The Intestinal Mycobiota in Wild Zebrafish Comprises Mainly Dothideomycetes While Saccharomycetes Predominate in Their Laboratory-Reared Counterparts

PubMed Central

Siriyappagouder, Prabhugouda; Kiron, Viswanath; Lokesh, Jep; Rajeish, Moger; Kopp, Martina; Fernandes, Jorge

2018-01-01

As an integral part of the resident microbial community of fish intestinal tract, the mycobiota is expected to play important roles in health and disease resistance of the host. The composition of the diverse fungal communities, which colonize the intestine, is greatly influenced by the host, their diet and geographic origin. Studies of fungal communities are rare and the majority of previous studies have relied on culture-based methods. In particular, fungal communities in fish are also poorly characterized. The aim of this study was to provide an in-depth overview of the intestinal mycobiota in a model fish species (zebrafish, Danio rerio) and to determine differences in fungal composition between wild and captive specimens. We have profiled the intestinal mycobiota of wild-caught (Sharavati River, India), laboratory-reared (Bodø, Norway) and wild-caught-laboratory-kept (Uttara, India) zebrafish by sequencing the fungal internal transcribed spacer 2 region on the Illumina MiSeq platform. Wild fish were exposed to variable environmental factors, whereas both laboratory groups were kept in controlled conditions. There were also differences in husbandry practices at Bodø and Uttara, particularly diet. Zebrafish from Bodø were reared in the laboratory for over 10 generations, while wild-caught-laboratory-kept fish from Uttara were housed in the laboratory for only 2 months before sample collection. The intestine of zebrafish contained members of more than 15 fungal classes belonging to the phyla Ascomycota, Basidiomycota, and Zygomycota. Fungal species richness and diversity distinguished the wild-caught and laboratory-reared zebrafish communities. Wild-caught zebrafish-associated mycobiota comprised mainly Dothideomycetes in contrast to their Saccharomycetes-dominated laboratory-reared counterparts. The predominant Saccharomycetes in laboratory-reared fish belonged to the saprotrophic guild. Another characteristic feature of laboratory-reared fish was the significantly higher abundance of Cryptococcus (Tremellomycetes) compared to wild fish. This pioneer study has shed light into the differences in the intestinal fungal communities of wild-caught and laboratory-reared zebrafish and the baseline data generated will enrich our knowledge on fish mycobiota. PMID:29559965

The Intestinal Mycobiota in Wild Zebrafish Comprises Mainly Dothideomycetes While Saccharomycetes Predominate in Their Laboratory-Reared Counterparts.

PubMed

Siriyappagouder, Prabhugouda; Kiron, Viswanath; Lokesh, Jep; Rajeish, Moger; Kopp, Martina; Fernandes, Jorge

2018-01-01

As an integral part of the resident microbial community of fish intestinal tract, the mycobiota is expected to play important roles in health and disease resistance of the host. The composition of the diverse fungal communities, which colonize the intestine, is greatly influenced by the host, their diet and geographic origin. Studies of fungal communities are rare and the majority of previous studies have relied on culture-based methods. In particular, fungal communities in fish are also poorly characterized. The aim of this study was to provide an in-depth overview of the intestinal mycobiota in a model fish species (zebrafish, Danio rerio ) and to determine differences in fungal composition between wild and captive specimens. We have profiled the intestinal mycobiota of wild-caught (Sharavati River, India), laboratory-reared (Bodø, Norway) and wild-caught-laboratory-kept (Uttara, India) zebrafish by sequencing the fungal internal transcribed spacer 2 region on the Illumina MiSeq platform. Wild fish were exposed to variable environmental factors, whereas both laboratory groups were kept in controlled conditions. There were also differences in husbandry practices at Bodø and Uttara, particularly diet. Zebrafish from Bodø were reared in the laboratory for over 10 generations, while wild-caught-laboratory-kept fish from Uttara were housed in the laboratory for only 2 months before sample collection. The intestine of zebrafish contained members of more than 15 fungal classes belonging to the phyla Ascomycota, Basidiomycota, and Zygomycota. Fungal species richness and diversity distinguished the wild-caught and laboratory-reared zebrafish communities. Wild-caught zebrafish-associated mycobiota comprised mainly Dothideomycetes in contrast to their Saccharomycetes-dominated laboratory-reared counterparts. The predominant Saccharomycetes in laboratory-reared fish belonged to the saprotrophic guild. Another characteristic feature of laboratory-reared fish was the significantly higher abundance of Cryptococcus (Tremellomycetes) compared to wild fish. This pioneer study has shed light into the differences in the intestinal fungal communities of wild-caught and laboratory-reared zebrafish and the baseline data generated will enrich our knowledge on fish mycobiota.

Social learning of an associative foraging task in zebrafish

NASA Astrophysics Data System (ADS)

Zala, Sarah M.; Määttänen, Ilmari

2013-05-01

The zebrafish ( Danio rerio) is increasingly becoming an important model species for studies on the genetic and neural mechanisms controlling behaviour and cognition. Here, we utilized a conditioned place preference (CPP) paradigm to study social learning in zebrafish. We tested whether social interactions with conditioned demonstrators enhance the ability of focal naïve individuals to learn an associative foraging task. We found that the presence of conditioned demonstrators improved focal fish foraging behaviour through the process of social transmission, whereas the presence of inexperienced demonstrators interfered with the learning of the control focal fish. Our results indicate that zebrafish use social learning for finding food and that this CPP paradigm is an efficient assay to study social learning and memory in zebrafish.

Susceptibility of zebrafish (Danio rerio) to a model pathogen, spring viremia of carp virus

USGS Publications Warehouse

Sanders, George E.; Batts, William N.; Winton, James R.

2003-01-01

To improve our understanding of the genetic basis of fish disease, we developed a pathogen model, using zebrafish (Danio rerio) and spring virema of carp virus (SVCV). Replicate groups of 10 fish were acclimated to 20 or 24°C, then were exposed to SVCV concentrations of 103 to 105 plaque-forming units per milliliter (PFU/ml) of water and observed daily. In a second trial, fish were acclimated to 15°C, and replicate groups of 10 fish were exposed to SVCV at a concentration of 105 PFU/ml; however, the temperature was raised 1°C/wk. Moribund fish were collected for histologic examination, and dead fish were assayed for virus by use of cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Mortality exceeded 50% in fish exposed to 105 PFU of SVCV/ml at the lower temperatures. Clinical signs of disease became evident seven days after viral exposure and were observed most consistently in fish of the 105 PFU/ml groups. Affected zebrafish were anorectic and listless, with epidermal petechial hemorrhages followed by death. Use of plaque assays and RT-PCR analysis confirmed presence of SVCV at titers ≥ 104 PFU/g of tissue. Histologic lesions included multifocal brachial necrosis and melanomacrophage proliferation in gills, liver, and kidneys. These results indicate that zebrafish are susceptible to infection by SVCV under conditions that mimic a natural route of exposure.

