邻苯二甲酸丁基苄酯（BBP）对斑马鱼的毒性效应研究

经急性毒性试验,求得邻苯二甲酸丁基苄酯（BBP）对斑马鱼96小时半致死浓度（LC50）为3.98 mg•L-1。在此基础上,设置4个浓度梯度：0、0.332、0.665、1.33 mg•L-1,研究了在不同的时间（7、14、21、28d）暴露下,BBP对斑马鱼超氧化物歧化酶（SOD）、乙酰胆碱酯酶（AChE）、过氧化氢酶（CAT）活性的影响。结果表明,随着时间的延长,SOD活性呈现先上升后下降的趋势,在14d时,各处理组SOD活性达到最高水平;实验期间（0-28d）,各处理组AChE活性被显著抑制,整体呈现下降趋势;CAT活性,在7d时,除0.665 mg•L-1浓度组有极显著抑制作用（p＜0.01）外,其余各浓度组CAT活性均与对照接近,无显著差异,14d开始,除了0.332 mg•L-1浓度组高于对照组,其他浓度组与对照组相比均呈现先下降后上升的趋势。BBP对斑马鱼SOD、AChE、CAT活性产生的影响,其变化可作为水体污染的分子生物标记物,为制订BBP水质标准及对其进行生态风险评价提供科学依据。     

Effects of Pseudoloma neurophilia infection on the brain transcriptome in zebrafish (Danio rerio)

Laboratory zebrafish are commonly infected with the intracellular, brain-infecting microsporidian parasite Pseudoloma neurophilia. Chronic P. neurophilia infections induce inflammation in meninges, brain and spinal cord, and have been suggested to affect neural functions since parasite clusters reside inside neurons. However, underlying neural and immunological mechanisms associated with infection have not been explored. Utilizing RNA-sequencing analysis, we found that P. neurophilia infection upregulated 175 and downregulated 45 genes in the zebrafish brain, compared to uninfected controls. Four biological pathways were enriched by the parasite, all of which were associated with immune function. In addition, 14 gene ontology (GO) terms were enriched, eight of which were associated with immune responses and five with circadian rhythm. Surprisingly, no differentially expressed genes or enriched pathways were specific for nervous system function. Upregulated immune-related genes indicate that the host generally show a pro-inflammatory immune response to infection. On the other hand, we found a general downregulation of immune response genes associated with anti-pathogen functions, suggesting an immune evasion strategy by the parasite. The results reported here provide important information on host–parasite interaction and highlight possible pathways for complex effects of parasite infections on zebrafish phenotypes.     

团头鲂和斑马鱼鳞片的发生发育过程比较研究

为探究鱼类鳞片的发生发育过程,以团头鲂(Megalobrama amblycephala)和斑马鱼(Danio rerio)为实验对象,结合形态学和组织学方法,对这两种鱼的鳞片发生发育过程进行了系统的比较研究。结果显示:团头鲂和斑马鱼分别在出膜后24 d和39 d开始出现鳞片,鳞片首次出现时的体长分别为16.3～19.7 mm和8.5～9.2 mm。鳞片分别于出膜后44 d和47 d,体长达到23.9～24.6 mm和12.1～12.5 mm时覆盖完全。团头鲂的鳞片最先出现于鳃盖后缘侧线处,随着体长的增加,沿着侧线向尾部逐渐覆盖,侧线下方的覆盖速度快于侧线上方。相比之下,斑马鱼的鳞片最先出现在尾柄侧线处,随着体长的增加,鳞片沿着侧线向头部逐渐覆盖,其水平覆盖的速度快于垂直覆盖的速度。组织学结果表明,团头鲂和斑马鱼鳞片的发生主要经历了形态发生早期、形态发生晚期、分化早期、分化晚期和折叠期,共5个时期。相关性分析结果表明,团头鲂、斑马鱼的体长和日龄均与鳞片的覆盖率有相关性,其中体长与鳞片的覆盖率相关性更高。     

