Polyploidy induction by hydrostatic pressure shock and embryo development of sea cucumber Apostichopus japonicus

The manipulation of the chromosome set for commercially valuable marine animals is important for enhancing aquacultural production. In this study, triploid and tetraploid sea cucumber Apostichopus japonicus were induced by hydrostatic pressure shock, and the conditions of appropriate induction were tested with different starting times, and hydrostatic pressure intensities and durations. The highest rate of triploid induction reached 20% and that of tetraploid was 60%. In consideration of the survival rate and hatch rate, the appropriate treatment for triploid was 55 Mpa of hydrostatic pressure for 5 min at 55 min after fertilization (a.f.), while for tetraploid it was 60 Mpa for 5 min at 61 min a.f. The triploid of the sea cucumber could survive through the pelagic larval stage and attachment stage, and develop like the control group of the experiment. The tetraploid, however, could not survive the attachment stage.     

Genetic variation in two sea cucumber （Apostichopus japonicus） stocks revealed by ISSR markers

Sea cucumber Apostichopus japonicus samples were collected in Changdao, Penglai (PL), 27 individuals, and Lingshandao, Qingdao (QD), 30 individuals, in the Shandong Peninsula, China. Ten SSR primers were used to assess the genetic variation and relationship between and within the two stocks. Respectively, for each stock, the percentage of polymorphic bands was 85.2% and 86.9%; the gene diver- sity was 0.360 5 and 0.342 8; and the Shannon’s information index was 0.515 0 and 0.499 0. At species level, the percentage of polymorphic bands was 92.2%, the total gene diversity was 0.378 9 and the Shannon’s information index was 0.550 4. The coefficient of overall genetic differentiation and the ge- netic distances between the stocks were also calculated to be 0.073 0 and 0.079 6 using the POPGENE program. Results show that the genetic diversity of the two stocks is still large but the genetic distance between the two stocks is close. A dendrogram was constructed for the 57 individuals from the two stocks, showing that the genetic structure was unitary for PL stock but complex for QD stock.     

Stress Responses to Rapid Temperature Changes of the Juvenile Sea Cucumber （Apostichopusjaponicus Selenka）

Activities of hexokinase （HK）, pyruvate kinase （PK）, superoxide dismutase （SOD） and catalase （CAT） and Hsp70 level were measured to evaluate the response of the commercially important sea cucumber （Apostichopus japonicus Selenka） to rapid temperature changes in laboratory. Animals were subjected to a higher temperature （from 10 to 20℃） （Tinc treatment） or to a lower temperature （from 20 to 10℃） （Tddec treatment） for 72 h. At 1, 3, 12, 24, 72 h of exposure, animals were removed and prepared for further analysis. Results showed that the effect of acute temperature changes on enzyme activities was significant. In Tinc treatment, activities of SOD and CAT increased immediately. The significant enhancement of SOD and CAT activities suggested that oxidative stress increases significantly when ambient temperature increasing from 10 to 20℃. The up-regulation of Flsp70 in Tinc and Tdec treatments indicated that Hsp70 was a bioindicator of thermal stress in the sea cucumber, and the expression pattern depended on the thermal treatment.     

CYTOLOGICAL COMPARISON OF GAMETOGENESIS OF SCALLOPS, ARGOPECTEN IRRADIANS AND CHLAMYS FARRERI

Histological characteristics of gametogenesis of two kinds of scallops, gonochoric Chinese scallop, Chlamys farreri and hermaphroditic bay scallop, Argopecten irradians were investigated in this study. Spermatogenesis in C. farreri has different developmental stages: spermatogonia, primary spermatocyte, second spermatocyte, spermatid and spermatozoon. A large number of same developmental stage spermatic cells converge at a definite area of the testis. Premeiotic, previtellogenic and vitellogenic oocytes can be found during oogenesis in C. farreri, where oocyte distribution is obviously irregular. The A. irradians gonad consists of two different parts in one individual: one part functions as testis, the other as ovary. Between these two parts is a special appearance area, where a large number of spermatic cells are bound with two layers of acellular substance with many oocytes in it.     

