Effects of water salinity on melatonin levels in plasma and peripheral tissues and on melatonin binding sites in European sea bass (Dicentrarchus labrax)

Sea bass is an euryhaline fish that lives in a wide range of salinities and migrates seasonally from lagoons to the open sea. However, to date, the influence of water salinity on sea bass melatonin levels has not been reported. Here, we evaluated the differences in plasma and tissue melatonin contents and melatonin binding sites in sea bass under four different salinity levels: seawater (36‰), isotonic water (15‰), brackish water (4‰) and freshwater (0‰). The melatonin content was evaluated in plasma, whole brain, gills, intestine and kidney, while melatonin binding sites were analyzed in different brain regions and in the neural retina. Plasma melatonin levels at mid-dark varied, the lowest value occurring in seawater (102 pg/mL), and the highest in freshwater (151 pg/mL). In gills and intestine, however, the highest melatonin values were found in the seawater group (209 and 627 pg/g tissue, respectively). Melatonin binding sites in the brain also varied with salinity, with the highest density observed at the lower salinities in the optic tectum, cerebellum and hypothalamus (30.3, 13.0, and 8.0 fmol/mg protein, respectively). Melatonin binding sites in the retina showed a similar pattern, with the highest values being observed in freshwater. Taken together, these results reveal that salinity influences melatonin production and modifies the density of binding sites, which suggests that this hormone could play a role in timing seasonal events in sea bass, including those linked to fish migration between waters of different salinities for reproduction and spawning.     

Effect of conjugated linoleic acid on dietary lipids utilization, liver morphology and selected immune parameters in sea bass juveniles (Dicentrarchus labrax)

Increased energy content in fish feeds has led to an enhanced fat deposition, particularly in European sea bass, concerning fish farmers. Inclusion of conjugated linoleic acid (CLA) could reduce fat deposition as in other vertebrates. To determine if dietary CLA affects fat deposition, lipid metabolism, lipid composition and morphology of different tissues, growth and selected immune parameters, European sea bass juveniles were fed 4 graded levels of CLA (0, 0.5, 1 and 2%). Growth and feed conversion were not affected by CLA, whereas feed intake was reduced (P < 0.05) by feeding 2% CLA. In these fish perivisceral fat was also reduced (P < 0.05), particularly reducing (P < 0.05) monounsaturated fatty acids. CLA has not affected tissue proximal composition, but reduced (P < 0.05) saturated and monounsaturated fatty acids and increased (P < 0.05) the n−3 and n−3 highly unsaturated fatty acids in muscle and increase (P < 0.05) CLA content in muscle, liver and perivisceral fat. A progressive reduction in lipid vacuolization of hepatocytes cytoplasm and regular-shaped morphology was found in fish fed increased CLA levels, together with a progressive increase in malic enzyme activity (only significant in fish fed 1% CLA). Finally, inclusion of CLA up to 1% increased (P < 0.05) plasma lysozyme activity and was positively correlated with alternative complement pathway.     

Glucose-6-phosphate dehydrogenase from Dicentrarchus labrax liver: kinetic mechanism and kinetics of NADPH inhibition

The kinetic mechanism of the reaction catalyzed by glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from Dicentrarchus labrax liver was examined using initial velocity studies,NADPH and glucosamine 6-phosphate inhibition and alternate coenzyme experiments. The results are consistent with a steady-state ordered sequential mechanism in which NADP⁺ binds first to the enzyme and NADPH is released last. Replots of NADPH inhibition show an uncommon parabolic pattern for this enzyme that has not been previously described. A kinetic model is proposed in agreement with our kinetic results and with previously published structural studies (Bautista et al. (1988) Biochem. Soc. Trans. 16, 903–904). The kinetic mechanism presented provides a possible explanation for the regulation of the enzyme by the [NADPH]/[NADP⁺] ratio.     

