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

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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.     

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.     

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.     

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

The gastric shield and underlying stomach epithelium of Chlymas ffarreri were examined at the light and ultrastructural levels.The results showed that the gastric shield consisted of two different size lobes joined together by a narrow middle piece,the thicker lobe was shaped like a funnel,but unclosed at the lateral side;the other lobe was irregularly triangular-shaped.The transverse section of the thicker lobe was obviously laminated and gradually decreased in thickness from the peak to the margins of the shield.The underlying epithelium bore numerous about 3μm diameter spherical processes formed by the apical plasmalemma of the epithelial cells becoming blunt pseudopodia.Microvilli and some interspersed cilia were present in the areas among the spherical processes regions where only microvilli existed.Rough endoplasmic reticulum,Golgi apparatus,different-sized electron-dense secretory granules and electron-lucent vacuoles as well las abundant mitochondria were present in the underlying epithelial cells.Fused droplets of the secretion from the underlying epithelial cells formed the gastric shield.     

Genetic diversity analysis with ISSR PCR on green algae <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis>

In the present study, genetic polymorphism and diversity in unicellular clones of Chlorella vulgaris Beijerinck and Chlorella pyrenoidosa Chick were studied with Inter Simple Sequence Repeats PCR （ISSR PCR）. Samples including four clones of C. vulgaris and three clones of C. pyrenoidosa were purified by single-clone-choice method. For four C. vulgaris unicellular clones, the total number of the bands scored for 18 primers was 298; and the number of the polymorphic bands was 118, of which 39.6% were polymorphic. The size of PCR products ranged from 200 to 2 500 bp. The total number of bands scored for 18 primers, the number of polymorphic bands and the percentage of three C. pyrenoidosa unicellular clones was 194.83 and 30.8%, respectively. POPGENE analysis show that the average Nei genetic diversity （h^＊） and Shannon index of diversity （I^＊） in the four C. vulgaris unicellular clones was 0.2181 and 0.3208, respectively, which is slightly higher than those of the three C. pyrenoidosa unicellular clones （0.190 3 and 0.274 8）, which agreed with the percentage of polymorphic bands in the mixed samples of the two species. The results suggest that ISSR is a useful method to Chlorella for intra-species genetic analysis.     

Immune response of <Emphasis Type="Italic">Pseudosciaena crocea</Emphasis> to the injection of <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

For the investigation of anti-infection immune response of Pseudosciaena crocea, 160 healthy fish samples were categorized into infected and control groups. Each individual fish in the infected group was injected intraperitoneally (i.p.) with 0.2 ml bacterial suspension of Vibrio alginolyticus in density of 2×10⁷ CFU/ml, while each individual in the control group was injected i.p. with 0.2 ml sterile saline solution (0.85%). It was observed that the artificial injection of V. alginolyticus significantly increased the number of erythrocytes, leucocytes, lymphocytes in peripheral blood as well as peripheral serum antibacterial activity and antibody titer of large yellow croaker, and significantly reduced the number of peripheral blood granulocytes as compared with those in the control group. No significant difference in acid phosphytase and superoxide dismutase activity of serum was detected between the two groups. It is suggested that non-specific immune factors including leucocytes and anti-bacteria substance in peripheral blood played important role at the initial stage of infection, and specific immune factors such as antibody then played important role in response to anti-infection at the latter stage. Supported by the National High Technology Research and Development Program of China (863 program, No. 2007AA09Z115) and Technology Program of Xiamen (No. 3502Z73019)     

Expression and localization of a novel phosducin-like protein from amphioxus <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>

A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri. Supported by the Pilot Projects of Knowledge Innovation Project of Chinese Academy of Sciences (No. KZCX2-YW-211-03 and MGE2008KG06)     

Non-specific immune response of bullfrog <Emphasis Type="Italic">Rana catesbeiana</Emphasis> to intraperitoneal injection of bacterium <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

