First description of a natural infection with spleen and kidney necrosis virus in zebrafish

Zebrafish has become a popular research model in the last years, and several diseases affecting zebrafish research facilities have been reported. However, only one case of naturally occurring viral infections was described for this species. In 2015, infectious spleen and kidney necrosis virus (ISKNV) was detected in zebrafish from a research facility in Spain. Affected fish showed lethargy, loss of appetite, abnormal swimming, distention of the coelomic cavity and, in the most severe cases, respiratory distress, pale gills and petechial haemorrhages at the base of fins. Cytomegaly was the most relevant histopathological finding in organs and tissues, sometimes associated to degenerative and necrotic changes. ISKNV belongs to the relatively newly defined genus Megalocytivirus, family Iridoviridae, comprising large, icosahedral cytoplasmic DNA viruses. This is the first case of naturally occurring Megalocytivirus infection in zebrafish research facilities, associated with morbidity. The virus has been identified based on both pathologic and genetic evidence, to better understand the pathogenesis of the infection in zebrafish and the phylogenetic relationship with other iridoviruses. Given the ability of megalocytiviruses to cross‐species boundaries, it seems necessary to implement stringent biosecurity practices as these infections may invalidate experimental data and have major impact on laboratory and cultured fish.     

基于Bootstrap的ELEFAN方法在评估方氏云鳚群体生长参数中的应用

渔业数据有限性是小型渔业资源评估所面临的常见问题。电子体长频率分析(electroniclengthfrequency analysis,ELEFAN)常用于年龄数据难以获取或缺失的渔业,但该方法的可靠性尚待检验。本研究根据2013―2018年春、秋季共11个航次的海州湾底拖网调查数据,分别使用传统的ELEFAN与结合Bootstrap的ELEFAN方法,比较了2013―2015年与2016―2018年两个时间段内海州湾方氏云鳚(Pholis fangi)群体von-Bertalanffy生长方程中参数之间的变化。结果显示,在海州湾海域,方氏云鳚的生长参数具有显著变化, 2013―2018年,群体的极限体长变小,生长速率加快,说明海州湾方氏云鳚群体近年来呈现小型化的趋势。相比传统的ELEFAN方法,结合Bootstrap的ELEFAN方法能够给出较为稳健的参数估计,受采样随机性的影响较小,可以较好地应用于数据缺乏的小型渔业中。本研究加深了对方氏云鳚种群动态的认识,并推动了基于体长频率的生长参数估算方法在数据有限资源评估中的应用。     

Detection of the parasitic nematode,Pseudocapillaria tomentosa,in zebrafish tissues and environmental DNA in research aquaria

Although zebrafish continue to increase in popularity as a vertebrate animal model for biomedical research, chronic infectious diseases in laboratory populations remain prevalent. The presence of pathogens such as Pseudocapillaria tomentosa, a parasitic nematode found in the intestine of infected zebrafish, can significantly influence experimental endpoints and negatively impact reproducibility of research findings. Thus, there is a need for screening tests for zebrafish with the sensitivity to detect even low levels of pathogens present in tissues. Assays based on the detection of DNA are commonly used for such screening tests. Newer technologies such as digital PCR provide an opportunity to improve the sensitivity and precision of these assays, so they can be reliably used to detect pathogen DNA in water, reducing the need for lethal testing. We have designed a qPCR-based assay with the sensitivity to detect less than 5 copies of the P. tomentosa SSU-rDNA gene in tissues of infected zebrafish and environmental DNA from aquarium water housing infected fish. In addition, we adapted this test to a dPCR platform to provide a precise quantification of P. tomentosa DNA and demonstrate the resistance of this assay to inhibitors commonly found in freshwater aquaria.     

A reference growth curve for nutritional experiments in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) and changes in whole body proteome during development

