Discovery of genes involved in defense/immunity functions in a haemocytes cDNA library from Fenneropenaeus chinensis by ESTs annotation

Crustacean haemocytes play important roles in the host immune response including recognition, phagocytosis, melanization, cytotoxicity and inter-cellular signal communication. Expressed sequence tags (ESTs) analysis is proved to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. In order to further understand the innate immune system and defense mechanisms of Chinese shrimp at molecular level, complementary DNA library is constructed from the haemocyte tissue of Fenneropenaeus chinensis. A total of 2371 cDNA clones are successfully sequenced and the average sequence length is 460 bp. About 50% are identified as orthologs of known genes from other organisms by BLASTx and BLASTn program. By sequences comparability and analysis, 34 important genes including 177 ESTs are identified that may be involved in defense or immune functions in shrimp based on the known knowledge. These genes are categorized into five categories according to their putative functions in shrimp immune system: 13 genes are different types of antimicrobial peptides (AMP, penaeidin, antilipopolysaccharide factor, etc.), and their proportion is about 3 8%; 11 genes belong to prophenoloxidase system (prophenoloxidase, serine proteinase, serine proteinase inhibitor, etc.), and their proportion is about 32%; five genes have high homology with clotting protein (lectin, transglutaminase, etc), and their proportion is about 15%; three genes may be involved in inter-cell signal communication (peroxinectin, integrin), and their proportion is about 9%; two genes have been identified to be chaperone proteins (Hsc70, thioredoxin peroxidase), and their proportion is about 6%. These EST sequences enrich our understanding of the immune genes of F chinensis and will help farther experimental research into immune factors and improve our knowledge of the immune mechanisms of shrimp. (c) 2007 Elsevier B.V. All rights reserved.

Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri

Histone H2A is reported to participate in host defense response through producing novel antimicrobial peptides (AMPs) from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not to our knowledge been reported in mollusca. In the present study, gene cloning, mRNA expression of H2A from scallop Chlamys farreri, and the recombinant expression of its N-terminus were conducted to investigate whether a similar mechanism exists in mollusca. The full-length DNA of H2A was identified by the techniques of homology cloning and genomic DNA walking, The full-length DNA of the scallop H2A was 696 bp long, including a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 228 bp with a stem-loop structure and a canonical polyadenylation signal sequence AATAAA, and an open reading frame of 375 bp encoding a polypeptide of 125 amino acids. The mRNA expression of H2A in the hemocytes of scallop challenged by microbe was measured by semi-quantitative RT-PCR. The expression of H2A was not upregulated after stimulation, suggesting that H2A did not participate in immunity response directly. The DNA fragment of 117 bp encoding 39 amino acids corresponding to the N-terminus of scallop H2A, which was homologous to buforin I in vertebrates, was cloned into Pichia pastoris GS115. The transformants (His(+) Mut(+)) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418. The peptide of 39 amino acids was expressed by induction of 0.5% methanol. The recombinant product exerted antibacterial activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria. The antibacterial activity toward G(+) bacteria was 2.5 times more than that against G(-) bacteria. The results elucidated that N-terminus of H2A was a potential AMP and provided a promising candidate for a new antibiotic screening. However, whether H2A is really involved in scallop immune response mechanisms needs to be further investigated. (C) 2006 Elsevier Ltd. All rights reserved.