Releasing of the European sea bass Dicentrarchus labrax (Linnaeus) in the Adriatic Sea: Large-volume versus intensively cultured juveniles

The main goals of this project were to evaluate if artificial reefs are suitable sites for releasing hatchery-reared sea bass and if intensively and extensive large-volume cultured sea bass are suitable to be released into the wild for stock enhancement purposes. Large-volume cultured bass were reared in lower densities compared to intensively cultured ones and, when fry were about 90 days old, were transferred into external ponds connected to the channels of the surrounding marsh, where that they could integrate pellet food sources with live prey. Intensively cultured bass were fed for 55 days with Artemia salina (Linnaeus, 1758) and then with pellets. Underwater visual census (UVC) and fishing sampling were carried out to verify the presence of the tagged specimens at the artificial reef. A low mortality rate after tagging was observed and good tag retention was recorded. Individuals dead after tagging were statistically smaller than survivors. A total of 45 tagged bass (42 large-volume and 3 intensively cultured) were returned by fishers and 16 specimens were observed during UVC (15 large-volume and 1 intensively cultured). The majority of recaptured bass concentrated in the surroundings of river mouths and harbours suggesting that, after release, sea bass migrated towards shallower and brackish waters. Subsequently, as they grew, they came back towards deeper waters and tended to aggregate around artificial structures. Analysis of stomach contents of returned individuals confirmed their dependence on hard-substrate food items. The study provides evidence to support the suitability of large-volume juveniles for restocking purposes, due to their ability to prey on wild food and their endurance to the stress caused by release operations.     

Saline preferendum for the European sea bass, Dicentrarchus labrax, larvae and juveniles: effect of salinity on early development and sex determination

The sea bass, Dicentrarchus labrax, is an eurhyaline marine fish. Juveniles of this species are known to frequent estuaries and lagoons where salinity is lower than in the open sea. Sex determination occurs during this phase of fish life and has been shown labile and sensitive to environmental factors. In this work, the effect of rearing salinity on sex determination and early development of the sea bass was investigated. An excess of males (87%) was found in all groups and the salinity level [(natural sea water salinity, mean: 37‰) vs. (15‰)], when maintained constant, had no effect on the sex-ratio. The transfer from low to high salinity at 93 days post-fertilization (p.f.) increased the percentage of males (93%) suggesting that sexual differentiation in this species may be influenced by such an osmotic stress. Growth was improved by a 15‰ salinity at the beginning of larval rearing (14 days p.f.) and at the end of pre-growing (234-458 days p.f.), periods during which low temperatures were applied. Survival during larval rearing and nursery were also improved in the groups reared at low salinity and so was swimbladder inflation. These results show that sea bass juveniles have a low saline preferendum, a finding that corresponds to the conditions they actually encounter in the wild during their juvenile ecophase; sex determination is not directly modulated by the salinity level but seems to be subjected to complex environmental regulations.     

Vaccination of sex reversed hybrid tilapia (Oreochromis niloticus × O. aureus) with an inactivated Vibrio vulnificus vaccine

Vibrio vulnificus causes disease in economically important aquaculture raised fish and is an opportunistic human pathogen. This study reports on the isolation of V. vulnificus from diseased hybrid tilapia (Oreochromis niloticus × O. aureus) cultured in a North American water reuse facility. Our objectives were to characterize the isolate using biochemical and molecular methods, develop a disease challenge model, and determine the ability of a formalin inactivated whole-cell vaccine to protect against V. vulnificus. The V. vulnificus isolate recovered was biotype 1, 16S rRNA type B, vcg type C, and vvhA type 2 and caused disease in tilapia held in static salt water (1.5 g/l sea salt). Fish vaccinated with the formalin inactivated whole-cell vaccine responded to vaccination with titers from vaccinated fish ranging from 32 to 64 and titers from non-vaccinated fish ranging from 4 to 8. In two trials, vaccinated tilapia exhibited relative percent survival (RPS) of 73 and 60% following homologous isolate challenge. In two additional trials, vaccinated tilapia exhibited RPS values of up to 88% following challenge with a heterologous isolate; the use of a mineral oil adjuvant enhanced protection. This vaccine may provide an effective means of preventing infections caused by biochemically and genetically diverse V. vulnificus.     

