Embryonic and larval development of honeycomb grouper Epinephelus merra Bloch

Spawning and successful rearing of larvae of honeycomb grouper Epinephelus merra Bloch 1793 upto juvenile stage was accomplished at the finfish hatchery of Mandapam Regional Centre of Central Marine Fisheries Research Institute during 2004. The fertilized eggs were free, spherical and buoyant with size ranging from 710 to 730 μm. Complete early embryonic development took place within 24–27 h and hatching occurred. The hatchlings measured 1.5 mm. Mouth opening (115 μm) appeared at 72 h when the larvae were 2.2 mm in size. Pectoral fin developed on the fifth day. Complete metamorphosis took place and by the 60th day the larvae transformed into juveniles (45 mm) and attained skin colouration and honeycomb pattern.     

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.     

Treatment with an aromatase inhibitor induces complete sex change in the protogynous honeycomb grouper (Epinephelus merra)

To study the mechanism of protogynous sex change in honeycomb grouper, the wild fish of both sexes and transitionals were collected for one year, and changes in gonadal structures and serum levels of sex hormones in each individual were examined. The onset of sex change was associated with low serum estradiol - 17β (E2) levels. In order to clarify whether E2 deprivation causes sex change, maturing females were implanted with the non-steroidal aromatase inhibitor (Fadrozole, AI). AI caused complete sex change from female to functional males in two and half months. The present results suggest that E2 is an important endogenous factor which regulates protogynous sex change in the honeycomb grouper.     

Natural spawning,embryonic and larval development of F2 hybrid grouper,tiger grouper Epinephelus fuscoguttatus × giant grouper E. lanceolatus

This study aims to reveal the first report of the natural spawning of F1 hybrid grouper (TGGG), a crossbreed between the tiger grouper, Epinephelus fuscoguttatus × giant grouper, E. lanceolatus, since its first production in 2006. This marks the completion of its full cycle after a 10-year period. In order to establish a seed rearing protocol for a novel F2 hybrid TGGG, natural spawning, embryonic and larval developments were thoroughly observed. Five batches of natural spawning were recorded with an average of 1.50–15.3 kg eggs collected, while fertilization and hatching rates were recorded at 85.3–97.6%, and 63.0–98.3%, respectively. F2 larvae hatched out at 17:50 hours with an average body size of 1.74 ± 0.01 mm, and a yolk sac volume of 0.85 ± 0.197 mm3. The first feeding was initiated 3 days after hatching, which coincided with the onset of functional feeding apparatus and active swimming behavior. Larval dorsal and pelvic spines were formed at 6 days AH coupled with dynamic feeding activity, as more food was found in the digestive tract. Meanwhile, the F2 hybrid grouper shifted habitat from pelagic to benthic as early as 25 days AH, and entered a juvenile stage at 35 days AH, attaining a skin coloration similar to that of the F1 juvenile. This study concluded that naturally spawned eggs of F2 hybrid TGGG were exceptionally high in quality, although larvae were small and fragile, and performed vigorous feeding activities and cannibalistic behavior. Thus, these findings can serve as primary data to further develop the optimal rearing protocol to enhance the overall rearing performance.     

Molecular cloning of gonadotropin cDNA in sevenband grouper, Epinephelus septemfasciatus

The cDNA for glycoprotein hormone (GPH) α-subunit, β-subunits of the FSH (GtH I) and LH (GtH II) were isolated, cloned, and sequenced from sevenband grouper pituitary using RACE PCR. These results show that sevenband grouper has two different types of gonadotropins FSH (GtH I) and LH (GtH II), as other teleosts.     

石斑鱼虹彩病毒病发生风险评估模型的建立和验证

为评估石斑鱼养殖过程虹彩病毒病发生的风险,实验结合Delphi法和层次分析法(analytic hierarchy process,AHP)构建新加坡石斑鱼虹彩病毒(SGIV)发生的风险评估模型.风险因素评估指标体系包括1个目标层(石斑鱼虹彩病毒病发生风险),5个准则层(水质、石斑鱼健康状况、饲养管理、养殖模式和养殖环...     