Zebrafish is one of the most used vertebrate model organisms in molecular and developmental biology, recently gaining popularity also in medical research. However, very little work has been done to assess zebrafish as a model species in nutritional studies in aquaculture in order to utilize the methodological toolbox that this species represents. As a starting point to acquire some baseline data for further nutritional studies, growth of a population of zebrafish was followed for 15 weeks. Furthermore, whole body proteome was screened during development by means of bi-dimensional gel electrophoresis and mass spectrometry. Fish were reared under best practice laboratory conditions from hatching until 103 days post-fertilization (dpf) and regularly fed ad libitum with Artemia nauplii from 12 dpf. A growth burst occurred within 9–51 dpf, reaching a plateau after 65 dpf. Fork length and body weight were significantly lower in males than in females from 58 dpf onwards. Proteomics analysis showed 28 spot proteins differently expressed through development and according to sex. Of these proteins, 20 were successfully identified revealing proteins involved in energy production, muscle development, eye lens differentiation, and sexual maturation. In summary, zebrafish exhibited a rapid growth until approximately 50 dpf, when most individuals started to allocate part of the dietary energy intake for sexual maturation. However, proteomic analysis revealed that some individuals reached sexual maturity earlier and already from 30 dpf onwards. Thus, in order to design nutritional studies with zebrafish fed Artemia nauplii, it is recommended to select a period between 20 and 40 dpf, when fish allocate most of the ingested energy for non-reproductive growth purposes.     

斑马鱼早期胚胎发育囊胚sphere时期的蛋白组学研究

斑马鱼(Danio rerio)早期胚胎发育囊胚时期是胚胎发育过程的关键时期,利用i TRAQ蛋白质质谱分析技术,检测斑马鱼早期胚胎发育过程中囊胚sphere时期的蛋白质表达情况,并分析该时期表达的蛋白质的相应功能和参与调控的生物过程。以野生型斑马鱼发育至sphere时期即4 hpf的胚胎为样本,利用i TRAQ标记与LC-MS/MS串联质谱技术,结合数据库比对,对该时期表达的蛋白质进行定性和定量的鉴定分析。检测结果共鉴定到的总蛋白数为1 178个,利用生物信息学进行功能分析,发现这些蛋白广泛参与了细胞信号传递、细胞运动和细胞骨架构建、细胞增殖、细胞分化、物质合成与代谢等各项重要的生命活动过程。研究表明,利用i TRAQ标记的方法可以对4hpf时期的斑马鱼胚胎中的蛋白质进行有效的分离和鉴定,并初步建立了斑马鱼早期发育关键阶段sphere时期的蛋白质组表达图谱,以期为斑马鱼胚胎发育过程中蛋白质组学和调控机制的研究提供参考。     

斑马鱼微卫星分子标记检测鲤鱼种间多态性

用6072对斑马鱼（Danio rerio）微卫星引物对黑龙江鲤（Cyprinus carpio haematopterus Temminck et Schlegel）、荷包红鲤（Cyprinus carpio var．wuyuanensis）和柏氏鲤（Cyprinus pellegrini Tchang）进行种间遗传差异分析，共发现563个斑马鱼微卫星标记在鲤鱼种间表现出多态性。从中随机抽选25个标记，对斑马伍和3种鲤鱼的遗传多样性进行分析，使用Phylip3．63软件按照Nei氏标准遗传距离计算种间遗传距离，再用MEGA3．0软件绘制NJ系统发生树，并进行1000次bootstrap检验系统树。结果显示，黑龙江鲤与荷包红鲤首先聚类，然后是柏氏鲤、斑马鱼。这些具有多态性的斑马鱼微卫星标记可用于鲤鱼种间种质鉴定，同时，借用模式生物斑马鱼的丰富遗传标记资源，增加同科的鲤鱼遗传连锁图谱上遗传标记的密度，必将对鲤鱼遗传育种进入分子育种时代起到促进作用。     

Evaluation of zebrafish (Danio rerio) as an animal model for the viral infections of fish

Zebrafish (Danio rerio) is a laboratory model organism used in different areas of biological research including studies of immune response and host–pathogen interactions. Thanks to many biological tools available, zebrafish becomes also an important model in aquaculture research since several fish viral infection models have been developed for zebrafish. Here, we have evaluated the possible use of zebrafish to study infections with fish viruses that have not yet been tested on this model organism. In vitro studies demonstrated that chum salmon reovirus (CSV; aquareovirus A) and two alloherpesviruses cyprinid herpesvirus 1 (CyHV‐1) and cyprinid herpesvirus 3 (CyHV‐3) are able to replicate in zebrafish cell lines ZF4 and SJD.1. Moreover, CSV induced a clear cytopathic effect and up‐regulated the expression of antiviral genes vig‐1 and mxa in both cell lines. In vivo studies demonstrated that both CSV and CyHV‐3 induce up‐regulation of vig‐1 and mxa expression in kidney and spleen of adult zebrafish after infection by i.p. injection but not in larvae after infection by immersion. CyHV‐3 is eliminated quickly from fish; therefore, virus clearing process could be evaluated, and in CSV‐infected fish, a prolonged confrontation of the host with the pathogen could be studied.     

