Assessing the anti-estrogenic activity of sodium pentachlorophenol in primary cultures of juvenile goldfish (Carassius auratus) hepatocytes using vitellogenin as a biomarker

Both pentachlorophenol and 2,3,7,8-tetrachlorodibenzo-p-dioxin （TCDD） had been studied widely because of their probable anti-estrogenic activity. Sodium pentachlorophenol （PCP-Na）, as a industrial product used in many fields, usually contains a trace of TCDD. The aim of this study was to assess the anti-estrogenic effect of PCP-Na in juvenile goldfish （Gurassius auratus） hepatocyte cultures using vitellogenin （VTG） as the biomarker. The ID50 of PCP-Na was investigated and then a series of concentrations （0.001 0.5 μg/ml） of PCP-Na were evaluated to estimate the anti-estrogenic activity. Results showed that PCP-Na was cytotoxic for hepatocytes even at very low concentration 〈1.21 μg/ml, and it could not induce VTG at any concentrations tested. Since it failed to stimulate VTG production, the possibility of its anti-estrogenic effect was tested, and a well-known anti-estrogenic compound-tamoxifen was used as positive control. PCP-Na caused a reduction in VTG synthesis in juvenile goldfish （Carassius auratus） hepatocytes at concentrations 〉0.1μg/ml when co-exposure with 1μg/ml 17β-estradiol （E2）, making its anti-estrogenic activity approximately as potent as tamoxifen. Our results indicate that PCP-Na can act as negative modulators of estrogenic function in juvenile goldfish （Carassius auratus） hepatocytes.

Transcription profiles of skin and head kidney from goldfish suffering hemorrhagic septicemia with an emphasis on the TLR signaling pathway

DEAR EDITOR, Hemorrhagic septicemia is an acute, highly fatal disease that affects goldfish (Carassius auratus). To gain a better understanding of related immune genes, the transcriptomes of the skin and head kidney of goldfish suffering hemorrhagic septicemia were sequeneed, assembled, and characterized.

The acute lethality and endocrine effect of 1,2,3,7,8-PeCDD in juvenile goldfish (Carassius auratus) in vivo

1,2,3,7,8-PeCDD was administrated to juvenile goldfish （Carassius auratus） by peritoneal injections to explore the acute lethality and endocrine effects of 1,2,3,7,8-PeCDD in vivo. The value of acute median lethal dosage （LD50） of 1,2,3,7,8-PeCDD was determined in acute lethality tests. The endocrine effect of 1,2,3,7,8-PeCDD, whose exposed concentrations were determined based on the LD50 （1.84 mg/kg）, was studied by measuring the plasma vitellogenin （Vtg） content in juvenile male goldfish with enzyme-linked immunosorbent assays （ELISA）. Due to its significant induction of the plasma Vtg after one week＇s exposure in vivo in the 1/2 LD50 and LD30 groups, 1,2,3,7,8-PeCDD might be one of the important contributors to the estrogenic effect of PCDDs in the environment. The values of 1/2 LD50 and LD30 were within the range of the effective dosages of 1,2,3,7,8-PeCDD, indicating that there was a certain relationship between the estrogenic effective dosages and the LD50.

LOCALIZATION OF A PUTATIVE EPIBOLY-DETERMINING FACTOR IN OOCYTES OF THE GOLDFISH (CARASSIUS AURATUS)

It is unknown whether cytoplasmic determinants in goldfish eggs are asymmetrically localized beforematuration. In this study fully grown goldfish oocytes with infact germinal vesicles (GVs) were ligated withbaby hair loops along desired planes into two parts, mature in vitro, and inseminated. Embryos developedfrom the animal halves with GV of oocytes ligated along the equator un unable to undergoepiboly, while those developed from the GV-containing parts of oocytes ligated along the animal-vegetal axiswere able to. Also, embryos developed from the animal halves obtained from fertilized eggs could undergo epiboly. From there results, it was postulated that the goldfish’s premature oocyte possessed a putativefactor in the vegetal hemisphere which was responsible for epiboly of the embryonic cells.

