EFFECTS OF CO—CULTURE AND SALINITY ON THE GROWTH AND AGAR YIELD OF GRACILARIA TENUISTIPITATA VAR LIUI ZHANG ET XIA

Gracilaria tenuistipitata var Liui were mono-cultivated and co-cultivated with Pinctada martensii under high(33) and low(21) salinity conditions in laboratory.The daily growth rate of the alga was determined.Tissue carbon and nitrogen contents,the yield and fractional composition of agar were analyzed.Results showed that:1.Gracilaria grew better under low salinity conditions,the daily growth rate was twice that under high salinity conditions.Co-cultivated algae grew faster than mono-culti-vated algae under low salinity conditions,the daily growth rate was about 37.6?higher.2.Compared with mono-cultivated algae,tissue nitrogen contents of co-cultivated algae were higher,while the C:N ratios were much lower.3.The agar yields of co-cultivated algae were much lower than those of mono-culti-vated algae.Agar yield was found to be negatively correlated to the tissue nitrogen contents,and positively correlated to the C:N ratios.4.The highest fractional yields obtained from co-cultivated algae were extracted with 40?ethanol,while from mono-cultivated algae,the highest fractional yields obtained were extracted with distilled water at room temperature.     

Properties of polysaccharides in several seaweeds from Atlantic Canada and their potential anti-influenza viral activities

To explore the polysaccharides from selected seaweeds of Atlantic Canada and to evaluate their potential anti-influenza virus activities, polysaccharides were isolated from several Atlantic Canadian seaweeds, including three red algae (Polysiphonia lanosa, Furcellaria lumbricalis, and Palmaria palmata), two brown algae (Ascophyllum nodosum and Fucus vesiculosus), and one green alga (Ulva lactuca) by sequential extraction with cold water, hot water, and alkali solutions. These polysaccharides were ana-lyzed for monosaccharide composition and other general chemical properties, and they were evaluated for anti-influenza virus activities. Total sugar contents in these polysaccharides ranged from 15.4% (in U. lactuca) to 91.4% (in F. lumbricalis); sulfation level was as high as 17.6% in a polysaccharide from U. lactuca, whereas it could not be detected in an alikali-extract from P. palmaria. For polysaccharides from red seaweeds, the main sugar units were sulfated galactans (agar or carrageenan) for P. lanosa, F. lumbricalis, and xylans for P. palmata. In brown seaweeds, the polysaccharides largely contained sulfated fucans, whereas the polysaccharides in green seaweed were mainly composed of heteroglycuronans. Screening for antiviral activity against influenza A/PR/8/34 (H1N1) virus revealed that brown algal polysaccharides were particularly effective. Seaweeds from Atlantic Canada are a good source of marine polysaccharides with potential antiviral properties.     

EFFECTS OF CO-CULTURE AND SALINITY ON THE GROWTH AND AGAR YIELD OF GRACILARIA TENUISTIPITATA VAR LIUI ZHANG ET XIA

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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(cox1). In artificial 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.     

Study on extraction of agaropectin from <Emphasis Type="Italic">Gelidium amansii</Emphasis> and its anticoagulant activity

Gelidium amansii agar was fractionated on DEAE-cellulose and four fractions were obtained sequentially. The yields of 1.0 mol/L NaCl fraction and 2.5 mol/L NaCl fraction were 2.80% and 2.03%. They are highly sulfated agar, and named as agaropectin with sulfate content being 22.8% and 32.5%, respectively. The anticoagulant experiment results show that agaropectin could effectively prolong the coagulation time in a dose-dependent manner in vitro. Agaropection could be absorbed and effectively prolong the plasma coagulation time in vivo. After intragastric administration at the doses of 100, 200, and 400 mg/kg-d in rats for 15 days, TT （thrombin time）, CT （coagulation time）, PT （prothrombin time）, and APTT （activated partial thromboplastin time） could be effectively prolonged and the plasma Fib level could be significantly lowered.     

