Fatty Acid Composition Analyses of the DCMU Resistant Mutants of Nannochloropsis oculata (Eustigmatophyceae)

1　Introduction　Nannochloropsissp .areoneofpotentialsourcesofpolyunsaturatedfattyacids ,especiallytheeicosapen taentaenoicacid (EPA ,C2 0∶5 ) ,whichisimportantforbothanimalsandhumans .　Thefattyacidcompositionofthisalgaisaffectedbyvariousenvironmentalfactors ,suchaslightintensity(Sukeniketal.,1989;Sukeniketal.,1990 ) ,imply ingthatafactorinfluencingthephotosyntheticprocessmayaffectcellularfattyacidsynthesisandtheirmetabolism .AselectionprogramofincreasingcellularEPAcontenthasbeencarriedo…     

Construction and characterization of a normalized cDNA library of Nannochloropsis oculata (Eustigmatophyceae)

We constructed and characterized a normalized cDNA library of Nannochloropsis oculata CS-179, and obtained 905 nonredundant sequences (NRSs) ranging from 431–1 756 bp in length. Among them, 496 were very similar to nonredundant ones in the GenBank (E ≤1.0e-05), and 349 ESTs had significant hits with the clusters of eukaryotic orthologous groups (KOG). Bases G and/or C at the third position of codons of 14 amino acid residues suggested a strong bias in the conserved domain of 362 NRSs (>60%). We also identified the unigenes encoding phosphorus and nitrogen transporters, suggesting that N. oculata could efficiently transport and metabolize phosphorus and nitrogen, and recognized the unigenes that involved in biosynthesis and storage of both fatty acids and polyunsaturated fatty acids (PUFAs), which will facilitate the demonstration of eicosapentaenoic acid (EPA) biosynthesis pathway of N. oculata. In comparison with the original cDNA library, the normalized library significantly increased the efficiencies of random sequencing and rarely expressed genes discovering, and decreased the frequency of abundant gene sequences.     

Effects of nutritional factors on the growth and heterotrophic eicosapentaenoic acid production of diatom <Emphasis Type="Italic">Nitzschia laevis</Emphasis>

The effects of several nutritional factors on the growth and eicosapentaenoic acid (EPA) production of diatom Nitzschia laevis were studied. 4 LDM (quadrupled concentration of the nutrient salt) was the optimal concentration of nutrient salt for the growth and EPA production of N. laevis. The growth of N. laevis was inhibited when the glucose concentration was either lower than 10 gL⁻¹ or higher than 15 gL⁻¹. Both sodium nitrate and urea were good nitrogen sources for the growth and EPA production, while ammonium chloride seriously decreased the dry cell weight (DW) and the EPA content. Silicate seriously influenced the growth of N. laevis. The maximum DW of 2.34 gL⁻¹ was obtained in the presence of 150 mgL⁻¹ Na₂SiO₃·9H₂O. The EPA content remained almost the same when the silicate concentration was lower than 150 mgL⁻¹; however, higher silicate concentrations resulted in a steady decrease of EPA content. Low medium salinity (⩽29) did not seem to influence the DW of N. laevis, and high salinity resulted in a decrease of DW. The highest EPA content (4.08%) and yield (110 mgL⁻¹) were observed at the salinity of 36 and 29, respectively.     

Monthly Changes of Glycogen, Lipid and Free Amino Acid of Oyster

Monthly difference of the chemical composition of oyster cultured along the eastern coast of Shandong Province was analyzed. The components analyzed included glycogen, fatty acid and free amino acid （FAA）. The content of glycogen was high in January and March （2.89 and 2.82 g（100 g） ^-1 on average, respectively） and low in October （2.07g（100g）^- 1 on average）. The low content of neutral lipids in October reflected a relatively poor nutritional value of oyster （1.42 g（ 100 g）^- 1 on average）. The main fatty acids of oyster were palmitic acid （16：0）, oleic acid （18： 1）, eicosapentaenoic acid （EPA, 20： 5（0 3） and docosahexaenoic acid （DHA, 22：6（0 3）. The major FAAs of oyster were Taurine, Glutamicacid, Glycin, Alanine, Arginine and Proline. Taurine was the most abundant FAA with its content ranging from 603 mg（100 g） ^-1 to 1 139 mg（ 100 g） ^-1. The high contents of glycogen, polyunsaturated fatty acid and FAA showed that oyster cultured along the eastern coast of Shandong Province was nutritionally good in January and March.     