Temperature Influences on the Expression of GFP Promoted by the Upstream Sequence of cpcB from Arthrospiraplatensis

In order to investigate the regulation mechanism of the phycocyanin gene,a series of functional analyses of the upstream sequence of cpcB gene from Arthrospira platensis were conducted in E. coli with green fluorescent protein encoding gene(gfp) as the reporter. Results showed that the gfp gene could express at a high level under the promotion of the upstream sequence,suggesting the existence of some strong promoter elements in it. The expression of GFP was influenced by temperature. Higher temperature led to higher expression level. The bioinformatics analyses followed by mutation analyses on the secondary structure of translation ini-tiation region(TIR) revealed that RNA thermosensor might account for the temperature regulation.     

In Silico Screening for Microsatellite Markers from Expressed Sequence Tags of Porphyra yezoensis （Bangiales,Rhodophyta）

The genomic resources of Porphyra yezoensis expressed sequence tags （ESTs） were utilized to identify simple sequence repeats （SSRs）, or microsatellites. This method took the advantage of using ESTs and microsatellites either for the establishment of gene identities or for the acquisition of high polymorphism. The microsatellites can be used as gene markers when microsatellites are tagged to genes. Revealed by bioinformatics analysis, 1162 out of 21954 ESTs contained microsatellites and cluster analysis indicated that 984 of these ESTs fell into 112 contigs, while the other 178 ESTs were singletons. A total of 290 unique SSR-containing genes were identified. The AAC SSRs were the most populous type of microsatellites. GC-rich microsatellites were predominant among all the microsatellites.     

Cylindrotheca closterium Is a Species Complex as Was Evidenced by the Variations of rbcL Gene and SSU rDNA

The genus Cylindrotheca consists of a small group of marine diatoms with a few species described. Eleven isolates of diatoms identified as Cylindrotheca closterium morphologically were obtained from Jiaozhou Bay with their nuclear-encoded small-subunit ribosomal RNA （SSU rDNA） and chloroplast-encoded rbcL gene sequences determined in this study. Interestingly, very high sequence divergences of SSU rDNA and rbcL gene were found among these isolates, and numerous nucleotide variation of rbcL gene caused relatively few variation of deduced amino acid sequence. Phylogenetic analyses based on SSU rDNA and rbcL gene, respectively, grouped the isolates into 6 clades. Phylogenetic tree of SSU rDNA placed all the Cylindrotheca isolates together, separating them into two lineages clearly. LineageⅠ was composed of the eleven C. closterium isolates obtained in this study together with another C. closterium isolate, but some clades were not well supported. LineageⅠwas contained two C. closterium isolates and one C. fusiformis isolate. Phylogenetic analysis of rbcL gene also separated the Cylindrotheca isolates into two well-defined lineages. The eleven C. closterium isolates formed a lineage and all clades were supported strongly. Statistical comparisons of SSU rDNA indicated that the average distance within lineageⅠwas significantly higher than that of other microalgae species （P 〈0.01）. These results suggested the existence of cryptic species within C. closterium.     

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

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

Competition Among Dinoflagellate Alexandrium Tam—arense, Raphidophyte Heterosigma Carterae and Diatom Skeletonema Costatum under Combinations ofTwo Temperatures and Five Salinities

Competition among HAB (Harmful Algal Bloom) species Dinoflagellate Alexandrium tamarense, Raphidophyte Heterosigma carterae, and Diatom Skeletonema costatum was studied in the laboratory. Experiments with these three major HAB species under combinations of different salinities (10, 18, 25, 30, 35) and temperatures (19℃, 25℃) were carried out. The results showed that S. costatum successfully competed with the other two species at salinities of 18, 25,30, and 35 at temperatures of 19℃ and 25℃. However, H. carterae showed its advantage at low salinity of 10 and became the single dominant species at salinity 10 and 25℃. A. tamarense could not compete successfully with the other two species especially at low salinities. However, it could remain at low density in the presence of higher densities of other algae.     

In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid to gill cell line of flounder Paralichthy olivaceus

In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid (IMI) to the gill cell line of flounder (FG) that collected in the gill of Paralichthys olivaceus, was examined by 3 widely used endpoint bioassays: NR (neutral red), MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and TCP (total cell protein). The result shows that the IMI increased at concentrations ≥0.5 μg/ml. The IC50 value of NR, MTT, and TCP was 41.86, 38.46, and 39.08 μg/ml, respectively. The ultrastructural observation revealed that the mitochondria of the cells exposed to 60 μg/ml IMI for 48 h were severely damaged, swollen or disrupted, while their nuclei and rough endoplasmic reticulum (RER) remained normal. This would suggest that the mitochondria are probably the primary target of IMI.     

