Preparation of polysaccharides in different molecular weights from Ulva pertusa Kjellm （Chorophyta）

As molecular weight controls the biological activities of polysaccharides, screening the optimal molecular weight of polysaccharides is important in drug research and application. In this study, hydrogen peroxide was employed as oxidant, and temperature, reaction time, and concentration of polysaccharides and hydrogen peroxide were examined for their effects on the preparation of polysaccharides in different molecular weights from Ulva pertusa. Our experiment suggested that the optimal degradation concentrations for polysaccharides and hydrogen peroxide were 2.5% (w/v) and 8.0% (v/v), respectively. The range of degradation measured by relative viscosity was mainly controlled by temperature and time. Results revealed that 35℃ was the optimal temperature for obtaining low-degradation samples, and 50℃ was the most favorable temperature to accelerate the reaction to yield highly-degraded samples. Four samples in different molecular weights A, B, C and D were finally prepared. The controllability was evaluated by the relative standard deviation (RSD) of relative viscosity, and the peak molecular weights and the polydispersity indexes (Mw/Mn) of molecular weights were measured by high performance gel permeation chromatography (HPGPC).     

ASPECTS OF IRON NUTRITION IN MACROALGAE ULVA PERTUSA （CHLOROPHYTA） UNDER IRON STRESS

Fe, Chlorophyll (Chl) and total nitrogen (TN) content in tissues were measured in Fe-deficient cultures ofUlva. pertusa over a period of 60 days. Photosynthetic carbon fixation rates were studied at the start of and 30 days after Fe-deficiency culture, when the effects of Fe-deficiency on the ultrastructure were also analyzed. The iron content in tissue decreased exponentially during Fe-deficiency (from 726.7 to 31.6 μg/gdw) and simultaneously Chl and TN content declined to 4.35% and 59.9% of their original levels respectively. Maximum carbon fixation rate (50–250 μmol/m² s) under Fe-deficiency decreased significantly compared with the control (p<0.01) and was 13.6 to 0.365 μg C/cm² h. Photosynthesis in Fe-deficient cells became light-saturated at lower irradiance than that in control. Ultrastructural observations of Fe-deficient cells showed reductions in chloroplast number, some degeneration of lamellar organization, an increase in vacuolar area, a decrease in mitochondrial matrix density, and variation in accumulation body number and morphology. During Fe-deficiency, the algae growth rate continued to decline and after 6 weeks of iron deficiency, no further growth was detectable. These suggested that the lower growth rate ofUlva. pertusa under Fe-deficiency could be due mainly to nitrogen utilization and inhibition of photosynthesis. This project No. 39725023 supported by NSFC for Talented Youths and the Project under the Major State Basic Research of China (Grant No. G1999012011).     

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.     

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.     

Observation of female gamete (Carpogonia) germination of Porphyra yezoensis and P. oligospermatangia

Several papers have reported that part or whole leafy thallus seemingly consisting of zygotospores can give rise to both blades and conchocelis in the same culture ofPorphyra. Study on samples of wild and cultivatedPorphyra yezoensis andP. oligospermatangia were conducted to clarify the origination of the young blades in the culture. It is confirmed that single cells on the blade of both species, which normally intermixed with zygotospores, germinated into young blades. TEM and SEM observation has shown that the single cells ofPorphyra yezoensis had typical features of female gamete (carpogonia) but archeospore. Therefore, the female gametes are responsible in developing leafy thalli. This project was sponsored by the NSFC (No. C-0205-05-39770593)     

Molecular identification of Prorocentrum (Dinophyceae) species

A fragment of a large sub-unit ribosomal DNA (LrDNA) of 12 strains ofProrocentrum species was amplified by polymerase chain reaction (PCR). The PCR products were digested by 3 restriction endonucleases (Cfo I, Hae III, and RSA I) and then resolved in agarose gels. Results show that different species had different RFLP patterns, except forP. arcuatum (ME 131), which had the same pattern toP. micans (ME160 and 04). The same fragment of 19 strains of the genus was also amplified and subjected to denaturing gradient gel electrophoresis (DGGE). 11 different patterns were resolved. Different cultures of a same species had the same pattern. The results of RFLP and DGGE analyses showed that eight newly isolated epibenthicProrocentrum species were different from each other, and also from other cultured ones examined in this study.P arcuatum (ME132) could not be differentiated fromP. micans (ME160 and 04), it was probably mis-identified, since they are quite different morphologically.P. redfieldii (ME138) could also not be distinguished formP. triestinium (ME132), it should be regarded as a synonym ofP. triestinium. Unexpectedly, a restriction site was found inP. micans, compared with previous sequence data. Project supported by National Basic Research Priorities Program (2001CB409701, 2001CB409710) and supported by NSFC (40376040, 40025614)     