Husbandry stress exacerbates mycobacterial infections in adult zebrafish, Danio rerio (Hamilton)

USGS Publications Warehouse

Ramsay, J.M.; Watral, Virginia G.; Schreck, C.B.; Kent, M.L.

2009-01-01

Mycobacteria are significant pathogens of laboratory zebrafish, Danio rerio (Hamilton). Stress is often implicated in clinical disease and morbidity associated with mycobacterial infections but has yet to be examined with zebrafish. The aim of this study was to examine the effects of husbandry stressors on zebrafish infected with mycobacteria. Adult zebrafish were exposed to Mycobacterium marinum or Mycobacterium chelonae, two species that have been associated with disease in zebrafish. Infected fish and controls were then subjected to chronic crowding and handling stressors and examined over an 8-week period. Whole-body cortisol was significantly elevated in stressed fish compared to non-stressed fish. Fish infected with M. marinum ATCC 927 and subjected to husbandry stressors had 14% cumulative mortality while no mortality occurred among infected fish not subjected to husbandry stressors. Stressed fish, infected with M. chelonae H1E2 from zebrafish, were 15-fold more likely to be infected than non-stressed fish at week 8 post-injection. Sub-acute, diffuse infections were more common among stressed fish infected with M. marinum or M. chelonae than non-stressed fish. This is the first study to demonstrate an effect of stress and elevated cortisol on the morbidity, prevalence, clinical disease and histological presentation associated with mycobacterial infections in zebrafish. Minimizing husbandry stress may be effective at reducing the severity of outbreaks of clinical mycobacteriosis in zebrafish facilities. ?? 2009 Blackwell Publishing Ltd.

The zebrafish embryo model in toxicology and teratology, September 2–3, 2010, Karlsruhe, Germany.

PubMed

Busch, Wibke; Duis, Karen; Fenske, Martina; Maack, Gerd; Legler, Juliette; Padilla, Stephanie; Strähle, Uwe; Witters, Hilda; Scholz, Stefan

2011-05-01

The use of fish embryos is gaining popularity for research in the area of toxicology and teratology. Particularly embryos of the zebrafish offer an array of different applications ranging from regulatory testing to mechanistic research. For this reason a consortium of two research centres and a company with the support of the COST Action EuFishBiomed has organised the Workshop “The zebrafish embryo model in toxicology and teratology”, in Karlsruhe, Germany, 2nd–3rd September 2010. The workshop aimed at bringing together experts from different areas of toxicology using the (zebra)fish embryo and stimulating networking between scientists and representatives from regulatory bodies, research institutions and industry. Recent findings, presented in various platform presentations in the area of regulatory toxicity, high throughput screening, toxicogenomics, as well as environmental and human risk assessment are highlighted in this meeting report. Furthermore, the constraints and possibilities of the model as discussed at the workshop are described. A follow up-meeting was appreciated by the about 120 participants and is planned for 2012.

Model-free information-theoretic approach to infer leadership in pairs of zebrafish.

PubMed

Butail, Sachit; Mwaffo, Violet; Porfiri, Maurizio

2016-04-01

Collective behavior affords several advantages to fish in avoiding predators, foraging, mating, and swimming. Although fish schools have been traditionally considered egalitarian superorganisms, a number of empirical observations suggest the emergence of leadership in gregarious groups. Detecting and classifying leader-follower relationships is central to elucidate the behavioral and physiological causes of leadership and understand its consequences. Here, we demonstrate an information-theoretic approach to infer leadership from positional data of fish swimming. In this framework, we measure social interactions between fish pairs through the mathematical construct of transfer entropy, which quantifies the predictive power of a time series to anticipate another, possibly coupled, time series. We focus on the zebrafish model organism, which is rapidly emerging as a species of choice in preclinical research for its genetic similarity to humans and reduced neurobiological complexity with respect to mammals. To overcome experimental confounds and generate test data sets on which we can thoroughly assess our approach, we adapt and calibrate a data-driven stochastic model of zebrafish motion for the simulation of a coupled dynamical system of zebrafish pairs. In this synthetic data set, the extent and direction of the coupling between the fish are systematically varied across a wide parameter range to demonstrate the accuracy and reliability of transfer entropy in inferring leadership. Our approach is expected to aid in the analysis of collective behavior, providing a data-driven perspective to understand social interactions.

Model-free information-theoretic approach to infer leadership in pairs of zebrafish

NASA Astrophysics Data System (ADS)

Butail, Sachit; Mwaffo, Violet; Porfiri, Maurizio

2016-04-01

Collective behavior affords several advantages to fish in avoiding predators, foraging, mating, and swimming. Although fish schools have been traditionally considered egalitarian superorganisms, a number of empirical observations suggest the emergence of leadership in gregarious groups. Detecting and classifying leader-follower relationships is central to elucidate the behavioral and physiological causes of leadership and understand its consequences. Here, we demonstrate an information-theoretic approach to infer leadership from positional data of fish swimming. In this framework, we measure social interactions between fish pairs through the mathematical construct of transfer entropy, which quantifies the predictive power of a time series to anticipate another, possibly coupled, time series. We focus on the zebrafish model organism, which is rapidly emerging as a species of choice in preclinical research for its genetic similarity to humans and reduced neurobiological complexity with respect to mammals. To overcome experimental confounds and generate test data sets on which we can thoroughly assess our approach, we adapt and calibrate a data-driven stochastic model of zebrafish motion for the simulation of a coupled dynamical system of zebrafish pairs. In this synthetic data set, the extent and direction of the coupling between the fish are systematically varied across a wide parameter range to demonstrate the accuracy and reliability of transfer entropy in inferring leadership. Our approach is expected to aid in the analysis of collective behavior, providing a data-driven perspective to understand social interactions.