斜带石斑鱼幼鱼的饲料精氨酸需求量

通过配制7种含不同水平精氨酸(2.13%、2.42%、2.71%、2.95%、3.20%、3.48%、3.74%干物质)的等氮、等脂的实验饲料,研究精氨酸对斜带石斑鱼(Epinephelus coioides)幼鱼生长、饲料利用和血清一氧化氮合酶等的影响。每组设3个重复,每个重复30尾鱼(7.52±0.02g),饲喂8周。结果显示,饲料精氨酸水平对各组存活率无显著影响(P0.05);精氨酸水平为2.95%的斜带石斑鱼获得最大增重率和特定生长率,显著高于其他各组(P0.05);2.95%组饲料系数显著低于2.13%、2.42%和2.71%组(P0.05);3.20%组体蛋白达到最大,显著高于2.13%组(P0.05)。随着饲料中精氨酸水平的增加,斜带石斑鱼幼鱼血糖含量呈逐渐降低的趋势,2.13%与2.42%组显著高于其他各组(P0.05)。2.95%组血清总蛋白和血清总一氧化氮合酶显著高于其他各组(P0.05)。研究表明,日粮中适宜的精氨酸水平不仅能促进斜带石斑鱼幼鱼生长和体蛋白的合成,还能提高机体免疫力。以特定生长率为判据,通过二次多项式回归分析表明,斜带石斑鱼幼鱼精氨酸的最适需要量是饲料干重的3.06%(饲料蛋白含量的6.07%)。     

Iron bioavailability of different sources in juvenile grouper Epinephelus coioides

A 70‐day experiment was implemented to study the iron (Fe) bioavailability of three Fe sources in Epinephelus coioides (initial weight, 12.19 ± 0.14 g). Treatments consisted of 0, 50, 90, 130, 170 and 210 mg supplemental Fe/kg from iron sulphate (FeSO₄), ferric citrate (Fe‐citrate) and hydroxy methionine analogue iron (Fe‐MHA). The results showed that the growth performance was not affected by Fe level regardless of Fe sources. Hepatic catalase activity (CAT), the haematocrit value (Hct), haemoglobin (Hb), mean corpuscular volume (MCV) and Fe content in whole body or liver firstly increased significantly and then plateaued as Fe level increased regardless of the Fe source, except significant decreasing of Hct in 210 mg Fe/kg group from FeSO₄. Manganese (Mn) content decreased significantly and then kept stable in the whole body and liver whatever the source was. The slope‐ratio method using FeSO₄ as the reference and Hb content, CAT activity, body and hepatic Fe concentration as the response parameter showed the average relative bioavailability of Fe‐citrate and Fe‐MHA was 103.25 ± 4.35 (n = 4) and 134.5 ± 15.67 (n = 4), respectively, with the maximum value of Fe‐MHA in any case. In conclusion, Fe‐MHA was the most effective to meet preferable haematological index, CAT activity and mineral deposition, with little difference between Fe‐citrate and FeSO₄, athough Fe levels with different sources failed to influence the growth in grouper.     