研究报告: 基于核酸适配体的胶体金比色法检测鳗弧菌

目的 鳗弧菌（Vibrio anguillarum）可引起鲑鱼、鳗鲡、鲈鱼和牙鲆等多种水产养殖动物的疾病,是水产养殖中的一种重要病原菌,对其进行快速检测是确保水产养殖安全和食品安全所必需的。方法 本文利用鳗弧菌与其核酸适配体之间较强的亲和力,通过鳗弧菌夺取胶体金颗粒表面的核酸适配体,使胶体金溶液的吸光度发生变化,从而建立一种可定量检测鳗弧菌的方法。结果 该方法对鳗弧菌的吸光度值显著高于对溶藻弧菌、铜绿假单胞菌、变形假单胞菌、嗜水气单胞菌和迟钝爱德华氏菌等非目标菌的吸光度值（P<0.01）,并在1~10⁵ CFU/ml的检测范围内呈现较好的线性关系。用该方法对不同盐度和鱼体组织样品进行加标回收检测,结果显示回收率和相对标准偏差等指标都符合相应检测标准。结论 该检测方法对鳗弧菌有较好的特异性,可用于水产品或食品中鳗弧菌的定量检测。     

黄花大苞姜花药发育qRT-PCR内参基因筛选

qRT-PCR技术具有定量准确、灵敏度高、重复性好等特点,被广泛用于基因表达分析。内参基因的稳定性对于准确分析实验结果非常重要。该研究以黄花大苞姜(Caulokaempferia coenobialis)花粉母细胞时期(PMC)、四分体时期(TET)、成熟花粉时期(MP)的花药组织为材料,基于3个阶段花药转录组表达谱数据以及常用传统内参基因,筛选出Glyceraldehyde 3-phosphate dehydrogenase(GAPDH)、Malate dehydrogenase(MDH)、α-tubulin3(TUA3)、β-tubulin7(TUB7)和Actin6(ACT6)作为候选内参基因,进行qRT-PCR分析;并运用BestKeeper、geNorm和Normfinder软件综合分析5个候选内参基因在黄花大苞姜花药发育过程中的表达稳定性。结果表明:MDH和TUB7的表达最稳定,ACT6的稳定性最差;分别以MDH和TUB7作为内参,分析GBE1在黄花大苞姜花药发育中的表达模式,并与该基因在花药转录组中的表达模式做相关系数分析,3种表达模式结果一致,进一步验证了MDH和TUB7的表达稳定性。这说明MDH和TUB7适合作为qRTPCR分析黄花大苞姜花药发育过程中相关基因表达模式的内参基因。该研究结果为黄花大苞姜花药发育分子机制相关研究奠定了基础,也为姜科花药发育相关内参基因的选择提供了参考。     

蚬壳花椒种子萌发时期内参基因的筛选与验证

为了解蚬壳花椒种子萌发的分子机制,需要筛选蚬壳花椒种子萌发时期表达稳定的内参基因。该研究通过赤霉素处理种子促进萌发,以不同萌发阶段的蚬壳花椒种子为材料,采用实时荧光定量PCR技术分析了6个候选内参基因GAPDH、ACT、18SrRNA、UBQ5、TUA和CYP在蚬壳花椒种子萌发时期的表达稳定性。结果表明:(1)α-淀粉酶基因、DELLA基因和异柠檬酸裂解酶基因分别反应了种子萌发阶段糖、激素和脂肪的代谢活动,因此选择蚬壳花椒异柠檬酸裂解酶基因(Unigene0032088)、α-淀粉酶基因(Unigene0033597)和DELLA基因(Unigene0058868)作为验证基因进行相对表达量验证。(2)综合geNorm、NormFinder和BestKeeper的分析结果显示在蚬壳花椒种子萌发过程中ACT表达稳定性最好,UBQ5次之。(3)以ACT、UBQ5基因为内参基因的结果显示验证基因的表达量与种子萌发生理状态一致,初步揭示了GA处理的种子易于萌发而清水处理的种子在萌发第3天容易腐败这一现象出现的可能原因。综上所述,ACT是蚬壳花椒种子萌发时期最合适的内参基因,其次是UBQ5。     