Bioaccumulation of Pb<Superscript>2+</Superscript> and its effects on growth,morphology and pigment contents of <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

A laboratory experiment was conducted to assess the bioaccumulation of Pb²⁺ and its effects on growth, morphology and pigment contents of Spirulina (Arthrospira) platensis. The specimen cultured in Zarrouk liquid medium was treated with various initial metal concentrations (0, 5, 10, 30, 50 and 100 μg mL⁻¹). The growth of S. platensis was adversely affected by Pb²⁺ at high concentrations (30, 50 and 100 μg mL⁻¹). However, at low concentrations (5 μg mL⁻¹), Pb²⁺ could stimulate its growth slightly. The pigment contents (chlorophyll α and β carotene) were decreased in a dose-dependent manner. The highest reductions (67% and 53% respectively in chlorophyll α and β carotene) were observed in 100 μg mL⁻¹ treatment group. The LC₅₀ (96 h) of Pb²⁺ was measured as 75.34 μg mL⁻¹. Apart from a few cases of filament breakages at elevated concentrations (50 and 100 μg mL⁻¹), morphological abnormalities are not specific. Metal bioaccumulation increased with Pb²⁺ concentrations, but decreased with exposure time. The maximum accumulated amount was 188 mg g⁻¹ dry weight. The bioconcentration factor (BCF) reached to a peak at day 2, followed by a gradual reduction for all the exposure concentrations. S. platensis is able to tolerate considerably high Pb²⁺ concentrations. Consequently it can be used as a potential species to remove heavy metal from contaminated waters.     

Seasonal variation in fatty acid composition of seston and the copepod Calanus sinicus (Brodsky, 1962) in Jiaozhou Bay and its trophic implications

The fatty acid compositions of seston and Calanus sinicus were investigated to study trophic relationships in Jiaozhou Bay. Principal component analysis was carried out to ordinate the fatty acid patterns of seston in stations and months. The results showed that diatoms were most abundant in the phytoplankton at station A5 (located in the northwest of the bay:36°9′N, 120°20′E) and least abundant at station D7 (located outside of the bay: 35°59′N, 120°26′E). By contrast, dinoflagellates were most abundant at station D7 and least abundant at station A5. According to the annual variations of 16:1ω7 and 18:4ω3/ 16:1ω7, diatoms flourished mainly in spring and summer, while dinoflagellates bloomed exclusively in summer. A distinctive feature of the fatty acid composition of C. sinicus was the prevalence of 20:5ω3 and 22:6ω3. The higher content of 16:1ω7 over 18:4ω3 in females indicated that diatoms contributed more than dinoflagellates to the diet of C. sinicus. The feeding intensity of C. sinicus on diatoms was higher in spring and autumn than in other seasons. The herbivorous indicators 20:1 and 22:1 were comparatively low, suggesting that besides phytoplankton, C. sinicus might feed on a wider range of particles including organic detritus, bacteria and small copepods.     

Effect of NH4-non the pigment content ofLaminaria japonica

Experiments withLaminaria japonica were conducted in Meidao Bay and Pier Bay, Qingdao, China, Nitrogen-starved plants were fertilized intermittently with 7.1 mM NH₄−N solution for 1 hour at 3 day intervals, after which chlorophyll a, fucoxanthin, chlorophyll c and β-carotene contents were analyzed. Photosynthetic and growth rates of plants and nitrogen content of seawater were determined. their contents of chlorophyll a, fucoxanthin, chlorophyll c and β-carotene were 1.65, 0.67, 0.33 and 0.06 mg.dm⁻² respectively, or 2.46, 2.03, 1.86 and 1.81 times those of the controls. The ratio of fucoxanthin to chlorophyll a in nitrogen-enriched plants was lower than that of the controls. Normal growth rate of the plants (2 cm·day⁻¹ in length) were reached when the chlorophyll a content exceeded 0.1 mg·g⁻¹ fresh wt., indicating that chlorophyll a content can serve as an indicator of normal growth. Experimental results show that seawater is regarded as fertile for the normal growth ofLaminaria if the total inorganic nitrogen (including ammonium salt, nitrate and nitrite) content in seawater is about 2 μm, and infertile if less than 1 μm, in which case fertilizer should be applied. Contribution No. 1703 from the Institute of Oceanology, Academia Sinica     