Embryonic Alcohol Exposure Impairs the Dopaminergic System and Social Behavioral Responses in Adult Zebrafish

PubMed Central

Fernandes, Yohaan; Rampersad, Mindy

2015-01-01

Background: The zebrafish is a powerful neurobehavioral genetics tool with which complex human brain disorders including alcohol abuse and fetal alcohol spectrum disorders may be modeled and investigated. Zebrafish innately form social groups called shoals. Previously, it has been demonstrated that a single bath exposure (24 hours postfertilization) to low doses of alcohol (0, 0.25, 0.50, 0.75, and 1% vol/vol) for a short duration (2 hours) leads to impaired group forming, or shoaling, in adult zebrafish. Methods: In the current study, we immersed zebrafish eggs in a low concentration of alcohol (0.5% or 1% vol/vol) for 2 hours at 24 hours postfertilization and let the fish grow and reach adulthood. In addition to quantifying the behavioral response of the adult fish to an animated shoal, we also measured the amount of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid from whole brain extracts of these fish using high-pressure liquid chromatograph. Results: Here we confirm that embryonic alcohol exposure makes adult zebrafish increase their distance from the shoal stimulus in a dose-dependent manner. We also show that the shoal stimulus increases the amount of dopamine and 3,4-dihydroxyphenylacetic acid in the brain of control zebrafish but not in fish previously exposed to alcohol during their embryonic development. Conclusions: We speculate that one of the mechanisms that may explain the embryonic alcohol-induced impaired shoaling response in zebrafish is dysfunction of reward mechanisms subserved by the dopaminergic system. PMID:25568285

Mayo Clinic Zebrafish Facility Overview.

PubMed

Leveque, Ryan E; Clark, Karl J; Ekker, Stephen C

2016-07-01

The zebrafish (Danio rerio) is a premier nonmammalian vertebrate model organism. This small aquatic fish is utilized in multiple disciplines in the Mayo Clinic community and by many laboratories around the world because of its biological similarity to humans, its advanced molecular genetics, the elucidation of its genome sequence, and the ever-expanding and outstanding new biological tools now available to the zebrafish researcher. The Mayo Clinic Zebrafish Facility (MCZF) houses ∼2,000 tanks annotated using an in-house, Internet cloud-based bar-coding system tied to our established zfishbook.org web infrastructure. Paramecia are the primary food source for larval fish rearing, using a simplified culture protocol described herein. The MCZF supports the specific ongoing research in a variety of laboratories, while also serving as a local hub for new scientists as they learn to tap into the potential of this model system for understanding normal development, disease, and as models of health.

EuFishBioMed (COST Action BM0804): A European Network to Promote the Use of Small Fishes in Biomedical Research

PubMed Central

Bally-Cuif, Laure; Kelsh, Robert; Beis, Dimitris; Mione, Marina; Panula, Pertti; Figueras, Antonio; Gothilf, Yoav; Brösamle, Christian; Geisler, Robert; Knedlitschek, Gudrun

2012-01-01

Abstract Small fresh water fishes such as the zebrafish (Danio rerio) have become important model organisms for biomedical research. They currently represent the best vertebrate embryo models in which it is possible to derive quantitative data on gene expression, signaling events, and cell behavior in real time in the living animal. Relevant phenotypes in fish mutants are similar to those of other vertebrate models and human diseases. They can be analyzed in great detail and much faster than in mammals. In recent years, approximately 2500 genetically distinct fish lines have been generated by European research groups alone. Their potential, including their possible use by industry, is far from being exploited. To promote zebrafish research in Europe, EuFishBioMed was founded and won support by the EU COST programme (http://www.cost.esf.org/). The main objective of EuFishBioMed is to establish a platform of knowledge exchange for research on small fish models with a strong focus on widening its biomedical applications and an integration of European research efforts and resources. EuFishBioMed currently lists more than 300 member laboratories in Europe, offers funding for short-term laboratory visits, organizes and co-sponsors meetings and workshops, and has successfully lobbied for the establishment of a European Zebrafish Resource Centre. To maintain this network in the future, beyond the funding period of the COST Action, we are currently establishing the European Society for Fish Models in Biology and Medicine. PMID:22537014

Automated Planar Tracking the Waving Bodies of Multiple Zebrafish Swimming in Shallow Water.

PubMed

Wang, Shuo Hong; Cheng, Xi En; Qian, Zhi-Ming; Liu, Ye; Chen, Yan Qiu

2016-01-01

Zebrafish (Danio rerio) is one of the most widely used model organisms in collective behavior research. Multi-object tracking with high speed camera is currently the most feasible way to accurately measure their motion states for quantitative study of their collective behavior. However, due to difficulties such as their similar appearance, complex body deformation and frequent occlusions, it is a big challenge for an automated system to be able to reliably track the body geometry of each individual fish. To accomplish this task, we propose a novel fish body model that uses a chain of rectangles to represent fish body. Then in detection stage, the point of maximum curvature along fish boundary is detected and set as fish nose point. Afterwards, in tracking stage, we firstly apply Kalman filter to track fish head, then use rectangle chain fitting to fit fish body, which at the same time further judge the head tracking results and remove the incorrect ones. At last, a tracklets relinking stage further solves trajectory fragmentation due to occlusion. Experiment results show that the proposed tracking system can track a group of zebrafish with their body geometry accurately even when occlusion occurs from time to time.

Automated Planar Tracking the Waving Bodies of Multiple Zebrafish Swimming in Shallow Water

PubMed Central

Wang, Shuo Hong; Cheng, Xi En; Qian, Zhi-Ming; Liu, Ye; Chen, Yan Qiu

2016-01-01

Zebrafish (Danio rerio) is one of the most widely used model organisms in collective behavior research. Multi-object tracking with high speed camera is currently the most feasible way to accurately measure their motion states for quantitative study of their collective behavior. However, due to difficulties such as their similar appearance, complex body deformation and frequent occlusions, it is a big challenge for an automated system to be able to reliably track the body geometry of each individual fish. To accomplish this task, we propose a novel fish body model that uses a chain of rectangles to represent fish body. Then in detection stage, the point of maximum curvature along fish boundary is detected and set as fish nose point. Afterwards, in tracking stage, we firstly apply Kalman filter to track fish head, then use rectangle chain fitting to fit fish body, which at the same time further judge the head tracking results and remove the incorrect ones. At last, a tracklets relinking stage further solves trajectory fragmentation due to occlusion. Experiment results show that the proposed tracking system can track a group of zebrafish with their body geometry accurately even when occlusion occurs from time to time. PMID:27128096

Ameliorative efficacy of bioencapsulated Chironomous larvae with Shilajit on Zebrafish (Danio rerio) exposed to Ionizing radiation.