Enhancing the culturability of bacteria from the gastrointestinal tract of farmed adult turbot Scophthalmus maximus

Eighteen agar media were tested for the culture of gut-associated bacteria from farmed adult turbot(Scophthalmus maximus),including 16 agar media with or without 1% gastrointestinal(GI)supernatant,or with 2% or 4% GI supernatant.A total of 1 711 colonies were analyzed and 24 operational taxonomic units(OTUs)were identifi ed.The greatest bacterial diversity was isolated on Zobell 2216E/Zobell 2216E+ agar media,whereas MRS/MRS+ agar media produced a low diversity of colonies.Agar media with GI supernatant(1%,2%,or 4%)showed increased diversity and yielded different profi les of OTUs from the corresponding original media,suggesting that GI supernatant provides substances that enhance the culture effi ciency of bacteria from the turbot GI tract.The large majority of the colonies(82%)were γ-Proteobacteria,whereas 15.6% and 2.4% of colonies were Firmicutes and Actinobacteria,respectively.At the genus level,49.4% of all colonies were assigned to Vibrio.Other potential pathogens,including Pseudomonas,Photobacterium,and Enterobacter,and potential probiotics,including Bacillus,Paenibacillus,and Pseudomonas,were also isolated on agar media.Most cultured bacteria belonged to species that were fi rst described in the turbot GI tract.The impact of these species on turbot physiology and health should be investigated further.     

The effect of salinity on Fucus ceranoides (Ochrophyta,Phaeophyceae) in the Mondego River (Portugal)

Algae(and their extracts) are increasingly important for pharmaceutical applications due to the diversity of useful compounds they contain. The genus Fucus contains one of the most studied species,Fucus vesiculosus. The species F. ceranoides differs from the others of the genus by presenting longitudinal air-vesicles and a capacity to survive at low salinities. It is an alga that inhabits the Mondego River estuary(Portugal), at the southern limit of its distribution, and can serve as a role model to understand the effect of a salt gradient on the production of bioactive compounds. We assessed the phenolic content and antioxidant activity of different F. ceranoides extracts(e.g. methanolic, aqueous and polysaccharide) prepared from samples harvested from two different zones to evaluate if the adaptation of F. ceranoides to different salinity levels influenced its chemical composition. The antioxidant activity of the extracts was determined using 1,2-diphenyl-picrylhydrazyl(DPPH) and 2.2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)radicals. These assays demonstrated that the methanolic extract of lyophilized F. ceranoides that grew at low salinities was the most bioactive, i.e. DPPH(IC_(50)=50.39 μg/mL) and ABTS(TEAC=2.42). The total phenolic content(Folin-Ciocalteu method) and the methanolic extract of the lyophilized F. ceranoides collected from a low salinity habitat exhibited the highest phenolic content(PGE=49.48 μg/mg of lyophilized extract)amongst those sampled. Thin layer chromatography(TLC) and Fourier transform infrared spectroscopy(FTIR) were used for the identification of compounds in the extracts. This characterization allowed confirmation that the various extracts contained almost the same compounds but with notable quantitative differences. Based on these results, we conclude that there were differences in the quantity of the compounds due to the effect of salinity. The drying methods used were also found to have influenced the quality of the extracted compounds.     

Genome size of <Emphasis Type="Italic">Alexandrium catenella</Emphasis> and <Emphasis Type="Italic">Gracilariopsis lemaneiformis</Emphasis> estimated by flow cytometry

Flow cytometry(FCM) technique has been widely applied to estimating the genome size of various higher plants. However, there is few report about its application in algae. In this study, an optimized procedure of FCM was exploited to estimate the genome size of two eukaryotic algae. For analyzing Alexandrium catenella, an important red tide species, the whole cell instead of isolated nucleus was studied, and chicken erythrocytes were used as an internal reference. The genome size of A. catenella was estimated to be 56.48 ± 4.14 Gb(1C), approximately nineteen times larger than that of human genome. For analyzing Gracilariopsis lemaneiformis, an important economical red alga, the purified nucleus was employed, and Arabidopsis thaliana and Chondrus crispus were used as internal references, respectively. The genome size of Gp. lemaneiformis was 97.35 ± 2.58 Mb(1C) and 112.73 ± 14.00 Mb(1C), respectively, depending on the different internal references. The results of this research will promote the related studies on the genomics and evolution of these two species.     