Quantitative l-lysine requirement of juvenile grouper Epinephelus coioides

An 8‐week feeding trial was conducted to determine the quantitative lysine requirement of juvenile grouper Epinephelus coioides (initial mean weight: 15.84 ± 0.23 g, mean ± SD) in eighteen 500‐L indoors flow‐through circular fibreglass tanks provided with sand‐filtered aerated seawater by feeding diets containing six levels of l ‐lysine ranging from 19.2 to 39.5 g kg^?1 dry diet in 4 g kg^?1 increments. The diets, in which 250 g crude protein kg^?1 diet came from fish meal and soybean protein concentrate, and 230 g kg^?1 from crystalline amino acids, were formulated to simulate the amino acid profile of 480 g kg^?1 whole chicken egg protein except for lysine. Each diet was assigned to three tanks in a completely randomized design. Grouper were fed to apparent satiation twice daily during the week and once daily on weekends. Weight gain and specific growth rate increased with increasing levels of dietary lysine up to 27.2 g kg^?1 (P < 0.05) and remained nearly the same thereafter (P > 0.05). Feed efficiency was the poorest for fish fed the lowest lysine diet (P < 0.05) and showed no significant differences among other treatments (P > 0.05). Survival could not be related to dietary treatments. Body composition remained relatively constant except for lipid contents in muscle and liver. Total essential amino acid contents in liver increased with dietary lysine level although there was a slight decline for fish fed the highest lysine level of diet. Plasma protein content increased with increasing dietary lysine level (P < 0.05), but cholesterol, triacylglycerol and glucose contents were more variable and could not be related to dietary treatments. Dietary lysine level significantly influenced morphometrical parameters (condition factor, hepatosomatic index and intraperitoneal fat ratio) of juvenile grouper (P > 0.05). Broken‐line analysis of weight gain indicated the dietary lysine requirement of juvenile grouper to be 28.3 g kg^?1 diet or 55.6 g kg^?1 dietary protein.     

七带石斑鱼染色体核型研究

采用PHA体内直接注射法制备了七带石斑鱼(Epinephelus septemfasciatus)头肾组织染色体标本并分析其核型。结果表明,七带石斑鱼二倍体染色体数为48,24对染色体均为端部着丝粒染色体,其染色体臂数(NF)为48,核型公式为:2n=48t,NF=48。未发现与性别相关的异型染色体,第24对染色体长度明显小于其他染色体。通过与其他22种石斑鱼染色体核型进行比较,发现七带石斑鱼具有石斑鱼属鱼类的原始核型特征,属于石斑鱼属鱼类的原始类群。     

Conditioning,maturation and year‐round natural spawning of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), in recirculating aquaculture system

The present experiment was aimed at studying the conditioning, maturation and natural spawning of orange‐spotted grouper, Epinephelus coioides, in a recirculatory aquaculture system (RAS). Thirty fish (n = 30; 3.35 ± 0.05 kg) were stocked in a circular tank of 125 m³ capacity fitted with an RAS for conditioning and broodstock development. After 15 days, 15 fish were implanted with 17 α methyl testosterone and letrozole at the rate of 5 mg and 0.2 mg/kg body weight, respectively, for conversion from female to male. The gonadal development started after 1 month, and by 90th day, 63.53 ± 3.78% and 2.07 ± 0.84% of the oocytes attained a size of 400–500 μm and 500–600 μm respectively. Natural spawning commenced in the RAS from 4th month onward after stocking and spawning continued round the year. The spawning pair showed courtship behaviour with a typical vertical burst of swimming just before release of gametes. The total number of eggs spawned during 1 year was 47.23 million with spawning frequency varying form 5 to 13 times per month. The association of spawning events with new moon day (lunar cycle) weakened as time progressed. The mean monthly fertilization and hatching rates varied from 77.80 ± 3.34% to 83.70 ± 1.76% and 82.80 ± 4.21% to 88.33 ± 1.39% respectively. The study proved that RAS is an efficient system that provides a stable, controllable and conducive environment for year‐round natural breeding of orange‐spotted grouper.     

七带石斑鱼的形态和生物学性状

2010年在广东省饶平县柘林湾进行了七带石斑鱼（Epinephelus septemfasciatus）海水网箱人工养殖研究,观察和研究了人工养成的七带石斑鱼的形态特征和生物学性状。通过观察、测量、解剖,对114尾七带石斑鱼的外部形态、可量比性状和内部结构进行了研究。结果表明,七带石斑鱼体呈椭圆形,侧扁,体侧具7条褐色横带,第1条横带仅伸达主鳃盖骨上方边缘,第6条横带分成2条,体侧横带具有不规则的白色斑纹,各鳍边缘白色,背鳍鳍棘部褐色和白色横带相间,鳍条部具有由白色横带延伸而成的3个白斑。胃呈卜型,幽门盲囊发达;肠道长度为体长的0.7～1.4倍,与七带石斑鱼为肉食性鱼类的特征相一致。     

Determination of sex and gonadal maturity in the honeycomb grouper, <Emphasis Type="Italic">Epinephelus merra</Emphasis>, through biopsy

Determination of the sex and gonadal maturity of fish is necessary for successful commercial aquaculture operations. Sex identification is problematic in grouper fish, because they change sex at certain ages or sizes. Here we represent an accurate and reliable method for sex determination of the live grouper, using 5–10 mg gonadal tissue samples, harvested using non-lethal gonadal biopsy. Sex and sexual maturity of biopsied individuals were determined using standard histological method. All biopsied fish survived and there were no serious infections resulting from the surgery. Therefore, this biopsy technique is a simple and cost-effective tool for grouper fishery management.     

