Effect of n?3 HUFA levels in rotifers and Artemia on growth and survival of larval black sea bream ( Sparus macrocephalus )

Requirement for dietary n−3 HUFA (n−3 highly unsaturated fatty acid) for growth and survival of black sea bream (Sparus macrocephalus) larvae was studied using rotifers andArtemia at various levels of n−3 HUFA. Five treatments with rotifers andArtemia differing in n−3 HUFA were prepared by enriching them with various oil emulsions. Results indicated that dietary n−3 HUFA significantly influence fish n−3 HUFA levels and are essential for growth and survival of black sea bream larvae. The results also indicated that the incorporation of n−3HUFA TG (triacylglycerols) into tissues of larval black sea bream was more effective from natural fish oil in comparison with n−3 HUFA fatty acid ethyl esters from ethyl-esterified oil. Project No. 39770588 supported by the NSFC.     

Effect of n-3 HUFA levels in rotifers and Artemia on growth and survival of larval black sea bream （Sparus macrocephalus）

Requirement for dietary n-3 HUFA (n-3 highly unsaturated fatty acid) for growth and survival of black sea bream (Sparus macrocephalus) larvae was studied using rotifers and Artemia at various levels of n-3 HUFA. Five treatments with rotifers and Artemia differing in n-3 HUFA were prepared by enriching them with various oil emulsions. Results indicated that dietary n-3 HUFA significantly influence fish n-3 HUFA levels and are essential for growth and survival of black sea bream larvae. The results also indicated that the incorporation of n-3HUFA TG (triacylglycerols) into tissues of larval black sea bream was more effective from natural fish oil in comparison with n-3 HUFA fatty acid ethyl esters from ethyl-esterified oil.     

Studies on the phosphorus requirement and proper calcium/phosphorus ratio in the diet of the black sea bream (Sparus macrocephalus)

An expriment on the phosphorus requirement and the proper Ca/P ratio in the diet of the black sea bream using the phosphorus gradient method (with casein as basic diet, sodium dihydrogen phosphate as source of phosphorus, and calcium lactate as source of calcium) showed that growth was greatly affected by the diet's phosphorus content and Ca/P ratio. Inadequate phosphorus in the diet resulted in slow growth and poor food conversion ratio (FCR). Analyses of the fish body showed it contained a high level of lipid but a low level of moisture, ash, calcium and phosphorus. The optimal values of phosphorus and Ca/P ratio in the black sea bream diet are 0.68% and 1∶2 respectively. Phosphorus in excess of this optimum value resulted in slow growth or even death. The results of this experiment clearly indicated that phosphorus is the principal mineral additive affecting black sea bream growth. Contribution No. 1972 from the Institute of Oceanology, Academia Sinica.     

Studies on the optimum protein content and the ratio of animal to plant protein in the diet of the black sea bream (Sparus macrocephalus)

An experiment on the optimum dietary protein content for the black sea bream using semi-purified diets showed the optimum is 50.19% by weight as inferred from the growth rate and food consumption of the fish. Another experiment on the proper ratio of animal to plant protein in its diet using sandeel as the animal protein source and peanut cake meal as plant protein source showed that the ratio had great effect on its growth and should be 1.00: 0.89–0.96 as judged from the growth rate and food consumption of the fish. Contribution No. 1973 from the Institute of Oceanology, Chinese Academy of Sciences.     

STUDIES ON THE PHOSPHORUS REQUIREMENT AND PROPER CALCIUM/PHOSPHORUS RATIO IN THE DIET OF THE BLACK SEA BREAM(SPARUS MACROCEPHALUS)

An experiment on the phosphorus requirement and the proper Ca/P ratio in the diet of the black sea bream using the phosphorus gradient method (with casein as basic diet, sodium dihydrogen phosphate as source of phosphorus, and calcium lactate as source of calcium) showed that growth was greatly affected by the diet's phosphorus content and Ca/P ratio. Inadequate phosphorus in the diet resulted in slow growth and poor food conversion ratio (FCR). Analyses of the fish body showed it contained a high level of lipid but a low level of moisture, ash, calcium and phosphorus. The optimal values of phosphorus and Ca/P ratio in the black sea bream diet are 0.68% and 1:2 respectively. Phosphorus in excess of this optimum value resulted in slow growth or even death. The results of this experiment clearly indicated that phosphorus is the principal mineral additive affecting black sea bream growth.     

