南方池养牙鲆全人工配合饲料饲养试验

采用全人工配合饲料(试验组饲料)与湿颗粒饲料 (对照组饲料 )对南方池养牙鲆进行为期 204d的饲养试验.结果表明,试验组与对照组牙鲆的尾相对增重率分别为 2 304%、2 297%,尾平均日增重差异不大,平均饲料系数分别为 1. 56、2. 58,牙鲆增重 1kg所需饲料成本分别为 10. 92元和 13. 55元,试验组比对照组节省饲料成本2. 63元 /kg鱼增重;试验组与对照组牙鲆平均内脏比 (分别为 6. 50%、6. 38% )、平均丰满度(分别为 1. 09、1. 07)、肌肉粗蛋白含量、粗脂肪含量等差异不显著(p>0. 05),10种必需氨基酸和 5种鲜味氨基酸的含量很接近.由此可见,南方池养牙鲆采用全人工配合饲料饲养是完全可行的.     

Cloning of the cDNA encoding adenosine 5′-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5’-UTR of 78 bp, a 3’-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) (P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) (P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.     

The toxic mechanism of high lethality of herbicide butachlor in marine flatfish flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder, Paralichthys Olivaceus, was analyzed by histopathological examination, antioxidant enzymes activities and ATP content assay. Histopathological examination of gill, liver and kidney of exposed fishes showed that gill was a target organ of butachlor. The butachlor seriously impaired the respiration of gills by a series of lesions such as edema, lifting and detachment of lamellar epithelium, breakdown of pillar cells, and blood congestion. The dysfunction of gill respiration caused suffocation to the exposed flounder with extremely high acute lethality. Antioxidant enzyme activity assay of the in vitro cultured flounder gill(FG)cells exposed to butachlor indicated that butachlor markedly inhibited the antioxidant enzyme activities of Superoxide dismutase(SOD), catalase(CAT)and glutathione peroxidase(GPX). Furthermore, along with the decline of antioxidant enzyme activities, ATP content in the exposed FG cells decreased, too. This infers that the oxidative stress induced by butachlor can inhibit the production of cellular ATP. Similar decrease of ATP content was also observed in the exposed flounder gill tissues. Taken together, as in FG cells, butachlor possibly induced a short supply of ATP in pillar cells by inhibiting the antioxidant enzyme activities and then affecting the contractibility of the pillar cells, which in turn resulted in the blood congestion and suffocation of exposed flounder.     

Analysis of new microsatellite markers developed from reported sequences of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

The expressed sequence tags (ESTs) of Japanese flounder, Paralichthys olivaceus, were selected from GenBank to identify simple sequence repeats (SSRs) or microsatellites. A bioinformatic analysis of 11111 ESTs identified 751 SSR-containing ESTs, including 440 dinucleotide, 254 trinucleotide, 53 tetranucleotide, 95 pentanucleotide and 40 hexanucleotide microsatellites respectively. The CA/TG and GA/TC repeats were the most abundant microsatellites. AT-rich types were predominant among trinucleotide and tetranucleotide microsatellites. PCR primers were designed to amplify 10 identified microsatellites loci. The PCR results from eight pairs of primers showed polymorphisms in wild populations. In 30 wild individuals, the mean observed and expected heterozygosities of these 8 polymorphic SSRs were 0.71 and 0.83 respectively and the average PIC value was 0.8. These microsatellite markers should prove to be a useful addition to the microsatellite markers that are now available for this species.     

Does heavy oil pollution induce bacterial diseases in Japanese flounder Paralichthys olivaceus?

As basic research for the effect of heavy oil on the fish immune system, in this study, the number of leukocyte was counted in Japanese flounder Paralichthys olivaceus, after exposure to heavy oil at a concentration of 30 g/8 L for 3 days. To compare the numbers of bacteria in the skin mucus between oil-exposed and control fish, viable bacteria were enumerated by counting colony forming unit (CFU). Compared with 5.79 ± 1.88 × 10⁷ leukocytes/mL in the controls, the exposed fish demonstrated higher counts, averaging 1.45 ± 0.45 × 10⁸ cells/mL. The bacterial numbers of control fish were 4.27 ± 3.68 × 10⁴ CFU/g, whereas they were 4.58 ± 1.63 × 10⁵ CFU/g in the exposed fish. The results suggest that immune suppression of the fish occurred due to heavy oil stressor, and bacteria could invade in the mucus, resulting in the increasing leukocyte number to prevent infectious disease.     