PubMed

Musthafa, M Saiyad; Athaullah, A; Anbumani, S; Ali, A Jawahar; War, Mehrajuddin; Paray, Bilal Ahmad; Al-Sadoon, Mohammad K; Muthiah, S S; Kembeeram, Palani; Harikrishnan, R

2017-10-01

Using Zebrafish (Danio rerio) as a model organism, we evaluated the radioprotective and antioxidant effects of the Indian traditional medicine Shilajit exposed to X-Ray. The Zebrafish were divided into three experimental groups and control group, each group containing ten fish. The three experimental fish groups, group I, group II and group III were fed with 3, 5 and 7ppm shilajit encapsulated Chironomous larvae and group IV served as a control fed with non- encapsulated larvae. After 60 days of feeding trial, fish were irradiated with X-Ray at a single acute dose of 1Gy. 72h of post-irradiation, each experimental fish were observed for its morphological, behavioral, clinical symptoms, antioxidant levels and DNA damage were evaluated. Among the experimental groups 5ppm shilajit encapsulated Chironomous larvae fed fish group shows the most significant radioprotective effects compared with control and other experimental fish groups. The present study indicates that shilajit have significant radioprotective and antioxidant enhancing capability. The humus substance of shilajit may be the factor responsible to react with radiation-derived or radiation related reactive species on zebrafish. Copyright © 2017 Elsevier Ltd. All rights reserved.

Making Waves: New Developments in Toxicology With the Zebrafish.

PubMed

Horzmann, Katharine A; Freeman, Jennifer L

2018-05-01

The laboratory zebrafish (Danio rerio) is now an accepted model in toxicologic research. The zebrafish model fills a niche between in vitro models and mammalian biomedical models. The developmental characteristics of the small fish are strategically being used by scientists to study topics ranging from high-throughput toxicity screens to toxicity in multi- and transgenerational studies. High-throughput technology has increased the utility of zebrafish embryonic toxicity assays in screening of chemicals and drugs for toxicity or effect. Additionally, advances in behavioral characterization and experimental methodology allow for observation of recognizable phenotypic changes after xenobiotic exposure. Future directions in zebrafish research are predicted to take advantage of CRISPR-Cas9 genome editing methods in creating models of disease and interrogating mechanisms of action with fluorescent reporters or tagged proteins. Zebrafish can also model developmental origins of health and disease and multi- and transgenerational toxicity. The zebrafish has many advantages as a toxicologic model and new methodologies and areas of study continue to expand the usefulness and application of the zebrafish.

Neurochemical measurements in the zebrafish brain

PubMed Central

Jones, Lauren J.; McCutcheon, James E.; Young, Andrew M. J.; Norton, William H. J.

2015-01-01

The zebrafish is an ideal model organism for behavioral genetics and neuroscience. The high conservation of genes and neurotransmitter pathways between zebrafish and other vertebrates permits the translation of research between species. Zebrafish behavior can be studied at both larval and adult stages and recent research has begun to establish zebrafish models for human disease. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that permits the detection of neurotransmitter release and reuptake. In this study we have used in vitro FSCV to measure the release of analytes in the adult zebrafish telencephalon. We compare different stimulation methods and present a characterization of neurochemical changes in the wild-type zebrafish brain. This study represents the first FSCV recordings in zebrafish, thus paving the way for neurochemical analysis of the fish brain. PMID:26441575

Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration.

PubMed

Schall, K A; Holoyda, K A; Grant, C N; Levin, D E; Torres, E R; Maxwell, A; Pollack, H A; Moats, R A; Frey, M R; Darehzereshki, A; Al Alam, D; Lien, C; Grikscheit, T C

2015-08-01

Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation. Copyright © 2015 the American Physiological Society.

Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration

PubMed Central

Schall, K. A.; Holoyda, K. A.; Grant, C. N.; Levin, D. E.; Torres, E. R.; Maxwell, A.; Pollack, H. A.; Moats, R. A.; Frey, M. R.; Darehzereshki, A.; Al Alam, D.; Lien, C.

2015-01-01

Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation. PMID:26089336

Zebrafish monosex population reveals female dominance in sex determination and earliest events of gonad differentiation.

PubMed

Tong, Sok-Keng; Hsu, Hwei-Jan; Chung, Bon-chu

2010-08-15

The zebrafish is a popular model for genetic analysis and its sex differentiation has been the focus of attention for breeding purposes. Despite numerous efforts, very little is known about the mechanism of zebrafish sex determination. The lack of discernible sex chromosomes and the difficulty of distinguishing the sex of juvenile fish are two major obstacles that hamper the progress in such studies. To alleviate these problems, we have developed a scheme involving methyltestosterone treatment followed by natural mating to generate fish with predictable sex trait. Female F1 fish that gave rise to all-female offspring were generated. This predictable sex trait enables characterization of gonadal development in juvenile fish by histological examination and gene expression analysis. We found the first sign of zebrafish sex differentiation to be ovarian gonocyte proliferation and differentiation at 10 to 12 days post-fertilization (dpf). Somatic genes were expressed indifferently at 10 to 17 dpf, and then became sexually dimorphic at three weeks. This result indicates clear distinction of male and female gonads derived independently from primordial gonads. We classified the earliest stages of zebrafish sex determination into the initial preparation followed by female germ cell growth, oocyte differentiation, and somatic differentiation. Our genetic selection scheme matches the prediction that female-dominant genetic factors are required to determine zebrafish sex. Copyright 2010 Elsevier Inc. All rights reserved.

Closed-loop control of zebrafish response using a bioinspired robotic-fish in a preference test

PubMed Central

Kopman, Vladislav; Laut, Jeffrey; Polverino, Giovanni; Porfiri, Maurizio

2013-01-01

In this paper, we study the response of zebrafish to a robotic-fish whose morphology and colour pattern are inspired by zebrafish. Experiments are conducted in a three-chambered instrumented water tank where a robotic-fish is juxtaposed with an empty compartment, and the preference of live subjects is scored as the mean time spent in the vicinity of the tank's two lateral sides. The tail-beating of the robotic-fish is controlled in real-time based on feedback from fish motion to explore a spectrum of closed-loop systems, including proportional and integral controllers. Closed-loop control systems are complemented by open-loop strategies, wherein the tail-beat of the robotic-fish is independent of the fish motion. The preference space and the locomotory patterns of fish for each experimental condition are analysed and compared to understand the influence of real-time closed-loop control on zebrafish response. The results of this study show that zebrafish respond differently to the pattern of tail-beating motion executed by the robotic-fish. Specifically, the preference and behaviour of zebrafish depend on whether the robotic-fish tail-beating frequency is controlled as a function of fish motion and how such closed-loop control is implemented. PMID:23152102

Zebrafish (Danio rerio) as a screen for attenuation of Lancefield group C streptococci and a model for streptococcal pathogenesis.