Induced sex reversal of dusky grouper, Epinephelus marginatus (Lowe)

The present paper describes sex reversal in dusky grouper, Epinephelus marginatus (Lowe). Over a 4-month feeding period, 5 mg kg^?1 of the male hormone 17-α methyltestosterone was combined with frozen fish as feed. After 2.5 months of treatment, the gonads of all six fish treated in the experiment presented tissues with oocytes and spermatogonia, and after 3 months, there were only a few remnants of oogonial tissue left in the gonads. All fish in the experiment gave sperm of varying quality after 4 months of the trial. The quantity of the hormone administered to the fish over these 4 months was 89-113 mg kg^?1. Spermatozoid motility varied from 95% to 40%, and lasted for about one hour in sea water. Using the combined sperm of three males, fertilization ranged from 50% to 95%. Fertilized eggs and hatched larvae continued to develop normally.     

斜带石斑鱼3种致病性弧菌的分子生物学鉴定

从患病斜带石斑鱼分离到3株病原菌EcGS020802、EcGS021001、EcGS020801,经常规生理生化鉴定均属于弧菌属的种类。为了进一步确定其分类地位,测定了3株病原菌的16SrRNA和HSP60(heat shock protein,HsP60)基因部分序列。16SrRNA基因系统进化分析表明,3株病原菌与哈维氏弧菌、溶藻弧菌、副溶血弧菌亲缘关系较近,相互之间同源性均大于98．6％,差异不明显。HSP60基因序列分析表明,菌株EcGS020802、EcGS021001、EcGS020801 HSP60基因序列分别与哈维氏弧菌(AF230934)、溶藻弧菌(Ab230931)、副溶血弧菌(AF230951)HSP60基因的同源性最高,依次为95．7％、99．8％和99．8％,而与其它弧菌HSP60基因的同源性均低于90．6％,3株病原菌相互之间的同源性低于91．0％。HSP60基因构建的系统进化树表明,EcGS020802、EcGS021001、EcGS020801分别与Vibrio harveyi、Vibrio alginolyticus、Vibrio parahaemolyticus聚类。综合上述结果,菌株EcGS020802、EcGS021001、EcGS020801可分别鉴定为哈维氏弧菌、溶藻弧菌、副溶血弧菌。结果表明,HSP60基因比16SrRNA基因更适合用于海水鱼致病性弧菌种问的分类研究。     

Salinity and temperature tolerance of brown-marbled grouper Epinephelus fuscoguttatus

Grouper have to face varied environmental stressors as a result of drastic changes to water conditions during the storm season. We aimed to test the response of brown-marbled grouper to drastic and gradual changes in temperature and salinity to understand the grouper’s basic stress response. The results can improve the culture of grouper. Brown-marbled grouper, Epinephelus fuscoguttatus (6.2 ± 0.8 g) were examined for temperature and salinity tolerances at nine different environmental regimes (10, 20, and 33 ‰ combined with 20, 26 and 32 °C), in which the fish were subjected to both gradual and sudden changes in temperature and salinity. The critical thermal maximum (50 % CT_MAX) and the upper incipient lethal temperature (UILT) were in the ranges of 35.9–38.3 and 32.7–36.5 °C, respectively. The critical thermal minimum (50 % CT_MIN) and the lower incipient lethal temperature (LILT) were in the ranges of 9.8–12.2 and 14.9–22.3 °C, respectively. The critical salinity maximum (50 % CS_MAX) and the upper incipient lethal salinity (UILS) were in the ranges of 67.0–75.5 and 54.2–64.8 ‰, respectively. Fish at temperature of 20 °C and a salinity of 33 ‰ tolerated temperatures as low as 10 °C when the temperature was gradually decreased. Fish acclimated at salinities of 10–33 ‰ and a temperature of 32 °C tolerated salinities of as high as 75–79 ‰. All fish survived from accumulating salinity after acute transfer to 20, 10, 5, and 3 ‰. But all fish died while transferred to 0 ‰. Relationships among the UILT, LILT, 50 % CT_MAX, 50 % CT_MIN, UILS, 50 % CS_MAX, salinity, and temperature were examined. The grouper’s temperature and salinity tolerance elevated by increasing acclimation temperature and salinity. On the contrary, the grouper’s temperature and salinity tolerance degraded by decreasing acclimation temperature and salinity. The tolerance of temperature and salinity on grouper in gradual changes were higher than in drastic changes.     