Polyphyletic Origin of Ornamental Goldfish

Mitochondrial DNA fragment of cytb was compared in all species Carassius auratus complex and three forms of ornamental goldfish. It is shown that the phylogenetic relationships between complex species correspond to the existing views, based on mtDNA data and geographical distribution. All forms of ornamental goldfish have a monophyletic origin from Chinese goldfish C. auratus auratus. The analysis showed that three nuclear genes (rps7, GH1 and Rh) in the two forms of ornamental goldfish (Oriental twintail goldfish and Chinese Ranchu) were almost identical C. auratus auratus genes, while all three gene in another more simple form of goldfish (common goldfish) were highly homologous to carp Cyprinus carpio nuclear genes. The obtained data suggested that in the history of aquarium goldfish breeding occurred the stage of distant hybridization between goldfish and common carp. Subsequently, the nuclear genomes of some ornamental forms could be enriched by goldfish genes (a relatively recent form as Oriental twintail goldfish and Chinese Ranchu) or common carp genes (the simplest and most ancient forms like common goldfish) as a result of multidirectional breeding and selection of aquarium goldfish various forms.

Preliminary Study on the Histology in the Head Sarcoma of Goldfish (Carassius auratus)

The sarcoma structure of goldfish was first analyzed by frozen section technique. The present study revealed the alkaline and acid phosphatase distribution in the head sarcoma of goldfish. And the histochemical staining in situ by calcium-cobalt method and lead nitrate method displayed a large amount of alkaline and acid phosphatases in the head sarcoma, with higher enzyme activity. The alkaline and acid phosphatases belong to hydrolases, which are widely present in various tissues. The results showed that the activity of external sarcoma is similar to the middle one, while there was more alkaline phosphatase near to the tissue sarcoma under the goldfish scales. And they have the strong activity. The study will lay the theoretical basis on enhancing the ornamental value of goldfish.

Penetrating Ocular Trauma in a Goldfish Model after Chloramphenicol or Traditional Medicine Angong Niuhuang Pill Treatment: An Analysis of Retinal Responses after 7 Days

Purpose: While there were numerous clinical examples of ocular damage, the histopathology of retinal responses was poorly characterized, and animal model research was limited. The consequences of retinal lesions with traditional medicine treatment were mostly unknown. In this work, goldfish was employed as a model for penetrating injury of the eyes to study the ocular trauma with antibiotic and traditional medicine treatments. Materials and Methods: The penetration of the eyes was performed at 2mm above the midline of the limbus in the superior quadrant with an 18-gauge sterile needle, all the way to the retina but avoiding the lens while the animals were anaesthetized in water containing 100 mg/L ethyl 3-aminobenzoate methanesulfonate (MS-222). The animals were divided into three groups and received treatments of saline, chloramphenicol, and Angong Niuhuang pill (Angong) eyedrops twice a day respectively. Angong after seven days, the animals were sacrificed. Immunohistochemical evaluation of proliferative cell nuclear antigen (PCNA), brain-derived neurotrophic factor (BDNF), catalase, and cleaved caspase-3, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) for apoptotic cells were performed on the fish’s superior quadrants of the retinae. The whole experiment was repeated two times. Result: It was evident that both the retinae treated with chloramphenicol and Angong had fewer apoptotic cells and more proliferative cells, particularly in the cases of the Angong treatment. BDNF was in the inner nuclear layer and ganglion cell layer of all groups. In the chloramphenicol and Angong groups, some cells in the outer nuclear layer displayed BDNF. Catalase reactions confirmed equivalent findings that the treated retinae with chloramphenicol or Angong treatment had less catalase. Conclusion: Angong Niuhuang pill is a traditional medicine composite formula with antibiotic and neuroprotective effects on the brain and the eyes, and this work also pointed out its possible pharmaceutical potential.

水丝蚓及人工饲料对草金鱼生长和体色的影响

为评价水丝蚓或人工饲料对草金鱼生长和体色的影响,选择初始体质量(7.82±1.84)g、体长(5.73±0.45)cm的草金鱼,分别投喂水丝蚓(SY组)和人工饲料(SL组),定期测定体质量和体色色度。结果显示,SY组试验鱼增质量率、特定生长率显著高于SL组(P<0.05),肥满度差异不显著(P>0.05);两组试验鱼体质量直线斜率差异不显著(P>0.05)。两组草金鱼体色L*(亮度)值与投喂时间呈正相关趋势,a*(+a*红,-a*绿)、b*(+b*黄,-b*蓝)值均呈负相关趋势;两组试验鱼色度值直线斜率(K)差异不显著(P>0.05);试验结束时,SY组试验鱼体表a*和b*值均显著高于SL组(P<0.05)。研究表明,草金鱼体质量与生长时间的线性相关性较强,投喂水丝蚓更容易被草金鱼接受,更能稳定促进其生长,但无论投喂水丝蚓或是人工饲料,其体表红色均会逐渐变淡。

