哈氏弧菌诱导布氏石斑鱼头肾提取物的抗菌活性研究

以致病性哈氏弧菌（Vibrio harveyi）腹腔注射布氏石斑鱼（Epinephelus bleekeri）18h后,取布氏石斑鱼头肾制备头肾粗提物,并对该粗提物的抗菌活性进行研究,结果表明：布氏石斑鱼经哈氏弧菌诱导后,其头肾粗提物不仅对金黄色葡萄球菌（Staphyloccocus aureus）等革兰氏阳性菌有良好抑制作用,而且对大肠杆菌D31（Escherichio coli D31）、哈氏弧菌和溶藻弧菌（V.alginolyticus）等革兰氏阴性菌也有良好抑制效果;利用电泳凝胶琼脂糖弥散法检测发现,头肾粗提物中具有多种对大肠杆菌D31有较强作用效果的蛋白类抗菌物质;切胶回收AU—PAGE凝胶上的抗菌组分,利用SDS—PAGE凝胶检测各抗菌活性组分的分子质量大小依次约为：10．9KD,10．0KD,8．1KD,7．2KD,6．3KD,由于这些抗菌活性成分可被蛋白酶破坏,且分子质量均接近或低于10KD,可初步将其认定为抗菌肽。     

口服当归多糖对点带石斑鱼非特异性免疫力的影响

[目的]研究口服当归多糖对点带石斑鱼非特异性免疫力的影响。[方法]将05、02、00、350和500 mg/kg当归多糖添加到基础饵料中饲喂点带石斑鱼28 d,研究血浆中酸性磷酸酶（ACP）、溶菌酶（LZM）、补体C3和C4含量、白蛋白/球蛋白比值（A/G）、血液白细胞数和白细胞吞噬能力。[结果]喂食当归多糖能提高血浆ACP、LZM活力、补体C3和C4含量,降低血浆A/G值。另外,喂食当归多糖能提高点带石斑鱼血液白细胞数和白细胞吞噬率。[结论]当归多糖能提高点带石斑鱼非特异性免疫力,饵料中当归多糖的建议添加量为50 mg/kg,连续喂食21 d。     

投喂频率对流水养殖鞍带石斑鱼生长、摄食及免疫酶活力的影响

在水温(28.59±1.74)℃下,选用冰鲜杂鱼饲养平均体质量为858.73g的鞍带石斑鱼,经过60d的生长实验,研究了不同投喂频率(1次/d、1次/2d、1次/3d)对流水养殖鞍带石斑鱼生长、摄食及免疫酶活力的影响.结果表明,投喂频率对鞍带石斑鱼存活率的影响差异不具有统计学意义(p0.05),实验期间各个实验组存活率在99.40%~99.80%之间;各实验组间的肥满度(CF=2.22~2.35)、体质量变异系数(SV=20.08~24.27)、饲料系数(FCR=2.70~3.01)和日增质量(7.50~12.51g/d)均无显著影响(p0.05),而投喂频率1次/d组和1次/2d组的特定生长率(SGR)和相对增质量(RWG)均显著高于1次/3d组(p0.05),投喂频率1次/d组的日摄食率和1次/2d组的饲料转化效率均显著高于1次/3d组(p0.05);在免疫酶活力方面,投喂频率对鞍带石斑鱼血浆中的碱性磷酸酶(AKP=0.083 5~0.089金氏单位/mL)、过氧化氢酶(CAT=7.96~8.19U/mL)及超氧化物歧化酶(SOD=32.40~34.45U/mL)均无显著影响(p0.05).在本实验条件下,流水模式养殖鞍带石斑鱼的适宜投喂频率为1次/d或1次/2d.     