斜带石斑鱼口服PELA-OmpK微球疫苗的示踪及免疫效果

采用可生物降解的合成高分子聚DL-乳酸-聚乙二醇共聚物(DL-polylactide-co-polyethylene glycol,PELA),包裹哈维氏弧菌(Vibrio harveyi,Vh)重组外膜蛋白OmpK后,制备成PELA-OmpK微球.微球粒径小于10μm,且94%粒径小于5μm,平均粒径为2.8μm.蛋白包裹率达79.4%.斜带石斑鱼(Epinephelus coioides)口灌四甲基异硫氰酸罗达明(TRITC)荧光素标记的OmpK-PELA微球后,第1天在其后肠观察到大量红色荧光微球,第3天荧光微球数量明显减少,第7天荧光微球基本消失.免疫组化研究结果表明,口服PELA-OmpK组在斜带石斑鱼后肠组织黏膜层可见较强的棕黄色阳性着色.口服PELA-OmpK组血清抗体效价(峰值2m)显著高于口服OmpK蛋白溶液组(峰值24)与对照组(P＜0.05),低于注射组(峰值212)(P＜0.05);口服PELA-OmpK组的黏液抗体效价显著高于口服OmpK蛋白组(P＜0.05).初次免疫30 d后用活菌攻击,口服PELA-OmpK组的相对保护率(62.5%)显著高于口服OmpK蛋白组(12.5%)(P＜0.05);低于注射组(87.5%)(P＜0.05).结论为采用可生物降解材料PELA作为鱼类口服疫苗的投递载体是可行的.     

Dietary nucleotide supplementation enhances growth and immune responses of grouper,Epinephelus malabaricus

Basal diet containing 0.5, 1.0, 1.5 and 2.0 g kg^?1 mixture of inosine monophosphate (IMP), adenosine monophosphate (AMP), guanosine monophosphate (GMP), uridine monophosphate (UMP) and cytidine monophosphate (CMP) (1 : 1 : 1 : 1 : 1) (mixed‐NT; Experiment 1) and 1.5 g kg^?1 from each nucleotides and mixed‐nucleotides (NT; Experiment 2) were fed to triplicate groups of grouper for 8 weeks. Basal diet without NT was used as control in both Experiments. In Experiment 1, fish fed the diet with 1.5 g mixed‐NT kg^?1 had higher (P < 0.05) weight gain (WG) than the control group. The superoxide anion (O₂^?) production ratio was higher in fish fed diets with 1.0–1.5 g mixed‐NT kg^?1 than the fish fed diets with ≤0.5 g mixed‐NT kg^?1. In Experiment 2, fish fed diets with nucleotides had higher WG than the control group. The O₂^? production ratio was higher in fish fed the diet with 1.5 g AMP kg^?1, followed by fish fed diets with 1.5 g UMP and mixed‐NT kg^?1, and lowest in the control group. These results suggest that growth and immune responses were enhanced in grouper fed diet with 1.5 g mixed‐NT kg^?1 diet. Diet with 1.5 g kg^?1 of AMP seems to be more beneficial on the immune responses in fish than other nucleotides.