Toxicogenomic analysis of immune system-related genes in Japanese flounder (Paralichthys olivaceus) exposed to heavy oil

Heavy oil contamination is one of the most important environmental issues. Toxicities of polycyclic aromatic hydrocarbons (PAHs), including immune toxicities, are well characterized, however, the immune toxic effects of heavy oil, as a complex mixture of PAHs, have not been investigated. In the present study, we selected Japanese flounder (Paralichthys olivaceus) as a model organism, and observed alteration of immune function by the exposure to heavy oil. To analyze the expression profiles of immune system-related genes, we selected 309 cDNAs from our flounder EST library, and spotted them on a glass slide. Using this cDNA array, alteration of gene expression profiles was analyzed in the kidneys of flounders exposed to heavy oil. Six Japanese flounders (mean body weight: 197 g) were acclimated to laboratory conditions at 19–20 °C. Three fish were exposed to heavy oil C (bunker C) at a concentration of 3.8 g/L for 3 days, and the others were kept in seawater without heavy oil and used as the control. After the exposure period, the fish were transferred into control seawater and maintained for 4 days, and then they were dissected and their kidneys were removed. Total RNA was extracted from the kidney samples to use in gene expression analyses. The microarray detected alteration of immune system-related genes in the kidneys of heavy oil-exposed flounders, including down-regulation of immunoglobulin light chain, CD45, major histocompatibility complex class II antigens and macrophage colony-stimulating factor precursor, and up-regulation of interleukin-8 and lysozyme. These results suggest that pathogen resistance may be weakened in heavy oil-exposed fish, causing a subsequent bacterial infection, and then proinflammatory genes may be induced as a defensive response against the infection. Additionally, we found candidate genes for use as biomarkers of heavy oil exposure, such as N-myc downstream regulated gene 1 and heat shock cognate 71 kDa proteins.     

外源激素对养殖牙鲆血浆睾酮和雌二醇含量的影响研究

雄性牙鲆单独注射促黄体激素释放激素类似物(LHRH-A)(每千克质量的注射量,下同)25μg/kg或人绒毛膜促性腺激素(hCG)1 000 IU/kg,血浆睾酮(T)含量在注射后6 h大幅度降低,12h时达到最低值,以后逐渐回升;LHRH-A与hCG联合注射[LHRH-A(12.5μg/kg)+hCG(500 IU/kg)],注射后6 h血浆T含量剧烈下降,24 h时达到最低点,以后缓慢回升;对照组雄鱼血浆T含量随着采样进行而呈现降低趋势.雌鱼单独LHRH-A后,血浆雌二醇(E₂)水平逐渐下降,24 h时达到最低值,30 h后略有回升;单独注射hCG后,血浆E₂含量缓慢下降,30 h达到最低值;LHRH-A与hCG联合注射,注射后6 h血浆E2含量逐渐下降,24 h时达到最低点,30 h时缓慢回升.对照组雌鱼血浆E₂水平也随着采样的进行而逐渐降低.结果表明:单独或联合注射LHRH-A和hCG降低了雌、雄牙鲆血浆E₂和T含量,与对照组结果相同.     

条斑星鲽早期发育生物学研究——受精卵的形态、生态和卵胚发育特征

条斑星鲽(Verasper moseri)的受精卵为圆球形、分离透明、无油球的悬浮卵。卵膜具粗波纹状的纹络。卵黄均匀,浅黄色或米黄色。卵径平均为1.70 mm±0.02 mm。卵黄囊径1.57 mm±0.025 mm。卵黄间隙狭窄,约为0.11 mm。受精卵的可孵化水温为6.0~11.0℃,最适孵化水温8.0~10.0℃。可孵化盐度为27.00~36.00,最适孵化盐度为33.00~35.00。受精卵在不同盐度的海水中,呈现不同的生态分布特征。在水温8.0~10.0℃,盐度33.00,光照500~6000 lx,pH值7.5~8.4的孵化条件下,受精卵约历时216 h孵化出膜,出膜方式以头部首先破膜为主。优质卵的孵化率可达90%以上。在条斑星鲽胚胎发育过程中,原肠期及原肠期以前的时期对温度的敏感性明显强于原口关闭后各个发育时期。卵胚卵裂期的不同步分裂现象要比其他硬骨鱼类提前,出现在第三次卵裂时期。多细胞期的裂球均一程度较低。在孵化育苗生产中,出现大量带有"空泡"的不正常的卵胚和前期仔鱼。该研究结果为比较鲆鲽鱼类早期发育过程提供了基础参考资料,同时揭示作为冷温性鱼类条斑星鲽受精卵发育过程中出现的特殊规律,从而为该鱼种进一步大规模苗种生产提供了理论依据。