大菱鲆仔鱼皮肤发育及超微结构

The histological development and ultrastructure of larval turbot skin were studied by both light and transmission electron microscopes.The early larvae showed a complete bisymmetry of skin.The skin of 1-25days old larvae developed slowly and was composed of the thin epidermis of 1-2layers epidermal cells and undeveloped dermis before the metamorphosis.But the skin developed significantly when the.metamorphosis began.Till the completion of metamorphosis(about 60days old),the skin contained 3-4layers epidermal cells and very developed collagenous strata.Wisth the process of metamorphosis,the ocular side skin and blind side skin became different in ultrastructure.Ultrastructural observation showed the epidermis of turbot contained three types of epidermal cells;filament-containing cells,mucus cells and chloride cells.The ocular side epidermis with looser structure consisted one type of filament-containing cells,but the blind side epidermis was structured densely and two types of filament-containing cells were observed in it.Melanophores,iridophores,fibroblasts and other cell types and tisssues were found in the spongiosum of dermis.The distribution of melanophores depended on the larval developing stage and the pigmentation of skin.The differentiation of skin seems to be an adaptation for the change of life style of turbot larvae from pelagic living to benthonic living after the metamorphosis.     

Transplantation of pigmented and non-pigmented scales into the ocular and blind sides of the Japanese flounder Paralichthys olivaceus,suggesting the presence of ocular-side characteristic inducer in pigmented scales

After metamorphosis, both eyes of the Japanese flounder Paralichthys olivaceus are located on its left side, with only the ocular side becoming pigmented. Staining, or hypermelanosis, occurs on the blind side at 2–3 months after metamorphosis, thereby lowering the market price of the fish. To understand the pigmentation expansion process, we performed scale transplantation between the blind and ocular sides of an individual. About 40 % of transplanted scales were successfully engrafted, regardless of donor or recipient site. When blind-side scales were transplanted to the ocular side, they became pigmented after 2 weeks, while no change was observed when the scales were transplanted to the blind side. Ocular-side scales did not lose pigment, regardless of the recipient site. However, after removal of transplanted ocular-side scales, pigmented scales regenerated after 3 weeks, even at blind-side sites. Identical results were obtained when the stained area on the blind side was used as the recipient location. When an ocular-side scale with skin tissue was inserted under blind-side scales, the scales immediately above the transplanted area became pigmented, whereas ocular-side scales stripped of tissue did not induce pigmentation. These results strongly suggest the presence of an ocular-side characteristic inducer in pigmented scale tissues.     

Influence of lipid composition of live feed on growth,survival and pigmentation of turbot larvae

The effect of different lipid compositions of live feed on the survival, growth rate and pigmentation success of turbot larvae, Scophthalmus maximus (L.), was investigated. Rotifers, Brachionus plicatilis, together with the algae Tetraselmis sp., were administered until day 12, and Artemia was fed until day 27. The experimentally treated live feeds were enriched with four formulated emulsions, resulting in a gradient in the relative contents of 3 HUFA (highly unsaturated fatty acids) and in DHA (docosahexaenoic acid, 22:6 3)/EPA (eicosapentaenoic acid, 20:5 3) ratios in both the rotifers and Artemia.There were no differences in larval growth rate, and only small differences in survival rate throughout the feeding experiment, probably because of satisfactory levels of 3 HUFA in the live feed to sustain growth and survival. A correlation was obtained between the percentage of completely pigmented 27 d old turbot and the DHA/EPA ratio in the total lipids of 12 d old larvae, which again was correlated with the corresponding ratio in the live feed used. The results suggest that normal pigmentation in turbot requires dietary DHA in the early larval feeding period, and that this requirement cannot be replaced by EPA.     

