Cadmium toxicity to embryonic-larval development and survival in red sea bream Pagrus major

At 18 degrees C and 33 psu, 24 and 48 h LC50 values of cadmium (Cd) for red sea bream Pagrus major embryos were 9.8 and 6.6 mg l(-1), respectively, while 24,48, 72, and 96 h LC50 values for larvae were 18.9,16.2, 8.0, and 5.6 mg l(-1), respectively, indicating that embryos were more sensitive to Cd toxicity than larvae. Cd concentrations at >= 0.8 mg l(-1) led to low hatchability (0-90% in >= 0.8 mg l(-1) solutions vs. 97-100% in lower ones), delay in time to hatch, high mortality (38-100% vs. 1-10%), morphological abnormality (42-100% vs. 1-10%), reduced length (3.55-3.60 vs. 3.71-3.72 mm) in the embryos and larvae. They were Cd concentration dependent and potential biological significant endpoints for assessing the risk of Cd to aquatic organisms. Heart beat and yolk absorption of the larvae were significantly inhibited at some high concentrations but they were not as sensitive as other endpoints to Cd exposure. (C) 2008 Elsevier Inc. All rights reserved.

Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO + 10% PG + 10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6 +/- 16.7% (mean +/- S.D.) and 77.8 +/- 15.5%, were achieved by the straw vitrifying method (20.5% DMSO + 15.5% acetamide + 10% PG, thawing at 43 degrees C and washing in 0.5 M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5 M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation. (C) 2007 Elsevier Inc. All rights reserved.

The complete amino acid sequence of growth hormone and partial amino acid sequence of prolactin and somatolactin from sea perch (Lateolabrax japonicus)

Growth hormone (GH), prolactin (PRL) and somatolactin (SL) were purified simultaneously under alkaline condition (pH 9.0) from pituitary glands of sea perch (Lateolabrax japonicas) by a two-step procedure involving gel filtration on Sephadex G-100 and reverse-phase high-performance liquid chromatography (rpHPLC). At each step of purification, fractions were monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting with chum salmon GH. PRL and SL antisera. The yields of sea perch GH, PRL and SL were 4.2, 1.0 and 0.28 mg/g wet tissue, respectively. The molecular weights of 19,200 and 20,370 Da were estimated by SDS-PAGE for sea perch GH and PRL, respectively. Two forms of sea perch SL were found: one (28,400 Da) is probably glycosylated, while the other one (23,200 Da) is believed to be deglycosylated. GH bioactivity was examined by an in vivo assay. Intraperitoneal injection of sea perch GH at a dose of 0.01 and 0.1 mug/g body weight at 7-day intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The complete sea-perch GH amino acid sequence of 187 residues was determined by sequencing fragments cleaved by chemicals and enzymes. Alignment of sea-perch GH with those of other fish GHs revealed that sea-perch GH is most similar to advanced marine fish, such as tuna, gilthead sea bream, yellowfin porgy, red sea bream, bonito and yellow tail with 98.4, 96.2%, 95.7%, 95.2%, 94.1% and 91% sequence identity, respectively. Sea-perch GH has low identity to Atlantic cod (76.5%), hardtail (73.3%), flounder (68.4%), chum salmon (66.3%), carp (54%) and blue shark (38%). Partial amino-acid sequences of 127 of sea-perch PRL and the N-terminal of 16 amino-acid sequence of sea-perch SL have been determined. The data show that sea-perch PRL has a slightly higher sequence identity with tilapia PRL( 73.2%) than with chum salmon PRL(70%) in this 127 amino-acid sequence. (C) 2001 Elsevier Science B.V. All rights reserved.

Molecular identification and expression analysis of natural resistance associated macrophage protein (Nramp) cDNA from Japanese flounder (Paralichthys olivaceus)

Natural resistance associated macrophage protein (Nramp) controls partially innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from Japanese flounder (Paratichthys olivaceus). The full-length cDNA of the Nramp is 3066 bp in length, including 224 bp 5' terminal UTR, 1662 bp encoding region and 1180 bp 3' terminal UTR. The 1662-nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of amino acid sequence indicated that Japanese flounder Nramp consists of 12 transmembrane (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of Japanese flounder had 77.30%, 82.71%, 82.67%, 79.64%, 80.72%, 90.97%, 91.16%, 60.14%, 71.48%, 61.69%, 72.37% identity with that of rainbow trout Nramp alpha and beta, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, striped sea bass Nramp, red sea bream Nramp, mouse Nramp 1 and 2, human Nramp 1 and 2, respectively. RT-PCR indicated that Nramp transcripts were highly abundant in spleen, head kidney, abundant in intestine, liver and gill, and less abundant in heart. The level of Nramp mRNA in embryos gradually increases during embryogenesis from 4 h (8 cell stage) to 80 h (hatched stage) after fertilization. (c) 2005 Elsevier Ltd. All rights reserved.

