Analysis of phospholipids in microalga Nitzschia closterium by UPLC-Q-TOF-MS

Precise structural identification of phospholipids in the microalga Nitzschia closterium has been established using ultra performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS) for direct analysis of total lipid extracts.Mass spectrometry was performed in reflective time-of-flight using electron spraying ionization in negative mode.Phospholipid molecular species identification was based on the characteristic product ions and neutral loss yie...     

Unraveling enhanced membrane lipid biosynthesis in Chlamydomonas reinhardtii starchless mutant sta6 by using an electrospray ionization mass spectrometry-based lipidomics method

The unicellular green alga Chlamydomonas reinhardtii, a well-established model organism, has been widely used in dissecting glycerolipid metabolism in oxygenating photosynthetic organisms. In previous studies, it has been found that shunting carbon precursors from the starch synthesis pathway can lead to a 10-fold increase in TAG content as compared to the wild type, but it is unknown whether inactivation of AGPase may affect membrane lipids biosynthesis. The study aims to investigate global changes in lipid metabolism and homeostasis in the starchless mutant C. reinhardtii sta6. By utilizing an electrospray ionization/mass spectrometry (ESI/MS)-based lipidomics approach, a total of 105 membrane lipid molecules of C. reinhardtii were resolved, including 16 monogalactosyldiacylglycerol (MGDG), 16 digalactosyldiacylglycerol (DGDG), 11 phosphatidylglycerol (PG), 6 sulfoquinovosyldiacylglycerol (SQDG), 49 diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), 2 phosphatidylethanolamine (PE), and 5 phosphatidylinositol (PI) molecules. The quantitative results indicated that the membrane lipid profiles were similar between the two C. reinhardtii strains grown under both low- and high-light conditions, but the cellular contents of a great number of lipids were altered in sta6 due to the defect in starch biosynthesis. Under low-light conditions, sta6 accumulated more PI, MGDG, DGDG but less amounts of DGTS as compared to WT. Under high light, sta6 cells contained higher content membrane lipids than cc-124, except for PG, which is more or less similar in both strains. Our results demonstrate that the cellular membrane lipid homeostasis underwent profound changes in the starchless mutant, and thereby its physiological impact remains to be explored.     

Phospholipid Compositions in Portunus trituberculatus Larvae at Different Developmental Stages

Phospholipids are used to improve the growth and survival of Portunus trituberculatus,a widely cultured crab species in China.However,only total phospholipids or several classes are applied in crab diets.In this study,we employed a targeted lipidomic method to investigate the comprehensive phospholipid composition in P.trituberculatus larvae and reveal the changing phospholipid profile over the larval development.Results showed that P.trituberculatus larvae contain 112 phospholipid species belonging to 10 phospholipid classes,in which phosphatidylcholine(PC)and phosphatidylethanolamine(PE)species are the most abundant,and PC,PE,phosphatidic acid,and phosphatidylserine(PS)are with high concentrations.The levels of all phospholipids significantly changed with larval development,which was highlighted by the downward parabolic changes in PE,phosphatidylglycerol,phosphatidylino-sitol,PS,lysophosphatidic acid,and sphingomyelin levels.In addition,nearly all phospholipid species were depleted at the M stage,which probably contributed to the mass mortality of crab larvae.These findings on the composition and alterations of phospholipids in P.trituberculatus larvae provide novel perspectives for the targeted supplementation of phospholipids in crab diets.Our work also highlights the use of targeted UHPLC-MS lipidomics in understanding the changes of phospholipids during crab development.     

Isolation of Chloroplasts from Marine Microalga Isochrysis galbana Parke for Their Lipid Composition Analysis