Discovery of the genes in response to white spot syndrome virus (WSSV) infection in Fenneropenaeus chinensis through cDNA microarray

We used microarray technology to study differentially expressed genes in white spot syndrome virus (WSSV)-infected shrimp. A total of 3136 cDNA targets, including 1578 unique genes from a cephalothorax cDNA library and 1536 cDNA clones from reverse and forward suppression subtractive hybridization (SSH) libraries of Fenneropenaeus chinensis, plus 14 negative and 8 blank control clones, were spotted onto a 18 x 18 mm area of NH2-modified glass slides. Gene expression patterns in the cephalothorax of shrimp at 6 h after WSSV injection and moribund shrimp naturally infected by WSSV were analyzed. A total of 105 elements on the arrays showed a similar regulation pattern in artificially infected shrimp and naturally infected moribund shrimp; parts of the results were confirmed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The up-regulated expression of immune-related genes, including heat shock proteins (HSP70 and HSP90), trehalose-phosphate synthase (TPS), ubiquitin C, and so forth, were observed when shrimp were challenged with WSSV. Genes including myosin LC2, ATP synthase A chain, and arginine kinase were found to be down-regulated after WSSV infection. The expression of housekeeping genes such as actin, elongation factor, and tubulin is not stable, and so these genes are not suitable as internal standards for semiquantitative RT-PCR when shrimp are challenged by WSSV. As a substitute, we found that triosephosphate isomerase (TPI) was an ideal candidate of interstandards in this situation.

PRELIMINARY STUDIES ON CRYOPRESERVATION OF SYDNEY ROCK OYSTER (SACCOSTREA GLOMERATA) LARVAE

In this study several parameters critical to the success of cryopreserving Sydney rock oyster (Saccostrea glomerata) larvae were investigated. They were: (1) cryoprotectants (10% dimethyl sulfoxide and 10% propylene glycol). (2) freezing protocols (with or without the seeding step). (3) larval concentrations (1,000, 3,000, 5,000, 10,000, 30,000 individuals mL(-1)). and (4) larval ages (6, 12, 24, 48 and 96 h old). The survival rates were determined as percentages of postthaw larvae performing active movements for the 6 and 12 h larvae or active cilia movement for the 24, 48 and 96 h larvae. Analyses showed that the difference in survival rates between different age classses was significant in all the experiments conducted, with the maximum survival rate being achieved in the 24-h-old larvae the postthaw survival rates of larvae cryopreserved with 10% dimethyl sulfoxide (93.1 +/- 0.2%) were significantly higher (P < 0.001) that those with 10% propylene glycol (81.5 +/- 0.4%). Differences in postthaw survival rates between different concentrations (1,000 30,000 individuals mL(-1)) were not significant within each of the three larval age classes (6-, 12-, and 24-h-old ) used.

Discovery of Late Cretaceous garnet two-pyroxene granulite in the southern Lhasa terrane, Tibet and its tectonic significances

The Lhasa terrane, located between the Bangonghu-Nujiang suture zone and the Indus-Yalung Tsangpo suture zone in the southern Tibetan Plateau, was considered previously as a Precambrian continental block. Mesozoic and Cenozoic tectonic evolution of the Lhasa terrane is closely related to the subduction of the Tethys ocean and the collision between the Indian and European continents; so it is one of the keys to reveal the formation and evolution of the Tibetan plateau. The garnet two-pyroxene granulite which was found at the Nyingtri rock group of the southeastern Lhasa terrene consists of garnet, clinopyroxene, orthopyroxene, labradorite, Ti-rich amphibolite and biotite, with a chemical composition of mafic rock. The metamorphic conditions were estimated to be at T = 747 similar to 834 degrees C and P = 0.90 similar to 1.35GPa, suggesting a formation depth of 45km. The zircon U-Pb dating for the garnet amphibolite and marble associated with the granulite give a metamorphic age of 85 similar to 90Ma. This granulite-facies metamorphic event together with a contemporaneous magmatism demonstrated that the southern Lhasa terrane has undergone an Andean-type orogeny at Late Mesozoic time.