鲑鱼生长激素基因及其工程菌表达产物对真鲷的促生长效应

将鲑鱼生长激素基因转入真鲷受精卵，研究其对真鲷生长的影响。用鲑鱼生长激素基因的表达产物浸泡两月龄的真鲷鱼苗，研究其对真鲷生长的影响。提取含“全鱼”生长因素基因元件的重组质粒pAFPcnGH，用电脉冲法将其导入真鲷受精卵。从包含体中提取GH基因表达产物，用它浸泡两月龄的真鲷鱼苗，实验组比对照组体长分别增加8.0％，9.8％，17.5％；体重分别增加21.1％，32.0％，36.0％。

真鲷(Pagrosomus major)胚胎冷冻保存损伤机理及冷冻精子遗传物质稳定性研究

本文对真鲷心跳期胚胎对5种常用渗透性抗冻剂（DMSO、甘油、甲醇、丙二醇、乙二醇）和3种非渗透性抗冻剂（PVP、PEG-8000、蔗糖）的耐受性进行了研究。渗透性抗冻剂分6个浓度梯度（5%；10%；15%；20%；25%；30%）和3个时间组（10min；30min；1h）。非渗透性抗冻剂中，PVP、PEG-8000分3个浓度梯度（5%、10%、15%）和2个时间组（10min、30min），蔗糖为4个浓度梯度（5%、10%、15%、20%）和2个时间组（10min、30min）。实验结果表明，在渗透性抗冻剂组中，浓度为5%的处理组的孵化率（>90%）与对照组差异均不显著，随着抗冻剂浓度增大及处理时间的延长，真鲷心跳期胚胎的孵化率显著下降（P<0.05），在最高浓度的最长处理时间中胚胎孵化率均降到了0。总体上，真鲷心跳期胚胎对五种渗透性抗冻剂的耐受性从小到大依次为：甲醇 < 甘油 < 乙二醇 < DMSO < 丙二醇。对影响胚胎孵化率的三个因素（抗冻剂、浓度、时间）进行的因素效应分析结果表明，三种因素对孵化率的影响显著（P<0.05），并且浓度效应 > 时间效应 > 抗冻剂效应。在非渗透性抗冻剂组中，蔗糖组胚胎孵化率未呈显著变化；PVP组随着浓度及时间的增大，孵化率显著下降（P<0.05）；PEG-8000组随着浓度增大孵化率显著下降（P<0.05），但在两个时间组间差异不显著。相同处理情况下PEG-8000对真鲷心跳期胚胎的毒性要小于PVP。因素效应分析比较结果表明仅时间效应不显著，且抗冻剂效应 > 浓度效应 > 时间效应。 对所用各种抗冻剂进行了渗透压测量，实验中使用的渗透性抗冻剂（5%-30%）的渗透压值在959-7980mOsm/kg之间，均高于使用海水的渗透压值（919mOsm/kg）；使用的非渗透性抗冻剂的渗透压值在316-1040mOsm/kg之间，除20%蔗糖渗透压值（1040mOsm/kg）高于海水外，其他非渗透性抗冻剂的渗透压值均要低于海水。对孵化率与相应的溶液渗透压值进行相关回归分析结果表明，渗透性抗冻剂的渗透压与孵化率呈显著的负相关（P<0.05），而非渗透性抗冻剂的渗透压与孵化率相关不显著。渗透性抗冻剂组的回归分析结果表明，二次方程的曲线拟合度最高，得到的回归方程分别为：Y10min = -2×10-8X2 10min - 6×10-5 X 10min + 1.5635 （R2 = 0.713），Y30min= 5×10-8X2 30min－0.0007 X 30min + 2.097（R2 = 0.681），Y1h = 7×10-8X2 1h－0.0008 X 1h+ 2.0397（R2= 0.725）。 