致病性假交替单胞菌的分离鉴定及药敏特性研究

[目的]分离鉴定患病的七带石斑鱼（Epinephelus septemfasciatus Thunberg）的致病菌,并研究其药敏特性。[方法]从患病的七带石斑鱼体内分离出1株致病菌BY0081。通过ATB微生物鉴定系统和菌体常规形态特征、生理生化反应指标测定以及16S rDNA测序分析等进行综合鉴定;通过人工感染水生模式动物剑尾鱼测定该菌株对其的半数致死量（LD50）;最后进行药敏试验确定其药敏特性。[结果]致病菌BY0081为革兰氏阴性菌,菌落呈米色,边缘光滑,中间略有隆起,为假交替单胞菌（Pseudoa lteromonas sp.）。其在较低的温度下仍有较强的毒力,其对水生模式动物剑尾鱼（Xiphophorus helleri）的LD50为2.82×105cfu/g鱼体重。该菌对菌必治、复方新诺明、氯霉素和氟哌酸等抗生素高度敏感,对复达欣、红霉素、丁胺卡那霉素和庆大霉素等药物中度敏感。[结论]该研究对防治由假交替单胞菌引起的鱼类疾病有积极意义。     

Influence of feeding regime on fatty acid composition in young red-spotted grouper Epinephelus akaara

ABSTRACT:   Young red-spotted grouper Epinephelus akaara with an average total length of 3.4 cm were divided into five groups in floating net-cages, and reared under satiated conditions with different feeding frequencies (0.5, 1, 2, 4 and 8 times per day) for 40 days. Total food intake increased with feeding frequency. Feeding four times per day resulted in the highest growth and food conversion efficiency. Total lipid accumulation in the muscle, liver and intraperitoneal fat body (IPF) increased with feeding frequency. Red-spotted grouper were found to contain a high proportion of docosahexaenoic acid (DHA) in the muscle. Fish fed 0.5 times per day were characterized as having a high proportion of DHA in triglycerides (TG) in muscle (80%), liver (69%) and IPF (18%). Increasing saturated fatty acids and a relative reduction of DHA were observed in the muscle TG as the feeding frequency increased. Frequent feeding accelerated the accumulation of eicosapentaenoic acid and saturated acids in the liver and IPF. Low saturated acids and a subsidiary proportional increase in DHA were the result of the low lipid reserves. A possible role for DHA and saturated acids are discussed with regard to lipid reserves.     

Effects of aromatizable and nonaromatizable androgens on the sex inversion of red-spotted grouper (Epinephelus akaara)

The effects of aromatizable 17α-methyltestosterone (MT) and non-aromatizable 17α-methyldihydrotestosterone (MDHT) on sex inversion in red-spotted grouper, Epinephelus akaara, were investigated. Fish were implanted with MT, MDHT and MT+AI (aromatase inhibitor, AI) respectively for one month. The results showed that the three treated groups turned into transitional stage with intersex gonads, which contained atretic oocytes and spermatogenic germ cells at all stages of spermatogenesis. The controls did not change sex. The gonads of more than half MT-implanted fish were in early transitional stages of sex inversion, whereas those of more than half MDHT and MT+AI-implanted fish were in late transitional stages of sex inversion. No difference in serum estradiol-17β (E₂) levels between the controls and the treated groups were observed, whereas 11-ketotestosterone (11-KT) and testosterone (T) levels increased in all treated groups. Significantly lower gonadosomatic index (GSI) and gonadal aromatase activity were observed in the treated groups, which were in accordance with the lower mRNA expression of P450aromA. However, P450aromB mRNA expression increased in the MT group, while it did not change in the MDHT group. These results suggest that the sex inversion of red-spotted grouper by MT and MDHT implantation might be due to the suppression of P450aromA gene expression, and resulting in both the decrease of the ovarian estrogen –secretion, as well as the increase in the 11-KT levels. Furthermore, the main reason for MT being less effective than MDHT might be due to partial aromatization of MT to estrogen.     