Tsengiella,a new genus of delesseriaceae—(Rhodophyta) from China

A medium-sized delesseriaceous red alga has been collected from the subtidal area of the northern Huanghai Sea coast. The characteristics of the specimens precludes their being placed in any genus defined by the currently accepted criteria.Tsengiella gen. nov., withT. spinosa sp. nov., as the type and only known species, is created and allied with theHypoglossum group of the Delesserioideae (Delesseriaceae, Ceramiales, Rhodophyta). Contribution No. 1286 Academia Sinica Institute of Oceanology, Qingdao.     

A comparative analysis of four kinds of polysaccharides purified from Furcellaria lumbricalis

Four kinds of polysaccharides (FA, FB, FC and FD) were extracted and purified from a red alga Furcellaria lumbricalis. Their monosaccharide composition, average molecular mass and structural feature were determined by gas chromatography (GC), high performance gel permeation chromatography (HPGPC) and Fourier transform infrared spectroscopy (FTIR), respectively. The results showed that FA and FB were composed of galactose (Gal), 3,6-anhydro-galactose (AnG) and 4-sulfated-galactose (G4S), which were structurally similar to β-carrageenan and κ-carrageenan. FC was composed of sulfated galactose and small amount of AnG, indicating that it was a hybrid-sulfated galactan. FD was composed of α-1,4-glucose and belonged to floridean starch. The respective yields of FA, FB, FC and FD were 4.84%, 33.74%, 8.76% and 13.5%, and the corresponding were 537 kD, 432 kD, 513 kD and 51 kD respectively. Owning to its high content (>60%) of polysaccharide, F. lumbricalis will be a good source of marine sulfated polysaccharide and shows its potential utilization in food and medicine.     

Structural studies on agar fractions extracted sequentially from chinese red sea weeds:Gracilaria sjeostedtii,G. textorii andG. salicornia using13C-NMR and IR spectroscopy

The polysaccharides of three species of ChineseGracilaria (Rhodophyta) were extracted by sequential solvent extraction and the chemical structures of the fractions obtained were investigated by using¹³C-NMR and IR spectroscopy. The¹³C-NMR spectra showed that the main extract, cold water fraction, fromG. sjeostedtii consisted of repeating disaccharides of agarobiose and (1→3)-β-D-galactopyranosy 1-(1→4)-α-L-galactopyranose 6-sulfate, the latter being a precursor to agarobiose and converted to agarobiose after alkali modification. The component of the 60% ethanol fraction fromG. textorii was composed mainly of agarose 6-sulfate, 6-O-methyl-agarose and some amount of agarose, whse structures remained unchanged after alkali treatment. The autoclave extract fromG. salicornia revealed a complex structure composed of 6-O-methyl-agarose, 2-O-methyl-agarose, branched 4-O-methyl-L-galactose and agarose 4-sulfate. All the substituents in these agaroses were stable against alkali treatment. The alkali extracts fromG. sjeostedtii andG. textorii showed the distinct spectra of amylopectin-like floridean starch, while that fromG. salicornia showed a mixture of agaroses and floridean starch. From the IR spectra the relative contents of total sulfate, 3,6-anhydro-galactose, 4-sulfate and 6-sulfate in some fractions of polysaccharides of the algae were qualitatively estimated by the ratios of absorption bands of 1250/2920, 930/2920, 850/2920 and 820/2920, respectively. Contribution No. 1489 from Institute of Oceanology, Academia Sinica     

Estimating areal carbon fixation of intertidal macroalgal community based on composition dynamics and laboratory measurements

The community dynamics and potential carbon ?xation of intertidal macroalgae were investigated monthly from April 2014 to April 2015 in the northwest coast of Yellow Sea. Seasonal variations in biomass and carbon ?xation were presented and showed close relationship with community structure.The carbon ?xation rate ranged from 0.48±0.13 mg C/(g FW ·d) to 4.35±0.12 mg C/(g FW ·d). Sargassum thunbergii, Chondrus ocellatus and Ulva intestinalis were three most in?uential species which contributed27%, 21.9% and 18.5% variation of carbon ?xation rate, respectively. Standing carbon stocks ranged from7.52 g C/m~2 to 41.31 g C/m~2, and estimated carbon stocks varied from 11.77 g C/m 2 to 96.49 g C/m~2. The larger dif ference between estimated and standing carbon stocks implied that more ?xed carbon was exported from the community in summer and autumn than in winter. This study suggested that intertidal macroalgal community could provide a potential function in carbon ?xation of coastal ecosystem.     