Preliminary analysis of grain-size populations with environmentally sensitive terrigenous components in marginal sea setting

Terrigenous components were separated from the bulk sediment of Core A7 from the Okinawa Trough and Core A37 from the Ryukru Trench and grain-size distributions of these sub-samples were analyzed. Based upon an analysis of the grain-size data of the two sedimentary sequences, grain-size populations are identified to be sensitive to sedimentary environmental changes. The modal values and size ranges of the two main grain-size populations in Core A7 are evidently different from those of Core A37, indicating the spatial variability of sediment sources and transport processes between the two places. The downcore variations in the content of the environmentally sensitive grain-size populations reveal that during the accumulation of sedimentary material the environment remained relatively stable at the site where Core A7 was collected, except for the apparent events for the formation of two turbidite layers and a volcanic ash layer. However, the sedimentary sequence of Core A37 shows six sedimentary cycles, indicating a highly variable sedimentary environment at this location.

A preliminary study on thermoluminescence characteristics of sediments from the North Pacific

Thirty-eight surficial deposit samples were collected from the equatorial North Pacific, and the natural thermoluminescence (TL) characteristics of both bulk and clay fraction samples ( < 2 mu m fractions) were studied by the FJ427 - Al automatic TL Dosimeter for the first time. With the measurements of clay mineral composition, element composition by XRD and ICP, the correlations between TL intensity and sedimentary environment proxies were analyzed, such as water depth, ratio of FeO to Fe2O3 contents, LOI, and major clay mineral concentration, and it was found the bulk sample's TL signal was stronger than the clay ones. Usually, increase in the clay components may result in the decrease of TL intensity. From the shape of TL curves, the pelagic sediments can be divided into two groups: the majority group has two glow peaks, in general, the first peak is broad and flat, but the second narrow and sharp; the minority group only has a single peak because the first is absent. The peak centers of TL curves are almost fixed, falling in the temperature sections 230 similar to 260 and 390 similar to 405 degrees C respectively. Lorentz model packed in the Origin 7.5 was chosen to deal with the TL curves. From the processing results, three parameters ( H, C and A), corresponding to the height, center, and kurtosis of TL curve, were obtained to describe the curve characteristics. The correlations between TL curve parameters and sedimentary environment proxies were also calculated. On the basis of the above work, the relationship between TL characteristics and sediment type, mineral composition, sedimentary environment of surface sediments was discussed in the study area, and a conclusion is: sediments from the environment of shallower water, higher organic contents and weaker reductivity have stronger TL signals.

A preliminary study on fingerprinting approach in marine sediment dynamics with the rare earth elements

Locating the quantitized natural sediment fingerprints is an important work for marine sediment dynamics study. The total of 146 sediment samples were collected from the Shelf of the East China Sea and five rivers, including Huanghe (Yellow), Changjiang (Yangtze), Qiantang, Ou and Min River. The sediment grain size and the contents of rare earth elements (REEs) were measured with laser particle size analyzer and ICP-MS technology. The results show that absolute REE content (Sigma REE) and the concentration ratio of light REEs to heavy REEs (L/HREE) are different in the sediments among those rivers. There are higher REE contents in being less than 2 m and 2-31 mu m fractions in the Changjiang Estuary surface sediments. The REE contents of bulk sediment are dominated by the corresponding values of those leading size-fractions. Sigma REE of sediment is higher close to the estuaries and declines seaward on the inner shelf of the East China Sea (ECS). The L/HREE ratio has a tendency of increase southward from 28 degrees N. Hydrodynamic conditions plays a predominate role on spacial distributions of the surficial sediment's REE parameters. In some situations, the currents tend to remove the coarser light grains from initial populations, as well as the deposit of the finer heavy mineral grains. In other situations, the currents will change the ratio of sediment constituents, such as ratio between silts and clays in the sediments. As a result, the various values of Sigma REE or L/HREE ratio in different bulk sediments are more affected by the change of size-fractions than source location. Under the long-term stable hydrodynamic environment, i.e., the East China Sea Shelf, new sediment transport model based on the size and density gradation concept may help to understand the spatial distribution patterns of REE parameters.