Does the gastrointestinal tract serve as the infectious route of Aeromonas hydrophila in crucian carp (Carassius carassius)?

Under experimental challenges, the gastrointestinal (GI) tract of fish has been proposed as an infectious route of several pathogenic bacteria. Is this also the case for diseased fishponds? A field research was conducted to verify this hypothesis. A crucian carp (Carassius carassius) reared fishpond with motile Aeromonas septicaemia outbreak was sampled in this study. A total of 62 strains of Aeromonas hydrophila were isolated and identified. The clonal relationship among these strains was determined by sequencing the gyrB gene, ERIC‐PCR, RAPD‐PCR, and the presence of seven virulence genes. Strains with identical genotypes were further confirmed as the same clone by multilocus sequence typing analysis. Experimental infection assays were also conducted in zebrafish (Danio rerio). The results show that the same clone strains identical to those in the blood of diseased fish existed in the intestinal digesta of diseased and uninfected fish. Regardless of their origins, all these strains were highly pathogenic to zebrafish. The result indicates that pathogenic strains of A. hydrophila had existed in the GI tract of fish before the infection occurred. This increases our knowledge on infectious route of A. hydrophila in crucian carp.     

Dietary taurine supplementation in plant protein based diets do not affect growth and reproductive performance of zebrafish

Taurine (Tau) has been regarded as a conditional essential nutrient for some fish species. Although its role has been extensively studied in higher vertebrates, limited results are reported with fish especially its role on reproductive performance and the ontogenic changes on Tau levels throughout the life cycle. Therefore, we designed a feeding trial using zebrafish as a model species to test whether Tau supplementation to plant protein diets would have a positive effect on growth and reproductive performance. Zebrafish were fed plant protein diets containing graded levels of Tau (0.2, 4.6, 5.9 and 13.7 g/kg diet) from 10 days post fertilization (dpf) to sexual maturity. An additional commercial diet was used as a positive control for performance. The trial followed a completely randomized design with five treatments (diets) and three replications. After 60 days of feeding, growth, Tau concentration in the body, redox status, lipid body composition, reproductive and offspring performances were analysed. Tau supplementation did not affect growth and/or reproductive performance; however, zebrafish seems to differently modulate Tau concentration according to the growth stage. Tau seemed to induce a hypolipidemic effect in zebrafish by reducing lipid accumulation in their bodies (p < .05). A trend to a more pro‐oxidant effect of Tau supplementation was observed by the decreased reduced glutathione levels. In sum, Tau does not affect growth and reproductive performance of zebrafish but it is important for normal lipid utilization and redox status.     

基于不同空间插值法的黄海和东海北部游泳动物多样性空间分布研究

物种多样性空间分布是生态学研究的重要内容之一,空间插值法在该领域应用广泛。本研究以2017年5月黄海和东海北部游泳动物多样性为例,选取种类数(S)、Shannon-Weaver多样性指数(H′)、Pielou均匀度指数(J)和Margalef丰富度指数(d)来衡量该海域物种多样性,应用反距离加权法(Inverse distance weighted, IDW)、径向基函数法(Radial basis function, RBF)、局部多项式法(Local polynomial interpolation, LPI)、普通克里格法(Ordinary kriging, OK)和协同克里格法(Ordinary cokriging, COK)对上述4种多样性指数进行插值,通过jackknife检验的方法选择适合每种多样性指数的空间插值法。结果表明,对于4种多样性指数,COK结果均最好,而其他4种方法精度较低,且差别不大。COK预测结果表明,S和d都呈现出东南部高,北部次之,中部最低的格局。H′和J也是中部最低,在南部和北部海域差异不大。4种多样性指数在32?N以南海域的高值区范围大致相同。COK可以作为黄海和东海北部游泳动物多样性空间插值的方法。     

Rearing zebrafish (Danio rerio) larvae without live food: evaluation of a commercial, a practical and a purified starter diet on larval performance