注射姜黄素对草金鱼生长性能、脂质代谢、消化酶活力及抗氧化能力的影响

试验旨在研究姜黄素对草金鱼脂质代谢的改善效果,摸索适宜添加量。选用初重(40.12±1.23)g、体长(10.34±0.08)cm的草金鱼360尾,随机分为6组,每组3个重复,每个重复20尾。各组分别注射不同浓度的(0、40、80、160、320、640μmol/L)姜黄素,养殖14 d。结果显示,注射姜黄素7、14 d后,160~640μmol/L姜黄素组草金鱼血清总胆固醇(TC)、甘油三酯(TG)含量显著低于对照组(P<0.05);320μmol/L姜黄素组和640μmol/L姜黄素组草金鱼肝胰脏谷草转氨酶(AST)和谷丙转氨酶(ALT)活性显著低于对照组(P<0.05);320μmol/L姜黄素组草金鱼肝胰脏过氧化氢酶(CAT)、总超氧化物歧化酶(T-SOD)活性显著高于对照组(P<0.05)。研究表明,注射姜黄素能够在一定程度上降低草金鱼血清TC和TG的含量,对肝胰脏具有一定修复作用,能够提升肝胰脏抗氧化能力。

Ecotoxicological effects of waterborne PFOS exposure on swimming performance and energy expenditure in juvenile goldfish (Carassius auratus)

The potential risks of perfluorooctane sulfonate （PFOS） are of increasing ecological concern. Swimming performance is linked to the fitness and health of fish. However, the impacts of PFOS on swimming performance remain largely unknown. We investigated the ecotoxicological effects of acute exposure to PFOS on the swimming performance and energy expenditure of juvenile goldfish （Carassius auratus）. The fish were exposed to a range of PFOS concentrations （0, 0.5, 2, 8 and 32 mg/L） for 48 hr. The spontaneous swimming activity, fast-start swimming performance, critical swimming speed （Ucrit） and active metabolic rate （AMR） of the goldfish were examined after exposure to PFOS. PFOS exposure resulted in remarkable effects on spontaneous activity. Motion distance was reduced, and the proportion of motionless time increased with increasing concentrations of PFOS. However, no significant alterations in the fast-start performance-related kinematic parameters, such as latency time, maximum linear velocity, maximum linear acceleration or escape distance during the first 120 msec after stimulus, were observed after PFOS exposure. Unexpectedly, although PFOS exposure had marked influences on the swimming oxygen consumption rates and AMR of goldfish, the U crit of the goldfish was not significantly affected by PFOS. This may result in a noteworthy increase in the energetic cost of transport. The overall results indicate that, in contrast to spontaneous activity, underlying swimming capabilities are maintained in goldfish after short-term exposure to PFOS, but energy expenditure during the process of swimming is dramatically aggravated.

Upregulation of the PPAR signaling pathway and accumulation of lipids are related to the morphological and structural transformation of the dragon-eye goldfish eye

Goldfish comprise around 300 different strains with drastically altered and aesthetical morphologies making them suitable models for evolutionary developmental biology.The dragon-eye strain is characterized by protruding eyes(analogous to those of Chinese dragons).Although the strain has been selected for about 400 years,the mechanism of its eye development remains unclear.In this study,a stable dragon-eye goldfish strain with a clear genetic background was rapidly established and studied.We found that upregulation of the PPAR signaling pathway accompanied by an increase in lipid accumulation might trigger the morphological and structural transformation of the eye in dragon-eye goldfish.At the developmental stage of proptosis(eye protrusion),downregulation of the phototransduction pathway was consistent with the structural defects and myopia of the dragon-eye strain.With the impairment of retinal development,cytokine-induced inflammation was activated,especially after proptosis,similar to the pathologic symptoms of many human ocular diseases.In addition,differentially expressed transcription factors were significantly enriched in the PAX and homeobox families,two well-known transcription factor families involved in eye development.Therefore,our findings reveal the dynamic changes in key pathways during eye development in dragon-eye goldfish,and provide insights into the molecular mechanisms underlying drastically altered eyes in goldfish and human ocular disease.