Structural analysis and anti-complement activity of polysaccharides extracted from Grateloupia livida (Harv.) Yamada

Polysaccharides were extracted from Grateloupia livida(Harv.) Yamada using hot water(extracted product denoted WGW) and then degraded in dilute sulfuric acid(degraded product denoted WGWD). The degraded mixture was then separated into four fractions through anion exchange chromatography on 2-diethylaminoethanol(DEAE)-Bio-Gel Agarose FF gel. Electrospray ionization collision-induced dissociation tandem mass spectrometry(ESI-CID-MS/MS) was performed to elucidate the structural features of all fractions. In combination with nuclear magnetic resonance spectroscopy(NMR)and infrared spectroscopy(IR) data, the major polysaccharide structures were concluded to be μ-carrageenan and κ-carrageenan. μ-Carrageenan usually has a backbone of alternating 1,3-linked β-D-galactopyranose residues sulfated at C-4 and 1,4-linked a-D-galactopyranose residues sulfated at C-6, while κ-carrageenan consists of alternating 1,3-linked β-D-galactopyranose residues sulfated at C-4 and 1,4-linked a-D-3,6-anhydrogalactopyranose residues. Trace v-carrageenan, composed of 1,3-linked β-D-galactopyranose residues sulfated at C-4 and 1,4-linked a-D-galactopyranose residues sulfated at C-2 and C-6, was also detected. Furthermore, the polysaccharide had a backbone comprising 1,3-linked β-D-galactopyranose and1,4-linked α-L-galactopyranose sulfated at C-6, which is the agarose precursor. The hydroxy groups in the galactopyranose were partially substituted by methyl and pyruvic acid acetal(PA) groups. The anticomplementary activities of WGW and its derivatives against classical pathways were measured. The native polysaccharides in WGW had higher activities, while the derivatives had much weaker activities. This indicated that the molecular weight and sulfate content were important factors affecting the anti-complement activity.     

A new species ofSolieria from China

A morphological study of a marine red alga from China, previously identified by various authors as “Solieria mollis (Harv.) Kylin”, has been shown to be an undescribed species ofSolieria. The present new speciesSolieria tenuis is characterized by the slender branches, the unbranched and nonseptate connecting filament and the discoid holdfast surrounded by prop branches resembling proproots of land plants. Contribution No. 989 from the Institute of Oceanology, Academia Sinica.     

Microalgae cultivation in a tubular bioreactor and utilization of their cells

In this study on the possiblities of microalgae technology as an option for CO₂ mitigation, many microalgae were isolated from seawater. Some species of the isolates,Chlamydomonas sp. strain YA-SH-1, which accumulates starch in cells under light and ferment ethanol in dark and anaerobic condition, was grown outdoors by using 50-L tubular bioreactors in batch cultivation and harvested. Using these cells, the performance of ethanol production was examined quantitatively in a 0.5-L scale fermentor. Another species,Tetraselmis sp. strain Tt-1, was cultivated in a semi-batch manner by a similar type of tubular bioreactor indoors and examined for its utilization. Tests showed these cells could be used as partial substitute for wood and kenaf pulp for processing into paper. With the idea of making microalgae produce cellulose by genetic engineering in their minds, the authors studied the structure of bacterial cellulose synthase genes and the low temperature-induced, reversible flocculation in a thermophilic blue green alga (Cyanobacterium),Synechocystis vulcanus in order to examine the feasibility of using these genes as gene source and the cynanobacterium as host.

