A Comparative Study of Genetic Variation betwccn Chinese Mitten Crab Eriocheir Sinensis and Hepu Mitten Crab E. Hepuensis

Horizontal starch gel electrophoresis was used to investigate the biochemical genetic structure of Chinese mittencrab Eriocheir sinensis and Hepu mitten crab E. hepuensis. Sixteen putative enzyme-coding loci were examined for forty-nineChinese 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 expectedheterozygosity 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 ge-netic distance between two species is 0.002 4.     

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.     

EFFECTS OF FISHES ON THE GROWTH AND SURVIVAL OF CHINESE MITTEN CRAB, ERIOCHEIR SINENSIS

This experiment on the effects of fishes (Pelteobagrus fulvidraco, Channa argus, Cyprinus carpio) on growth and survival of juvenile Chinese mitten crab,Eriocheir sinensis was conducted in cages placed in Bao'an Lake. T-tests showed that effects of fishes on the carapace width growth were not significant as compared with the control (no fish). But the effects of these fishes on crab survival were negative. The causes of negative effects of fishes on crab survival were simply discussed on the basis of food habits. This research was financially supported by the Department of Agriculture (96-008-02-03), P. R. China, and Chinese Academy of Sciences (Key Project: KZ951-A1-102).     

Isolation of protoplasts from <Emphasis Type="Italic">undaria pinnatifida</Emphasis> by alginate lyase digestion

The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28°C for 2h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5g fresh thalline with NaCl 50 and at the shaking speed of 150 r min⁻¹ during digestion. The protoplast yield can reach 2.62±0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca²⁺ and slightly enhanced by Fe²⁺ and Mn²⁺ at concentrations of 0.05, 0.08 and 0.10 mol L⁻¹.     

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.     

Isozyme analysis of spotted halibut Verasper variegatus temminck et schlegel

To investigate the tissue-specificities of isozymes and the genetic structure of wild spotted halibut (Verasper variegates) population, horizontal starch gel electrophoresis was performed on 45 individuals collected in part of the Yellow Sea. The performances of 17 isozymes in 8 kinds of tissues or organs were screened preliminarily in a TC-7.0 buffer system. The results showed that the screened isozymes displayed remarkable tissue-specificities. Finally, 14 enzymes (AAT, ADH, EST, GPI, G3PDH, IDHP, LAP, LDH, MDH, MPI, PGDH, PGM, SDH and SOD) and 4 kinds of tissues (eye, skeleton muscle, liver and heart) were selected for genetic analysis. Fourteen isozymes are encoded by 20 loci, and 9 of them are polymorphic. The polymorphic loci are AAT-1*. GPI-2* G3PDH*, IDHP-1*, LDH*, MPI*, PGM-1*. PGM-2* and SDH*, and the proportion of polymorphic loci is 0.4500 (P0.99). The mean values of observed and expected heterozygosities are 0.027 8 and 0.026 5, respectively and the average effective number of alleles is 1.067 5.     

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.     

Notes on Two Marine Ciliates from the Yellow Sea,China： Placus salinus and Strombidium apolatum（Protozoa, Ciliophora）

The living morphology and infraciliature of two rare marine ciliates, Placus salinus Dietz, 1964 and Strombidium apolatum Wilbert and Song, 2005, collected from the coastal waters near Qingdao, China, were investigated by in vivo observation and protargol impregnation technique. The improved diagnosis for Placus salinus is as follows: medium-sized marine Placus, in vivo (50–60)μm×(30–40)μm; cell elliptical to barrel-shaped; 28–31 somatic kineties; single macronucleus usually ellipsoid and one micronucleus located in the indention of the macronucleus; one contractile vacuole posteriorly positioned. Strombidium apolatum is characterized by: marine strombidium (40–60)μm×(30–45)μmm in vivo, cordiform in shape with somewhat pointed posterior end and conspicuous apical protrusion; extrusomes prominent, about 15μm in length and evenly arranged along the circle kinety; about 16 collar and 5–6 buccal membranelles; one elongate macronucleus and one micronucleus; circle and ventral kineties consisting of about 53 and 45 dikinetids respectively.     