湛江沿海五种石斑鱼线粒体DNA的RFLP分析

In this paper, genetic diversity of intraspecies, and genetic relationship of interspecies in Epinephelus spp. (E. merra, E. fario, E. awoara, E. akaara and E. septemfasciatus) were assessed by using mitochondrial DNA restriction fragment length polymorphisms (mtDNA RFLPs).The samples were collected from the coastal area of Zhanjiang,Guangdong province. MtDNA was extracted from the fresh liver tissue by applying a difference centrifugation procedures. Using 17 restriction enzymes with 5-or 6-bp recognition sites, the purified mtDNA was cleaved by single enzymes. These enzymes included BamH Ⅰ，Bgl Ⅰ，Bgl Ⅱ，Dra Ⅰ，EcoR Ⅰ，EcoR Ⅴ，Hind Ⅲ，Kpn Ⅰ，Mlu Ⅰ，Pst Ⅰ，Pvu Ⅱ，Sal Ⅰ，Sca Ⅰ，Sma Ⅰ，Sty Ⅰ，Xba Ⅰ and Xho Ⅰ. The phylogenetic analysis was done using the Neighbor joining(NJ) method and Unweighted pairgroup method with arithmetic mean(UPGMA) method. Genetic diversity indices such as haplotype diversity (h), average genetic distance between haplotypes (P) and nucleotide diversity (π) were calculated using Nei and Li's segment method to quantify the genetic diversity within species. There were 8, 5, 8, 5 and 2 haplotypes detected within E. merra, E. fario, E.awoara, E.akaara and E. septemfasciatus, respectively. The haplotype diversity (h) was 0.8943, 0.6186, 0.9242, 0.6927 and 0.1820,respectively. The average genetic distance between haplotypes (P) was 0.62%±0.31%, 0.64%±0.37%, 1.12%±0.55%,0.72%±0.42% and 0.45%, respectively. And the nucleotide diversity (π) was 0.22%, 0.13%, 0.46%, 0.17% and 0.04%, respectively. The wild groupers in the Zhanjiang Coastal Area exhibited a relative higher level of genetic diversity. The net genetic distance between species (P_net) was 0.0694（E. merra - E. fario）,0.1337（E. merra - E. awoara）,0.1090 E. merra -E. akaara）, 0.1286（E. merra - E. septemfasciatus）,0.1590（E. fario -E. awoara）,0.0825（E. fario -E. akaara）,0.1153（E. fario - E. septemfasciatus）,0.1131 E. awoara - E. akaara）,0.0724（E. awoara -E. septemfasciatus） and 0.1336（E. akaara - E. septemfasciatus）. Both NJ and UPGMA methods yielded an identical phylogenetic tree for the five species. The E. merra and E. fario first clustered together, then joined with E. akaara, and finally clustered with E. awoara and E. septemfasciatus.     

Histological observations of seasonal reproductive and lunar-related spawning cycles in the female honeycomb grouper Epinephelus merra in Okinawan waters

Seasonal reproductive and lunar-related spawning cycles of the female honeycomb grouper Epinephelus merra inhabiting Okinawan waters were examined by histological observation of ovaries. Gonadosomatic index (GSI) increased beginning in May and peaked in June. Histological observations revealed that many oocytes laden with yolk were present in the ovaries from May to August. From September to October, ovaries were occupied by immature oocytes. These results suggest that the reproductive season of E.   merra lasts for 4 months from May through August in Okinawan waters. When the fish were collected according to the lunar cycle, GSI increased with the approach of the full moon. Oocytes at various development stages were observed from the first quarter to the full moon. Fresh ovulatory follicles were present in the ovaries around the last quarter moon. These results suggest that E.   merra has a lunar spawning cycle and spawns between the full moon and the last quarter moon. Ovarian features of the fish collected around the last quarter moon were different among individuals; some fish had many oocytes at the tertiary yolk stage in the ovaries, while the ovaries of the others were occupied by the oocytes at the peri-nucleolus and the oil droplet stages. This observation suggests that a minor release of eggs occurs in this species before or after a major spawning lunar day. Oocytes at the migratory nucleus and the maturation stages were not observed in any ovarian samples. This may mean that maturation of oocytes is related to the spawning behavior of this species and makes rapid progress in the process of aggregation at the spawning sites.     

Isolation and characterization of a regulator of G protein signaling (RGS16) gene homologue in yellow grouper Epinephelus awoara

Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAP) which act as modulators of G protein-coupled receptors. We isolated a RGS16 homologue in yellow grouper (Epinephelus awoara) spleen using suppression subtractive hybridization and RACE-PCR. The nucleotide sequence of yellow grouper RGS16 full-length cDNA was 700 bp and contained an open reading frame of 537 bp, encoding a putative protein of 178 amino acids. The encoded protein shows 47–61% identities to other homologues. RT-PCR analysis demonstrated that RGS16 was expressed in yellow grouper spleen and up-regulated in kidney heart, liver, and anterior kidney by lipopolysaccharide. This study will help towards validating the specific function of RGS in marine fish.