The effects of the dietary biotin on zebrafish Danio rerio reproduction

An experiment was conducted to investigate the effects of dietary biotin on zebrafish Danio rerio reproduction. Biotin and avidin (biotin antagonist) were added to two isonitrogenous and isocaloric purified diets to provide molar avidin:biotin ratios of 0:1 (biotin‐sufficient diet) and 120:0 (biotin‐unsufficient diet). Each diet was fed to a group of males mean initial mass 0.262 g) and a group of females (mean initial mass 0.285 g) for 99 days. Following this feeding period, males fed biotin‐sufficient diet exhibited higher gonado‐somatic index, sperm density, sperm motility and sperm viability than those fed biotin‐unsufficient diet (P < 0.05). In the presence of biotin‐sufficient males, biotin‐sufficient females spawned more eggs (222.2 eggs) than biotin‐deficient females (18.8 eggs) (P < 0.05). The same pattern was observed with biotin‐deficient males (7.6 vs. 1.8 eggs) (P < 0.05). Biotin‐sufficient males generated a higher percentage of fertilized eggs (90% vs. 42%), hatching rate (62% vs. 27%), larvae survival (98% vs. 37%) and larvae length at 7 days post fertilization (4.4 mm vs. 4.2 mm) than biotin‐deficient males (P < 0.05) . Biotin status of the male is of high consideration for successful breeding in zebrafish, because it significantly impacts the reproductive performances of the female.     

玻璃化冻存对斑马鱼卵母细胞的影响

采用比色法测定了在不同玻璃化冻存液中冻存1、2、4、8d时斑马鱼卵母细胞中谷胱甘肽含量和三磷酸腺苷酶的活性,以研究玻璃化冻存对卵母细胞谷胱甘肽含量和线粒体三磷酸腺苷酶活性的影响。试验结果显示,V4组斑马鱼(玻璃化液组分是:1.5mol/L甲醇、6.0mol/L乙二醇、0.25mol/L蔗糖)卵母细胞冻存1、2、4、8d后存活率最高[分别为(48.89±2.58)%、(39.35±1.34)%、(24.18±2.32)%和(14.56±1.64)%],显著高于其他组。经玻璃化冻存后,斑马鱼卵母细胞谷胱甘肽含量和线粒体三磷酸腺苷酶活性均随冻存天数的增加而显著下降(P0.05)。V4组的谷胱甘肽含量[(13.37±0.59)mg/g]和线粒体三磷酸腺苷酶活性较高,线粒体Ca~(2+)-三磷酸腺苷酶[(3.823±0.144)U/mg]、Mg~(2+)-三磷酸腺苷酶[(4.503±0.144)U/mg]和Na~+/K~+-三磷酸腺苷酶[(7.520±0.198)U/mg]活性最高。各组细胞的活性随着冻存天数的增加而逐渐下降。试验结果表明,玻璃化冻存不能完全避免低温和冷冻保护剂的毒性和对卵母细胞的损伤。     

苯硫脲对斑马鱼黑色素生成及早期发育的影响

为探索苯硫脲抑制斑马鱼黑色素生成的最佳浓度及苯硫脲对斑马鱼早期发育的影响,用0.05～2.O mmol/L苯硫脲处理28-体节期斑马鱼胚胎,用1.0～15 mmol/L苯硫脲处理受精后2、10、24 h的斑马鱼胚胎.结果表明,在不同的发育时期,抑制黑色素生成的苯硫脲最佳浓度是不同的;且发育时期越早,斑马鱼对苯硫脲毒性越敏感;苯硫脲低于0.2 mmol/L时对斑马鱼孵化、存活和早期发育无显著影响;0.2 mmol/L苯硫脲导致斑马鱼孵化率降低;0.5～2.0 mmol/L苯硫脲导致斑马鱼的孵化率和存活率均降低,并引起斑马鱼畸形.     