Development and validation of 89 novel expressed sequence tag-derived microsatellite markers in blood clam, <Emphasis Type="Italic">Tegillarca granosa</Emphasis>

Blood clam, Tegillarca granosa, is an important shellfish in Chinese mariculture industry. Investigative research in this species, such as genetic linkage mapping, requires a large panel of molecular markers. In present study, a total of 89 polymorphic microsatellite markers were developed in T. granosa using the sequence database of Life Sciences Technology 454 next generation sequencing technology. All 89 loci were characterized in 20 individual clams from a natural population inhabiting Yueqing Gulf, Zhejiang Province, China. The number of alleles per polymorphic locus varied between 2 and 15, while the observed heterozygosity, expected heterozygosity and polymorphic information content varied between 0.000 and 1.000, 0.102 and 0.921, and 0.048 and 0.886, respectively. Of the 89 loci identified, 32 loci deviated significantly from Hardy-Weinberg equilibrium following Bonferroni correction. Thirty nine markers, which were shown to be polymorphic in a full-sibling family, were tested in Mendelian segregations. As expected, 32 loci were co-dominantly segregated in a Mendelian fashion. These novel developed microsatellite markers represent useful research tools for investigation of population genetic structure and genetic diversity in this species.     

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.     

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.     

Preliminary analysis of population genetic diversity of cultivated <Emphasis Type="Italic">Laminaria japonica</Emphasis> sporophyte via AFLP technique

The amplified fragment length polymorphic DNA (AFLP) technique was adopted to estimate the population genetic polymorphism among 30 sporophytes of Laminaria japonica collected from a cultivating farm in Rongcheng, China. Three methods were used for genomic DNA extraction from Laminaria japonica sporophyte and only the products obtained using the improved genomic DNA extraction kit method proved qualified for AFLP analysis. The parameters of the method were optimized. Samples of forty milligrams and the cell lysis time of 120 min were suggested to replace the parameters recommended by the manufacturer. Thirty individuals of Laminaria japonica from the same cultivating site were investigated using one pair of selective primers. A total of 21 loci were obtained and 17 of them were polymorphic. The mean percent age of polymorphic loci of this population was 80.95%. The Nei’s gene diversity (H) within this population was 0.3028 and the average Shannon’s Information index (I) was 0.4498. A genetic distance matrix among different individuals was constructed as well. Through this study, an applicable AFLP genetic analysis working system for Laminaria japonica sporophyte was established. The results of this research also revealed a high level of genetic diversity within the studied population.     

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)     

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.     

Biodegradation of <Emphasis Type="Italic">Enteromorpha</Emphasis> polysaccharides by intestinal micro-community from <Emphasis Type="Italic">Siganus oramin</Emphasis>

Micro-communities are supposed to have more potential functions of biodegradation of polysaccharides than single strain; however, the intestinal micro-communities involved in the biodegradation of Enteromorpha polysaccharides (EP) were seldom reported. In order to obtain the EP-degrading micro-community, the intestines of Siganus oramin was obtained to isolate the micro-communities, which were enriched by 0.3% of EP as the sole carbon source. A stable micro-community with EP degradative capability was achieved after seven generations of subculture, named H1. Results showed that H1 was able to degrade 75% of EP within 24 hours, and the activity of EP lyases reached 500 U mL^?1 in 32 hours. With denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis, ten bacteria closely related to Marinomonas pontica, Microbacterium sp., Leucobacter chironomi, Cyclobacterium sp., Algoriphagus winogradskyi, Pseudoalteromonas sp. and Vibrio sp. were determined. Furthermore, compared with the DGGE bands sequence and the clone library analysis, the dominant bacteria of the EP-biodegrading micro- community were Pseudoalteromonas sp. and Vibrio sp., with the respective proportion of 38% and 46%, and they should play an important role in EP degradation together with other degrading bacteria in the micro-community H1.     