Immunotoxicity effect of benzo[α]pyrene on scallop Chlamys farreri

The toxic effects of benzo[α]pyrene (B[α]P) at different concentrations (0.1, 0.5, 1, 2.5 and 7.5 μgL⁻¹) on scallop (Chlamy farreri) immune system were studied. The results showed that B[α]P had significant toxic effects on the haemocyte counts, neutral red uptake, phagocytosis, bacteriolytic and antibacterial activity (P<0.05), while the seawater control and acetone control had no significant differences. The haemocyte counts, neutral red uptake, phagocytosis and bacteriolytic activity in all B[α]P treatment groups as well as antibacterial activity in groups of 0.5, 1, 2.5 and 7.5 μgL⁻¹ B[α]P decreased significantly (P<0.05). Some of these indices tended to be stable on the sixth day and others on the ninth day, and the indices showed clear time- and concentration-response to B[α]P. Bacteriolytic activity in 0.1μgL⁻¹ B[α]P treatment group and antibacterial activity in 0.1 μgL⁻¹ and 0.5 μgL- B[α]P treatment groups increased at the beginning of exposure and reached their peaks on day 1 and day 6, respectively. Following that, both activities decreased gradually and became stable after day 9. When all the indices reached stability, they were significantly lower than those in control group (P<0.05), except for antibacterial activity in 0.1 μgL⁻¹ B[α]P treatment group (P>0.05). Thus, B[α] has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immunomodulation in aquatic organisms.     

STUDIES ON THE OPTIMUM PROTEIN CONTENT AND THE RATIO OF ANIMAL TO PLANT PROTEIN IN THE DIET OF THE BLACK SEA BREAM(SPARUS MACROCEPHALUS)

An experiment on the optimum dietary protein content for the black sea bream using semi-purified dietsshowed the optimum content is 50.19% by weight as inferred from the growth rate and food consumptionof the fish. Another experiment on the proper ratio of animal to plant protein in its diet using sandeel as theanimal protein source and peanut cake meal as plant protein source showed that the ratio had great ef-fect on its growth and should be 1.00:0.89—0.96 as judged from the growth rate and food consumptionof the fish.     

Effect of artificial regulations ofArtemia n−3 HUFA content on growth and survival of black seabream (Sparus macrocephalus) larvae

The requirement for dietary n−3 HUFA (highly unsaturated fatty acid) for growth and survival of black seabream (Sparus macrocephalus) larvae was tested usingArtemia with various levels of n−3 HUFA. Four treatments withArtemia differing in their n−3 HUFA were prepared by feeding them various oil emulsions. The results indicated that dietary n−3 HUFA significantly influences fish n−3 HUFA levels and are necessary for good growth and survival of black seabream larvae. The proper value of n−3 HUFA level and DHA, EPA level inArtemia should be 4.273% and 0.873% (wet weight) for good growth and survival in black seabream larvae respectively. Contribution No. 3044 from the Institute of Oceanology, Chinese Academy of Sciences. Project 39470562 supported by NSFC.     

大口黑鲈抗菌肽hepcidin cDNA序列和结构分析

以大口黑鲈为材料,提取肝脏总RNA,经RT-PCR扩增出hepcidin cDNA的开放阅读框(ORF)及3′端非编码区序列,应用5′RACE方法得到大口黑鲈hepcidin cDNA5′末端。将所获得的两个片段分别克隆到T载体后进行测序,并拼接成大口黑鲈hepcidin全长cDNA。序列分析表明:大口黑鲈hepcidin全长cDNA为564bp,含有一个258bp的ORF,编码86个氨基酸残基,由信号肽(24个残基)、前肽(42个残基)和成熟肽(20个残基)3部分组成hepcidin前体。在前肽部分具有前肽转化酶典型的RX(K/R)R基元,成熟肽部分含有8个保守的半胱氨酸残基,可形成四个链内二硫桥,使β-折叠结构保持稳定。大口黑鲈Hepcidin与其他鱼类的同源性在29.7%~90.5%间,尤其是信号肽区域,与鳜、尼罗罗非鱼、真鲷、花鲈、黑鯛、金眼狼鲈仅有2~3个氨基酸的差别。     