DEHP对斑马鱼仔鱼HPG和HPA轴相关功能基因表达的影响

为了探讨邻苯二甲酸二(2-乙基)己酯(DEHP)对斑马鱼早期生命阶段下丘脑-垂体-性腺轴(HPG)和下丘脑-垂体-肾上腺轴(HPA)相关功能基因表达的影响,将斑马鱼的受精卵暴露于10、30和300μg/L DEHP溶液中,同时设置空白对照,96 h后,利用实时荧光定量PCR技术,测定斑马鱼仔鱼HPG和HPA轴上12个功能基因的表达。结果显示,与对照组相比,30μg/L DEHP处理组的Cyp19b、GnRH3、Era和ERβ基因表达量显著升高,GnRH2、Pomc、Mr、Mc2r、Crh、Gr和Crhbp基因在各DEHP处理组表达量显著降低,FSH-β和Crhbp基因表达量无显著变化。以上研究结果表明：DEHP对斑马鱼胚胎/仔鱼阶段的2个主要内分泌分子通路产生了显著影响,将会导致内分泌功能的改变,从而可能影响斑马鱼的后期生长发育。     

Effects of nutritional status and diet composition on fatty acid transporters expression in zebrafish (Danio rerio)

This study investigated the expression of zebrafish (Danio rerio) fatty acid transporters, such as scavenger receptor CD36, plasma membrane fatty acid‐binding protein (FABPpm), and fatty acid transport protein family (FATPs), in responses to nutritional status and diet composition. For diet composition study, three isonitrogenous (43% crude protein) purified diets were formulated to contain 6%, 12% and 18% crude lipid levels. Meanwhile five isonitrogenous and isoenergetic purified diets were formulated to contain different lipid sources including fish oil (FO; rich in n‐3 HUFA), palmitic acid (PA; rich in C16:0), olive oil (OO; rich in C18:1n‐9), sunflower oil (SO; rich in C18:2n‐6) or perilla oil (PO; rich in C18:3n‐3) as the sole lipid source. Results showed that liver CD36, FABPpm‐a, FABPpm‐b, FATP1‐a and FATP6, intestine CD36, FABPpm‐a and FABPpm‐b, and muscle CD36 and FATP1‐b were significantly increased during postprandial period. During starvation, liver CD36, FABPpm‐a, FABPpm‐b and FATP1‐a, intestine FABPpm‐a, FABPpm‐b, FATP4 and FATP6, and muscle FATP1‐b, FATP4 and FATP6 were significantly increased. The expression of some fatty acid transporters (including liver CD36 and FABPpm‐b, intestine FABPpm‐a, and muscle CD36, FABPpm‐a, FATP1‐a, FATP2 and FATP4) was up‐regulated by refeeding, while the expression of other fatty acid transporters (liver FABPpm‐a and intestine FATP1‐a, FATP4 and FATP6) was down‐regulated by refeeding. The expression of several fatty acid transporters (liver FATP2 and FATP4, and intestine CD36, FATP2 and FATP4) was significantly increased by high‐lipid diet, whereas CD36, FATP1‐b, FATP2 and FATP4 expression in the muscle were reduced by increasing dietary lipid level. Compared with FO group, the muscle of fish fed the diet with OO had a higher CD36 mRNA abundance, while the muscle of fish fed the diet with OO and PO had a higher FATP1‐b expression. Compared with the FO group, the hepatic FATP2 mRNA was significantly increased in the PA group, while the intestine FATP2 mRNA was significantly increased in the SO and PO groups. In addition, intestine FATP4 expression in the PA and OO groups was lower compared with the FO group. The results indicate that zebrafish fatty acid transporters play a central role in coordinating the mobilization of fuel substrates in responses to nutritional status and diet composition.     

双酚A和壬基酚长期暴露对斑马鱼繁殖的影响

内分泌干扰物的研究目前正逐渐成为国际研究新的热点，但长期暴露条件下对生殖情况的研究目前较少。本文研究了环境类雌激素双酚A（BPA）以及和壬基酚（NP）联合的作用，对暴露一个世代的斑马鱼（F1）（Danio rerio）的生殖情况、子代质量的影响。将健康的受精卵48h后暴露于200，500，1000μg·L^-1 BPA以及三个浓度与30μg·L^-1 NP的联合，同时设置空白对照和30μg·L^-1 NP对照。130dph将成鱼置于清水喂养20d，统计每天的产卵量、产卵次数、畸形率、孵化率、卵径、破膜时间以及耐饥时间。以此为终点评估暴露后斑马鱼的生殖能力和子代质量。与对照相比NP，BPA可以抑制斑马鱼的生殖，两种类雌激素联合作用可以极显著地抑制斑马鱼的生殖（P〈0．01）；BPA各浓度组导致F1代畸形率上升（P〈0．05）；高浓度BPA抑制产卵量（P〈0．05）；BPA200，500μg·L^-1会延长F1代的破膜时间和耐饥时间（P〈0．01），而BPA1000μg·L^-1则产生相反的效果（P〈0．05）。双酚A对斑马鱼的生殖有明显的抑制作用，双酚A和壬基酚有协同效应。长时间暴露于双酚A会影响斑马鱼的生殖能力和子代质量。     