PubMed

Borst, L B; Patterson, S K; Lanka, S; Suyemoto, M M; Maddox, C W

2013-05-01

Group C streptococci are highly contagious pyogenic bacteria responsible for respiratory tract, lymph node, urogenital tract, and wound infections. Wild-type strains of Streptococcus equi ssp equi (S. equi) and Streptococcus equi ssp zooepidemicus (S. zoo) as well as a commercially available modified live vaccine strain of S. equi were evaluated for virulence in zebrafish. Survival times, histologic lesions, and relative gene expression were compared among groups. Based on the intramuscular route of infection, significantly shorter survival times were observed in fish infected with wild-type strain when compared to modified live vaccine and S. zoo strains. Histologically, S. zoo-infected fish demonstrated a marked increase in inflammatory infiltrates (predominantly macrophages) at the site of infection, as well as increased cellularity in the spleen and renal interstitium. In contrast, minimal cellular immune response was observed in S. equi-injected fish with local tissue necrosis and edema predominating. Based on whole comparative genomic hybridization, increased transcription of positive acute-phase proteins, coagulation factors, and antimicrobial peptides were observed in S. equi-injected fish relative to S. zoo-injected fish, while mediators of cellular inflammation, including CXC chemokines and granulin, were upregulated in S. zoo-injected fish relative to S. equi-injected fish. In a screen of 11 clinical isolates, S. equi strains with a single nucleotide deletion in the upstream region of szp, a known virulence factor of streptococci, were found to be significantly attenuated in zebrafish. These collective findings underscore the value of the zebrafish as a model of streptococcal pathogenesis.

Role of brain-derived neurotrophic factor during the regenerative response after traumatic brain injury in adult zebrafish.

PubMed

Cacialli, Pietro; Palladino, Antonio; Lucini, Carla

2018-06-01

Several mammalian animal models of traumatic brain injury have been used, mostly rodents. However, reparative mechanisms in mammalian brain are very limited, and newly formed neurons do not survive for long time. The brain of adult zebrafish, a teleost fish widely used as vertebrate model, possesses high regenerative properties after injury due to the presence of numerous stem cells niches. The ventricular lining of the zebrafish dorsal telencephalon is the most studied neuronal stem cell niche because its dorso-lateral zone is considered the equivalent to the hippocampus of mammals which contains one of the two constitutive neurogenic niches of mammals. To mimic TBI, stab wound in the dorso-lateral telencephalon of zebrafish was used in studies devoted to fish regenerative properties. Brain-derived neurotrophic factor, which is known to play key roles in the repair process after traumatic brain lesions, persists around the lesioned area of injured telencephalon of adult zebrafish. These results are extensively compared to reparative processes in rodent brain. Considering the complete repair of the damaged area in fish, it could be tempting to consider brain-derived neurotrophic factor as a factor contributing to create a permissive environment that enables the establishment of new neuronal population in damaged brain.

A Model of Superinfection of Virus-Infected Zebrafish Larvae: Increased Susceptibility to Bacteria Associated With Neutrophil Death

PubMed Central

Boucontet, Laurent; Passoni, Gabriella; Thiry, Valéry; Maggi, Ludovico; Herbomel, Philippe; Levraud, Jean-Pierre; Colucci-Guyon, Emma

2018-01-01

Enhanced susceptibility to bacterial infection in the days following an acute virus infection such as flu is a major clinical problem. Mouse models have provided major advances in understanding viral-bacterial superinfections, yet interactions of the anti-viral and anti-bacterial responses remain elusive. Here, we have exploited the transparency of zebrafish to study how viral infections can pave the way for bacterial co-infections. We have set up a zebrafish model of sequential viral and bacterial infection, using sublethal doses of Sindbis virus and Shigella flexneri bacteria. This virus induces a strong type I interferons (IFN) response, while the bacterium induces a strong IL1β and TNFα-mediated inflammatory response. We found that virus-infected zebrafish larvae showed an increased susceptibility to bacterial infection. This resulted in the death with concomitant higher bacterial burden of the co-infected fish compared to the ones infected with bacteria only. By contrast, infecting with bacteria first and virus second did not lead to increased mortality or microbial burden. By high-resolution live imaging, we showed that neutrophil survival was impaired in Sindbis-then-Shigella co-infected fish. The two types of cytokine responses were strongly induced in co-infected fish. In addition to type I IFN, expression of the anti-inflammatory cytokine IL10 was induced by viral infection before bacterial superinfection. Collectively, these observations suggest the zebrafish larva as a useful animal model to address mechanisms underlying increased bacterial susceptibility upon viral infection. PMID:29881380

Towards an alternative for the acute fish LC(50) test in chemical assessment: the fish embryo toxicity test goes multi-species -- an update.

PubMed

Braunbeck, Thomas; Boettcher, Melanie; Hollert, Henner; Kosmehl, Thomas; Lammer, Eva; Leist, Erik; Rudolf, Mark; Seitz, Nadja

2005-01-01

After its standardisation at the national level in Germany (DIN 38415-6, 2001, 2001), the 48 h sewage testing assay with zebrafish (Danio rerio) embryos has been submitted for standardisation to ISO. As an alternative to the conventional acute (96 h) fish test, a modified fish embryo test will be submitted to the OECD for chemical testing in late 2005. For this, a protocol originally designed for zebrafish was adapted to fit also the requirements of other OECD species, namely medaka (Oryzias latipes) and fathead minnow (Pimephales promelas). Results document that the transfer of the protocol is possible with only minor modifications. Data obtained from embryo tests with the three species are comparable. Statistical analysis of existing zebrafish embryo toxicity data resulted in the conclusions (1) that there is a reliable correlation between the fish embryo test and the acute fish test, (2) that the confidence belt of the regression line was relatively small, but that the prediction range was relatively wide. The regression thus seems appropriate to describe the relationship between acute fish and embryo LC(50) with good confidence, but is less appropriate as a prediction model. Investigations into oxygen requirements of zebrafish embryos reveal that they adapt to a broad range of oxygen levels and survive at concentrations of 2 mg/l without malformations. Zebrafish embryos can thus be exposed in very small toxicant volumes (100 microl), which is of particular interest for the testing of metabolites. Dechorionation studies with 48 h old zebrafish embryos indicate that the barrier function of the chorion increases with the lipophilicity of the test compound. Finally, examples are given as to how additional endpoints can be incorporated into the fish embryo test protocol to extend its scope, e.g. to sediment toxicity assessment or genotoxicity and mutagenicity testing.

Comparative mapping for bighead carp (Aristichthys nobilis) against model and non-model fishes provides insights into the genomic evolution of cyprinids.

