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.     

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.     

The Gastric Shield and the Underlying Epithelium of Chlamys Farreri:Morphological, Histological and Histochemical Characterizations

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Histological and Histochemical Analyses of the Cuttlebone Sac of the Golden Cuttlefish Sepia esculenta

The secretion function of mantle is closely related to shell formation in some bivalves and gastropods. Up to now, few researches have been reported for cuttlebone formation in the class Cephalopoda. In this study, the structure and secretion function of cuttlebone sac of the golden cuttlefish Sepia esculenta was analyzed using the histological and histochemical methods. The results showed that high and columnar cells located in sac epithelium, and flat cells existed near the base membrane. A lot of fibroblasts were found in the lateral mantle collective tissue. Some mucus, mucopolysaccharide and alkaline phosphatase (ALP) were found in the sac. The ultrastructural characteristics of Quasi-connective-tissue-calcium cells (QCTCC) were observed using a transmission electron microscope (TEM). The relationship between cuttlebone sac secretion function and shell formation was discussed.     

Effects of benzo(a)pyrene exposure on the antioxidant enzyme activity of scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis>

Scallop Chlamys farreri was exposed to different concentrations of benzo(a)pyrene (BaP) (0.5 μg/L, 1.0 μg/L, 10.0 μg/L and 50.0 μg/L) for 30 days in seawater. The 7-ethoxyresorufin O-deethylase (EROD) activity was significantly induced, and increased with the increasing BaP concentration. The glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), Glutathione peroxidase (GPx) activities increased in short time at low concentration of BaP, and was significantly depressed at high concentrations. Scallop gill was more sensitive to BaP than the digestive gland, and the digestive gland was the main tissue to deal with oxyradicals. The contents of malondialdehyde (MDA) increased with the exposure time and there was a positive correlation (concentration-effect) between the MDA content and the concentration of BaP. The biomarkers determined in this experiment had important roles in detoxification, and showed great potential as biomarkers for oxidative stress. Controlled laboratory experiments designed to simulate field exposure scenarios are particularly useful in ascertaining biomarkers suitable for use with complex contaminant mixtures in the marine environment. Supported by the Technology Development Program of Shandong (No. 2008GG1005010) and the Program of Introducing Talents of Discipline of Universities (111 Project, No. B08049)     

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).     

A Fast and Indriect Fluorescent Antibody Assay for the Vibro in Large Yeelow Croaker Pseudosciaena Crocea (Richardson)

A fast and indirect fluorescnet antibody assay for the Vibrio alginolyticus and V.Parahaemolyticus infecting the large yellow croaker has been developed.The specific antisera for the two strains of vibrio were prepared with New Zealand rabbit and the antiserum and cross-reactive efficacy was tested by coagulation in tube.It showed that the goat anti-rabbit IgG had been labeled by fuorescence isothiocyanate(FITC).The results showed that positive reactions were 100% for the large yellow croaker Pseudosciaena crocea with typical symptom of vibrio infection,while the positive reaction to the pathogen in healthy yellow croakers reached 40%,but seemed negative for aquaculter water.The results demonstrated that this fast and indirect fluorescent antibody assay can be used not only to test the vibrio pathogen in diseased yellow croaker but also in infected animals with no symptom.     

Isozymes Analysis of the Golden Cuttlefish Sepia esculenta（Cephalopoda ： Sepiidae）

Thirty-nine isozymes in four tissues (mantle muscle, buccal bulb muscle, eye and liver) of Sepia esculenta were screened for enzymatic analysis using starch gel electrophoretic technique. Eighteen enzymes (G3PDH, LDH, MDH, MEP, IDHP, PGDH, GRS, NP, AAT, CK, AK, EST, ALP, ACP, FBP, MPI, GPI and PGM) show strong activities and good convergence in zymogram. They are proved to be suitable genetic markers in Sepia esculenta. Among the tissues used, mantle muscle is the best for electrophoretic analysis of isozymes. Eye and liver are fairly good for some special enzymes, such as LDH, EST, MPI, etc. Twenty-six loci are detected. The proportion of polymorphic loci is 0.115 in the Qingdao sample and 0.153 in the Rizhao sample (P<0.99). The mean values of the observed and expected heterozygosity per locus of Qingdao sample are 0.016 and 0.017, while those of the Rizhao sample are 0.023 and 0.025 respectively.     

NEW OBSERVATIONS ON THE MEIOTIC PROCESS IN THE MARINE DINOFLAGELLATE NOCTILUCA SCINTILIANS （NOCTILUCALES, DINOPHYCEAE）

The meiotic process in Noctiluca scintillans were observed under light microscope.Some abnormal cell divisions, incompletely separated “zoospores” and the changes of the zoospores are described in this paper. Together with the fmdings of field samplings and the previous results by other researchers, the process of meiosis in N. scintillans was supposed to be a pathway to reduce the extra high density of NH3-N within the cell in order to ensure normal population growth.     

Acute Toxicity of Formalin to the Red-tide Dinoflageilate Prorocentrum minimum Schiller （Protozoa, Mastigophora）

The acute toxic levels of formalin to a marine red-tide dinoflagellate Prorocentrum minimum Schiller (Mastigophora, Dinoflagellida) were determined by linear regression analysis. The data obtained revealed that there is a close regression relationship between logarithmic concentrations of formalin and mortality probit scales of the organism when exposure times are 1, 2, 6, 12, 24 h. The median lethal concentrations (LC50 values) obtained from correlation analysis for these exposure times (with 95% confidence intervals), were 11.83, 6.76, 4.37, 4.27 and 3.98mgL^-1 respectively. A toxicity curve was obtained to connect the exposure time and LC50 value from the correlation between them. The results indicated that P. minimum could be killed or fixed by formalin in the concentration range of 4-12mgL^-1 within 1-24h.     