Uncertainties in Stock Assessment and the Precautionary Management Suggestions for the Anchovy （Engraulisjaponicus） Stock in the Yellow Sea

Anchovy(Engraulis japonicus) is an abundant fish species in the Yellow Sea,and its natural stock is decreasing rapidly in recent years. Based on the stock-recruitment(SR) data from 1987 to 2002 published in Zhao et al.(2003),the criterion BIC(Bayesian Information Criterion) is applied to selecting a suitable model from six normal and lognormal error structured SR statisti-cal models,the age-structured model is used to calculate the biological reference points(BRPs),and the precision of the SR parame-ters and BRPs are calculated using bootstrap method. The results indicate that the anchovy fishery resource in the Yellow Sea is in an over-fished state. The precaution management principle requires that the fishery should be closed immediately.     

The Antitumor Components from Marine-derived Bacterium Streptoverticillium luteoverticillatum 11014 Ⅱ

Eight known compounds were isolated from a marine-derived bacterium Streptoverticillium luteoverticillatum 11014 using bioassay-guided fractionations. Their structures were identified by spectral analysis as bis （4-hydroxybenzyl） ether （1）, p-hydroxyphenylethyl alcohol （2）, N-（4-hydroxyphenethyl） acetamide （3）, indole-3 carboxylic acid methyl ester （4）, dibenzo[b,e] [1,4]dioxine （5）, thymine （6）, cytosine deoxyribonucleoside （7） and 2, 3-butanediol （8）. These compounds were evaluated for their cytotoxic activities against K562 cell line with the SRB method for the first time. Compounds 2 and 4 showed cytotoxcities with IC50 values of 101.1 and 165.3 μolL^-1, respectively. All compounds were isolated from S. luteoverticillatum 11014 for the first time.     

Selection of Yarrowia lipolytica Strains with High Protein Content from Yeasts Isolated from Different Marine Environments

A total of 78 Yarrowia lipolytica yeast strains from seawater, sediments, mud of salterns, the guts of marine fish, and marine algae were obtained. After the crude protein of the yeasts was estimated by the method of Kjehldahl, we found that seven strains of the marine yeasts grown in soy bean cake hydrolysate with 20 g L-1 of glucose for 48 h at 28℃ contained more than 41.0 g protein per 100 g of cell dry weight and the cell dry weight was more than 4.4 g per L of the culture. Among them, strain SWJ-1b contained the highest crude protein. The results of Biolog identification and molecular methods further confirmed that they indeed belonged to Y. lipolytica.     

AGE STRUCTURES OF MODULES OF CLONAL PEATLAND SEDGE Carex middendorffii

Age structure of a plant population carries important information on population dynamics. The traditional age classification of individuals by development phases could not explain the generation relationship neither between individuals nor between modules, and it could not accurately predict the future of population or the tendency of peatland evolution. In a peatland of the Xiao Hinggan Mountains, China, at the middle of the growth season, the age structures of 3 modules, ramets, active buds and rhizomes of a Carex middendorffii clonal population were investigated, with the method of classifying age classes of ramets and active buds by counting generation quantity of tiller nodes, and classifying age classes of rhizomes by their real survival time. The quantity of vegetative ramets was dominant. Tiller nodes of ramets can propagate vegetatively for a maximum of 3 generations. The population of ramets consisted of 3 age classes of ramets at the middle of the growth season, and showed a stable age structure. In the two sampling events, there was no significant difference between quantities and age structure of the population. The maximum age of an excavated rhizome was 12 years old. Rhizomes were classified in 8 age classes, and age classes 4-6 contributed most to the total biomass. There was no significant difference in total length and total biomass per unit area, or in biomass per unit length in rhizomes between the two samplings. Four age classes of active buds were recognized, and their number increased from July to August. The Carex middendorffii clonal population achieved regeneration by budding from the tiller nodes of ramets. The age structures of the 3 modules suggested that the Carex middendorffii clonal population could persist in the early development phase of the oligotrophic peatland in the Xiao Hinggan Mountains, but it could not be dominant. It also faces the risk to disappear from the community as the peatland develops further.     