金鱼Caspase-8多克隆抗体的制备

为研究环境压力对金鱼细胞凋亡外在途径的影响,设计特异性引物扩增Caspase-8基因片段(编码第305—763位氨基酸),然后进行原核表达,并通过动物免疫实验制备Caspase-8多克隆抗体,检测其效价、金鱼不同组织表达水平和相关蛋白互作.结果表明:制备的Caspase-8多克隆抗体与抗原结合的最大稀释倍数为1∶256 000,显示出良好的免疫原性;Caspase-8在金鱼不同组织中有不同程度表达;Caspase-8与E3泛素连接酶cullin3存在相互作用.综上,制备的Caspase-8多克隆抗体有望应用于金鱼细胞凋亡蛋白Caspase-8表达水平及相关蛋白互作的研究中.

Causal gene identification and desirable trait recreation in goldfish

Goldfish(Carassius auratus) have long fascinated evolutionary biologists and geneticists because of their diverse morphological and color variations.Recent genome-wide association studies have provided a clue to uncover genomic basis underlying these phenotypic variations,but the causality between phenotypic and genotypic variations have not yet been confirmed.Here,we edited proposed candidate genes to recreate phenotypic traits and developed a rapid biotechnology approach which combines gene editing with high-efficiency breeding,artificial gynogenesis,and temperature-induced sex reversal to establish homozygous mutants within two generations(approximately eight months).We first verified that low-density lipoprotein receptorrelated protein 2B(lrp2a B) is the causal gene for the dragon-eye variation and recreated the dragon-eye phenotype in side-view Pleated-skirt Lion-head goldfish.Subsequently,we demonstrated that the albino phenotype was determined by both homeologs of oculocutaneous albinism type II(oca2),which has subfunctionalized to differentially govern melanogenesis in the goldfish body surface and pupils.Overall,we determined two causal genes for dragon-eye and albino phenotypes,and created four stable homozygous strains and more appealing goldfish with desirable traits.The developed biotechnology approach facilitates precise genetic breeding,which will accelerate re-domestication and recreation of phenotypically desirable goldfish.

Estrogenic effects of water from the Yangtze River (Nanjing section) on goldfish (Carassius auratus) after an early life stage exposure

Effects of river water from the Yangtze River （Nanjing section） on fish development, vitellogenin （VTG） induction, gonado-somatic index （GSI） and sex ratio were investigated by exposing goldfish （Carassius auratus） in the early life stage （from fertilization to 28 days post-hatch） to water samples （25%, 50% and 100%） collected from three representative sections. The results showed that there was no significant effect on hatching success for any of the exposure groups, but survival was significantly reduced when compared with the control （P 0.05）. Body lengths, weights of all treated fish did not differ significantly from those of the control. Condition factors （CF） of larval fish exposed to 50% and 100% river water from the Jiangxinzhou section and 100% river water from the Daqiao section were significantly lower than that of the control （P 0.05）. VTG inductions were significant in larval fish exposed to all the dilution series of river water. No significant difference in CF value was observed in any exposure group after 150 days of depuration. VTG was fully eliminated after 75 days of depuration. For both female and male, GSI did not significantly differ between exposure groups and the control after 150 days of convalescence. The highest female：male ratios were observed in response to the treatment with 50% or 100% river water from the Jiangxinzhou section and 100% river water from the Daqiao section （53：47, 56：44 and 54：46, respectively）, but no significant difference in sex ratio was observed in any treated group when compared to the control. The results showed that early life stage exposure of river water from the Yangtze River （Nanjing section） had adverse effects on goldfish development and reproductive health, and the effects on CF and VTG were reversible after depuration in clean water.