锦鲤酪氨酸酶基因序列分析及在不同锦鲤品系的组织表达

为了解色素控制基因与体色分布的关系,实验用分子克隆技术得到了锦鲤酪氨酸酶(tyrosinase, Tyr) cDNA 基因序列, 并利用半定量PCR的方法分析了酪氨酸酶基因在锦鲤不同品系和组织中的表达差异。实验得到了长约1 779 bp锦鲤酪氨酸酶cDNA基因, 其包括长1 608 bp开放阅读框, 编码535个氨基酸。cDNA、蛋白水平序列分析和系统发育分析都表明酪氨酸酶在鱼类间的保守性要高于鱼类与其他脊椎动物间的保守性。半定量分析研究显示,酪氨酸酶基因在皮肤、肝、心、脑和眼中都有表达, 其中眼部和黑色皮肤表达最高, 其次是脑, 红色和黄色皮肤, 肝和心的表达最低。不同品系同组织间眼、肝、心、脑的酪氨酸酶基因表达差异不显著, 但是在皮肤中有差异表达, 黑色皮肤表达最高, 其次是红色和黄色皮肤, 在白色皮肤中也有少量表达。酪氨酸酶基因在非黑色组织中有较高表达可能与存在多种形式酪氨酸酶有关, 也有可能非黑色素细胞能转化为黑色素细胞。     

Ocular bacterial signatures of exophthalmic disease in farmed turbot (Scophthalmus maximus)

As is the case at other sites in the body (e.g. the gut, skin and mouth), the ocular microbiota plays a crucial role in their host, as disturbances of the composition and function of the ocular microbiota are known to be associated with ocular disorders. Exophthalmic disease (ED) is a significant cause of high mortality in fish species, including farmed turbot (Scophthalmus maximus). However, the relationship between alterations in the ocular microbiota and ED in turbot is unclear. In this work, we collected turbot samples from farmed ponds with ED and healthy samples to understand changes in the ocular microbiota of turbot suffering from ED. We compared the structural and metabolic differences of ocular bacterial communities from farmed turbot with exophthalmic disease and those of healthy controls. Besides less microbial diversity found in turbot with ED regarding the control group, we also found that Aeromonas was the dominant bacteria both in controls and ED samples, but the abundance of Aeromonas was significantly greater in ED individuals. Moreover, the results of correlation test further suggest that Aeromonas overgrowth was correlated with the progress of the disease and shifts in ocular microbiota functional pathways in turbot. These findings emphasize that an increased abundance of Aeromonas serves as an ocular bacterial signature associated with ED in turbot, which provide basic information useful for diagnoses, prevention and treatment of ocular diseases occurring in cultured fish.     

Comparative composition and shelf-life of fillets of wild and cultured turbot (Scophthalmus maximus) and Atlantic halibut (Hippoglossus hippoglossus)

Turbot and Atlantic halibut are highly valued fish species. However,very little is known about fillet shelf-life characteristics associated withboth species. Thus, fillet -tocopherol content and proximate compositionof wild turbot (1.5 kg) and Atlantic halibut (1.1 kg)caught off the south coast of Ireland and the north-west coast of Iceland,respectively, were investigated. In addition, the susceptibility of fillets, storedunder retail conditions, to lipid oxidation and colour change was studied.Proximate composition analysis showed that turbot had significantly highermoisture (P < 0.001) and lower protein (P < 0.001) contents compared toAtlantic halibut. Atlantic halibut incorporated significantly higher (P <0.001) levels of -tocopherol into fillets than turbot. Over 14 days ofstorage on ice, fillets from Atlantic halibut exhibited significantly lower (P =0.020) levels of lipid oxidation than those of turbot. However, malondialdehyde(MDA) concentrations were generally very low, never exceeding 0.6 gg^–1 fillet. Turbot maintained a significantly higher (P< 0.001) pH over the storage period. The lightness (L^* values) offillets from both species increased over 14 days of storage, but wassignificantly higher (P < 0.001) in Atlantic halibut than in turbot. Turbotdeveloped a relatively intense yellow colour during storage (decrease in hueangle and increase in b^* values), whereas this was not the case forAtlantic halibut. The results of this study demonstrate that fillets of wildAtlantic halibut stored on ice, were less prone to lipid oxidation anddiscolouration than those of wild turbot. However, quality changes in turbotwere very small showing that both fish have tremendous shelf-life capacities interms of lipid oxidation. These findings are considered in the context of knownmaterial for farmed fish.     