Cloning and phylogenetic analysis of a fatty acid elongase gene from Nannochloropsis oculata CS179

Nannochloropsis oculata CS179, a unicellular marine microalga, is rich in long-chain polyunsaturated fatty acids (LCPUFAs). Elongase and desaturase play a key role in the biosynthesis of PUFAs. A new elongase gene, which encodes 322 amino acids, was identified via RT-PCR and 5′ and 3′ RACE. The sequence of the elongase gene was blast-searched in the NCBI GenBank and showed a similarity to those of the cryptosporidium. But the NJ-tree revealed that the N. oculata CS179 elongase clustered with those of the microalgae Phaeodactylum tricornutum, Ostreococcus tauri and Thalassiosira pseudonana.     

Effects of sodium nitrate and sodium acetate concentrations on the growth and fatty acid composition of <Emphasis Type="Italic">Brachiomonas submarina</Emphasis>

One isolate of Brachiomonas submarina was tested for its ability to grow heterotrophicly on 5 different organic compounds. Sodium acetate and glucose were found to be effective in supporting the growth. Sodium acetate was chosen as the organic nutrient to test the combined effects of organic and inorganic solutions on the growth and fatty acid composition of Brachiomonas submarina. The best growth rates were achieved at 3 mmol L⁻¹ CH₃COONa and 0.88 mmol L⁻¹ NaNO₃ in heterotrophic condition, and 4 mmol L⁻¹ CH₃COONa and 3.52 mmol L⁻¹ NaNO₃ in mixotrophic condition. The differences between fatty acid contents were significant. The total polyunsaturated fatty acids (T. P. U. F. As) varied from 55.79% to 67.72% in heteritrophic growth and from 52.39% to 65.55% in mixotrophic growth. It is concluded that CH₃COONa and NaNO₃ at 3 mmol L⁻¹ and 3.52 mmol L⁻¹ should respectively be used in order to achieve the highest growth rate and fatty acid content.     

Genetic transformation of <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> with a bacterial phleomycin resistance gene as dominant selective marker

The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2×10⁷ cells mL^?1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS188. The construct was transferred into the protoplasts through electroporation (1 kV, 15 μS). The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL^?1 zeocin. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.     

Salicylic acid and heat acclimation pretreatment protects Laminaria japonica sporophyte (Phaeophyceae) from heat stress

Possible mediatory roles of heat acclimation and salicylic acid in protecting the sporophyte of marine macroalga Laminaria japonica (Phaeophyceae) from heat stress were studied. Heat stress resulted in oxidative injury in the kelp blades. Under heat stress significant accumulation of hydrogen peroxide (H₂O₂) and malonaldehyde (MDA), a membrane lipid peroxidation product, and a drastic decrease in chlorophyll a content were recorded. Activity of the enzymatic antioxidant system was drastically affected by heat stress. The activity of superoxide dismutase (SOD) was significantly increased while peroxidase (POD), catalase (CAT) and glutathione peroxidase (GPX) were greatly inhibited and, simultaneously, phenylalanine ammonia-lyase was activated while polyphenol oxidase (PPO) was inhibited. Both heat acclimation pretreatment and exogenous application of salicylic acid alleviated oxidative damage in kelp blades. Blades receiving heat acclimation pretreatment and exogenous salicylic acid prior to heat stress exhibited a reduced increase in H₂O₂ and MDA content, and a lower reduction in chlorophyll a content. Pretreatment with heat acclimation and salicylic acid elevated activities of SOD, POD, CAT, GPX and PPO. Considering these results collectively, we speculate that the inhibition of antioxidant enzymes is a possible cause of the heat-stress-induced oxidative stress in L. japonica, and enhanced thermotolerance may be associated, at least in part, with the elevated activity of the enzymatic antioxidant system.     