Variations of ion-transport enzyme activities during early development of the shrimps <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> and <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

The ion-transport enzyme activities were studied in the nauplii 1–2, zoea 1–3, mysis 1–3, and postlarva 1–7 of the shrimps Marsupenaeus japonicus and Fenneropenaeus chinensis. The results showed that total ATPase, Na⁺-K⁺-ATPase, and V-ATPase (V-type H⁺-ATPase) activities increased during the early development for both the species, from zero in nauplii to stable levels after zoea stage. The enzyme activities of the latter species were significantly higher than those of the former species after zoea stage (F>F _0.05). The contributions of Na⁺-K⁺-ATPase, V-ATPase and HCO₃ ⁻-ATPase to the total ATPase activity of the two species varied in different developmental stages and accounted for 40%–70%, 22%–46% and 2%–13% in M. japonicus from zoea to postlarva stage, whilst the shares of them were 42%–69%, 28%–44% and 2.5%–22%, respectively in F.chinensis. These findings suggest the possible culture of the two species with varying water quality, especially the salinity and pH.     

Analysis of the genetic diversity and differentiation of <Emphasis Type="Italic">Fenneropenaeus penicillatus</Emphasis> populations using AFLP technology

Fenneropenaeus penicillatus (redtail shrimp) is an important marine commercial animal in China. Recently, its resources have been depleted rapidly as a result of, for example, over-exploitation and environmental degradation of spawning grounds. Therefore, we analyzed the genetic diversity and differentiation of nine wild populations of F. penicillatus of China (Ningde, Lianjiang, Putian, Xiamen, Quanzhou, Zhangpu, Dongshan, Nanao, and Shenzhen populations) by amplified fragment length polymorphism (AFLP) technology, to provide genetic information necessary for resource protection, rejuvenation, artificial breeding, and sustainable use of the resource. Eight AFLP primer pairs were used for amplification, and 508 bands were detected among the populations. The results show that the percentage of polymorphic loci (P) ranged from 41.34% to 63.58%; the Nei’s gene diversity (H) of the populations was 0.119 4-0.230 5; and Shannon’s Information Index (I) was 0.184 1-0.342 5. These genetic data indicate that the genetic diversity of F. penicillatus was high. The genetic differentiation coefficient (GST=0.216 2) and gene flow (Nm=1.812 4) show that there was a high level of genetic differentiation and a moderate level of gene flow among populations. More studies on the genetic differentiation mechanism of F. penicillatus along the south-eastern coast of China need to be conducted to find more effective scientific protection strategies for the conservation of F. penicillatus genetic resources.     

Microsatellite DNA polymorphisms and the relation with body weight in sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

The relationship between microsatellite polymorphism and body weight of captive bred Chinese sea cucumber Apostichopus japonicus was investigated in two local populations in Dalian. Among ten loci discovered, nine show changes except for AJ07 loci. Seven loci were found highly polymorphic in both populations. For each locus in two populations, the average number of alleles is 6.428 6 and 6.285 7, the average observed heterozygosity at 0.225 7 and 0.245 9, the expected heterozygosity at 0.776 8 and 0.748 8, the polymorphism information content (PIC) at 0.709 2 and 0.674 6, respectively. Further analysis show significant correlation between A. japonicus body weight and occurrence markers AJ02 and AJ04. The findings of the relation may be helpful for molecular breeding, as well as the marker-assisted selection of sea cucumbers. Supported by the National High Technology Research and Development Program of China (863 Program, No. 2006AA10A411), the Natural Science Foundation of Liaoning Province (No.20072139), the Grant of Dalian Fisheries University, the Key Laboratory Foundation of the Educational Department of Liaoning Province (No.2009S024)     

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.     

Partial sequence analysis of mitochondrial COI gene of the chinese shrimp, <Emphasis Type="Italic">fenneropenaeus chinensis</Emphasis>

Eight hundred and thirty eight base pair fragment of mitochondrial COI gene of wild and cultured populations (CP1, CP4, CP5 and CP6) of Fenneropenaeus chinensis was amplified and sequenced. The A, T, G and C contents of the sequence were 235 bp (28.0%), 307 bp (36.6%), 138 bp (16.5%) and 158 bp (18.9%), respectively. Furthermore, 556 bp fragment of the sequence was used to discuss the phylogenetic relationship among 14 Penaeidae species using Alpheus armillatus as the outgroup. From the molecular phylogenetic tree constructed by neighbor-joining method, we obtained three large shrimp groups: Farfantepenaeus, Litopenaeus and Fenneropenaeus group. The results also indicated that there were a closer genetic relationships between F. aztecus and F. paulensis, L. schmitti and L. setiferus, F. indicus and F. merguiensis, and the genus Farfantepenaeus was closer to Litopenaeus.