大菱鲆子二代家系白化与正常幼鱼生长及形态学差异

白化现象是大菱鲆(Scophthalmus maximus)生产中存在的主要问题之一.2009年,采用大菱鲆育种标准化苗种培育方案培育大菱鲆子二代全同胞家系32个,选取其中白化较为严重的16个全同胞家系(包括6对半同胞家系组合)作为样本家系,抽样测定统计各家系85日龄(dph)幼鱼的形态指标,比较了各全同胞家系白化和正常鱼的形态特征,以及全同胞家系间生长的差异.大菱鲆幼鱼体色分为完全正常、局部白化和完全白化3个类型,白化率统计时将部分白化幼鱼归于白化幼鱼一类.16个全同胞家系的白化率处于17.2％～86.7％范围内,6对半同胞家系组合的白化率差别较大,姊妹家系间白化率差值0 ～ 46％,因此认为白化可能受到母系遗传或卵子质量的影响.多数家系的白化与正常幼鱼的全长、体长、总高、体高、全重差异不显著(P＞0.05).16个全同胞家系样本中,除2个家系外,其余家系的白化与体色正常鱼的可比性状差异不显著(p＞0.05).将各全同胞家系抽样的全部90尾鱼的全重平均值作为指标进行比较,发现全重的生长差异在全同胞家系间表现较为明显.因此,白化鱼与原色鱼的生长和身体形态整体上不存在显著差异.本研究还获得速生候选家系1个,高体型候选家系3个.根据研究结果分析大菱鲆白化现象发生的原因,认为虽然遗传不是大菱鲆白化与否的决定因素,但却是一个重要因素.     

半滑舌鳎酪氨酸酶基因(TYR)和多巴色素异构酶基因(DCT)的克隆表达与分析

作为我国鲆鳎鱼类中的一种主要养殖品种,半滑舌鳎(Cynoglossus semilaevis)有时会发生无眼侧黑化(Melanism)、有眼侧白化(Albinism)的体色异常现象。本研究克隆了半滑舌鳎体色相关的酪氨酸酶基因(TYR)和多巴色素异构酶基因(DCT)的cDNA序列,并对这2个基因进行系统发育分析和时空表达分析。实验获得了TYR基因编码区cDNA序列长度为1620 bp,编码539个氨基酸;DCT基因编码区cDNA序列长度为1551 bp,编码516个氨基酸。结果显示,TYR和DCT在20日龄前的鱼苗体内表达量较高,尤其是在变态关键时期(15~20日龄)表达量最高,30日龄时的表达量锐减到很低水平;在其他时期的皮肤组织中,这2个基因在有眼侧正常皮肤和无眼侧黑化皮肤的表达量最高,在有眼侧白化皮肤和无眼侧正常皮肤中表达量极低;在其他时期的其他组织中,在眼睛中的表达量最高,其次是肝脏,在脾脏和肌肉中表达量极低。研究表明,TYR和DCT基因是半滑舌鳎无眼侧黑化发生和有眼侧体色维持的关键基因。本研究为查明半滑舌鳎体色异常机制提供了重要依据和参考。     

A sandwich ELISA to detect VHSV and IPNV in turbot

The recent demonstration that reared turbot (Scophthalmus maximus L) is a natural host for salmonid rhabdoviruses has made their rapid detection relevant to these fish species. A unique protocol to select and use non-competitive monoclonal antibodies (Mabs) for two high-sensitivity sandwich ELISAs has been developed to detect both infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) in turbot kidney extracts to assess the possibility of using them in field diagnosis. For maximum sensitivity, turbot kidney extracts can be two-fold diluted with high-ionic strength buffers and assayed for the presence of the major viral proteins (VMS rhabdovirus nucleoprotein N/Nx and/or IPN birnavirus protein VP3). The use of control plates coated with irrelevant mouse antibodies (IgG1 and IgG2a) in parallel ELISAs allows for a precise estimation of possible false positives. Turbot kidney extracts with low levels of virus might now be assayed directly without using cell culture, with high precision and in a short time during the acute phase of these viral diseases in reared turbot.     

