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.     

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.     

Characterization of SNAP-25 gene from marine teleostean, Lateolabrax japonicus

The t-SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) plays an essential role in regulating fusion between the vesicle and plasma membranes during exocytosis. To clone and characterize SNAP-25 gene, the first step in the functional study of SNARE proteins in marine teleostean, was to obtain the cDNA of sea perch SNAP-25 (SPsn25) by RT-PCR and RACE-PCR amplification of a Japanese sea perch. The full-length cDNA of 831bp contains a CDS of 615 bp, coding 204 amino acid residues, and a 5′UTR of 219bp. Bioinformatic analysis revealed that SPsn25 corresponds with SNAP-25a isoform and shares 91.1% identity with SNAP-25a of a goldfish and a zebrafish. The SPsn25 expression in both mRNA and protein levels in the Japanese sea perch had been identified through semi-quantitative RT-PCR and Western Blot assay. Together, these data again confirmed the nerve tissue specificity of the fish SNAP-25 gene expression.     

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.     

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.     

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.     

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.     

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.     

<Emphasis Type="Italic">GST</Emphasis> (<Emphasis Type="Italic">phi</Emphasis>) gene from Macrophyte <Emphasis Type="Italic">Lemna minor</Emphasis> is involved in cadmium exposure responses

Reactive oxygen species (ROS) scavengers, including ascorbate peroxidase, superoxide dismutase, catalase and peroxidase, are the most commonly used biomarkers in assessing an organisms’ response to many biotic and abiotic stresses. In this study, we cloned an 866 bp GST (phi) gene in Lemna minor and investigated its characteristics, expression and enzymatic activities under 75 μmol/L cadmium concentrations in comparison with other ROS scavengers. GST (phi) gene expression patterns were similar to those of other scavengers of ROS. This suggests that GST (phi) might be involved in responding to heavy metal (cadmium) stress and that its expression level could be used as a bio-indicator in monitoring cadmium pollution.     

Low temperature stress on the hematological parameters and <Emphasis Type="Italic">HSP</Emphasis> gene expression in the turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis>

To study the effect of low temperature stress on hematological parameters and HSP gene expression in the turbot (Scophthalmus maximus), water temperature was lowered rapidly from 18 to 1°C. During the cooling process, three individuals were removed from culture tanks at 18, 13, 8, 5, 3, and 1°C. Blood samples and tissues were taken from each individual, hematological indices and HSP gene expression in tissues were measured. The red blood cell count, white blood cell count, and hemoglobin concentration decreased significantly (P < 0.05) as temperature decreased. Enzyme activities of plasma alanine transaminase and creatine kinase increased as temperature decreased, whereas aspartic transaminase and γ-glutamyl transpeptidase activities displayed no obvious changes above 1°C and lactate dehydrogenase activity increased first and then decreased. Blood urea nitrogen and uric acid levels were highest at 8°C, and creatinine concentration was highest at 3°C. The concentrations of plasma cortisol, cholesterol, and triglyceride all increased significantly (P < 0.05) as temperature decreased. The serum glucose concentration increased first and then decreased to the initial level. The HSP70 mRNA expression showed various patterns in different tissues, whereas HSP90 mRNA expression showed the same tendency in all tissues. Overall, these results indicate that temperature decreases in the range of 8 to 5°C may induce a stress response in S. maximus and that temperature should be kept above 8°C in the aquaculture setting to avoid damage to the fish.     

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.     

The characteristics of <Emphasis Type="Italic">vasa</Emphasis> gene from Japanese sea bass (<Emphasis Type="Italic">Lateolabrax japoni</Emphasis>cas) and its response to the external hormones

The RNA helicase Vasa is an important regulator of primordial germ cell development. Its function in mature fish, especially the hormone-related differences in maturing male fish has seldom been documented. In this study, a full length cDNA sequence of the vasa gene was cloned from Japanese sea bass, Lateolabrax japonicas, and it was named jsb-vasa. Homology analysis showed that jsb-vasa was closely related to its teleost homologs. The spatial distribution of jsb-vasa indicated that it was only highly expressed in testis, showing its germ cell-specific expression pattern. During the testicular development cycle, jsb-vasa was highly expressed during early period of spermatogenesis, and reduced when spermatogenesis advanced. In addition, the jsb-vasa gene expression was significantly inhibited at 6 h, 12 h and 24 h after injecting hCG (human chorionic gonadotropin) and GnRHa (Gonadotropin-releasing hormone analogue), indicating that jsb-vasa gene may play an important role in spermatogenesis of Japanese sea bass, and be under the regulation of external sex hormones.     

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.     

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.     

Tissue Expression and Stock Variation of Isozymes of Stone Flounder （Kareius bicoloratus）

Tissue expression and stock variation of isozymes of stone flounder (Kareius bicoloratus) were analyzed with horizontal starch gel electrophoresis. For the fourteen enzymes assayed, 31 loci were recorded. The results indicated that all the isozymes ex- amined were obviously tissue-specific. The expressions of SOD*, GDH*, G3PDH-2* and ADH-2* were detected only in liver, SDH-1*, MDH-1* and ADH-1* only in muscle, and LDH-B* and LDH-C* only in eyes. In comparison, MDH-2*, GPI-3* and SDH-2* were detected in all tissues examined. Other loci examined were detected in a variety of tissues. Muscle and liver were se- lected to detect the isozyme variation of the two geographic stocks of Qingdao and Weihai, Shandong Province, China. The percent- ages of polymorphic loci (P0.99) were 29.17% and 25.00%, the observed heterozygosities (H0) were 0.028 ± 0.014 and 0.040 ± 0.019, and the expected heterozygosities (He) were 0.039 ± 0.017 and 0.052 ± 0.022 in Qingdao and Weihai stock, respectively. The coeffi- cient of gene differentiation (Fst) and genetic distance (D) between the two stocks was 0.012 and 0.0011, respectively, indicating that the genetic differentiation is low between them. Compared with other species of Pleuronectiformes, both the percentage of polymor- phic loci and the mean heterozygosity of K. bicoloratus were at a middle level.     

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.     

Identification and characterization of pathogen to bacterial septicaemia in cultured turbot, Scophthalmus maximus

Bacteria-infected turbots Scophthalmus maximus with septicaemia were examined between 2001 and 2004 in aspects of the conditions of disease occurrence, clinical syndromes and pathological changes. The phenotypic information of pathogenic bacteria was studied, including morphology, physiological and biochemical characteristics, and the mol% G C of the DNA. In addition, representative strains (S010623-1, LH031120-1) were selected for molecular identification by partial 16S rRNA gene sequencing. The results show that the isolates (LH031120-1 to LH031120-6, HT040308-1 to HT040308-6, HT040620-1 to HT040620-6) from three farms were identified as Edwardsiella tarda. The isolates (S010610-1 to S010610-10, S010623-1 to S010623-20) from one farm were identified as Listonella anguillarum. We conducted studies on the pathogenicity of isolates by artificial infection, and revealed all infected groups in morbidity and mortality. The septicaemia infected turbot showed a syndrome similar to that of the naturally infected fish. Antibiotic sensitivity showed that of 37 antimicrobial agents, E. tarda was sensitive to 27 agents, and L. anguillarum was sensitive to 21 agents.