Marine microalga Isochrysis galbana is an important feed species with a high nutritional value.Different from other uni-cellular algae,its cell contains two chloroplasts which are the major sites for lipid synthesis.Here,we optimized a chloroplast isola-tion approach suitable for the isolation of I.galbana chloroplasts and determined the purity and integrity of the isolated chloroplasts through microscopic observations and enzyme activity assay.The chloroplast lipids were analyzed with a ultrahigh-performance li-quid chromatography-Q Exactive Orbitrap-mass spectrometry.This newly developed isolation approach is simple and reliable to isolate chloroplasts with high integrity and purity.The average yield of intact chloroplasts was 15.3%±0.1%.Glycolipids and acyl-glycerols were the main chloroplast lipids.Glycolipids accounted for 56.6%of chloroplast lipid.Digalactosyldiacylglycerol(DGDG),monogalactosyldiacylglycerol(MGDG)and sulfoquinovosyldiacylglycerol(SQDG)were the main glyceroglycolipids.The fatty acyl R1/R2 were mostly 18:4/16:1,18:3/16:1 and 18:4/18:5 in DGDGs,14:0/18:4,18:4/18:5,18:4/18:4 and 18:3/18:4 in MGDGs and 16:0/14:0,16:0/18:3,and 18:4/18:3 in SQDGs.In addition,diacylglycerol(DAG)was the most abundant acylglycerols;the content of 22:6/18:4-DAG was the highest.There was a little amount of glycosphingolipid(GSL)in chloroplast.Digalactosylmonoglyceride(DGMG),monogalactosylmonoglyceride(MGMG),sulfoquinovosylmonoacylglycerol(SQMG),monoglyceride(MAG),phospholi-pids(PLs),ceramide(Cer)and betaine lipids were nearly undetectable in chloroplast.The fatty acid proportions of DGDGs,MGDGs,SQDGs,DAGs,triglycerides(TAGs)and GSLs were either higher or lower than or similar to those of whole-cell.Collectively,our isolation approach is applicable to many aspects of chloroplast biology,and may offer a reference for the isolation of chloroplasts from other marine microalgae.     

Comparison of lipids in organs of the starfish Asterias amurensis associated with different treatments

Lipids were extracted from organs of the starfish Asterias amurensis associated with different treatments(raw-control,boiling and heating),and then analyzed for lipid content,lipid oxidation index,lipid classes and fatty acid composition.Results showed that boiling softened the hard starfish shells,thus facilitating the collection of starfish organs.As compared with raw organs,the boiled organs had lower water content and higher lipid content,possibly due to the loss of water-holding capacity caused by protein denaturation.Both boiling and heating increased the peroxide value(PV),thiobarbituric acid(TBA) value and carbon value(CV) of lipids.Despite slight increases in the content of complex lipids,associated lipid composition had no substantial variations upon boiling and heating.For simple lipids,the content of 1,2-diglyceride decreased in boiled and heated organs,with free fatty acids observed on thin layer chromatography(TLC).However,neither boiling nor heating significantly changed the fatty acid compositions of simple or complex lipids in starfish organs,suggesting that these two treatments had no significant effects on complex lipids in starfish organs.Together,our results indicated that boiling of starfish soon after capture facilitated the handling and extraction of useful complex lipids consisting of abundant glucosylceramide and eicosapentaenoic acid(EPA)-bounded phospholipids.     

Effects of chilled storage and cryopreservation on sperm characteristics,antioxidant enzyme activities,and lipid peroxidation in Pacific cod <Emphasis Type="Italic">Gadus microcephalus</Emphasis>

The present study evaluated the effects of chilled storage and cryopreservation on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod Gadus macrocephalus. Sperm motility and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (Gr), and lipid peroxidation (measured via malondialdehyde (MDA) content) were determined after the milt was stored at 4°C for 12 h, cryopreserved without cryoprotectant in 12% propylene glycol (PG), cryopreserved in 12% PG+0.1 mol/L trehalose, or cryopreserved in 12% PG spermatozoa but centrifuged to decant the supernatant prior to cryopreservation (only sperm cells were cryopreserved). After chilled storage or cryopreservation, the SOD, CAT and GPx activities were reduced in sperm cells and increased in seminal plasma in almost all treatments; sperm motility parameters were also decreased. However, the addition of trehalose into the cryoprotectant could significantly improve the postthaw sperm quality as revealed by the sperm average path velocity. This improvement might be attributed to the function of trehalose in scavenging reactive oxygen species. Chilled storage and cryopreservation had significant effects on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod.     