Non-specific immune response of bullfrog Rana catesbeiana to pathogenic Aeromonas hydrophila was studied to 60 individuals in two groups. Each bullfrog in bacterium-injected group was injected intraperitoneally （i.p.） with 0.2 ml bacterial suspension at a density of 5.2 × 10^6 CFU/ml, while each one in control group injected i.p. with 0.2 ml sterile saline solution （0.85%, w/v）. Three bullfrogs in both groups were sampled at 0, 1, 3, 7, 11, 15 and 20 days post-injection （dpi） for the evaluation of non-specific immune parameters. It was observed that intraperitoneal injection of A. hydrophila significantly increased the number of leucocytes and that of NBT-positive cells in peripheral blood. Significant increases in serum bactericidal activity and serum acid phosphatase activity were also observed in the bacterium-injected frogs when compared with those in the control group. However, a significant reduction was detected in vitro in phagocytosis activity of peripheral blood phagocytes. No significant difference in changes in the number of peripheral erythrocytes, serum superoxide dismutase （SOD） activity, and lysozyme activity was detected between the two groups. It is suggested that bullfrogs may produce a series of non-specific immune reactions in response to the A. hydrophila infection.     

A Taxonomic Study of the Genus Tinocladia （Chordariales,Phaeophyta） in China Coasts I. Two new species, T. eudesmoides sp.nov, and T. zhangii sp.nov.

Two new species of the genusTinocladia Kylin (1940)T. eudesmoides Ding et Lu sp. nov. andTinocladia zhangii Ding et Lu sp. nov., are described.T. eudesmoides is mainly characterized by its longitudinally and transversely produced sub-cortical filaments from the medulla, its short uniseriate assimilating filaments, usually composed of 6–10 cells with slightly swollen and narrow ultimate cells, and its unilocular sporangia (60–115)×55–70μm, variable in size.T. zhangii is mainly characterized by its very solid frond, not rip apart by hands, its evident longer primary branches, its short and slender secondary branches, with very evident sub-cortical layer, and filaments divided repeatedly, and its large and small unilocular sporangia, with the latter borne on an evidently swollen pedicel. Project B80981816 supported by the NSFC. Contribution No. 4443 from the Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.     

Report of Pontoniinae Shrimps （Crustacea： Decapoda） Collected by Joint Chinese-German Marine Biology Expeditions to Hainan Island, South China Sea. Ⅲ. PericHmenes

The present paper reports 12 species of genus Periclimenes, subfamily Pontoniinae, collected from Hainan Island, South China Sea, by the Joint Chinese-German Marine Biology Expeditions, in which, Periclimenes consobrinus and P. tenuipes are new records from Chinese waters, and Periclimenes amymone, P. holthuisi, P. soror, and P. toloensis are recorded for the first time from Hainan Island.     

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.     

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)     

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.     

Two New Species of the Genus finocladia Kylin （Chordaria-les, Phaeophyta） from Yellow Sea Coasts of China

Two new species of the genus Tinocladia Kylin, T. gracilis sp. nov. and T. microsporangii sp. nov,, are described. T. gracilis is characterized by its slender frond, variable size, infrequently branched medullary filaments, and short assimilating filaments with 2 - 7 cells. T. microsporangii is characterized by its medullary cells which are slightly larger than sub-cortical ones; narrow sub-cortical layer, usually consisting of 4- 6 cells and very small unilocular sporangia. The holotypes are deposited at the Herbarium of the Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.     

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.     

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.     

A comparative study of genetic variation between Chinese mitten crab <Emphasis Type="Italic">ericheir sinensis</Emphasis> and Hepu mitten crab <Emphasis Type="Italic">E. hepuensis</Emphasis>

Horizonal starch gel electrophoresis was used to investigate the biochemical genetic structure of Chinese mitten crab Eriocheir sinensis and Hepu mitten crab E. hepuensis. Sixteen putative enzyme-coding loci were examined for forty-nine chinese mitten crabs and thirty-eight Hepu mitten crabs. Nine loci, AAT-1 ^*, AAT-2 ^*, G3PDH ^*, GPI ^*, IDHP-1 ^*, IDHP-2 ^*, MDH-1 ^*, MDH-2 ^* and PGM ^*, are polymorphic in Chinese mitten crab, and seven, AAT-1 ^*, AAT-2 ^*, GPI ^*, IDHP-1 ^*, MDH-1 ^*, MDH-2 ^* and PGM ^*, are polymorphic in Hepu mitten crab. The proportion of polymorphic loci and the expected heterozygosity are 0.562 5 and 0.080 3 for Chinese mitten crab, and 0.437 5 and 0.075 4 for Hepu mitten crab. The Nei’s genetic distance between two species is 0.002 4.     

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.