytoplankton. Pectenotoxin-2 (PTX2), trace amounts of azaspiracid (AZA1 or AZA40), and 13-desmethyl spirolide C (13-DesMe-C) were detected in phytoplankton samples, while PTX2 and gymnodimine (GYM) were detected in shellfish samples. The toxin content in shellfish samples was much lower than the regulatory limit or values reported previously. Results suggest that lipophilic marine toxins should have low risk in coastal waters of Weihai in autumn. Based on the data of high throughput sequencing, the OTUs were assigned to 5 identified species of Alexandrium, including A. ostenfeldii capable of producing 13-DesMe-C and GYM. Two OTUs were found closely related to the toxic species in genus Dinophysis, but it is impossible to assign them to any identified species due to the low resolving power of the V4 region for Dinophysis. The OTUs could not be assigned to any identified species in the genus Azadinium, suggesting the existence of unidentified species in this region.

     

Two new species of Sellaphora (Bacillariophyta) from a deep oligotrophic plateau lake, Lake Fuxian in subtropical China

We describe two new species of Sellaphora from Lake Fuxian, Yunnan Plateau, China. Based on both light and scanning electron microscopy, these species are described as S. yunnanensis sp. nov. and S. sinensis sp. nov. The primary features of S. yunnanensis are: elliptical to linear-elliptical valves with broadly rounded ends, straight filiform raphe, almost straight central endings and small, slightly expanding central pores, small central area, symmetrical or slightly asymmetrical central nodule. The primary features of S. sinensis are: elliptical valves, obtusely rounded ends, similar raphe and axial area, transapically less expanded central area, larger, elliptical central nodule. We compare these species to those of a similar shape and morphology.     

Inhibitory activity of an extract from a marine bacterium Halomonas sp. HSB07 against the red-tide microalga Gymnodinium sp. （Pyrrophyta）

In recent years, red tides occurred frequently in coastal areas worldwide. Various methods based on the use of clay, copper sulfate, and bacteria have been successful in controlling red tides to some extent. As a new defensive agent, marine microorganisms are important sources of compounds with potent inhibitory bioactivities against red-tide microalgae, such as Gymnodinium sp. （Pyrrophyta）. In this study, we isolated a marine bacterium, HSB07, from seawater collected from Hongsha Bay, Sanya, South China Sea. Based on its 16S rRNA gene sequence and biochemical characteristics, the isolated strain HSB07 was identified as a member of the genus Halomonas. A crude ethyl acetate extract of strain HSB07 showed moderate inhibition activity against Gymnodinium sp. in a bioactive prescreening experiment. The extract was further separated into fractions A, B, and C by silica gel column chromatography. Fractions B and C showed strong inhibition activities against Gymnodinium. This is the first report of inhibitory activity of secondary metabolites of a Halomonas bacterium against a red-tide-causing microalga.     

Colloid‘s influences on microalgae growth as a potential environmental factor

The role of colloid as “colloid pump”in the ocean is well known. The important influence of colloid in seawater on the growth of microalga was found in our 1999--2000 study. Colloid concentrates were obtained by employing a cross-flow filtration system to ultrafilter seawater(which had been pre-filtrated by 0.45 μm acetate cellulose membrane) successively with different membranes. Ultrafiltration retentions (we called them colloid concentrates) together with control sample (seawater without colloid) were then inoculated with two species of microalgae and cultivated in selected conditions. Monitoring of microalgae growth during cultivation showed that all colloid concentrates had obvious influence on the growth of the microalgae studied. Addition of Fe(OH)3 colloid or organic colloid (protein or carbohydrate) to the control sample enhanced the microalgae‘s growth.     

A new species and new recorded species of Lumbrineridae Schmarda, 1861 (Annelida: Polychaeta) from China

Based on material deposited in the Marine Biological Museum of the Chinese Academy of Sciences(Qingdao),a new species,Lumbrineris sinensis sp.nov.,as well as Augeneria albidentata(Ehlers,1908),which is recorded for the first time from coastal water of China,are reported in the present paper.The specimens examined were collected during 1958 to 1960 from the Yellow Sea,East China Sea and South China Sea at depths of 4 to 182 m.     

Construction of a new gene integration platform system for theSynechococcus sp. PCC7942

According to the known sequence of iron stress-induced gene (isiAB operon), we cloned its 1.5 kb fragment by PCR, and used this fragment as integration homologous fragment. After several steps of subcloning donor DNA into theisiAB fragment, a donor plasmid pZL which could be integrated into the chromosomal DNA ofSynechococcus sp. PCC7942 was constructed. In order to express the heterologous gene at a high level through the integration platform system, we constructed the donor DNA by the following steps. We cloned the strong promoter (240 bp) of heat shock genegroESL operon fromSynechococcus sp. PCC7942 by PCR. Then subcloned the multiple cloning sites (MCS),rbcS polyA into the downstream of thegroESL promoter. The kanamycin resistance gene, as the marker gene, was also subcloned into the donor DNA. Thus, in the donor plasmid pZL, the integration homologous fragment and several expression elements, such asgroESL promoter, MCS,rbcS polyA terminator and kanamycin resistance gene, were all included.