拓展性学习资源的开发与教学尝试

运用金鱼杂交实验开发出系列拓展性的学习资源,从互补性、新颖性和综合性三个方面,开展“孟德尔的豌豆杂交实验(一)”的教学尝试,激发学生学习兴趣,促进学生在解决问题中提升创新思维和科学探究能力。

STUDIES ON ULTRASTRUCTURE OF MATURE ERYTHROCYTE IN GOLDFISH(Carassius auratus)

Weinreb first reported the results of transmission electron microscopy observation on ultrastructure of goldfish erythrocyte in 1963. In recent years, there are few reports about the ultrastructure of blood cells in fish For further understanding the fine structure of mature erythrocyte in teleosts, using goldfish (Carassius auratus) as the experimental material We made an observation of the morphology of its mature erythrocyte by means of scanning electron microscopy as well as a study on the ultrastructure by freeze etching technique. Since there are none of reports concerning the study on erythrocyte in fish by using freeze etching technique, the present note will provide here a practical method and the data of studying about ultrastructure of erythrocyte in fish.

Preliminary study on search for genes for early embryonic development in goldfish by mRNA differential display

MATERNAL RNAs stored in eggs play an essential role during the initial stage of embryonic de-velopment in animals and they are degraded as the development proceeds. In some animals (e.g. fish and amphibian), zygotic genes begin to transcribe new mRNA only when the embryosdevelop to about midblastula stage. This degradation of maternal RNA and synthesis of

vsx1 3' untranlated region-mediated translation difference at different developmental stages of goldfish embryos

Visual system homeobox-1 （vsxl） is important in retinal progenitor proliferation, differentiation, and function maintenance of bipolar cells in vertebrates. Recent study in Xenopus laevis has shown that vsxl 3＇ untranslated region （3＇ UTR） can mediate cell-specific translation of vsxl mRNA in the bipolar cells of the retinal inner nuclear layer （INL）. vsxl is also transcribed at the early developmental stages prior to eye formation and its spatiotemporal expression patterns are conserved in all the examined vertebrates. In order to determine whether the vsxl 3＇ UTR has a role in regulating the spatiotemporal expression of vsxl during early embryogenesis, we constructed a vsxl UTR-controlled green fluorescent protein （GFP） reporter gene system and examined the GFP expression pattern in goldfish, Carassius auratus, at different developmental stages, Our results indicated that both the vsxl 5＇ UTR and the vsxl 3＇ UTR remarkably repressed GFP expression at transcription level but did not regulate tissue-specific translation at early developmental stages. GFP protein was ubiquitously expressed in the embryos injected with GFP-sensors containing vsxl UTRs before 60 h post-fertilization （hpf）. From hatching stage （72 hpf） onwards, however, GFP protein was specifically expressed in the bipolar cells of the retinal 1NL in the vsxl 3＇UTR-GFP-sensor embryos, but was still ubiquitously expressed in the embryos injected with GFP-sensor lacking vsxl 3＇ UTR. These observations showed a significant difference of vsxl 3＇ UTR-mediated translation between early and late developmental stages and suggested that vsxl 3＇ UTR might not be involved in regulating the spatiotemporal expression of vsxl until hatching stage during embryogenesis.

Assessment of dietary chaste tree (Vitex agnus-castus) fruit extract on growth performance, hemato-biochemical parameters, and mRNA levels of growth and appetite-related genes in goldfish (Carassius auratus)

In this study,the effect of oral administration of chaste tree fruit(CTF)extract was evaluated on growth performance,hematological indices,serum biochemical parameters,and the expression of growth and appetite-related genes in goldfish(Carassius auratus).The fry(N=300;2.40±0.12 g)were randomly dispersed into 12 aquaria and fed with different levels of CTF extract(0(control),0.5%,1.0%,and 1.5%)for 56 days.At the end of the trial,the highest body weight gain and the lowest feed conversion ratio were observed in the fish fed with 1.5%CTF extract(P<0.05).The group fed with CTF extract at 1.5%showed the highest levels of red blood cell,hemoglobin,hematocrit,and serum albumin(P<0.05),while no significant changes were observed for mean corpuscular volume,mean hemoglobin,and mean corpuscular hemoglobin concentration among all groups(P>0.05).The levels of aspartate transaminase and lactate dehydrogenase were decreased by increasing the supplementation levels of CTF extract and the lowest values were obtained in 1.5%CTF extract diet(P<0.05).The expression of growth hormone,insulin-like growth factor 1,and ghrelin genes was significantly up-regulated in the fish fed with 1.5%CTF extract compared to other treatments(P<0.05).Overall,the enrichment of goldfish diet with 1.5%CTF extract could interfere positively in the growth-related traits and hematological parameters.