Freeze-drying of live attenuated Vibrio anguillarum mutant for vaccine preparation

Vibrio anguillarum MVAV6203 is a mutant strain as a candidate of live attenuated vaccine. In vaccine preparation, the freeze-drying conditions of the strain were investigated to improve the survival after freeze-drying, including the protectant, rehydration medium, freezing temperature, and initial cell concentration. Vibrio anguillarum MVAV6203 is sensitive to freeze-drying and the viability was only 0.03% in the absence of protectant. Of the tested protectants, 5% trehalose with 15% skimmed milk gave the highest viability of 34.2%. Higher cell survival was obtained by quick freezing at -80 degrees C than slow freezing at -20 degrees C. Initial cell concentration was another important factor, preferable for 1-3 x 10(10)CFU/ml. The supplementation of 10% skimmed milk in rehydration medium improved obviously freeze-drying viability. The combination of the optimal conditions achieved 51.4% cell viability after freeze-drying.     

A survey of quantitative real-time polymerase chain reaction internal reference genes for expression studies in Brassica napus

Eight reference genes of Brassica napus were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) data, focusing on vegetative tissues and developing embryos. Analyses of expression stability indicated that UP1, UBC9, UBC21, and TIP41 were the top four choices as stably expressed reference genes for vegetative tissues, whereas ACT7, UBC21, TIP41, and PP2A were the top four choices for maturing embryos. In addition, radiolabeling of overall messenger RNA (mRNA) of maturing embryos indicated that the expression patterns of the top four ranked reference genes reflected the overall mRNA content changes in maturing embryos.     

基于转录组测序的姬松茸镉胁迫下内参基因筛选

[目的] 筛选不同实验条件下,尤其是镉胁迫下的姬松茸内参基因,为研究姬松茸镉富集相关基因功能研究奠定基础。[方法] 根据不同浓度Cd（0、2、5 mg/L）胁迫下菌丝中转录组表达量数据,筛选出18个候选基因,设计了30对引物;根据引物扩增的特异性、扩增效率初步筛选出来自不同基因的5对引物;运用qRT-PCR检测了5个基因在不同浓度Cd胁迫下的菌丝样品、原基、菌柄和菌盖中的C_t值,利用geNorm、NormFinder和BestKeeper对这些基因的表达稳定性进行评价。[结果] SGT2和STK是菌丝阶段Cd胁迫下最稳定的2个候选内参基因,GAPDH是不同组织中最稳定的内参基因,SGT2、STK和GAPDH是所有实验条件下最稳定的3个内参基因。[结论] 在姬松茸镉胁迫条件下,本研究筛选出的SGT2和STK比常用的内参基因表现出更强的稳定性。     

Development of Encarsia bimaculata (Heraty and Polaszek) (Hymenoptera: Aphelinidae) in Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) nymphs

Encarsia bimaculata was recently described from India as a potentially useful parasitoid of Bemisia tabaci. Its developmental biology was studied in the laboratory at 25–30 °C and 70–75% RH. Results showed that E. bimaculata is a solitary, arrhenotokous, heteronomous, autoparasitoid. Mated females laid eggs internally in B. tabaci nymphs that developed as primary parasitoids. Males developed as hyperparasitoids, either in females of their own species or in other primary aphelinid parasitoids. Superparasitism was common under cage conditions. Both sexes have an egg, three larval instars, prepupal, and pupal stages. Development from egg to adult took 12.70 ± 2.10 days for females and 14.48 ± 2.60 days for males. Individual B. tabaci nymphs were examined for E. bimaculata parasitization using three isozymes: esterase, malate dehydrogenase, and xanthine dehydrogenase. All three isozymes showed differential banding patterns that identified E. bimaculata parasitized or unparasitized B. tabaci nymphs.     