双酚A和壬基酚长期暴露对斑马鱼繁殖的影响

内分泌干扰物的研究目前正逐渐成为国际研究新的热点,但长期暴露条件下对生殖情况的研究目前较少.本文研究了环境类雌激素双酚A(BPA)以及和壬基酚(NP)联合的作用,对暴露一个世代的斑马鱼(F1)(Danio rerio)的生殖情况、子代质量的影响.将健康的受精卵48 h后暴露于200,500,1 000 μg·L-1 BPA以及三个浓度与30 μg·L-1 NP的联合,同时设置空白对照和30 μg·L-1 NP对照.130 dph将成鱼置于清水喂养20 d,统计每天的产卵量、产卵次数、畸形率、孵化率、卵径、破膜时间以及耐饥时间.以此为终点评估暴露后斑马鱼的生殖能力和子代质量.与对照相比NP,BPA可以抑制斑马鱼的生殖,两种类雌激素联合作用可以极显著地抑制斑马鱼的生殖(P＜0.01);BPA各浓度组导致F1代畸形率上升(P＜0.05);高浓度BPA抑制产卵量(P＜0.05);BPA200,500 μg·L-1会延长F1代的破膜时间和耐饥时间(P＜0.01),而BPA1 000 μg·L-1则产生相反的效果(P＜0.05).双酚A对斑马鱼的生殖有明显的抑制作用,双酚A和壬基酚有协同效应.长时间暴露于双酚A会影响斑马鱼的生殖能力和子代质量.     

Effect of age on the mercury sensitivity of zebrafish (Danio rerio) sperm

The effect of age on the sensitivity of zebrafish sperm against mercury exposure was investigated in the present study. Although results of the use of sperm from mature individuals for toxicity tests have been published, there is no information about the exact age of the fish in some cases, which can affect the results. During the experiments, pooled sperm was stripped from males of 7, 12, or 18 months of age, divided into 5 sub-groups, diluted with different concentrations of Hg (0, 0.5, 1, 2.5, and 5 mg/L Hg), and incubated for 240 min. The motility parameters of sperm (progressive motility (%), curvilinear velocity (VCL)) were measured by a computer-assisted sperm analysis system, at 30, 120, and 240 min of exposure. Regarding the age, significant differences were found in PMOT (p?=?0.0267) as well as in VCL (p?=?0.0004) among the three different age groups. The different concentrations of Hg also caused significant differences. The most significant differences in PMOT were between the 7- and 18-month-old groups; these differences were observed at 0.5, 1 and 2.5 mg/L Hg at 30 min, at 0.5 and 1 mg/L at 120 min, as well as at 0.5 mg/L at 240 min. In VCL the most significant differences were found between the 7- and 12-month-old groups; significant differences were found at each tested concentration at 30 min as well as at 0.5 and 2.5 mg/L at 240 min. According to the results, the age of zebrafish negatively influences the sensitivity of its sperm. This may concern not only toxicology tests but many techniques in fish breeding where the sperm is treated before use (cryopreservation, pressure shock, etc.).

     

Effects of prebiotic mannan oligosaccharide on the growth,survival, and anxiety-like behaviors of zebrafish (Danio rerio)

A study was designed to investigate the possible positive prebiotic effects of mannan oligosaccharides (MOS) on growth and reduction of anxiety behaviors in zebrafish (Danio rerio). The experimental diets were supplemented with 0, 2, 4, 6, or 8 g MOS kg^?1 and were fed to juvenile fish (0.47 ± 0.017 g) in triplicates for six weeks. Significant differences were found in the final body weight, body weight intake, percent body weight intake, and specific growth rate among treatments. The regression analyses showed that the optimum MOS level was 4–4.1 g MOS kg^?1 diet. Fish fed 4 g MOS kg^?1 feed showed higher survival than the other treatments. Also, using the novel tank test, fish fed with 4 g MOS kg^?1 diet showed lower anxiety by swimming in the upper portion of the tank. The other behavior parameters remained unaffected by the prebiotic. The results indicated the beneficial effect of the prebiotic MOS on growth and partly on welfare of zebrafish juveniles.     