Co-Production of Lipids,Eicosapentaenoic Acid,Fucoxanthin, and Chrysolaminarin by Phaeodactylum tricornutum Cultured in a Flat-Plate Photobioreactor Under Varying Nitrogen Conditions

The marine diatom Phaeodactylum tricornutum is a polymorphological, ecologically significant, and well-studied model of unicellular microalga. This diatom can accumulate diverse important metabolites. Herein, we cultured P. tricornutum in an internally installed tie-piece flat-plate photobioreactor under 14.5 m mol L~(-1)(high nitrogen, HN) and 2.9 m mol L~(-1)(low nitrogen, LN) of KNO_3 and assessed its time-resolved changes in biochemical compositions. The results showed that HN was inductive to accumulate high biomass(4.1 g L~(-1)). However, the LN condition could accelerate lipid accumulation in P. tricornutum. The maximum total lipid(TL) content under LN was up to 42.5% of biomass on day 12. Finally, neutral lipids(NLs) were 63.8% and 75.7% of TLs under HN and LN, respectively. The content of EPA ranged from 2.3% to 1.5% of dry weight during the growth period under the two culture conditions. Peak volumetric lipid productivity of 128.4 mg L~(-1)d~(-1) was achieved in the HN group(on day 9). The highest volumetric productivity values of EPA, chrysolaminarin, and fucoxanthin were obtained in the exponential phase(on day 6) under HN, which were 9.6, 93.6, and 4.7 mg L~(-1)d~(-1), respectively. In conclusion, extractable amounts of lipids, EPA, fucoxanthin, and chrysolaminarin could be obtained from P. tricornutum by regulating the culture conditions.     

Simultaneous identification and quantification of tetrodotoxin in fresh pufferfish and pufferfish-based products using immunoaffinity columns and liquid chromatography/quadrupole-linear ion trap mass spectrometry

In this study,we established a comprehensive method for simultaneous identification and quantification of tetrodotoxin(TTX) in fresh pufferfish tissues and pufferfish-based products using liquid chromatography/quadrupole-linear ion trap mass spectrometry(LC-QqLIT-MS).TTX was extracted by 1% acetic acid-methanol,and most of the lipids were then removed by freezing lipid precipitation,followed by purification and concentration using irnmunoaffinity columns(IACs).Matrix effects were substantially reduced due to the high specificity of the IACs,and thus,background interference was avoided.Quantitation analysis was therefore performed using an external calibration curve with standards prepared in mobile phase.The method was evaluated by fortifying samples at 1,10,and 100 ng/g,respectively,and the recoveries ranged from 75.8%-107%,with a relative standard deviation of less than 15%.The TTX calibration curves were linear over the range of 1-1 000 ng/L,with a detection limit of 0.3 ng/g and a quantification limit of 1 ng/g.Using this method,samples can be further analyzed using an informationdependent acquisition(IDA) experiment,in the positive mode,from a single liquid chromatography-tandem mass spectrometry injection,which can provide an extra level of confirmation by matching the full product ion spectra acquired for a standard sample with those from an enhanced product ion(EPI) library.The scheduled multiple reaction monitoring method enabled TTX to be screened for,and TTX was positively identified using the IDA and EPI spectra.This method was successfully applied to analyze a total of 206 samples of fresh pufferfish tissues and pufferfish-based products.The results from this study show that the proposed method can be used to quantify and identify TTX in a single run with excellent sensitivity and reproducibility,and is suitable for the analysis of complex matrix pufferfish samples.     

Identification of dissolved and particulate carbonyl compounds produced by marine harmful algal bloom species

Carbonyl compounds,especially polyunsaturated aldehydes(PUAs),are increasingly reported as teratogenic to grazers and deleterious to phytoplankton.While PUAs have been considered to be mainly produced by diatoms after cell wounding,little is known about whether microalgae other than diatoms,particularly harmful algal bloom(HAB)-forming species,produce carbonyl compounds.In this study,we analyzed the carbonyl compounds from eight common HAB-forming species(Akashiwo sanguinea,Karenia mikimotoi,Karlodinium veneficum,Margalefidinium polykrikoides,Prorocentrum donghaiense,P.minimum,Scrippsiella trochoidea,and Heterosig,ma akashiwo)using gas chromatography-mass spectrometry in full scan and selected ion monitoring(SIM)modes.Our results show a ubiquitous presence of carbonyl colmpounds in both dissolved and particulate forms in all species we examined.In the full scan mode,133 chromatographic peaks were detected from 51 samples altogether.Both the varieties of carbonyl compounds and their quantities were algal species dependent,although most of the carbonyl compounds could not be fully identified according to the mass spectral database only due to the unavailability of enough standards for all analytes.Aided with nine standards in SIM mode,we further identified and quantified all nine aldehydes(2-methyl-2-pentenal,trans-2-nonenal,cis-6-nonenal,2,6-dimethyl-5-heptenal,trans-2-hexenal,trans-2-decenal,2,4-heptadienal,trans-trans-2,4-octadienal,and trans-trans-2,4-decadienal).Four of these nine aldehydes were detected in particulate form,which confirmed that trans-2-nonenal could be produced by K.mikimotoi,Karl.veneficum,P.donghaiense,P.minimum,S.trochoidea,and H.akashiwo,2,4-heptadienal and trans-trans-2,4-decadienal by A.sanguinea,M.polykrikoides,and S.trochoidea,and trans,trans-2,4-octadienal by S.trochoidea,respectively.We proved that some dinoflagellate and raphidophyte species could contribute to the pool of carbonyl compounds including PUAs in a marine ecosystem.Some carbonyl compounds,particularly those in high cell quota and/or dissolved concentration,may be related to fishkilling or allelopathy which needs further identification and quantification.     