Effects of dietary β-glucan and glycyrrhizin on non-specific immunity and disease resistance of the sea cucumber (<Emphasis Type="Italic">Apostichopus japonicus</Emphasis> Selenka) challenged with <Emphasis Type="Italic">Vibrio splendidus</Emphasis>

Sea cucumbers, Apostichopus japonicus Selenka, were fed diets containing non-immunostimulant (basal diet), 0.2% β-glucan and 0.02% glycyrrhizin in a recirculatory water system for 45 days, and subsequently challenged with Vibrio splendidus by injection at 1.0×10⁸ cfu / sea cucumber for 15 days. Phagocytic capacity (PC), intracellular superoxide anion production (ISAP), lysozyme (LSZ) activity and superoxide dismutase (SOD) activity in the coelomic fluid were analyzed on the 0^th, 5^th, 10^th and 15^th days after injection. Results showed that after the 45-day feeding period, PC, ISAP, LSZ activity and SOD activity in sea cucumbers fed with dietary β-glucan or glycyrrhizin were significantly higher than in those fed with the basal diet. On the 5th day after infection, all the immune parameters examined in the sea cucumbers injected with V. splendidus decreased in value significantly. On the 15^th day, PC, ISAP and LSZ activity returned to levels similar to those on the 0^th day. For the sea cucumbers injected with saline, there were no significant differences in all the immune parameters examined and in the cumulative morbidity during the 15-day challenging trial. After injecting with V. splendidus, the cumulative morbidity of sea cucumbers fed with the basal diet was significantly higher than those fed with dietary β-glucan or glycyrrhizin when challenged with V. splendidus challenged sea cucumber fed with the basal diet was significantly higher than those fed with dietary β-glucan or glycyrrhizin. There was no significant difference in cumulative morbidity between the dietary β-glucan and glycyrrhizin treatments over time.     

The effects of recombinant eel growth hormone,methyltestosterone and L-thyroxine on the growth of red sea bream,Pagrosomus major

the effects of recombinant eel growth hormone (reGH), methyltestosterone (MT) and L-thyroxine (T4) on the growth of red sea bream,Pagrosomus major, were investigated. Administration of reGH to fry by immersion at 2 mg/1 for 2 h every 5 days resulted in significant increase in both weight and length, but the condition factor (CF) diminished relative to that of similarly treated controls over the 37 day treatment period. Immersion in 0.1 mg/l T4 also resulted in significant increase in both weight and length and higher survival rate of test fry compared to the controls. Immersion in MT had less effect on growth and high-dose resulted in high mortality. In the second study, injection of 2 μg reGH/(g·wk) caused a significant increase in the specific growth rate (SGR) of test red sea bream fingerlings relative to that of the controls during the 4—week treatment period and maintained the increasing trend over the post-treatment period (weeks 4–6). Injection of MT at a dosage of 1μg/(g·wk) resulted in a significant increase in SGR during the 4—week treatment period. Intramuscular injection of 0.1 μg T4/(g·wk) also resulted in a significant increase in weight. Injection of high-doses of MT and T4 inhibited growth and resulted in darkening skin, bulging eyes and thinning body. In muscle chemical composition, the treated groups had no significant differences compared to the controls, but the high-dose MT and T4 groups showed significant increases in lipid content. Potential practical methods for hormone application in fish culture are discussed.     

Analysis of IL-2-1ike factor in lymphocyte culture supernatant of olive flounder, Paralichthys oliveaceus

To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 (IL-2)-like factor was detected in the supernatant of plant haemoglutinin (PHA)-stimulated lymphocyte culture from peripheral blood, spleen and head kidney of olive flounder,Paralichthys olivaceus. The quantities of IL-2-like factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes (P<0.01). The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2, (rhIL-2) was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor (IL-2R) on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25(IL-2R) was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%. This work was supported by National “973” Project G1999012003, G19999012006.     