Performance of Albino and Normal Channel Catfish (Ictalurus punctatus) in Different Water Temperatures

Experiments to determine effects of skin colour on growth in different water temperatures were conducted with albino and normally pigmented channel catfish (Ictalurus punctatus). In Experiment 1, fish were reared in 29°C, 27°C, or 22°C water from hatching to 40 weeks of age. Two additional groups were initially reared in 27°C water for either 12 or 16 weeks and in 22°C water for either the remaining 28 or 24 weeks, respectively. Results indicated that: (1) albino fish reared in 29°C water were superior in growth to normal fish; (2) albino fish did not differ from normal fish in growth when reared in either 27°C or 22°C water; (3) albino fish were inferior in growth to normal fish when reared in 27°C water for either 12 or 16 weeks of the 40-week growth period; (4) genetic differences between families and the age of fish strongly influenced relative growth of both colour types; and (5) both albino and normal catfish were able to compensate in growth at a later age. Experiment 2 began when fish were 48 weeks old and included both albino and normal fish which were either full-siblings or not genetically related. Results indicated that: (1) albinos and normal catfish did not respond differently to a 5°C water temperature change; (2) genetically related and randomly selected albino and normal fish produced consistent results concerning water temperature, skin pigmentation, and sex effects on growth; (3) increasing water temperature from 22°C to 27°C for 19 weeks resulted in increased weight and length of the fish by 144 and 80%, respectively; and (4) growth increase during the first 19-week period resulting from the higher water temperature was followed by a significant increase in the subsequent 10-week growth when the fish were in the lower water temperature.     

Lipid and fatty acid compositions of muscle from farmed and wild adult turbot

The aim of this study was to compare the total lipid, neutral lipid, phospholipid contents, phospholipid class distribution and fatty acid composition of these lipids in muscle of wild and farmed turbot (Scophthalmus maximus) of similar body size. The results showed that muscle of farmed turbot had a higher lipid content than that of wild turbot (1.06% versus 0.64%) because they contained more neutral lipids (0.52% versus 0.24%) and more phospholipids (0.54% versus 0.40%).Distribution of phospholipid classes in muscle of both wild and farmed turbot was similar with 67–70% phosphatidyl choline, 22–25% phosphatidyl ethanolamine and 4.4–5.2% phosphatidyl inositol plus phosphatidyl serine and 2.4–2.9% sphingomyeline.Compared to wild turbot, all of the lipid classes from farmed turbot contained lower proportions of long chain -3 PUFA and 20:4 -6 and conversely higher proportions of 20:1 and 22:1. The ratio between -3 and -6 polyunsatured fatty acids was higher in all the lipid fractions of wild turbot than in those of farmed fish (8.8 versus 4.2 in total lipids, 11.0 versus 3.3 in neutral lipids, 7.9 versus 4.7 in phospholipids). © Rapid Science Ltd. 1998     

Protein kinase C in the spleen of the turbot (Scophthalmus maximus L.)

PKC activity was detected in spleen extracts from the turbot, Scophthalmus maximus, a teleost flatfish that is farmed commercially in several countries, in assays with the substrate EGF- R651–658 as phosphate acceptor. The activity was purified about 700-fold by a three-step chromatographic procedure (DEAE-cellulose, phenyl-Sepharose and threonine-Sepharose). Maximal activity was obtained in the presence of the typical PKC cofactors Ca2+ (0.1 mM) PtdS (20 g ml–1) and either DAG (2 g ml–1) or PMA (2 g ml–1). Activity was dose-dependently inhibited by H7 and by the PKC-specific inhibitors PKC19–36 and N-myristoylated PKC19–31. The rate of phosphorylation was highest with the PKC-specific substrate MARCKS161–175. In immunoblotting, MC5 (a mouse monoclonal antibody raised against bovine PKC) recognized bands of 80 and 100 kDa. Immunoblotting with antibodies raised against mouse PKC isozymes (, , , , , , and ) indicated the presence of all these isozymes in turbot spleen.     