PubMed

Zhu, Chuankun; Tong, Jingou; Yu, Xiaomu; Guo, Wenjie

2015-08-01

Comparative mapping provides an efficient method to connect genomes of non-model and model fishes. In this study, we used flanking sequences of the 659 microsatellites on a genetic map of bighead carp (Aristichthys nobilis) to comprehensively study syntenic relationships between bighead carp and nine model and non-model fishes. Of the five model and two food fishes with whole genome data, Cyprinus carpio showed the highest rate of positive BLAST hits (95.3 %) with bighead carp map, followed by Danio rerio (70.9 %), Oreochromis niloticus (21.7 %), Tetraodon nigroviridis (6.4 %), Gasterosteus aculeatus (5.2 %), Oryzias latipes (4.7 %) and Fugu rubripes (3.5 %). Chromosomal syntenic analyses showed that inversion was the basic chromosomal rearrangement during genomic evolution of cyprinids, and the extent of inversions and translocations was found to be positively correlated with evolutionary relationships among fishes studied. Among the five investigated cyprinids, linkage groups (LGs) of bighead carp, Hypophthalmichthys molitrix and Ctenopharyngodon idella exhibited a one-to-one relationship. Besides, LG 9 of bighead carp and homologous LGs of silver carp and grass carp all corresponded to the chromosomes 10 and 22 of zebrafish, suggesting that chromosomal fission may have occurred in the ancestor of zebrafish. On the other hand, LGs of bighead carp and common carp showed an approximate one-to-two relationship with extensive translocations, confirming the occurrence of a 4th whole genome duplication in common carp. This study provides insights into the understanding of genome evolution among cyprinids and would aid in transferring positional and functional information of genes from model fish like zebrafish to non-model fish like bighead carp.

Behavorial assessments of larval zebrafish neurotoxicology

EPA Science Inventory

Fishes have long been a popular organism in ecotoxicology research, and are increasingly used in human health research as an alternative animal model for chemical screening. Our laboratory incorporates a zebrafish (Danio rerio) embryo/larval assay to screen chemicals for developm...

The Behavioral Space of Zebrafish Locomotion and Its Neural Network Analog.

PubMed

Girdhar, Kiran; Gruebele, Martin; Chemla, Yann R

2015-01-01

How simple is the underlying control mechanism for the complex locomotion of vertebrates? We explore this question for the swimming behavior of zebrafish larvae. A parameter-independent method, similar to that used in studies of worms and flies, is applied to analyze swimming movies of fish. The motion itself yields a natural set of fish "eigenshapes" as coordinates, rather than the experimenter imposing a choice of coordinates. Three eigenshape coordinates are sufficient to construct a quantitative "postural space" that captures >96% of the observed zebrafish locomotion. Viewed in postural space, swim bouts are manifested as trajectories consisting of cycles of shapes repeated in succession. To classify behavioral patterns quantitatively and to understand behavioral variations among an ensemble of fish, we construct a "behavioral space" using multi-dimensional scaling (MDS). This method turns each cycle of a trajectory into a single point in behavioral space, and clusters points based on behavioral similarity. Clustering analysis reveals three known behavioral patterns-scoots, turns, rests-but shows that these do not represent discrete states, but rather extremes of a continuum. The behavioral space not only classifies fish by their behavior but also distinguishes fish by age. With the insight into fish behavior from postural space and behavioral space, we construct a two-channel neural network model for fish locomotion, which produces strikingly similar postural space and behavioral space dynamics compared to real zebrafish.

The Behavioral Space of Zebrafish Locomotion and Its Neural Network Analog

PubMed Central

Girdhar, Kiran; Gruebele, Martin; Chemla, Yann R.

2015-01-01

How simple is the underlying control mechanism for the complex locomotion of vertebrates? We explore this question for the swimming behavior of zebrafish larvae. A parameter-independent method, similar to that used in studies of worms and flies, is applied to analyze swimming movies of fish. The motion itself yields a natural set of fish "eigenshapes" as coordinates, rather than the experimenter imposing a choice of coordinates. Three eigenshape coordinates are sufficient to construct a quantitative "postural space" that captures >96% of the observed zebrafish locomotion. Viewed in postural space, swim bouts are manifested as trajectories consisting of cycles of shapes repeated in succession. To classify behavioral patterns quantitatively and to understand behavioral variations among an ensemble of fish, we construct a "behavioral space" using multi-dimensional scaling (MDS). This method turns each cycle of a trajectory into a single point in behavioral space, and clusters points based on behavioral similarity. Clustering analysis reveals three known behavioral patterns—scoots, turns, rests—but shows that these do not represent discrete states, but rather extremes of a continuum. The behavioral space not only classifies fish by their behavior but also distinguishes fish by age. With the insight into fish behavior from postural space and behavioral space, we construct a two-channel neural network model for fish locomotion, which produces strikingly similar postural space and behavioral space dynamics compared to real zebrafish. PMID:26132396

simUfish: An Interactive Application to Teach K-12 Students About Zebrafish Behavior.

PubMed

Mwaffo, Violet; Korneyeva, Veronika; Porfiri, Maurizio

2017-10-01

As the zebrafish is rapidly becoming a species of choice in preclinical research, several efforts are being placed toward creating educational programs for K-12 students based on this promising model organism. However, as any other model organisms, the use of zebrafish in classroom settings requires additional experimental resources and poses ethical challenges related to animal use. To mitigate these factors, we have developed an application (app), simUfish, which implements a mathematical model of zebrafish behavior for generating multiple fish trajectories and animating their body undulations. simUfish is developed using a multiplatform game engine and is expected to promote the knowledge of zebrafish behavior to both K-12 students and the general public. Specifically, it demonstrates basic principles of fish individual and social behaviors, including environment interaction; fear response toward a predator; shoaling; and attraction toward a stimulus, which can be a food source or simply a finger placed on the touch screen. The effectiveness of the app as an accessible experimental tool for learning was tested in an outreach activity on middle school students from the New York City school system. The results from this activity show an immediate, tangible improvement of students' satisfaction and willingness to learn about key concepts on zebrafish behavior, accompanied by high level of interest in life sciences.

On the relevance of genotoxicity for fish populations I: effects of a model genotoxicant on zebrafish (Danio rerio) in a complete life-cycle test.