Influence of dietary probiotic Bacillus TC22 and Prebiotic fructooligosaccharide on growth, immune responses and disease resistance against Vibrio splendidus infection in sea cucumber Apostichopus japonicus

The effects of probiotic Bacillus TC22 (isolated from intestine of infected sea cucumber) and prebiotic fructooligosaccharide (FOS) on growth, immunity and disease resistance in sea cucumber Apostichopus japonicus were studied. Six experimental diets were formulated with combinations of three levels of TC22 (0, 107 and 109 CFU g-1 diet) and two levels of FOS (0 and 0.5%) in a 3 × 2 factorial experiment. At the end of the 8-week feeding trial,animals were challenged by injecting Vibrio splendidus. The results revealed that the specific growth rates (SGR) of sea cucumbers were not affected by TC22 and FOS, or the interaction between TC22 and FOS (P > 0.05). However,there were significant interactions between TC22 and FOS for immune response and disease resistance in sea cucumbers (P < 0.05). When sea cucumbers were fed with TC22 at 109 CFU g?1 feed and 0.5% FOS alone or in combination, the phagocytosis, respiratory burst and phenoloxidase activity of sea cucumber coelomocytes were significantly enhanced;the disease resistance against V. splendidus infection was also increased significantly (P < 0.05).However, dietary combination of TC22 at 109 CFU g-1 diet and 0.5% FOS did not yield significantly higher efficiency than either TC22 at 109 CFU g-1 diet or 0.5% FOS used alone (P > 0.05). Therefore, further studies should examine the effects of combinations of other levels of FOS(> 0.5% or < 0.5%) and TC22 on the immunity and disease resistance of sea cucumbers.     

南海区4种鲷鱼精子的适盐性比较

研究了盐度对黄鳍鲷SparuslatusHouttuyn、平鲷Rhabdosargussarba（Forskal）、黑鲷Sparusmacrocephalus（Basilewsky）和真鲷Pagrsomusmajor（TemmincketSchlegel）4种鲷鱼精子活力的影响。对南海区4种鲷鱼精子的适盐性及其活力进行了比较,黄鳍鲷、平鲷、黑鲷和真鲷精子激活所需的最低盐度分别为8、10、＞8、＞10;激活的最适盐度则分别为21、22、25、25,与之相应的精子活动（涡动）时间分别为14．7、17．7、17．2、18．0min;在高盐条件（盐度40左右）下,黄鳍鲷精子的适应性最强,精子活力最好,其次是黑鲷、真鲷的精子,平鲷精子对高盐条件的适应性最差。     

Effects of carboxymethyl-chitosan on wound healing in vivo and in vitro

Effects of carboxymethyl-chitosan with different molecular weights on wound healing were investigated in vivo and in vitro.A second degree burn model was performed in rats and the accelerative effects of carboxymethyl-chitosan on wound repair were observed.Contents of transforming growth factor(TGF)-β1,interleukin(IL)-6 and matrix metalloproteinase(MMP)-1 in wounds were determined by enzyme-linked immunosorbent assay(ELISA).In vitro study evaluated the influence of carboxy-methyl-chitosan on cytokines secretion of fibroblasts and macrophages.In vivo results showed that carboxymethyl-chitosan effec-tively accelerated the wound healing process in burned rats(P<0.05).Levels of TGF-β1,IL-6 and MMP-1 in carboxymethyl-chitosan groups were significantly elevated,compared with control group(P<0.05).In vitro results indicated that carboxymethyl-chitosan significantly promoted the proliferation of fibroblasts and stimulated its IL-8 and IL-10 secretion at different incubation time,but it did not affect collagen secretion of fibroblasts.Carboxymethyl-chitosan enhanced phagocytosis ability of macrophages,and in-creased its tumor necrosis factor(TNF)-α secretion.In conclusion,carboxymethyl-chitosan promoted wound healing by activating macrophages,accelerating fibroblasts growth and exerting considerable effects on the secretion of a series of cytokines.     