Standing crop and primary production of benthic microalgae on the tidal flats in Yueqing Bay

The standing stock and primary production of benthic microalgae on tidal flats were measured seasonally at 3 transects (Puqing, Dahengchuang and Puqi) in Yueqing Bay during 2002 2003. The results showed that the integral chlorophyll a (Chl a) concentration in tidal flat mud exhibited a seasonal variation with the order of magnitude: winter (14.0 4.2 mg m-2) > spring (13.0 6.3 mg m-2) > autumn (7.7 5.9 mg m-2) > summer (4.6 3.2 mg m-2). The primary production showed an order of magnitude: spring (270.5 224.9 mgC m-2 d-1)>winter (238.7 225.5 mgC m-2 d-1)>autumn (214.1 56.2 mgC m-2 d-1)>summer (71.6 44.6 mgC m-2 d-1). Both chlorophyll a and primary production showed maximum values in the surface layer of sediment, and decreased rapidly with increasing depth due to sun light limitation. The results of variance analysis indicated that seasonal variation and tidal flat condition affected Chl a greatly, but had no significant effect on primary production. The annual primary production of benthic microalgae on tidal flats in Yueqing Bay was estimated at 16143 tons carbon, which is sufficient to support 1.02×105 tons shellfish production. The environmental factors affecting chlorophyll and primary production on the tidal flats in Yueqing Bay were discussed. By comparing with other bays on China’s coast, it was observed that Yueqing Bay is a region with high benthic microalgae standing crop and primary production, which may be related to the type of its sediment.     

Effects of temperature, salinity, light intensity, and pH on the eicosapentaenoic acid production of Pinguiococcus pyrenoidosus

The effects of temperature, light intensity, salinity, and initial pH on the growth and fatty acid composition of Pinguiococcus pyrenoidosus 2078 were studied for eicosapentaenoic acid (EPA) production potential. The fatty acid composition was assayed by gas chromatography-mass spectrometry, which indicated that the main fatty acids were C14:0, C16:0 and EPA. The highest EPA percentage 20.83% of total fatty acids was obtained at 20℃ with the temperature being set at 20, 24, and 28℃. Under different salinities and light intensities, the highest percentages of total polyunsaturated fatty acids (PUFAs) and EPA were 17.82% and 31.37% of total fatty acids, respectively, which were achieved at salinity 30 and 100μmol photon m-2s-1 illumination. The highest percentages of total PUFAs and EPA were 38.75% and 23.13% of total fatty acids, respectively, which were reached at an initial pH of 6 with the test range being from 5.0 to 9.0.     

Comparative studies of inorganic carbon utilization inEmiliania huxleyi and some non-calcifying marine microalgae