An efficient procedure for isolating microsatellite DNAs from sea cucumber (Apostichopus japonicus)

The construction of enrichment library proves to be one of the efficient approaches for isolating microsatellites in this study. The genomic DNA of sea cucumber was digested with HaeIII and size-selected DNA fragments (250–700 bp) were ligated to an adaptor. Microsatellite-containing sequences were captured by using a combination of GA and CA probes, which were attached to a nylon membrane. The microsatellite enrichment library constructed in this study consisted of approximately 700 clones. Two hundred and thirty-two clones reacted positively after the library screening procedure. Of the 50 clones sequenced, all contained at least one microsatellite and one duplicate clone was found. Approximately 86% of the sequenced fragments permitted to design primers for sequence tagged microsatellite site (STMS).     

Preliminary Study on the Responses of Three Marine Algae,Ulva pertusa （ Chlorophyta ） , Gelidium amansii （ Rhodophyta ）and Sargassum enerve （ Phaeophyta）, to Nitrogen Source and Its Availability

1　Introduction　Inrecentyears ,eutrophicationhasbecomeaworld wideenvironmentalprobleminthecoastalarea.Aqua culturebecomesaseriouspollutionsourcelikesewagedischarge .Wasteproductsfromfishfarmsconsistma inlyofnitrogen ,phosphorusandcarbondioxide .Inpen bas…     

Assessing the germplasm of Laminaria (phaeophyceae) with random amplified polymorphic DNA (RAPD) method

Eighteen gametophytes includingL. japonica, L. ochotensis andL. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains ofLaminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species ofLaminaria. Contribution No. 4389 from the Institute of Oceanology, Chinese Academy of Sciences. This research was supported by the State's High-tech Project of China, Marine 863 project (No. 819-03-02) and (2001AA621090).     

Immunosuppressive activities of fucoidan from Laminaria japonica

Effects of fucoidan fromLaminaria japonica on 2,4-dinitrochlorobenzene induced delayed-type hypersensitivity (DTH) reaction and the serum levels of IgG, IgM, complement C3 and C4 were investigated in the present study. Results showed that oral administration of fucoidan at dose of 150 and 300 mg/(kg· d) for 9 days before the hapten challenge significantly inhibited 2,4-dinitrochlorobenzene induced delayed-type hypersensitivity reaction; and also inhibited the humoral immunity. Serum C3 and C4 levels were markedly reduced by fucoidan at dose of 150 and 300 mg/kg; and serum IgG and IgM levels were reduced by fucoidan at dose of 300mg/kg. The inhibitory effects of fucoidan on delayed-type hypersensitivity suggested that it may be potential medication for chronic inflammatory diseases in the future. This study was supported by National Foundation for New Drug Research & Development (NFND) No.144.     

Isolation and pharmacological activities of bromophenols from Rhodomela confervoides

Four bromophenols were isolated from the extract of marine red alga,Rhodomela confervoides by column chromatography and HPLC methods. By means of spectroscopic methods inclding IR, MS, 1D, and 2D-NMR techniques, their structures were elucidated as (1) 3-bromo-4,5-dihydroxy benzoic acid methyl ester; (2) bis (2,3-dibromo-4,5-dihydroxybenzyl) ether; (3) 3,4-dibromo-5-(methoxymethyl)-1,2-benzenediol and (4) 3-bromo-4,5-dihydroxy-benzaldehyde. Compound 1 was first isolated from the algae in nature, and 1, 4 were found to have selective cytotoxic activities against KB, Bel 7402 and A549 cells, 2 showed powerful antibacterial activities againstStaphylococcus aurens andPseudomonas aeruginosa. Supported by National “863” Program (No. 2004AA625030, 2001AA620503), Quingdao marine sciences project (No. 04-2-NN-26) and Key Knowledge Innovative Project of the Chinese Academy of Sciences (KZCX3-SW-215).     