Owing to the increasing importance of zebrafish as a vertebrate model in many fields of research, efforts must be made to breed and maintain this species in laboratory. Zebrafish larvae are traditionally reared on cultured live paramecia during the first 9 days of exogenous feeding, followed by a combination of paramecia and artemia nauplii until day 21, making larval rearing expensive, labour intensive and unpredictable. Thus, a trial was conducted with zebrafish larvae in order to evaluate the suitability of artificial diets as an alternative to live food during the first 21 days of exogenous feeding. Five dietary treatments were tested: (1) artemia nauplii; (2) a commercial; (3) a purified; (4) a practical diet, all delivered continuously; (5) the same practical diet delivered manually. The best overall larval performance was achieved in the group fed artemia nauplii (86% survival, 14.3 mm standard length, 46.1 mg wet weight). Compared with existing results obtained with the traditional live food schedule, our results suggest that paramecia might not be the most suitable first food for zebrafish, and that artemia nauplii could be used as the only live food. The present work demonstrates that zebrafish larvae can be reared without live food with a significant growth and a high survival, provided that an appropriate artificial diet is presented in a continuous way. Among the diets tested, the practical diet, if continuously delivered, led to the best performance assuring a mean standard length of 72% of that obtained with artemia and a similar survival rate. Moreover, the purified diet, supporting over 50% survival and an appreciable growth, could be useful in some toxicological studies in which a well‐defined diet is needed.     

微囊藻毒素急性暴露对斑马鱼卵巢的损伤效应

为了探究蓝藻(Cyanobacteria)大量爆发产生的微囊藻毒素(microcystins,MCs)对鱼类的生殖毒性,以斑马鱼(Danio rerio)为实验对象,采取腹腔注射毒性最强的microcystin-LR(MC-LR)方式,研究MC-LR对斑马鱼卵巢的损伤效应及其作用机制。对性成熟雌性斑马鱼腹腔注射50μg/kg和200μg/kg MC-LR,在注射3、9、24、48 h后取卵巢分析其生理活性指标的变化。结果显示,染毒24 h后,200μg/kg剂量组斑马鱼性腺指数(gonad somatic index,GSI)14.14与对照组性腺指数16.98相比显著降低(P<0.05),其他组别无显著变化;卵巢发生卵母细胞空泡化、卵母细胞膜与滤泡细胞层连接组织缺失等病理现象;MC-LR显著抑制了斑马鱼卵巢蛋白磷酸酶(protein phosphatase 2A,PP2A)活性,并激活促成熟因子(maturation promoting factor,MPF)活性;MC-LR处理后,斑马鱼卵巢内丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)家族中的p 38MAPK、ERK1/2的转录水平显著上调,JNK未发生显著变化。研究表明,MC-LR抑制斑马鱼卵巢PP2A活性,并激活MPF活性与MAPK信号通路中ERK1/2与p 38MAPK的转录水平,进而干扰其卵母细胞的发育进程并产生生殖毒性。     

基于Bytetrack的多目标跟踪算法在斑马鱼毒性行为识别中的应用

利用计算机视觉技术识别斑马鱼(Danio rerio)在不同污染物暴露下的行为变化是水质毒性评价的常用方法之一,但传统方法存在效率低、面对遮挡和复杂环境时性能差等缺陷。针对这些问题,本研究使用基于Bytetrack的多目标跟踪算法追踪斑马鱼在4种污染物(Zn、Pb、Cr和苯酚)暴露2 h后的行为变化,对斑马鱼在4种浓度梯度中的平均速度、最大速度、最低速度、平均碰撞次数和行为轨迹等指标进行分析。结果显示,算法的追踪精度、漏检率和检测时间(每300帧)分别能达到90.26%、16.33%和0.19 min,检测时间和精度相比于传统目标检测方法有较大提升。同时,根据污染物不同,该方法能准确识别特定污染物环境中斑马鱼相应的运动状态及轨迹变化,可实现精确识别和实时响应,在鱼类毒性行为识别领域具有重要参考意义。     

长三角地区罗氏沼虾成虾主要疾病的病原研究

2009年8月-2010年8月在长三角地区开展了罗氏沼虾(Macrobrachium rosenbergii)成虾主要疾病的流行病学调查.结果显示:所采集病样按其症状分为肌肉白浊病(以上海和浙江病样为主)、蓝体病(以江苏病样为主)、红体病(以浙江病样为主)三类.从采集的罗氏沼虾病样中分离到优势细菌11株,人工感染健康罗...     