     

Hypoglossum fujianensis sp. nov. (Delesseriaceae, Rhodophyta) from Fujian Coast, China

Hypoglossum fujianensis sp. nov. is an epiphytic alga in the intertidal zone. Plants are light red, 0.9–2 cm high. Margin of branches gives rise to uniseriate hair-like rhizoids, formed outward from the fusion of the second-and third-order cells. Blades are single layered (except the midrib) and uncorticated. Tetrasporangial sori are formed on the middle part of blades. The globular tetrasporangia are developed from lateral pericentral cells. Project 39391800 supported by the NSFC.     

The genetic diversity,individual relatedness and possible mating system of an isolated population of the Cyprinid species Megalobrama pellgrini in upper reaches of the Changjiang (Yangtze) River,China

Megalobrama pellegrini is a cyprinid fish endemic to upper reaches of the Changjiang(Yangtze) River, China, which is also an important economic species in the local area. In recent years,resources of this species have decreased sharply and its conservation has drawn great attention. In the present study, we collected 120 individuals from the Longxi River, a tributary isolated from the main channel of the Changjiang River, where M. pellegrini is still relatively abundant. Using two different molecular markers, mitochondrial cytochrome b(cyt b) gene, and nuclear microsatellite(simple sequence repeat,SSR), we analyzed the genetic diversity of this isolated population. The results show that sequence genetic diversity was low(H_d:0.290 and P_i: 0.000 77 for cyt b gene),while the SSR genetic diversity was high(H_o:0.824 4±0.147 2,H_e: 0.823 510.145 1). Analysis indicated that this population had experienced a bottleneck,with inbreeding and small effective population size(around 50). Based on SSR data, relatedness analyzing revealed that the 120 samples were grouped into 10 completely independent clusters. It was inferred that the mating system of M. pellegrini was polygamy. We suggested that the low genetic diversity could be induced by the overfishing and inbreeding depression. Therefore, we suggested that the urgent conservation measures should be taken to control the overfishing and give better conditions for the fish to grow and spawn, then to restore population size.     

A comparison of different Gracilariopsis lemaneiformis (Rhodophyta) parts in biochemical characteristics,protoplast formation and regeneration

Gracilariopsis lemaneiformis is a commercially exploited alga. Its filaceous thallus can be divided into three parts, holdfast, middle segment and tip. The growth and branch forming trend and agar content of these three parts were analyzed, respectively, in this study. The results showed that the tip had the highest growth rate and branched most, although it was the last part with branch forming ability. The holdfast formed branches earliest but slowly. Holdfast had the highest agar content. We also assessed the difference in protoplast formation and regeneration among three parts. The middle segment displayed the shortest enzymolysis time and the highest protoplast yield; whereas the tip had the strongest vitality of protoplasts formation. Juvenile plants were only obtained from the protoplasts generated from the tip. These results suggested that the differentiation and function of G. lemaneiformis was different.     

On the ultrastructure and classification of the hemocytes of penaeid shrimp,Penaeus vannamei (Crustacea, Decapoda)

The ultrastructure of hemocytes ofPenaeus vannamei was examined by transmission electron microscope. Three types of hemocytes were identified: hyaline cell, small-granule cell and large-granule cell. Hyaline cells are the smallest of the hemocytes, lack electron-dense granules, have average size of 11.11 μm (SE±0.50,n=10)×6.96μm (SE±0.55,n=10), comprise 25.8% (SE±2.87,n=4×10) of the hemocytes; small granule cells are the most abundant type of hemocytes, comprise 58.1% (SE±3.40,n=4×10) of the hemocytes, contain small electron—dense granules, have average size of 10.78 μm (SE±0.60,n=10)×8.63 μm (SE±0.44,n=10); and large-granule cells comprise 16.1% (SE±2.55,n=4×10) of the hemocytes, are filled with large electron dense granules, have average size of 12.51 μm (SE±0.63,n=10)×8.99μm (SE±0.71,n=10). This research was supported by the National Climbing Project B, PDB6-6-3. Contribution No. 3448 from the Institute of Oceanology, Chinese Academy of Sciences.