不确定性对北部湾二长棘鲷渔业资源评估影响的模拟研究

利用蒙特卡罗模拟方法研究了生物学参考点F0.1、Fmax和当前捕捞死亡系数(current fishing mortality rate,Fcur)估计中的不确定性对北部湾二长棘鲷渔业资源评估的影响。结果表明:二长棘鲷渔业资源状态评估受到F0.1、Fmax、Fcur估计中的不确定性和管理者选取的决策置信水平的影响,二长棘鲷渔业被确定为过度捕捞的概率随着F0.1、Fmax、Fcur估计中不确定性和置信水平的增加而下降;F0.1和Fmax估计中的不确定性主要由生长参数的不确定性引起,自然死亡系数的不确定性对F0.1和Fmax估计中的不确定性的影响是有一定限度的。相比Fmax,将F0.1作为二长棘鲷渔业的管理目标更为合适。     

Construction of cDNA library from intestine, mesentery and coelomocyte of Apostichopus japonicus Selenka infected with Vibrio sp. and a preliminary analysis of immunity-related genes

The aquaculture of sea cucumber Apostichopus japonicus (Echinodermata, Holothuroidea) has grown rapidly during recent years and has become an important sector of the marine industry in Northern China. However, with the rapid growth of the industry and the use of non-standard culture techniques, epidemic diseases of A. japonicus now pose increasing problems to the industry. To screen the genes with stress response to bacterial infection in sea cucumber at a genome wide level, we constructed a cDNA library from A. japonicus Selenka (Aspidochirotida: Stichopodidae) after infecting them with Vibrio sp. for 48 h. Total RNA was extracted from the intestine, mesentery and coelomocyte of infected sea cucumber using Trizol and mRNA was isolated by Oligotex mRNA Kits. The ligated cDNAs were transformed into DH5α, and a library of 3.24×105 clones (3.24×105 cfu mL-1) was obtained with the sizes of inserted fragments ranging from 0.8 to 2.5 kb. Sequencing the cDNA clones resulted in a total of 1106 ESTs that passed the quality control. BlastX and BlastN searches have identified 168 (31.5%) ESTs sharing significant homology with known sequences in NCBI protein or nucleotide databases. Among a panel of 25 putative immunity-related genes, serum lectin isoform, complement component 3, complement component 3-like genes were further studied by real-time PCR and they all increased more than 5 fold in response to Vibrio sp. challenge. Our library provides a valuable molecular tool for future study of invertebrate immunity against bacterial infection and our gene expression data indicates the importance of the immune system in the evolution and development of sea cucumber.     

AVAILABILITY OF PHOSPHORUS FROM SELECTED INORGANIC PHOSPHATES TO JUVENILE ABALONE, HALIOTIS DISCUS HANNAI INO

The availability of phosphorus to juvenile abalone,Haliotis discus hannai from primary, secondary and tertiary calcium phosphate, primary sodium or potassium phosphate separately or in combination was determined in a 120-day feeding trial. Seven semi-purified diets were formulated to contain relatively constant dietary phosphorus, ranging from 0.78%–0.82%. The concentrations of dietary phosphorus were presumed to be slightly below the requirement for abalone. A brown alga,Laminaria japonica, was used as a control diet. Abalone juveniles of silimar size (average weight 1.18 g; average shell length 18.74 mm) were distributed in a single-pass, flow-through system using a completely randomized design with eight treatments and three replicates each treatment. The survival ranged from 71.1% to 81.1%, and was not significantly (P>0.05) related to dietary treatment. However, the weight gain rate (41.72%–65.02%), daily increment in shell length (36.87–55.07 μm) and muscle RNA-DNA ratio (3.44–4.69) were significantly (P<0.05) affected by dietary treatment. Soft body alkaline phosphatase activity (10.9–19.8 U/g wet tissue) and carcass levels of lipid (7.71%–9.33%) and protein (46.68%–49.35%) were significantly (P<0.05) responsive to available phosphorus of the diets. Dietary treatment had significant effect (P<0.05) on concentrations of phosphorus in the whole body (WB) and soft body (SB). Apparent digestibility coefficients (45%–97%) of phosphorus were also significantly (P<0.05) different between dietary treatments. The results indicated that among these inorganic compounds, primary calcium, potassium and sodium phosphates separately or in combination could be utilized effectively by juvenile abalone as dietary phosphorus sources (availability ranging from 87%–97%). However, secondary and tertiary calcium phosphates were found to be low in availability, the values being 45% and 77%, repectively. Project 39670572 supported by the Natural Science Foundation of China (NSFC).     