Molecular phylogenetic analysis of attached Ulvaceae species and free-floating <Emphasis Type="Italic">Enteromorpha</Emphasis> from Qingdao coasts in 2007

Based on the sequence data of the nuclear ribosomal DNA internal transcribed spacer (ITS) 1, 5.8 S, and ITS 2, the molecular phylogeny was analyzed on Ulvaceae species collected from Qingdao coasts in summer of 2007, including 15 attached Ulva and Enteromorpha samples from 10 locations and 10 free-floating Enteromorpha samples from seven locations. The result supported the monophyly of all free-floating Enteromorpha samples, implying the unialgal composition of the free-floating Enteromorpha, and the attached Ulvaceae species from Qingdao coasts were grouped into other five clades, suggesting that they were not the biogeographic origin of the free-floating Enteromorpha in that season. Supported by NSFC (40506030); the Innovative Key Project of the Chinese Academy of Sciences (KZCX2-YW-209); Science & Technology Project of Qingdao City (06-2-2-12-JCH)     

Effects of different enzymatic hydrolysis methods on the bioactivity of peptidoglycan in Litopenaeus vannamei

The effects of different hydrolysis methods on peptidoglycan(PG) were assessed in terms of their impact on the innate immunity and disease resistance of Pacific white shrimp,Litopenaeus vannamei.PG derived from Bifidobacterium thermophilum was prepared in the laboratory and processed with lysozyme and protease under varying conditions to produce several different PG preparations.A standard shrimp feed was mixed with 0.05% PG preparations to produce a number of experimental diets for shrimp.The composition,concentration,and molecular weight ranges of the soluble PG were analyzed.Serum phenoloxidase and acid phosphatase activity in the shrimp were determined on Days 6-31 of the experiment.The protective activity of the PG preparations was evaluated by exposing shrimp to white spot syndrome virus(WSSV).Data on the composition of the PG preparations indicated that preparations hydrolyzed with lysozyme for 72 h had more low-molecular-weight PG than those treated for 24 h,and hydrolysis by protease enhanced efficiency of hydrolysis compared to lysozyme.SDS-PAGE showed changes in the molecular weight of the soluble PG produced by the different hydrolysis methods.Measurements of serum phenoloxidase and acid phosphatase activity levels in the shrimp indicated that the PG preparations processed with enzymes were superior to the preparation which had not undergone hydrolysis in enhancing the activity of the two serum enzymes.In addition,the preparation containing more low-molecular-weight PG enhanced the resistance of the shrimp to WSSV,whereas no increased resistance was observed for preparations containing less low-molecular-weight PG.These findings suggest that the immunity-enhancing activity of PG is related to its molecular weight and that increasing the quantity of low-molecular-weight PG can fortify the effect of immunity enhancement.     

Occurrence and diversity of marine yeasts in Antarctica environments

A total of 28 yeast strains were obtained from the sea sediment of Antarctica.According to the results of routine identi-fication and molecular characterization,the strains belonged to species of Yarrowia lipolytica,Debaryomyces hansenii,Rhodotorula slooffiae,Rhodotorula mucilaginosa,Sporidiobolus salmonicolor,Aureobasidium pullulans,Mrakia frigida and Guehomyces pullu-lans,respectively.The Antarctica yeasts have wide potential applications in biotechnology,for some of them can produce b-galactosidase and killer toxins.     