在真鲷胚胎对抗冻剂耐受性实验的基础上，挑选五种抗冻剂--10%DMSO、5%甘油、10%甲醇、20%丙二醇、10%乙二醇，浸泡真鲷心跳期胚胎30min后，分别以超速（130℃/min）、快速（20℃/min）、慢速（3℃/min）的速度降温并使用低温显微镜进行观察，依次记录Toif（油球结冰）、Teif（胚胎外部结冰）、Tiif（胚胎内部结冰）等结冰点，Toif值在-9~-23℃之间；Teif值在-21~-35℃之间；Tiif值在-21~-52℃之间。结冰顺序为先油球结冰，然后胚胎外部结冰随之内部马上瞬间变黑形成内部冰晶。随着降温速度的提高，各结冰温度值显著下降。各抗冻剂之间的Teif及Tiif值不同，Toif值之间没有显著差异。对两种玻璃化冷冻方法进行模拟观察，发现胚胎冰晶形成的顺序与非玻璃化过程不同--先内部结冰然后逐渐蔓延至外部形成外部冰晶，而且模拟玻璃化的内部结冰温度Tiif值（-52.56℃）显著（P<0.05）低于使用低浓度的同种抗冻剂超速降温组的Tiif值（-40.11℃）。在快速及慢速降温组中，20%丙二醇组的Tiif要显著的低于其他组（P<0.05）；在超速降温中，甲醇组的Tiif值要显著的低于其他组（P<0.05）。在Tiif小于30℃的实验组中获得形态完整胚胎的比例平均仅有30.77%；在Tiif大于30℃的实验组中获得形态完整胚胎的平均比例高达70.37%，模拟玻璃化组达到100%。各抗冻剂之间，复温后胚胎形态完整率10%甲醇组最高（77.78%）；其次依次为10%乙二醇（66.67%）、20%丙二醇（55.56%）和10%DMSO（55.56%）；5%甘油组最低（11.11%）；推测甲醇的对胚胎的渗透效果要好于其他组。综上推测：使用丙二醇、甲醇作为抗冻剂以及玻璃化冷冻保存方法对真鲷心跳期胚胎超低温保存也许较为合适。 我们对低温保存的真鲷精子核DNA损伤进行了研究以期为下一步胚胎遗传物质稳定性研究提供参考依据。研究方法为单细胞凝胶电泳（SCGE），针对研究对象，在实验过程中对传统的碱性单细胞凝胶电泳在铺胶方法、电泳条件等进行了改进。对精子细胞进行预处理，在碱性电泳液中使核DNA双链解链变性后电泳，EB染色lOmin后，在荧光显微镜下观察，每次随机观察50个左右的核DNA。结果表明，对荧光显微镜下观察到的精子核按彗尾长度及荧光强度划分等级，出现损伤的精子核DNA的损伤程度主要为轻度损伤和中度损伤，很少见有完全损伤的真鲷精子核。经5%、10%、18%、20%、25%、30%DMSO冷冻保存后的精子彗星率分别为33.47％ ± 8.95％; 35.91％ ± 19.44％; 48.95％ ± 8.90％; 43.33％ ± 11.19％; 55.80％ ± 38.94％。鲜精彗星率为31.43 ％ ± 2.68％。对比真鲷冷冻精液与新鲜精液的精子DNA的损伤状况，表明仅用30％ DMSO冷冻精子DNA损伤状况与鲜精差异显著(P<0.05)。 综上所述，渗透性抗冻剂对胚胎的毒性与其渗透压值呈显著的负相关关系。丙二醇对真鲷心跳期胚胎毒性最小，甲醇较其他抗冻剂能更好的渗透入胚胎；玻璃化方法能显著降低Tiif值并能更好的保持超低温保存后胚胎的形态完整性，因此，使用丙二醇、甲醇作为抗冻剂以及玻璃化冷冻保存方法对真鲷心跳期胚胎超低温保存也许较为合适。常规使用的用于超低温保存真鲷精子的DMSO（浓度<15%）不会对精子核物质稳定性造成明显影响。由于胚胎较精子结构要复杂许多，对于真鲷胚胎损伤机理的研究还有大量工作可以开展。

Fluorescence Characterization of Dissolved Organic Matter in the East China Sea after Diatom Red Tide Dispersion