An extended variable-grid global ocean circulation model and its preliminary results of the equatorial Pacific circulation

To investigate the interaction between the tropical Pacific and China seas a variable-grid global ocean circulation model with fine grid[(1/6)degrees] covering the area from 20degreesS to 50degreesN and from 99degrees to 150degreesE is developed. Numerical computation of the annually cyclic circulation fields is performed. The results of the annual mean zonal currents and deep to abyssal western boundary currents in the equatorial Pacific Ocean are reported. The North Equatorial Current,the North Equatorial Countercurrent, the South Equatorial Current and the Equatorial Undercurrent are fairly well simulated. The model well reproduces the northward flowing abyssal western boundary current. From the model results a lower deep western boundary current east of the Bismarck-Solomon-New Hebrides Island chain at depths around 2 000 in has been found. The model results also show that the currents in the equatorial Pacific Ocean have multi-layer structures both in zonal currents and western boundary currents, indicating that the global ocean overturning thermohaline circulation appears of multi-layer pattern.

A preliminary study on the mechanism of harmful algal bloom mitigation by use of sophorolipid treatment

in order to investigate a new method of mitigating the deleterious effect of harmful algal blooms (HABs), the inhibition of the glycolipid biosurfactant sophorolipid on three common harmful algae Alexandrium tamarense, Heterosigma akashiwo and Cochlodinium polykrikoides was studied. The optimum preparation condition for sophorolipid, the inhibition capability of sophorolipid and the interaction mechanism of sophorolipid on the three algal species were investigated. Results showed that sophorolipid prepared by extraction with ethyl acetate exhibited the most prominent inhibition effect and that storage time of one year had little influence on the inhibition effect of sophorolipid. The optimum concentration of 10-20 mg/l sophorolipid inhibited the motility of about 90% of the tested harmful algal cells without recovery. Investigation of the algicidal process revealed that sophorolipid induced ecdysis of A. tamarense, quick lysis of H. akashiwo and swelling of C. polykrikoides in a relatively short time. Investigation of the nucleotides showed that more than 15% of the nucleotides were released from the cytoplasm under the effect of 10-20 mg/l sophorolipid, indicating the irreversible damage on the cellular membrane, which resulted in the disintegration of the harmful algal cells. (C) 2004 Elsevier B.V. All rights reserved.

Preliminary study of the dynamic origin of the distribution pattern of bottom sediments on the continental shelves of the Bohai Sea, Yellow Sea and East China Sea

The bottom sediment types in the Bohai Sea, Yellow Sea and East China Sea (BYECS) are diversified, and their distribution pattern is very complicated. However, the bottom sediment types can be simplified to be sandy sediment, clayey sediment and mixed sediment, which comprise the complicated distribution pattern of bottom sediment in the BYECS. The continental shelves of the BYECS are broad, with shallow water depths and tidal currents which are permanent and dominate the marine dynamics in the BYECS. Based on numerical simulation of tidal elevations and currents in the BYECS, the rates of suspended load transport and bed load transport during a single tidal cycle for sediments of eight different grain size ranges are calculated. The results show that any sediment, whose threshold velocity is less than that of tidal current, has the same transport trend. Suspended load transport rare, bed load transport rate, and the ratio of the former to the latter decrease with grain size becoming coarser and coarser. The erosion/accretion patterns of sediments with different grain sizes are determined by the sediment transport rate divergences, and the results show that the patterns are the same for sediments with different grain sizes. Three main bottom sediment types, i.e. sandy sediment mainly composed of fine sand, clayey sediment mainly composed of silty clay, and mixed sediment mainly composed of fine sand, silt, and clay, are obtained by computation. The three bottom sediment types and their distribution pattern are consistent not only with sediment transport field and the sea bed erosion/accretion pattern obtained by simulation, but also with field data of bottom sediment types and divisions. In the BYECS, sand ridges form mainly in the areas with strong rectilinear tidal currents, sand sheets form mainly in the areas dominated by strong rotatory tidal currents, and clayey sediments, i.e. mud patches, form mainly in the areas with weak tidal currents. Hence, not only the sandy sediments but also the clayey sediments in the BYECS are formed under the control of the whole tidal current field of the BYECS. The three main bottom sediment types are not isolated respectively-in fact, they constitute a whole tidal depositional system. Under the condition with no cyclonic cold eddy, the clayey sediments in the BYECS can form in weak tidal current environments. Therefore, a cold eddy is not necessary for the deposition of clayey sediments in the BYECS. (C) 2000 Academic Press.