Axenic culture of free-living conchocelis of<Emphasis Type="Italic">Porphyra yezoensis</Emphasis> and<Emphasis Type="Italic">Porphyra haitanensis</Emphasis>

After discarding marine microorganisms from conchocelis of Porphyra yezoensis and Porphyra ha/tanens/s, their axenic cultures were obtained through treatment with antibiotics. Antibiotic disc tests were carried out to determine the effectiveness of each antibiotic in eliminating contaminating microorganisms. Five of 12 antibiotics tested were selected and used to produce the axenic cultures in this study, which showed that 200 μg/mL streptomycin, 250 μg/mL penicillin, 252 μg/mL kanamycin, 30 μg/mL neomycin, 200 μg/mL chloramphenicol were effective concentrations for eliminating microorganisms from conchocelis when antibiotics were added singly step by step; whereas simultaneous combination of 150 μg/mL streptomycin, 250 (or 350) μg/mL penicillin, 150 (or 250) μg/mL kanamycin, 70 μg/mL neomycin and 200 μg/mL chloramphenicol was also effective for producing the axenic cultures. However,it seemed that the treatments with antibiotics applied individually were more feasible than those with all antibiotics added at the same time. This may be due to the combined inhibiting effect of antibiotics on the growth and development of conchocelis.     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

The wild Porphyra yezoensis collected from the Qingdao coast was used to prepare protoplasts by enzyme digestion. The pure line was constructed by cultivating the protoplasts. The 18S rDNA of the P. yezoensis pure line was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed using the neighbor-joining method. The results revealed a high diversity of 18S rDNA sequences in genus Porphyra and the considerable variation of 18S rDNA sequences in different strains of the same species P. yezoensis and P. tenera. Significant difference of 18S rDNA sequence was observed between P. yezoensis from Qingdao, China, and the two strains of P. yezoensis from Japan, but the three strains of P. yezoensis formed a stable clade in the phylogenetic tree. These results indicate the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.     

Synedra ulna var. repanda,a new variety of Synedra （Bacillariophyta） from Xinjiang,China

Synedra ulna var, repanda Q. X. Wang ＆ Q. M. You, a new variety of Synedra （Bacillariophyta） from Xinjiang, China, is described and illustrated, and the characteristic of the variety： includes undulate-linear valves and straight pseudoraphe, differs from other species of Synedra.     

The Complete Sequence of Mitochondrial COⅡ Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit Ⅱ （COⅡ） gene of Penaeinae shrimp Fenneropenaeus chinensis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus Japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A＋T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COⅠ and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COⅡ gene has a faster evolutionary rate than that of the COⅠ gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

Preliminary Study on the Water Drag of Schlegel＇s Black Rockfish Sebastes schlegeli

Drag forces acting on Schlegel‘s black rockfish Sebastes schlegeli were studied. A new drag force transducer was designed and used to measure the water drag on Schlegel‘s black rockfish in a vertical recirculating flume tank. Fourteen individuals were investigated, yielding two mean drag coefficients referred to the cross-sectional area and volume^2/3 respectively at water velocities ranging from 0.3 to 1.0 ms^-1 The drag coefficients can be used for estimating the drag forces acting on Sebastes schlegeli in water.     

The antitumor effect of bromophenol derivatives <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">Leathesia nana</Emphasis> extract <Emphasis Type="Italic">in vivo</Emphasis>

To investigate the antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo, six bromophenol derivatives 6-(2,3-dibromo-4,5-dihydroxybenzyl)-2,3-dibromo-4,5-dihydroxy benzyl methyl ether (1), (+)-3-(2,3-dibromo-4,5-dihydroxyphenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroisobenzofuran (2), 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)-5-methoxymethyl-pyrocatechol (3), 2,2′,3,3′-tetrabromo-4,4′,5,5′-tetrahydroxy-diphenylmethane (4), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (5), 2,2′,3-tribromo-3′,4,4′,5-tetrahydroxy-6′-ethyloxymethyldiphenylmethane (6) were isolated from brown alga Leathesia nana, and their cytotoxicity were tested by MTT assays in human cancer cell lines A549, BGC-823, MCF-7, B16-BL6, HT-1080, A2780, Bel7402 and HCT-8. Their inhibitory activity against protein tyrosine kinase (PTK) with over-expression of c-kit was analyzed also by ELISA. The antitumor activity of ethanolic extraction of Leathesia nana (EELN) was evaluated on S₁₈₀-bearing mice. All compounds showed very potent cytotoxicity against all of the eight cancer cell lines with IC₅₀ below 10 μg/mL. In PTK inhibition study, all bromophenol derivatives showed moderate inhibitory activity and compounds 2, 5 and 6 showed significant bioactivity with the inhibition ratio of 77.5%, 80.1% and 71.4%, respectively. Pharmacological studies reveal that EELN could inhibit the growth of Sarcoma 180 tumor and increase the indices of thymus and spleen to improve the immune system remarkably in vivo. Results indicated that the bromophenol derivatives and EELN can be used as potent antitumor agents for PTK over-expression of c-kit and considered in a new therapeutic strategy for treatment of cancer. Supported by the National High Technology Research and Development Program of China (863 Program, No. 2007AA09Z410) and Knowledge Innovation Program of Chinese Academy of Sciences (No. KZCX2-YW-209)     

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.