After naturally transformed and introduced the donor plasmid pZL intoSynechococcus sp. PCC7942, as in the pZL, the donor DNA sequence is flanked by two DNA fragments (0.4 kb and 0.7 kb) homologous to theisiAB fragment ofSynechococcus sp. PCC7942, the homologous DNA can recombine with the chromosomal DNA. After screening by kanamycin, the transformants which integrated the heterologous DNA were selected. The efficiency of transformation is about 1×10⁻⁶. By southern blot analysis, it was confirmed that the donor DNA had been integrated into the chromosomal DNA ofSynechococcus sp. PCC7942, located on the site of theisiAB gene, and can be replicated with the chromosomal DNA.

     

Algicidal activities of secondary metabolites of marine macroalgal-derived endophytic fungi

Red tide occurrs frequently and causes signi?cant damage to the environment and human health. As a result, development of new effcient and environment friendly red-tide microalgae inhibitors has gained increasing attention in recent times. Algicolous endophytic fungi with unique habitats are promising sources for active agents owing to their abundant secondary metabolites and distinguished activities. In this study, the algicidal activities of 49 marine macroalgal-derived endophytic fungi against phytoplankton Alexandrium tamarense, Prorocentrum donghaiense, Heterosigma akashiwo, and Chattonella marina were examined using 96-well microplate. Four fungal strains, including Aspergillus wentii(pt-1), A. ustus(cf-42),and A. versicolor(dl-29, pt-20), exhibited potent algicidal activities. A total of 32 pure compounds isolated from these fungi were noted to possess dif ferent degrees of algicidal activities. Of those, 11 compounds comprising ?ve anthraquinones, two terpenoids, and four steroids showed high 24-h inhibition rates for the four red tide algae, with 24 h EC_(50) values ranging from 0.01 to 14.29 μg/mL. Among them, compound1(1,5-dihydroxy-3-methoxy-7-methylanthraquinone) presented the strongest activity against H. akashiwo,and could decrease its chlorophyll a(Chl a) and superoxide dismutase contents and increase the soluble protein, malondialdehyde, and peroxidase contents. These results suggested that the identi?ed anti-algal compound might inhibit the growth of red tide algae by weakening photosynthesis(reducing Chl a content),destroying cell membrane, and damaging the antioxidant system.     

Oncholaimus zhangi sp. nov. (Oncholaimidae,Nematoda) from the intertidal zone of the East China Sea

A new species of free-living marine nematode is described from intertidal sediment of the East China Sea.The new species belongs to the Oncholaimidae family.Oncholaimus zhangi sp.nov.is characterized by males having a large ventral swelling situated just posterior to the middle of the tail,no precloacal papilla,and a row of 6–7 ventral genital setae.The tails of both males and females are similar,being conico-cylindrical in shape.The new species is distinguished from other closely related species by the presence of a prominent ventral swelling at the tail of males and a similar tail shape in both sexes.About 110valid species in this genus have been recorded to date.     

Isolation and characterization of phosphate-solubilizing bacteria from seagrass rhizosphere soil

Phosphate-solubilizing bacterial strains(6 Nos.) were isolated from the rhizosphere soils of two seagrasses(Halophila ovalis(R.Br.) Hook and Halodule pinifolia(Miki) Hartog) in the Vellar estuary.Experimental studies found that the strain PSSG6 was effective in phosphate solubilization with Phosphate Solubilization efficiency index E = 375 ± 8.54,followed by the strain PSSG5 with Phosphate Solubilization efficiency index E = 275 ± 27.3.Of the 6 strains isolated,the strains PSSG4 and PSSG5 be-longed to the genus Bacillus,and PSSG1,PSSG2 and PSSG3 were identified as Citrobacter sp.,Shigella sp.,and Klebsiella sp.,respectively,by conventional method,and PSSG6 was identified as Bacillus circulans using conventional and molecular methods.     

Identification of suitable reference genes for quantitative gene expression analysis in clam Cyclina sinensis under salinity stress and Vibrio infection

The appropriate reference gene is a prerequisite for accurate normalization of gene expression level, and research on suitable reference genes in clam Cyclina sinensis is scarce. To improve the situation, we selected five commonly used housekeeping genes, including β-actin, Elongation factor 1-α (EF1-α), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 40S ribosomal protein S18 (RPS18), and Tubulin α (TUB-α), then evaluated their expression stability in different adult tissues and under different experimental treatments (salinity stress and Vibrio parahaemolyticus infection). Their expression stability was analyzed by three frequently used programs, geNorm, NormFinder, and BestKeeper. This analysis indicated that multiple genes should be used for normalization, and we concluded that the reference gene combination GAPDH-RPS18-β-actin, should be used for qRT-PCR analysis in different tissues of C. sinensis under normal physiological conditions. For the clams under salinity stress and Vibrio infection, EF1-α-GAPDH-RPS18 was recommended as the gene combination for qRT-PCR normalization. TUB-α was generally poorly ranked by all programs, and should not be used in future studies. This study should provide fundamental support for accurate quantitative gene expression analysis of this species.