Effect of dietary phosphorus on the growth and body components of juvenile Synechogobius hasta

The effect of dietary phosphorus on the growth and body components of juvenile Synechogobius hasta was determined. Different percentages of dietary phosphorus (0.63, 0.77, 0.93, 1.06, 1.22 and 1.36) were tested by feeding the fish (body weight, 15.81 g ± 0.32 g; 20 individuals each group; 3 groups each percentage) at a surplus of 5%–10% above satiation for 35 d. Dietary phosphorus did not significantly affect the specific growth rate, feed intake, feed conversion ratio and protein efficiency rate. Nitrogen retention was found to be the highest in fish fed the diet containing 1.06% of phosphorus; however, this was not significantly different from other diets. Fish fed the diet containing 0.93% of phosphorus showed the highest phosphorus retention; similar phosphorus retention rates were found in fish fed the diets containing 0.77% and 1.06% of phosphorus. Fish fed the diet containing the highest percentage of dietary phosphorus were found to contain the least whole body lipid, lower than fish fed other diets (P<0.05). The protein content increased from 18.59% to 19.55% (although not significant) with the decrease of body lipid content (P>0.05). The contents of the whole body ash, whole body phosphorus and vertebrae phosphorus increased with dietary phosphorus percentage up to 1.06 (P<0.05), reaching a plateau after that. Dietary phosphorus did not significantly influence the muscle components (protein, lipid and moisture). Condition factor and hepatosomatic index were the highest in fish fed the diet containing 0.63% of dietary phosphorus; however, this was not significantly different from those of other diets. The second-order polynomial regression of phosphorus retention against dietary phosphorus identified a breakpoint at 0.88% of dietary phosphorus. However, the dietary requirement of phosphorus for maintaining maximum phosphorus storage determined by broken-line analysis of the contents of whole body phosphorus, and ash and vertebrae phosphorus was 1.06% of the diet.     

Localization and dynamic expression of a 27.8 kDa receptor protein for lymphocystis disease virus infection in sea bass (<Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>) tissues

Lymphocystis disease virus (LCDV) infects target cells by attaching to a 27.8 kDa receptor (27.8R) protein in flounder Paralichthys olivaceus, and anti-27.8R monoclonal antibodies (MAbs) have been developed. However, the 27.8R existence in tissues of sea bass (Lateolabrax japonicus) and its role in LCDV infection have remained unclear. In this study, the results of western blotting demonstrated that the same 27.8R was shared by flounder and sea bass. LCDV-free sea bass individuals were intramuscularly injected with LCDV, and viral copies were detected in tissues from 3 h post infection and showed a time-dependent increase during 9 days infection. Distribution and synthesis of 27.8R in sea bass tissues were investigated by using anti-27.8R MAbs as probes. It was found that 27.8R was distributed in all the tested tissues. The levels of 27.8R protein were highest in gill and skin, then a bit lowly in stomach, head kidney and heart, followed by spleen, intestine, blood cells, gonad and liver, and least in kidney and brain in healthy sea bass. Upon LCDV infection, 27.8R synthesis was up-regulated in each tissue, and higher in the tissues with higher LCDV copies. The 27.8R and LCDV were detected in some peripheral blood leukocytes but not in red blood cells. These results suggested that 27.8R was widely distributed in sea bass tissues, and it served as a receptor and correlated with tissue tropism of LCDV infection. Furthermore, leukocytes had the potential of being a LCDV carrier and were responsible for a systemic infection of LCDV in sea bass.