Fluorescence excitation-emission spectroscopy (EEMS) was employed to analyze the 3-dimensional fluorescence of dissolved organic matter in the East China Sea after diatom red tide dispersion. The relationships between fluorescence peak intensity, and salinity and chlorophyll-a were discussed. The centers of protein-like fluorescence peaks dispersed at Ex(max)/Em(max) = 270-280/290-315 nm (Peak B), 220-230/290-305 nm (Peak D), 230-240/335-350 nm(Peak S)and 280/320 nm(Peak T). Two humic-like peaks appeared at 255-270/435-480 nm (Peak A) and 330-350/420-480 nm(Peak C). High tyrosine-like intensity was observed in diatom red tide dispersion area, and tryptophan-like fluorescence was also found which was lower. High FIB/FIS showed that diatom red tide produced much tyrosine-like matter during dispersion. Peaks S, A and C had positive correlation with one another, and their distributions were similar, which decreased with distance increasing away from the shore. Good negative correlations between peaks S, A and C and salinity suggested that Jiangsu-Zhejiang coastal water was the same source of then-L Correlations between fluorescence peak intensity and chlorophyll-a were not remarkable enough to clear the relationship between fluorescence and living algal matter. It was supposed that the living algal matter contributed little to the fluorescence intensity of algal dispersion seawater.

Seismic stratigraphy of the Qiongdongnan deep sea channel system, northwest South China Sea

Based on more than 4000 km 2D seismic data and seismic stratigraphic analysis, we discussed the extent and formation mechanism of the Qiongdongnan deep sea channel. The Qiongdongnan deep sea channel is a large incised channel which extends from the east boundary of the Yinggehai Basin, through the whole Qiongdongnan and the Xisha trough, and terminates in the western part of the northwest subbasin of South China Sea. It is more than 570 km long and 4-8 km wide. The chaotic (or continuous) middle (or high) amplitude, middle (or high) continuity seismic facies of the channel reflect the different lithological distribution of the channel. The channel formed as a complex result of global sea level drop during early Pliocene, large scale of sediment supply to the Yinggehai Basin, inversion event of the Red River strike-slip fault, and tilted direction of the Qiongdongnan Basin. The large scale of sediment supply from Red River caused the shelf break of the Yinggehai Basin to move torwards the S and SE direction and developed large scale of prograding wedge from the Miocene, and the inversion of the Red River strike-slip fault induced the sediment slump which formed the Qiongdongnan deep sea channel.

Phylogenetic position and morphology of thecae and cysts of Scrippsiella (Dinophyceae) species in the East China Sea

Resting cysts of the marine phytoplanktonic dinoflagellate Scrippsiella spp. are encountered in coastal habitats and shallow seas all over the world. Identification of Scrippsiella species requires information on cyst morphology because the plate pattern of the flagellated cell is conserved. Cysts from sediments of the East China Sea were identified based on traits from both the cysts and the thecal patterns of germinated cells. Calcareous cysts belonged predominantly to S. trochoidea (F. Stein) A. R. Loebl., S. rotunda J. Lewis, and S. precaria Montresor et Zingone. The former two species also produced smooth and noncalcified cysts in the field. A new species, S. donghaienis H. Gu sp. nov, was obtained from six noncalcified cysts with organic spines. These cysts are spherical, full of pale white and greenish granules with a mesoepicystal archeopyle. The vegetative cells consist of a conical epitheca and a round hypotheca with a plate formula of po, x, 4', 3a, 7 '', 6c (5c + t), 6 s, 5''', 2'''' and are morphologically indistinguishable from S. trochoidea. Results of internal transcribed spacer (ITS) sequence comparisons revealed that S. donghaienis was distinct from the S. trochoidea complex and appeared nested within the Calciodinellum/Calcigonellum clade. Culture experiments showed that the presence of a red body in the cyst and the shape of the archeopyle were constant within cell lines from one generation to the next, while the morphological features of the cyst wall, such as calcification and spine shape, appeared to be phenotypically plastic.