Reproductive Performance of Pigmented and Albino Female Channel Catfish Induced to Spawn with HCG or Ovaprim

Reproductive performance of albino and pigmented channel catfish ( Ictalurus punctarus ) females was compared when spawning was induced with HCG (human chorionic gonadotropin) or Ovaprim ³ (domperidone and [d-Ala⁶, Pro⁹ NEt]-luteinizing hormone-releasing hormone). In 1985, only HCG was used to induce spawning. Albino females required more injections, had a longer latency period from time of first injection to spawning, and had lower spawning success (30%) than pigmented females (77%). No differences were observed between albino and pigmented females for time-of-day of spawning, number of eggs obtained by manual stripping, and number of eggs/kg body weight. In 1988, HCG, Ovaprim, and saline (control) were used to induce spawning. HCG induced spawning in 80% of albino and 71% of pigmented females. Ovaprim induced 67% of pigmented females to spawn, but only 2 of 5 albino females spawned. One of four pigmented females injected with saline spawned. Albino females yielded 40–50% fewer eggs/kg body weight than pigmented females. Other performance characteristics for albino and pigmented females were similar whether injected with HCG or Ovaprim. High temperatures encountered during the 1985 spawning season my have had a differential effect on albino and pigmented fish and may account for some differences in spawning success.     

正常和白化褐牙鲆皮肤和肌肉中DHA和EPA含量的比较

利用气相色谱法对相同养殖条件下生长的正常和白化褐牙鲆(Paralichthys olivaceus)的皮肤和肌肉组织中的DHA和EPA含量进行了比较分析。结果显示，正常和白化褐牙鲆的皮肤和肌肉中EPA含量相近，但白化个体皮肤中DHA含量显著低于正常个体，约为后者的1／2。而两者肌肉中的DHA含量则没有显著差异。同时，白化个体皮肤和肌肉组织中的DHA／EPA比值略低于正常个体。这一结果证明白化褐牙鲆皮肤蓄积或利用DHA的能力要低于正常褐牙鲆。关于鲆鳔类白化现象同鱼体对饵料中脂肪酸类物质的利用率之间的关系有待深入研究。     

半滑舌鳎(Cynoglossus semilaevis) 2种视黄酸受体RARα和RARγ克隆及组织表达特性

利用RT-PCR和RACE方法获得了半滑舌鳎(Cynoglossus semilaevis) 2种视黄酸受体RARα和RARγ的cDNA全长序列,并采用定量PCR技术分析了其组织表达特性。半滑舌鳎RARα cDNA序列全长为1823 bp,编码443个氨基酸;RARγ cDNA序列全长为1959 bp,编码489个氨基酸。同源性分析显示,半滑舌鳎RARα和RARγ同源性高达60.8%,与牙鲆(Paralichthys olivaceus)同源性高达97.0%,具有较强的进化保守性。系统进化分析显示,半滑舌鳎RARα和RARγ分别归属于单独的分支,且与其他鱼类聚合成簇。组织表达分析显示,RARα mRNA在肾中表达量最高,而RARγ mRNA在脾中表达量最高,RARα和RARγ mRNA在其他组织中均有表达,表明半滑舌鳎2种RAR都可能参与多种生理过程调控。半滑舌鳎2种RAR mRNA在有眼侧皮肤、无眼侧黑化皮肤和无眼侧正常皮肤中的表达量依次升高,且发现RARα在正常有眼侧皮肤的表达高于RARγ,而RARγ在无眼侧正常皮肤中的表达显著高于RARα,无眼侧黑化皮肤中RARα表达高于RARγ。RAR基因在有眼侧和无眼侧皮肤组织中的差异表达可能和RA/RAR系统调节体色有关。     

Microbially matured water: a technique for selection of a non-opportunistic bacterial flora in water that may improve performance of marine larvae