Effects of the wood extractive betulinol and 17beta-oestradiol on reproduction in zebrafish, Danio rerio (Hamilton)--complications due to a bacterial infection

Zebrafish were exposed to the wood extractive betulinol (5 microg L(-1)) and to 17beta-oestradiol (E2, 0.27 microg L(-1)) for 8 weeks in an attempt to study the possible endocrine-disrupting activity of betulinol. Females exposed to betulinol showed increased spawning intensity, while males exposed to betulinol and E2 had increased incidences of structural alterations in the testes. However, histological examination of the fish revealed that they were infected by acid-fast bacteria suspected to be Mycobacterium sp. despite a careful examination of their health state prior to the onset of the experiment. Fish exposed to betulinol and E2 showed more serious consequences of the bacterial infection than control fish indicating that the test chemicals had weakened the immune defence of the fish. When the exposure was repeated with healthy fish, an increase in the proportion of spermatogonia was seen in the testes of betulinol-treated males. A similar alteration, although not statistically significant, was also seen in the first experiment. However, no increase in the incidences of structural alterations in the testes was seen in betulinol- and E2-treated fish in the second experiment. Our study indicates that betulinol might have an endocrine-disrupting effect in zebrafish, but the increase in incidences of structural alterations in the testes might have been caused by a synergistic action between the test compounds and the bacterial infection. Our study stresses the importance of carefully checking the health of experimental fish, not only prior to the onset of an experiment but also upon termination of the experiment, in order to avoid misinterpretation of the results.     

三氯生对斑马鱼卵巢抗氧化和凋亡相关基因表达的影响

为了探讨三氯生(TCS)对硬骨鱼类性腺损伤作用的分子机制,通过半静态水体暴露法对斑马鱼(Danio rerio)进行染毒,将斑马鱼暴露在2种不同TCS浓度(0.034 mg/L和0.068 mg/L)溶液中,同时设置空白对照组,42 d后,利用实时荧光定量PCR(RT-qPCR)技术,探究TCS对斑马鱼卵巢抗氧化及凋亡相关基因表达的影响。结果发现:与空白对照相比,SOD、CAT和GPx1a基因在0.068 mg/L TCS处理组表达水平显著下调,GPx1和MT-2基因在0.034 mg/L浓度组表达显著上调;Bcl-2基因在0.068 mg/L浓度组表达显著下调,p53基因在0.034 mg/L浓度组表达极显著上调,Bax在TCS处理组中均显著上调。以上研究结果表明:较高浓度TCS显著影响了斑马鱼卵巢抗氧化和凋亡相关基因的表达,从而产生了氧化损伤和加速细胞凋亡的发生。     

A toxicity bioassay study concerning the effect of un-ionized ammonia on the mucus cells response originating from the gills of zebrafish Danio rerio

Ammonia is one of the most common and pervasive pollutants in the aquatic habitat affecting the health of aquatic animals. It affects the number and morphology of mucus producing cells, resulting in excessive and irregular secretion and alterations in quantity of mucus, which can lead to complications in fish health. A toxicity test on the mucus cells localized on the gills (primary lamellae) and inner operculum epithelial lining of zebrafish Danio rerio at different periods of exposure (24, 48 and 72 h) followed by recovery periods was studied using histopathology, scanning electron microscopy and anterior gradient 2 homologue (agr2) gene expression techniques. Fish samples subjected to low, medium and high external toxic ammonia concentrations (L: 17.21 mg/l, M: 25.81 mg/l and H: 38.91 mg/l NH₃-N) were observed to display a higher mucus layer production and active secretion compared with the control. Gill cellular alterations were more severe at 48 and 72 h. A high expression of agr2 was detected at 48 h (L and M) recovery periods and a (H) exposure and recovery period indicating an increase in quantity in newly proliferated alcian blue stained mucus cells and excessive secretion observed by histopathology. Such expression decreased at 72 h resulting in a decrease in mucus cell density and secretion.