PubMed

Diekmann, Markus; Hultsch, Veit; Nagel, Roland

2004-05-28

Genotoxicity may be detected in surface waters by means of various genotoxicity assays. In order to enable an ecotoxicological assessment of the consequences of such genotoxic potential for fish populations, a complete life-cycle test with zebrafish (Danio rerio) and the model genotoxicant 4-nitroquinoline-1-oxide (NQO) was conducted. Zebrafish (f1) were continuously exposed to NQO (i.e. 0.1, 0.3, 1.1, 2.9, and 14.6 microg/l, respectively) from fertilised eggs until sexual maturity. In addition to reproduction studies in the f1-generation, f2-fish were exposed to NQO during the first 6 weeks of development. Up to 2.9 microg/l NQO, fish did not display differences in survival and growth (P < 0.05). A NQO concentration of 14.6 microg/l, however, was lethal. Female fish exposed to all NQO concentrations up to 2.9 microg/l displayed a significant reduction in egg production (P < 0.05). A mathematical simulation revealed that exposure to weak concentrations of NQO is leading to an elevated extinction risk. Copyright 2004 Elsevier B.V.

A Retrospective Study of the Prevalence and Classification of Intestinal Neoplasia in Zebrafish (Danio Rerio)

PubMed Central

Paquette, Colleen E.; Buchner, Cari; Tanguay, Robert L.; Guillemin, Karen; Mason, Timothy J.; Peterson, Tracy S.

2013-01-01

Abstract For over a decade, spontaneous intestinal neoplasia has been observed in zebrafish (Danio rerio) submitted to the ZIRC (Zebrafish International Resource Center) diagnostic service. In addition, zebrafish displayed preneoplastic intestinal changes including hyperplasia, dysplasia, and enteritis. A total of 195 zebrafish, representing 2% of the total fish submitted to the service, were diagnosed with these lesions. Neoplastic changes were classified either as adenocarcinoma or small cell carcinoma, with a few exceptions (carcinoma not otherwise specified, tubular adenoma, and tubulovillous adenoma). Tumor prevalence appeared similarly distributed between sexes and generally occurred in zebrafish greater than 1 year of age, although neoplastic changes were observed in fish 6 months of age. Eleven lines displayed these preneoplastic and neoplastic changes, including wild-types and mutants. Affected zebrafish originated from 18 facilities, but the majority of fish were from a single zebrafish research facility (hereafter referred to as the primary facility) that has submitted numerous samples to the ZIRC diagnostic service. Zebrafish from the primary facility submitted as normal sentinel fish demonstrate that these lesions are most often subclinical. Fish fed the diet from the primary facility and held at another location did not develop intestinal lesions, indicating that diet is not the etiologic agent. PMID:23544991

DNA Microarray-based Ecotoxicological Biomarker Discovery in a Small Fish Model Species

EPA Science Inventory

This paper addresses several issues critical to use of zebrafish oligonucleotide microarrays for computational toxicology research on endocrine disrupting chemicals using small fish models, and more generally, the use of microarrays in aquatic toxicology.

Involvement of the α1-adrenoceptor in sleep-waking and sleep loss-induced anxiety behavior in zebrafish.

PubMed

Singh, A; Subhashini, N; Sharma, S; Mallick, B N

2013-08-15

Sleep is a universal phenomenon in vertebrates, and its loss affects various behaviors. Independent studies have reported that sleep loss increases anxiety; however, the detailed mechanism is unknown. Because sleep deprivation increases noradrenalin (NA), which modulates many behaviors and induces patho-physiological changes, this study utilized zebrafish as a model to investigate whether sleep loss-induced increased anxiety is modulated by NA. Continuous behavioral quiescence for at least 6s was considered to represent sleep in zebrafish; although some authors termed it as a sleep-like state, in this study we have termed it as sleep. The activity of fish that signified sleep-waking was recorded in light-dark, during continuous dark and light; the latter induced sleep loss in fish. The latency, number of entries, time spent and distance travelled in the light chamber were assessed in a light-dark box test to estimate the anxiety behavior of normal, sleep-deprived and prazosin (PRZ)-treated fish. Zebrafish showed increased waking during light and complete loss of sleep upon continuous exposure to light for 24h. PRZ significantly increased sleep in normal fish. Sleep-deprived fish showed an increased preference for dark (expression of increased anxiety), and this effect was prevented by PRZ, which increased sleep as well. Our findings suggest that sleep loss-induced anxiety-like behavior in zebrafish is likely to be mediated by NA's action on the α1-adrenoceptor. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Fishing the Molecular Bases of Treacher Collins Syndrome

PubMed Central

Weiner, Andrea M. J.; Scampoli, Nadia L.; Calcaterra, Nora B.

2012-01-01

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, and mutations in the TCOF1 gene are responsible for over 90% of TCS cases. The knowledge about the molecular mechanisms responsible for this syndrome is relatively scant, probably due to the difficulty of reproducing the pathology in experimental animals. Zebrafish is an emerging model for human disease studies, and we therefore assessed it as a model for studying TCS. We identified in silico the putative zebrafish TCOF1 ortholog and cloned the corresponding cDNA. The derived polypeptide shares the main structural domains found in mammals and amphibians. Tcof1 expression is restricted to the anterior-most regions of zebrafish developing embryos, similar to what happens in mouse embryos. Tcof1 loss-of-function resulted in fish showing phenotypes similar to those observed in TCS patients, and enabled a further characterization of the mechanisms underlying craniofacial malformation. Besides, we initiated the identification of potential molecular targets of treacle in zebrafish. We found that Tcof1 loss-of-function led to a decrease in the expression of cellular proliferation and craniofacial development. Together, results presented here strongly suggest that it is possible to achieve fish with TCS-like phenotype by knocking down the expression of the TCOF1 ortholog in zebrafish. This experimental condition may facilitate the study of the disease etiology during embryonic development. PMID:22295061

Fishing the molecular bases of Treacher Collins syndrome.

PubMed

Weiner, Andrea M J; Scampoli, Nadia L; Calcaterra, Nora B

2012-01-01

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, and mutations in the TCOF1 gene are responsible for over 90% of TCS cases. The knowledge about the molecular mechanisms responsible for this syndrome is relatively scant, probably due to the difficulty of reproducing the pathology in experimental animals. Zebrafish is an emerging model for human disease studies, and we therefore assessed it as a model for studying TCS. We identified in silico the putative zebrafish TCOF1 ortholog and cloned the corresponding cDNA. The derived polypeptide shares the main structural domains found in mammals and amphibians. Tcof1 expression is restricted to the anterior-most regions of zebrafish developing embryos, similar to what happens in mouse embryos. Tcof1 loss-of-function resulted in fish showing phenotypes similar to those observed in TCS patients, and enabled a further characterization of the mechanisms underlying craniofacial malformation. Besides, we initiated the identification of potential molecular targets of treacle in zebrafish. We found that Tcof1 loss-of-function led to a decrease in the expression of cellular proliferation and craniofacial development. Together, results presented here strongly suggest that it is possible to achieve fish with TCS-like phenotype by knocking down the expression of the TCOF1 ortholog in zebrafish. This experimental condition may facilitate the study of the disease etiology during embryonic development.