Before transfer to larval incubators, water was membrane filtered to remove >95% of the bacteria and then transiently maintained in a biofilter that promoted recolonization of the water by non-opportunistic bacteria. The process is termed microbial maturation of the water. Hypothetically the bacterial flora in the matured water should protect the marine larvae from colonization and proliferation by opportunistic bacteria. Testing of the hypothesis demonstrated 76% higher survival of yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus) in matured than in membrane filtered water. Proliferation of opportunistic bacteria was observed in the rearing water after hatching of turbot eggs (Scophthalmus maximus), but to a less extent in the microbially matured water. In the early phase of first feeding of turbot larvae, the matured water induced qualitative differences in the gut microflora. Significantly higher initial growth rate of the turbot larvae in the matured water affected 51% higher average weight of 13 days old larvae than in membrane filtered water. Algal addition to the matured water enhanced the larval growth further. The experiments conducted supported the proposed hypothesis that microbial maturation selects for non-opportunistic bacteria, which protects the marine larvae from proliferation of detrimental opportunistic bacteria.     

圆斑星鲽仔稚鱼色素细胞发育和体色变化

为研究圆斑星鲽早期发育过程中色素细胞的形态和分布,实验对1~72日龄的圆斑星鲽鱼苗进行了连续的显微观察,并绘制了早期发育生长曲线。结果显示,圆斑星鲽从20日龄开始进入快速的增长期,在水温12~19.5°C时完成变态发育需要50 d,黑色素细胞最早出现,数量最多,黄色素细胞次之,虹彩细胞最后发育,数量最少。变态前黑色素细胞密度先升高后降低,在9日龄密度最大为1390个/mm~2,色素细胞逐渐密集鱼体两侧对称分布;变态开始后鱼体两侧色素不对称,有眼侧体表黑色素细胞逐渐溶解消退,由成体黑色素细胞代替,体色变深,黑色素细胞密度稳定在150个/mm~2;无眼侧黑色素细胞逐渐消溶退化,体色逐渐变白。白化个体在变态开始后出现,有眼侧不能形成成体黑色素细胞,形成白化。     

Survival,persistence and proliferation of Vibrio anguillarum in juvenile turbot,Scophthalmus maximus (L.), intestine and faeces

The aim of this study was to determine whether orally administered V. anguillarum cells could survive passage through the intestinal tract of feeding turbot, and thereafter, proliferate in the released faeces. In addition, the growth of the pathogen in turbot faeces in the presence of a Carnobacterium sp. with inhibitory effects against a number of bacterial fish pathogens was studied. It was found that V. anguillarum cells survived the acidic environment of the stomach for several hours, persisted in the intestine and readily proliferated in the released faeces. The antagonistic Carnobacterium sp. inhibited the growth of V. anguillarum during co-culture in turbot faecal extracts. From the results, it was concluded that the turbot intestinal tract and faeces can serve as an enrichment site for V. anguillarum, and the use of intestinal bacteria with antagonistic activity against vibrios may be used to reduce the load of fish pathogenic vibrios in turbot hatcheries.     

牙鲆tyrp1a和tyrp1b的鉴定及tyrp1a与mmu-miR-143-5p_R+2的调控关系

为了研究牙鲆白化发生过程中的分子调控机制,本研究在获得正常和白化牙鲆转录组及micro RNA(mi RNA)深度测序数据的基础上,对tyrosinase related protein 1(tyrp1)和mmu-mi R-143-5p_R+2(mmu-143)进行了表达模式、靶基因预测及验证分析。首先通过RACE方法克隆得到白化相关基因tyrp1的2个转录本,进化树分析表明这2个转录本分别是tyrp1a和tyrp1b,利用RNAhybrid软件预测到mmu-143可能与tyrp1a基因存在互作关系,通过双荧光素酶实验初步验证了这一靶向关系。进一步的荧光定量结果显示,tyrp1a基因在正常牙鲆皮肤中的表达量显著高于白化牙鲆皮肤的表达量,正常牙鲆皮肤中mmu-143的表达量显著低于白化牙鲆皮肤中的表达量。本研究发现,牙鲆tyrp1存在2个转录本,分别是tyrp1a和tyrp1b。双荧光素酶实验和定量PCR分析初步证实,tyrp1a是mmu-143的靶基因,mmu-143是通过调控tyrp1a基因的表达来影响牙鲆白化的。此研究结果为深入揭示牙鲆白化发生的分子机制提供重要的基础资料。