Inorganic carbon utilization in the non-calcifying marine microalgae,Nannochloropsis oculata, Phaeodactylum tricornutum andPorphyridium purpureum was compared with high- and low-calcifying strains ofEmiliania huxleyi grown in artificial seawater medium aerated with either air (0.03% V/V CO₂) or CO₂-free air. For high-calcifying strain ofE. oculata andP. tricornutem, similar growth patterns were observed in air-and CO₂-free air-grown cultures.P. purpureum showed a less final cell density in CO₂-free air than in air-grown culture. However, low-calcifying strain ofE. huxleyi was able to grow only in air-grown culture, but not in CO₂-free air-grown culture. Measurements of alkalinity, pH, concentration of dissolved inorganic carbon (DIC) and free CO₂ showed different patterns of DIC utilization. WithN. oculata, P. tricornutum andP. purpureum the pattern of DIC utilization was characterized by an increase of pH and a decrease of DIC but a constant alkalinity in the cultures aerated with air or CO₂-free air, suggesting that bicarbonate utilization was concomitant with an efflux of OH⁻. Both alkalinity and pH were maintained rather constant in air-grown culture of low-calcifying strain ofE. huxleyi, suggesting that diffusive entry of CO₂ could meet the requirement of DIC for its photosynthesis and growth. High-calcifying strain ofE. huxleyi, however, showed a pattern of decrease of alkalinity and DIC but an almost constant pH, indicating that bicarbonate was the major form of inorganic carbon utilised by this organism and bicarbonate uptake is unlikely to be accompanied by an efflux of OH⁻. The final pH values reached byN. oculata, P. tricornutum andP. purpureum in a closed system were 10.75, 10.60 and 9.85 respectively, showing that bicarbonate utilisation is concomitant with an efflux of OH⁻. While the final pH of 8.4 in high-calcifyingE. huxleyi suggests that bicarbonate utilization was not accompanied by an efflux of OH⁻. Contribution No. 3557 from the Institue of Oceanology, Chinese Academy of Sciences. This work was supported by NERC grant GRE3/7853 U. K. and partly supported by Bio-Engineering Center, SSTC 96-C01-05-01.     

The study on fishing ground of neon flying squid,Ommastrephes bartrami,and ocean environment based on remote sensing data in the Northwest Pacific Ocean

The relationships between the neon flying squid, Ommastrephes bartrami, and the relative ocean environmental factors are analyzed. The environmental factors collected are sea surface temperature (SST), chlorophyll concentration (Chl-a) and sea surface height (SSH) from NASA, as well as the yields of neon flying squid in the North Pacific Ocean. The results show that the favorable temperature for neon flying squid living is 10°C–22°C and the favorite temperature is between 15°C–17°C. The Chl-a concentration is 0.1–0.6 mg/m³. When Chl-a concentration changes to 0.12–0.14 mg/m3, the probability of forming fishing ground becomes very high. In most fishing grounds, the SSH is higher than the mean SSH. The generalized additive model (GAM) was applied to analyze the correlations between neon flying squid and ocean environmental factors. Every year, squids migrate northward from June to August and return southward during October–November, and the characteristics of the both migrations are very different. When squids migrate to the north, most relationships between the yields and SST are positive. The relationships are negative when squids move to southward. The relationships between the yields and Chl-a concentrations are negative from June to October, and insignificant in November. There is no obvious correlation between the catches of squid and longitude, but good with latitude. Supported by the National High Technology Research and Development Program of China (863 Program, No. 2003AA607030); National Key Technology Research and Development Program (No. 2006BAD09A05)     

Effects of polypeptide from<Emphasis Type="Italic">Chlamys farreri</Emphasis> on amino acid content in guinea pig skin irradiated by chronic ultraviolet A and B

We examined the effects of polypeptide fromChlamys farreri (PCF) on the amount of hydroxyproline in guinea pig skin irradiated by chronic ultraviolet A (UVA) and ultraviolet B (UVB) radiation. PCF was applied locally before repeated exposure of guinea pig to UVA and UVB. The contents of hydroxyproline and other amino acids in guinea pig skin were determined by automatic amino acid analyzer. Our results showed that: (1) long-time UVA and UVB radiation can reduce dramatically the amounts of hydroxyproline, aspartic acid, threonine, glycine, phenylalanine and lysine in guinea pig skin in comparison with the control group (P<0.05); (2) Compared with model group, pre-treatment with 5% and 20% PCF prior to UVA and UVB radiation can inhibit the decline of amino acids content in guinea pig skin in a dose-dependent manner (P<0.05). As the decrease of hydroxyproline, glycine and lysine contents in the skin directly reflexes type I collagen degeneration, our results indicated that the chronic application of PCF can protect skin type I collagen against UV radiation, and thus protect skin from photoaging. Project No. 39970638 supported by the NSFC, and also supported by the Science and Technology Bureau of Qingdao (No: 2001-28-50).     