Energy flow in Branchiura sowerbyi (Oligochaeta: tubificidae) in a shallow macrophyte-dominated lake, Biandantang Lake

The energy flow ofBranchiura sowerbyi was studied for the first time in China in a shallow macrophytic lake, Biandantang Lake, Hubei Province. The energy flow was calculated from the measurement of flesh production (12.5241kJ/m²a), egestion (517.7302kJ/m²a), metabolism (38.3273 kJ/m²a), and excretion (4.3798kJ/m²a). The net growth efficiency of the species is about 22.7%, which accords well with the generally reported value for oligochaetes. In addition, the relationship between starvation respiration (R, mgO₂/ind·d), wet weight (Ww, mg) and temperature (T, °C) were also measured, with the regression function beingR=0.008Ww^0.736 e^0.050T. Project supported by NSFC (30270278, 3960019), the foundation of the government of Hubei Province (No. 2000J109), and the foundation of Ecological Station, CAS in the Institute of Hydrobiology.     

Comparison of growth and survival of larvae among different shell color stocks of bay scallop Argopecten irradians irradians (Lamarck 1819)

In order to improve production and breed new broods of bay scallopArgopecten irradians irradians, different-colored orange, purple and white lines were established by two mating methods of self-fertilization and mass spawning at a commercial scallop hatchery in spring, 2002. And then larval growth and survival of different lines was compared to test whether there is a relationship between shell color and growth and survival at early developmental stage. Both growth and survival have no significant differences among different experimental larvae of self-fertilization or mass spawning. Results are as following in the order of orange, purple and white shell stock. For the self-fertilization, growth rates of larvae were 6.174, 6.412, and 6.599 μm/d, respectively. Survival rates of larvae at Day 3 were 74.41%, 76.86%, and 82.05%; Day 6 were 49.14%, 65.63%, and 52.79%; and Day 9 were 25.06%, 20.80%, and 26.47%, respectively. For the mass spawning, the growth rates were 7.836, 7.941, and 7.878 μm/d, respectively. Survival rates at Day 3 were 93.05%, 91.95%, and 92.50%; Day 6 were 79.17%, 78.05%, and 82.50%; and Day 9 were 34.72%, 36.67%, and 38.33%, respectively. The absence of any relationship between shell color and growth and survival at the larval stage may be resulted from their common genetic basis. This work was financially supported by Project of Scientific Innovation, Chinese Academy of Sciences (ZKCX2-211).     

Effects of blue light on gametophyte development of Laminaria japonica (Laminariales, Phaeophyta)

Laminaria gametophyte was greatly influenced by light in its growth and development. Using light-emitting diodes (LED) as blue and red light sources, we analyzed the light effect on gametophytes development ofLaminaria japonica Aresch. The gametophytes were obtained from zoospores collected in April, May, July, 2003 and September, 2004. We found that the growth of gametophytes was stimulated by increasing intensity of blue light (BL) and red light (RL) illumination, of which BL was obviously stronger than that of RL. The fertilization of gametophytes depended largely on BL, and only sufficient BL illumination could take the reproductive effect. In addition, we noticed that there was a significant difference in light responses for gametophytes developed from zoospore collected in different times. For zoospores released in April, under BL₁ (73.90 μmol photons/m·s), the unicellular female gametophytes and multi-cellular male gametophytes produced eggs and sperms respectively, and further developed towards sporophytes. However, for gametophytes developed in May, July or September, they became multi-cellular and never formed oogonia or antheridia. It is believed that theLaminaria sporangium maturation stage could affect the gametophytes reaction to BL under laboratory culture conditions. Therefore, cryptochrome- or phototropin-like BL photoreceptors is probably involved in BL-induced development ofLaminaria gametophytes. This research was supported by the NSFC (No.40376049) and Japan Society for the Promotion of Science (The Invitation Fellowship for Research in Japan 2002).     

Purification and characterization of an alkaline protease from Acetes chinensis

An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca²⁺ could enhance the proteolytic activity of the protease, while Cu²⁺, EDTA and PMSF could inhibit its activity.     