蜜环菌(Armillaria mellea)内参基因的筛选

[背景]蜜环菌属(Armillaria)是一类营腐生或寄生生活的药食两用型真菌,研究其功能基因表达具有重要意义。[目的]筛选并获得蜜环菌(Armillaria mellea)最稳定的内参基因。[方法]以蜜环菌(A.mellea)541为研究对象,以马铃薯琼脂糖培养基培养的菌丝和菌索为对照组,以添加还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶抑制剂氯化二亚苯基碘鎓(diphenyleneiodonium chloride,DPI)培养的菌丝和菌索为抑制剂组,以添加木屑培养的菌丝和菌索为诱导剂组,利用RT-qPCR技术和BestKeeper程序系统评估候选内参基因ACT-1、α-TUB、β-TUB 1、γ-TUB、UBQ、EF-lγ、18S rRNA biogenesis protein基因(18S rRNA BP)和GAPDH的表达量稳定性。[结果]内参基因EF-1γ在对照组、DPI抑制剂组和木屑诱导剂组中的表达量稳定性最好。[结论]EF-1γ为蜜环菌(A.mellea)的最佳内参基因,为蜜环菌属真菌功能基因表达研究提供了参考。     

藜科植物藜和灰绿藜实时荧光定量PCR内参基因的选择

选择合适的内参基因是实时荧光定量PCR(qRT-PCR)研究的关键,目前对藜科耐盐(盐生)植物胁迫相关基因的表达分析中所用内参基因的报道较为有限.该研究利用GeNorm、NormFinder和BestKeeper 3个内参基因分析软件,对已选择过的β-TUBULIN、β-ACTIN、GAPDH 3个常用候选内参基因进行了比较分析,筛选出在NaCl和PEG胁迫下藜和灰绿藜中表达相对稳定的内参基因.结果表明:GeNorm、NormFinder内参软件分析在NaCl和PEG胁迫下,GAPDH是藜和灰绿藜中均共同稳定表达的内参基因,同时在藜和灰绿藜中也有各自表达较稳定的内参基因,β-ACTIN在藜中稳定表达,β-TUBULIN则在灰绿藜中稳定表达.对相同科不同种的植物内参基因表达差异进行比较,内参基因在相同科中具有相同稳定表达的内参;对相同胁迫下两种不同植物内参基因表达稳定性进行分析,内参基因的选择需根据实际的实验材料和实验条件而定.基于3个分析软件对以上3个常用内参基因的分析结果,初步确定了在藜科植物藜和灰绿藜中相对稳定的内参基因,为藜和灰绿藜胁迫相关基因的定量表达分析提供了参考依据.     

Evaluation of a highly discriminating multiplex multi-locus variable-number of tandem-repeats (MLVA) analysis for Vibrio cholerae

Vibrio cholerae is the etiological agent of cholera and may be used in bioterror actions due to the easiness of its dissemination, and the public fear for acquiring the cholera disease. A simple and highly discriminating method for connecting clinical and environmental isolates of V. cholerae is needed in microbial forensics. Twelve different loci containing variable numbers of tandem-repeats (VNTRs) were evaluated in which six loci were polymorphic. Two multiplex reactions containing PCR primers targeting these six VNTRs resulted in successful DNA amplification of 142 various environmental and clinical V. cholerae isolates. The genetic distribution inside the V. cholerae strain collection was used to evaluate the discriminating power (Simpsons Diversity Index = 0.99) of this new MLVA analysis, showing that the assay have a potential to differentiate between various strains, but also to identify those isolates which are collected from a common V. cholerae outbreak. This work has established a rapid and highly discriminating MLVA assay useful for track back analyses and/or forensic studies of V. cholerae infections.