Biological activity of a red-tide alga - A-tamarense under co-cultured condition with bacteria

The relationship between Alexandrium tamarense (Lebour) Balech, one of red-tide alga, and two strains of marine bacteria, Bacillius megaterium(S-7) and B. halmapulus(S-10) isolated from Xiamen Western Sea, was investigated by evaluating the growth state of A. tamarense and the variation of P-glucosidase activity in co-culture system. The results showed the growth and multiplication of the alga were related with the concentration, genus speciality of the bacteria, and growth stage of the alga itself. The growth of A. tamarense was obviously inhibited by S7 and S, at high concentration. Either inhibition or promotion contributed much more clearly in earlier than in later stage of the growth of the alga. Furthermore, there was a roughly similar variation trend of the activity of extra-cellular enzyme, beta-glucosidase, in the water of the separately co-cultured bacteria S-7 and S-10 with the alga. The beta-glucosidase activity (beta-GlcA) rapidly increased during the later algal growth accompanying the increase of the lysis of the alga cells. The obvious inhibition of A. tamarense by marine bacteria at high concentration and evident increase of beta-GlcA in co-colture system would help us in better understanding the relationship between red-tide alga and bacteria, and also enlightened us the possible use of bacteria in the bio-control of red-tide.

Microbial modulation in the biomass and toxin production of a red-tide causing alga

The effect of S-10, a strain of marine bacteria isolated from sediment in the Western Xiamen Sea, on the growth and paralytic shellfish poison (PSP) production in the alga Alexandrium tamarense (A. tamarense) was studied under controlled experimental conditions. The results of these experiments have shown that the growth of A. tamarense is obviously inhibited by S-10 at high concentrations, however no evident effect on its growth was observed at low concentrations. Its PSP production was also inhibited by S 10 at different concentrations, especially at low concentrations. The toxicity of this strain of A. tamarense is about (0.9512.14) x 10(-6) MU/cell, a peak toxicity value of 12.14 x 10(-6) MU/cell appeared on the 14th day, after which levels decreased gradually. The alga grew well in conditions of pH 6-8 and salinities of 20-34 parts per thousand. The toxicity of the alga varied markedly at different pH and salinity levels. Toxicity decreased as pH increased, while it increased with salinity and reached a peak value at a salinity of 30 parts per thousand, after which it declined gradually. S-10 at a concentration of 1.02 x 10(9) cells/ml inhibited growth and the PSP production of A. tamarense at different pH and salinity levels. S-10 had the strongest inhibitory function on the growth of A. tamarense under conditions of pH 7 and a salinity of 34 parts per thousand. The best inhibitory effect on PSP production by A. tamarense was at pH 7, this inhibitory effect on PSP production did not relate to salinity. Interactions between marine bacteria and A. tamarense were also investigated using the flow cytometer technique (FCM) as well as direct microscope counting. S-10 was identitied as being a member of the genus Bacillus, the difference in 16S rDNA between S-10 and Bacillus halmapalus was only 2%. The mechanism involved in the inhibition of growth and PSP production of A. tamarense by this strain of marine bacteria, and the prospect of using it and other marine bacteria in the biocontrol of red-tides was discussed. (c) 2005 Elsevier Ltd. All rights reserved.

Fatty acids of some algae from the Bohai Sea

The fatty acid compositions of 22 species of marine macrophytes, belonging to the Ceramiales, Cryptonemiales, Nemalionales, Laminariales, Chordariales, Scytosiphonales, Desmarestiales, Dictyosiphonales, Fucales, Dictyotales and Ulvales and collected from the Bohai Sea, were determined by capillary gas chromatography. The contents of polyunsaturated fatty acids (FAs) in the Bohai Sea algae, in comparison with the same species from the Yellow Sea were found to be lower. Red algae had relatively high levels of the acids 16:0, 18:1(n-7), 18:1(n-9), 20:5(n-3) and 20:4(n-6), and those examined were rich in C-20 PUFAs, these chiefly being arachidonic and eicosapentaenoic acids. The major FAs encountered in the Phaeophyta were 14:0, 16:0, 18:1(n-9), 18:2(n-6), 18:3(n-3), 18:4(n-3), 20:4(n-6) and 20:5(n-3). C18PUFAs are of greater abundance in the brown algae than in the red algae examined. All three green algae from the Ulvales had similar fatty acid patterns with major components, 16:0, 16:4(n-3), 18:1(n-7), 18:2(n-6), 18:3(n-3), and 18:4(n-3). They contained 16:3(n-3) and more 16:4(n-3), were rich in C18PUFAs, chiefly 18:3(n-3) and 18:4(n-3) and had 18:1(n-7)/18:1 (n-9) ratios higher than 1. (C) 2002 Elsevier Science Ltd. All rights reserved.