Properties of proteolytic toxin of Vibrio anguilolarum from diseased flounder

Extracellular products (ECP) produced byVibrio anguillarum strain M3 originally isolated from diseased flounder (Paralichthys olivaceus) were prepared. ECP of M3 showed gelatinase, casinase, amylase and haemolytic activity on agarose plates. High protease activity against azocasin was detected. Bacterium M2 showed highest growth and protease activity at 25°C. The protease present in ECP showed maximal activity at pH 8 and 55°C; was completely inactivated by application of 80°C heat for 30 min; was completely inhibited by EDTA and HgCl₂, and was partially inhibited by PMSF, SDS, MnCl₂ and iodoacetic acid; but not inhibited by CaCl₂ and MgCl₂. The ECP was toxic to flounder fish at LD₅₀ values of 3.1 μg protein/g body weight. The addition of HgCl₂ and application of heat at 50°C decreased the lethal toxicity of ECP. When heated at 100°C, ECP lethality to flounder was completely inhibited. After intramuscular injection of ECP into flounder, it showed evident histopathological changes including necrosis of muscle, extensive deposition of haemosiderin in the spleen, dilated blood vessels congested with numerious lymphocytes in the liver. These results showed that ECP protease was a lethal factor produced by the bacteriumV. anguillarum M3. Contribution No. 4213 from the Institute of Oceanology, Chinese Academy of Sciences. Funded by projects under the Major State Basic Research Development Program, Grant G1999012003.     

Oxygen consumption and ammonia-N excretion of Meretrix meretrix in different temperature and salinity

Effects of temperatures and salinities on oxygen consumption and ammonia-N excretion rate of clamMeretrix meretrix were studied in laboratory from Oct. 2003 to Jan. 2004. Two schemes were designed in incremented temperature at 10, 15, 20, 25°C at 31.5 salinity and in incremented salinity at 16.0, 21.0, 26.0, 31.5, 36.0, and 41.0 at 20°C, all for 8–10 days. From 10 to 25°C, both respiration and excretion rate were increased. One-way ANOVA analysis demonstrated significant difference (P<0.01) in physiological parameters in this temperature range except between 15 and 20°C. The highestQ ₁₀ thermal coefficient value (12.27) was acquired between 10 and 15°C, and about 1 between 15 and 20°C, indicatingM. meretrix could well acclimate to temperature changes in this range. Salinity also had significant effects on respiration and excretion rate (P<0.05). The highest values of respiration and excretion rate ofM. meretrix were recorded at 16.0 salinity (20°C). These two physiological parameters decreased as salinity increased until reached the minimumQ ₁₀ value at 31.5 (20°C), then again, these parameters increased with increasing salinity from 31.5 to 41.0.M. meretrix can catabolize body protein to cope with osmotic pressure stress when environmental salinity is away from its optimal range. No significant difference was observed between 26.0 and 36.0 in salinity (P>0.05), suggesting that a best metabolic salinity range for this species is between 26.0 and 36.0. This work is supported by National High-Tech R & D Program of China. (863 Program) (2002AA603014).     

The changes of three components in coelomic fluid of Urechis unicinctus exposed to different concentrations of sulfide

The changes in heme (associated with hemoglobin), hemoglobin and hematin in the coelomic fluid of marine worm,Urechis unicinctus, exposed to different concentrations of sulfide, were investigated using biochemical techniques. When exposed to different sulfide concentrations for up to 96 h, the relative amounts of the three components changed in a regular pattern suggesting that the coelomocytes play an important role in the worm's tolerance to sulfide. The possible roles of heme compounds in sulfide tolerance of this species are discussed on the basis of our experimental data. This research was supported by the NSFC-KOSEF scientific cooperation program and NSFC (No. 30271039)     

Effect of n?3 HUFA levels in rotifers and Artemia on growth and survival of larval black sea bream ( Sparus macrocephalus )

Requirement for dietary n−3 HUFA (n−3 highly unsaturated fatty acid) for growth and survival of black sea bream (Sparus macrocephalus) larvae was studied using rotifers andArtemia at various levels of n−3 HUFA. Five treatments with rotifers andArtemia differing in n−3 HUFA were prepared by enriching them with various oil emulsions. Results indicated that dietary n−3 HUFA significantly influence fish n−3 HUFA levels and are essential for growth and survival of black sea bream larvae. The results also indicated that the incorporation of n−3HUFA TG (triacylglycerols) into tissues of larval black sea bream was more effective from natural fish oil in comparison with n−3 HUFA fatty acid ethyl esters from ethyl-esterified oil. Project No. 39770588 supported by the NSFC.