Preliminary study on different nutrient pools supplies for the phytoplankton growth in the Jiaozhou Bay in China in the fall of 2004

The source and significance. of two mitrients, nitrogen. and phosphorous, were investigated by a modified dilution method performed on seawater samples from the Jiaozhou Bay in autumn 2004. This modified dilution method accounted for the phytoplankton growth rate, microzooplankton grazing mortality rate, the external nutrient pools, as well as nutrient supplied through remineralization by microzooplankton. The results indicated that the phytoplankton net growth rate increased in turn from inside the bay, to outside the bay, to in the Xiaogang Harbor. The phytoplankton, maximum growth rates and microzooplankton grazing mortality rates were 1.14 and 0.92 d(-1) outside the bay, 0.42 and 0.32 d(-1) inside the bay and 0.98 and 0.62 d(-1) in the harbor respectively. Outside the bay, the remineralized nitrogen (K-r = 24.49) had heavy influence on the growth of the phytoplankton. Inside the bay, the remineralized phosphorus(K-r = 3.49) strongly affected the phytoplankton growth. In the harbor, the remineralized phosphorus (K-r = 3.73) was in larger demand by phytoplankton growth. The results demonstrated that the different nutrients pools supplied for phytoplankton growth were greatly in accordance with the phytoplankton community structure, microzooplankton grazing mortality rates and environmental conditions. It is revealed that, nutrient remineralization is much more important for the phytoplankton growth in the Jiaozhou Bay than previously believed.

Polysaccharides from Ulva pertusa (Chlorophyta) and preliminary studies on their antihyperlipidemia activity

Polysaccharides from Ulva pertusa were isolated and prepared by extraction in hot water and precipitation by ethanol. The water-soluble polysaccharides were chemically well defined, containing 47.0% total carbohydrate, 23.2% uronic acids, 17.1% sulfate groups, 1.0% N and 29.9% ash. Gas chromatography analysis demonstrated that the neutral sugars were mainly composed of rhamnose, xylose and glucose and smaller amounts of mannose, galactose and arabinose. The FTIR and C-13-NMR spectra indicated that basic repeating units of the polysaccharides were (beta-D-GlcpA-(1->4)-alpha-L-Rhap 3S) and (alpha-L-IdopA-(1->4)-alpha-L-Rhap 3S). Fifty ICR mice were used to study the effect of water-soluble polysaccharides from Ulva pertusa on the level of plasma lipids, with inositol niacinate as positive control. The results indicated that the polysaccharides significantly lowered the contents of plasma total cholesterol, low-density lipoprotein cholesterol, triglyceride and markedly increased the contents of serum high-density lipoprotein cholesterol, compared with the hyperlipidemia control group (p<0.01). Moreover, administration of polysaccharides significantly decreased the atherogenic index. The present results suggest that the polysaccharides from Ulva pertusa have great potential for preventing ischemic cardiovascular and cerebrovascular diseases.

A preliminary genetic linkage map of the pacific abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F, family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (alpha = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.

Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO + 10% PG + 10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6 +/- 16.7% (mean +/- S.D.) and 77.8 +/- 15.5%, were achieved by the straw vitrifying method (20.5% DMSO + 15.5% acetamide + 10% PG, thawing at 43 degrees C and washing in 0.5 M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5 M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation. (C) 2007 Elsevier Inc. All rights reserved.