Isolation and characterization of an enoyl-acyl carrier protein reductase gene from microalga Isochrysis galbana

In most bacteria,plants and algae,fatty acid biosynthesis is catalyzed by a group of freely dissociable proteins known as the type II fatty acid synthase(FAS II) system.In the FAS II system,enoylacyl carrier protein reductase(ENR) acts as a determinant for completing the cycles of fatty acid elongation.In this study,the cDNA sequence of ENR,designated as IgENR,was isolated from the microalga Isochrysis galbana CCMM5001.RACE(rapid amplification of cDNA ends) was used to isolate the full-length cDNA of IgENR(1 503 bp),which contains an open reading frame(ORF) of 1 044 bp and encodes a protein of 347 amino acids.The genomic DNA sequence of IgENR is interrupted by four introns.The putative amino acid sequence is homologous to the ENRs of seed plants and algae,and they contain common coenzymebinding sites and active site motifs.Under different stress conditions,real-time quantitative polymerase chain reaction(RT-qPCR) showed the expression of IgENR was upregulated by high temperature(35℃),and downregulated by depleted nitrogen(0 mol/L).To clarify the mechanism of lipids accumulating lipids,other genes involved in lipids accumulation should be studied.     

Lipid and fatty acid compositions of cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>), haddock (<Emphasis Type="Italic">Melanogrammus aeglefinus</Emphasis>) and halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

This study was conducted to compare lipid and fatty acid composition of cod, haddock and halibut. Three groups of cod (276 g ± 61 g), haddock (538 g ± 83 g) and halibut (3704 g ± 221 g) were maintained with commercial feeds mainly based on fish meal and marine fish oil for 12 weeks prior to sampling. The fatty acid compositions of muscle and liver were determined by GC/FID after derivatization of extracted lipids into fatty acid methyl esters (FAME). Lipids were also fractionated into neutral and polar lipids using Waters silica Sep-Pak?. The phospholipid fraction was further separated by high-performance thin-layer chromatography (HPTLC) and the FAME profile was obtained. Results of the present study showed that cod and haddock were lean fish and their total muscle lipid contents were 0.8% and 0.7%, respectively, with phospholipid constituting 83.6% and 87.5% of the total muscle lipid, respectively. Halibut was a medium-fat fish and its muscle lipid content was 8%, with 84% of the total muscle lipid being neutral lipid. Total liver lipid contents of cod, haddock and halibut were 36.9%, 67.2% and 30.7%, respectively, of which the neutral lipids accounted for the major fraction (88.1%–97.1%). Polyunsaturated fatty acids were the most abundant in cod and haddock muscle neutral lipid. Monounsaturated fatty acid level was the highest in halibut muscle neutral lipid. Fatty acid compositions of phospholipid were relatively constant. In summary, the liver of cod and haddock as lean fish was the main lipid reserve organ, and structural phospholipid is the major lipid form in flesh. However, as a medium-fat fish, halibut stored lipid in both their liver and muscle.     

Brachiopods from China seas

A collection of brachiopods by the Institute of Oceanology, Academia Sinica (Qingdao), contains eight species from seven genera. Six of the species have been recorded previously from China seas—Lingula adamsi, L. anatina, Discinisca stella, Pelagodiscus atlanticus, Campages mariae, Terebratalia coreanica. Two species (Terebratulina hataiana andFrenulina sanguinolenta) have been described from other parts of the Pacific area. The apparent absence of any endemic species is a noteworthy feature of Chinese in comparison with Japanese faunas.     

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.     

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.     

The cytochemistry of oocytes of Chinese shrimpPenaeus orientalis

In the growth of oocytes ofPenaeus orientalis Kishinouye, five stages were distinguished. Histochemical tests showed the presence of DNA in the chromatin and nucleolus of the cell. The cytoplasm at the previtellogenetic stage and the nucleolus are rich in RNA and the proteins abounding with cysteine, tyrosine and tryptophan. The yolk consists mainly of proteins and phospholipids. The 1,2-glycol groups of carbohydrate occur in the cytoplasm at stage II, and aggregate mostly into cortical rods at stages IV and V. Neutral lipid droplets, and protein containing disulfides, appear in the cytoplasm at stages III and IV respectively. The proteins in the cortical rod differ from those in other components of the cell in the presence of cystine and absence of arginine.