Lipid oxidation and fatty acid composition in salt-dried yellow croaker (<Emphasis Type="Italic">Pseudosciaena polyactis</Emphasis>) during processing

Lipid oxidation in salt-dried yellow croaker (Pseudosciaena polyactis) was evaluated during processing with commonly used analytical indices, such as the peroxide value (POV), the thiobarbituric acid reactive substances (TBARS) value, and oxidative-relative lipoxygenase (LOX) activity. Additionally, fatty acids were analyzed using gas chromatography-mass spectrometry. Both POV and TBARS increased significantly (P < 0.05) at the rinsing stage. POV reached its peak value of 3.63 meq O₂ per kg sample at the drying stage, whereas TBARS constantly increased from 0.05 to 0.20 mg MDA per kg sample. Processing of salt-dried yellow croaker had an extremely significant (P < 0.01) effect on LOX activity. Twenty-six fatty acids were identified. Combined eicosapentaenoic acid (EPA; C20:5n3) and docosahexaenoic acid (DHA; C22:6n3) content varied between (19.20 ± 0.37) mg g^?1 and (23.45 ± 1.05) mg g^?1. The polyunsaturated fatty acid/saturated fatty acid (PUFA/SFA) ratio in yellow croaker was 0.73–1.10, and the n-6/n-3 PUFA ratio was approximately 0.13–0.20. The contents of most fatty acids varied significantly (P < 0.05) during the different processing stages, and these differences were caused by lipid oxidation. C18:0, C16:1n7, C19:0, and C22:6n3 showed clear changes in principle component one of a principle components analysis. These fatty acids are potential markers for evaluating lipid oxidation in fish muscle because there was a significant correlation between these markers and TBARS and LOX activity (P < 0.05) with Pearson’s coefficients > 0.931.     

Chemical constituents of marine medicinal mangrove plant <Emphasis Type="Italic">Sonneratia caseolaris</Emphasis>

Twenty-four compounds including eight steroids (1–8), nine triterpenoids (9–16, 24), three flavonoids (20–22), and four benzenecarboxylic derivatives (17–19, 23) were isolated and identified from stems and twigs of medicinal mangrove plant Sonneratia caseolaris. The structures of the isolated compounds were determined by extensive analysis of their spectroscopic data. Among these metabolites, compounds 1, 4–20 and 22–24 were isolated and identified for the first time from S. caseolaris. In the in vitro cytotoxic assay against SMMC-7721 human hepatoma cells, compound 21 (3′,4′,5,7-tetrahydroxyflavone) exhibited significant activity with IC₅₀ 2.8 μg/mL, while oleanolic acid (14), 3,3′-di-O-methyl ether ellagic acid (18), and 3,3′,4-O-tri-O-methyl ether ellagic acid (19) showed weak activity. None of these compounds displayed significant antibacterial activites. Supported by the National Natural Science Foundation of China (No. 30770234); Knowledge Innovation Program of Chinese Academy of Sciences (KZCX2-YW-211-04); Department of Science and Technology of Shandong Province (No.2006GG2205023)     

A comparative analysis of fatty acid composition and fucoxanthin content in six <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> strains from diff erent origins

Phaeodactylum tricornutum is a potential livestock for the combined production of eicosapentaenoic acid (EPA) and fucoxanthin. In this study, six marine diatom strains identified as P. tricornutum were cultured and their total lipid, fatty acid composition and major photosynthetic pigments determined. It was found that the cell dry weight concentration and mean growth rate ranged between 0.24–0.36 g/L and 0.31–0.33/d, respectively. Among the strains, SCSIO771 presented the highest total lipid content, followed by SCSIO828, and the prominent fatty acids in all strains were C16:0, C16:1, C18:1, and C20:5 (EPA). Polyunsaturated fatty acids, including C16:2, C18:2, and EPA, comprised a significant proportion of the total fatty acids. EPA was markedly high in all strains, with the highest in SCSIO828 at 25.65% of total fatty acids. Fucoxanthin was the most abundant pigment in all strains, with the highest in SCSIO828 as well, at 5.50 mg/g. The collective results suggested that strain SCSIO828 could be considered a good candidate for the concurrent production of EPA and fucoxanthin.