Observations on shell growth and morphology of the bivalve Ruditapes philippinarum

The bivalve Ruditapes philippinarum is not only one of the main mollusc species in the west Pacific Ocean, but also one of the main species for aquaculture in China. Knowledge of growth of the R. philippinarum shell will improve our understanding of the shell as an environmental archive. It is also useful for the aquaculture of R. philippinarum. In this research, a hanging box culture method was introduced in the culture of R. philippinarum. The bivalves were cultured for 126 days, from March 31 to August 3, 2002. The average growth rates of shell width, height, and thickness were 0.069, 0.046, and 0.032 mm/d, respectively. The mean increase of average individual wet mass was 0.028 g/d. The largest growth rates of both shell and average individual wet mass occurred in June, indicating that water temperature and bivalve reproduction were 2 important factors. The shell morphology underwent significant changes with shell growth. The ratio of height to thickness (value of B/C) shifted 1.58 in the first 60 days to 1.54 in the last 30 days, which was resulted from the change in major shell growth direction. Periodic changes in the B/C ratio led to corrugated shell form, which could be used to determine the age of the shell.     

Distribution of Bacteria Injected in Body of Giant Black Shrimp,Penaeus Monodon

Distribution of injected Vibrio anguillarum in body of P,,naeus monodon was studied with immunohistochemical method. Bacteria could be detected throughout the experiment in some individuals; however in lymphoid tissue, gill, heart and haemolymph of all vibrio injected shrimp, the bacteria could be observed only 5 min after injection. The bacteria density in haemolymph, haemolymph of the hepatopancreas and gills decreased with time. In the lymphoid organ and heart, the bacteria density was the highest 48 h after injection, then decreased. Nodules could be formed in the heart, lymphoid organ and injection site.     

Optimization of medium and cultivation conditions for enhanced exopolysaccharide yield by marine Cyanothece sp. 113

Cyanothece sp. 113 is a unicellular, aerobic, diazotrophic and photosynthetic marine cyanobacterium. The optimal medium for exopolysaccharide yield by the strain was 70.0 g/L of NaCl, and 0.9 g/L of MgSO4 based on the modified F/2 medium for cultivation of marine algae. The optimal cultivation condition for exopolysaccharide yield by this cyanobacterial strain was 29°C, aeration, and continuous illumination at 86.0 μE/M2/S. Under the optimal conditions, over 18.4 g/L of exopolysaccharide was produced within 12 days. This was so far the highest exopolysaccharide yield produced with strains of Cyanothece sp. obtained.     

Long interspersed elements in three species of Gracilaria （Gracilariaceae, Rhodophyta）

In order to find out whether long interspersed elements (LINEs) existed in macro-algae ge- nomes or not, we tested the LINE homologues in representative families (species): Gracilaria (G. eucheumoides Harv., G. tenuistipitata Chang et Xia, and G. textorii (Sur) De-Toni), Laminaria (L. longis- sima Miyabe and L. japonica Aresch.), and Ulva (U. lactuca L. and U. pertusa Kjellm.) during 2004 to 2005. Polymerase Chain Reaction (PCR) was carried out with degenerate oligonucleotide primers de- signed from LINEs of rice homologues and Cin4 of maize. Cloning and nucleotide sequencing of the PCR products revealed that 4 clones that derived from 3 species of Gracilaria have LINE homologues. The nucleotide sequences of the 4 LINE homologues diverged greatly, but the amino acid sequences deduced from them were relatively conserved. The endonuclease regions of the LINE homologues greatly di- verged from that of other plants, but they had closer phylogenetic relationship to Zepp elements in Chlor- ella sp., which indicated that sequence divergence by vertical transmission has been a major influence on the evolution of algal LINEs.     

Positive effect of porphyrans on the lifespan and vitality of Drosophila melanogaster

The effects of degraded porphyran (P1) and natural porphyran (P) on the lifespan and vitality of Drosophila melanogaster are studied. The porphyrans, added daily to the food medium at 0.2% and 1% concentrations, can significantly increase the lifespan in average of 55.79 and 58.23 d in 0.2% P1 diet females and 1% P1 diet males, extending by 12.29% and 8.60% over the corresponding controls, respectively. The effects of porphyrans on D. melanogaster in heat-stress condition were also examined, and found a remarkable increase in survival time. The results which are consistently associated with the use of porphyrans are related to their free radical scavenger action. Considerable increase in vitality demonstrated that vitalities of middle-aged fly (assessed by measuring their mating capacity) was observed after porphyrans addition. Therefore, porphyrans are effective in reducing the rate of aging, and P1 in low molecular weight is better than natural P.