Establishing a zebrafish transgenic line expressing tilapia lysozyme with enhanced antibacterial activity

In recent years, infectious disease caused by Streptococcus agalactiae has increased in aquaculture, threatening the healthy development of tilapia and resulting in substantial economic losses. Cultivating disease‐resistant tilapia by transgenic technology is one strategy that has been developed to address this issue. Here, we used a transposon system to investigate gene expression and tissue lytic activity in transgenic zebrafish expressing the tilapia lysozyme. The transpose vector contained the Tgf2 transposon skeleton, tilapia heat shock protein 70 promoter (Hsp70) and two target genes (tilapia C‐type lysozyme 3 [lysozyme‐C3] and green fluorescent protein [GFP]). The transgenic zebrafish F₀ generation was obtained by microinjecting the donor vector and transposase mRNA into fertilized zebrafish eggs. There was 45% green fluorescence in the zebrafish F₀ generation, which was spread over the entire body based on microscope observation. The F₀ generation was reared to sexual maturity, at which point individuals were mated with wild‐type zebrafish to produce the F₁ generation. Tilapia lysozyme‐C3 expression was detected in the liver of F₁ generation by RT‐PCR and western blotting, but was not detected in the skin, intestine, or muscle. Significantly higher liver bacteriolysis activity was detected in transgenic zebrafish compared with wild‐type zebrafish. Therefore, transgenic zebrafish may have an increased potential for bacterial resistance.     

Gonadotropins and their paracrine signaling network in the zebrafish ovary

Pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), play fundamental roles in vertebrate ovarian development and function. However, there has been an increasing body of evidence that the actions of FSH and LH are mediated or modulated by a variety of locally produced peptide or protein factors, which form an intimate regulatory network within and between the ovarian follicles. In the past few years, a variety of growth factors have been identified and characterized in the zebrafish ovary including activin and epidermal growth factor (EGF), which are important components of the intraovarian communication network. To understand how this local network interacts with the gonadotropins from the pituitary, we have recently cloned and characterized all the subunits of zebrafish FSH and LH from the pituitary as well as their receptors (FSHR and LHR) from the ovary. Using the Chinese hamster ovary (CHO) cells as the bioreactor, we have produced recombinant zebrafish FSH and LH with biological activities. With the recombinant hormones available, the functions of zebrafish FSH and LH in the ovary and their interactions with the local factors will be an important issue to address in the future. This review briefly summarizes some recent work from our laboratory and others on both gonadotropins and their potential intraovarian signaling factors in the zebrafish.     

鱼类生殖细胞移植的研究进展及应用前景

生殖细胞移植是指将供体的生殖细胞移植到同种或异种受体体内,供体生殖细胞嵌合到受体性腺,经过增殖、分化并最终发育为功能性配子的过程。作为辅助生殖技术,它不仅为珍稀濒危动物的繁育和保护提供了新途径,同时也为生殖干细胞的功能研究提供了有效手段。鱼类生殖细胞移植研究首先在模式鱼类斑马鱼中开展,经过十多年的发展,取得了一系列突破性的进展:主要包括先后建立了以胚胎、仔鱼和成鱼为受体的生殖细胞移植体系,精原和卵原干细胞的发现拓宽了供体生殖细胞的选择,受体的选择与制备方法的完善。该技术在缩短鱼类性成熟周期、性控育种、珍稀濒危鱼类保护等方面具有巨大的应用前景,已成功在多种淡水和海水鱼类中开展了研究和应用。本文结合作者的研究实践和经验,系统地梳理和总结了鱼类生殖细胞移植的研究进展,指出了该技术实践应用的关键问题,并探讨了其应用前景。     

Atrial natriuretic peptide. II. Pathophysiology and possible clinical significance. Review

The heart atrium, as well as under certain pathophysiological conditions the ventricle, synthesize and release ANP. Exerting natriuretic, diuretic and vasorelaxant effects, this peptide plays an important role in the body's blood volume and blood pressure homeostasis. Whereas the pharmacological actions of ANP have been quite convincingly demonstrated, its physiological and pathophysiological role is less well defined. ANP plasma levels tend to be increased in diseases with salt and water retention, such as essential hypertension, congestive heart failure, renal failure or liver cirrhosis. With regard to its hemodynamic effects, ANP seems to be beneficial in patients with hypertension. ANP appears to have little therapeutic potential as a diuretic in patients with congestive heart failure and liver cirrhosis, possibly due to the decreased renal responsiveness to ANP in these diseases. However, ANP might to be a valuable therapeutic agent in acute renal failure.