Electronic individual identification of zebrafish using radio frequency identification (RFID) microtags.

PubMed

Cousin, Xavier; Daouk, Tarek; Péan, Samuel; Lyphout, Laura; Schwartz, Marie-Elise; Bégout, Marie-Laure

2012-08-15

Although individual electronic tagging using passive integrated acoustic (PIT) tags is established, it is mainly for fish >60 mm in length and is unsuitable for fish of <30 mm, like zebrafish. We used radio frequency identification (RFID) microtags (1 mm in diameter and 6 mm in length, with a mass of ~10 mg) to individually identify juvenile zebrafish (length 16-42 mm, mass 138-776 mg) for the first time, and studied the effects of intracoelomic implantation on fish survival and microtag loss, growth, spawning and exploratory behaviour. After 5.5 months, both high survival (82%) and low microtag loss (11%) were achieved. The smallest surviving fish weighed 178 mg, and success in microtag reading was 73% for the size class 350-450 mg (26 mm). Greater success was achieved when fish were larger at the time of tagging but no negative effects on growth were observed for any size class and some tagged fish spawned. No significant differences in behavioural responses could be detected between tagged fish and untagged controls after 2 months. Overall, the results suggest that the tagging method is highly suitable for fish as small as zebrafish juveniles. We think this method will provide significant advances for researchers of the ever-growing fish model community and more generally for all small-fish users. Tagging is essential when one needs to identify fish (e.g. particular genotypes with no external cue), to run longitudinal monitoring of individual biological traits (e.g. growth) or to repeat assays with the same individual at discrete points in time (e.g. behaviour studies). Such a method will find applications in physiology, genetics, behaviour and (eco)toxicology fields.

Chondroitin sulfate and keratan sulfate are the major glycosaminoglycans present in the adult zebrafish Danio rerio (Chordata-Cyprinidae).

PubMed

Souza, Aline R C; Kozlowski, Eliene O; Cerqueira, Vinicius R; Castelo-Branco, Morgana T L; Costa, Manoel L; Pavão, Mauro S G

2007-12-01

The zebrafish Danio rerio (Chordata-Cyprinidae) is a model organism frequently used to study the functions of proteoglycans and their glycosaminoglycan (GAG) chains. Although several studies clearly demonstrate the participation of these polymers in different biological and cellular events that take place during embryonic development, little is known about the GAGs in adult zebrafish. In the present study, the total GAGs were extracted from the whole fish by proteolytic digestion, purified by anion-exchange chromatography and characterized by electrophoresis after degradation with specific enzymes and/or by high-performance liquid chromatography (HPLC) analysis of the disaccharides. Two GAGs were identified: a low-molecular-weight chondroitin sulfate (CS) and keratan sulfate (KS), corresponding to approximately 80% and 20% of the total GAGs, respectively. In the fish eye, KS represents approximately 80% of total GAGs. Surprisingly, no heparinoid was detected, but may be present in the fish at concentrations lower than the limit of the method used. HPLC of the disaccharides formed after chondroitin AC or ABC lyase degradation revealed that the zebrafish CS is composed by DeltaUA-1-->3-GalNAc(4SO4) (59.4%), DeltaUA-1-->3-GalNAc(6SO4) (23.1%), and DeltaUA-1-->3-GalNAc (17.5%) disaccharide units. No disulfated disaccharides were detected. Immunolocalization on sections from zebrafish retina using monoclonal antibodies against CS4- or 6-sulfate showed that in the retina these GAGs are restricted to the outer and inner plexiform layers. This is the first report showing the presence of KS in zebrafish eye, and the structural characterization of CS and its localization in the zebrafish retina. Detailed information about the structure and tissue localization of GAGs is important to understand the functions of these polymers in this model organism.

Natural mixtures of persistent organic pollutants (POP) increase weight gain, advance puberty, and induce changes in gene expression associated with steroid hormones and obesity in female zebrafish.

PubMed

Lyche, Jan L; Nourizadeh-Lillabadi, Rasoul; Almaas, Camilla; Stavik, Benedicte; Berg, Vidar; Skåre, Janneche Utne; Alestrøm, Peter; Ropstad, Erik

2010-01-01

In the present study, developmental and reproductive effects of lifelong exposure to environmental relevant concentrations of two natural mixtures of persistent organic pollutants (POP) were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot (Lota lota) liver originating from freshwater systems in Norway: one mixture with high levels and one mixture with background levels of polybrominated diphenyl ethers (PBDE), polychlorinated biphenyls (PCB), and dichlorodiphenyltrichloroethane metabolites (DDT). The concentration of POP measured in the zebrafish ranged from levels detected in wild fish from Lake Mjøsa to concentrations reported in human and wildlife populations, indicating that the experimental fish were exposed to concentrations comparable with wild fish. Phenotypic effects observed in both exposure groups included earlier onset of puberty, increased male/female sex ratio, and differences in body weight at 5 mo of age. Interestingly, genome-wide transcription profiling showed changes in regulation of genes involved in endocrine signaling and growth. The transcriptomics changes include key regulator genes for steroid hormone functions (ncoa3), and growth (c/ebp, ncoa3). The effects observed in the experimental zebrafish model raise the question whether chemical pollution represents a risk to reproductive health of wild fish inhabitating the freshwater system.

The toxicological application of transcriptomics and epigenomics in zebrafish and other teleosts.

PubMed

Williams, Tim D; Mirbahai, Leda; Chipman, J Kevin

2014-03-01

Zebrafish (Danio rerio) is one of a number of teleost fish species frequently employed in toxicology. Toxico-genomics determines global transcriptomic responses to chemical exposures and can predict their effects. It has been applied successfully within aquatic toxicology to assist in chemical testing, determination of mechanisms and environmental monitoring. Moreover, the related field of toxico-epigenomics, that determines chemical-induced changes in DNA methylation, histone modifications and micro-RNA expression, is emerging as a valuable contribution to understanding mechanisms of both adaptive and adverse responses. Zebrafish has proven a useful and convenient model species for both transcriptomic and epigenetic toxicological studies. Despite zebrafish's dominance in other areas of fish biology, alternative fish species are used extensively in toxico-genomics. The main reason for this is that environmental monitoring generally focuses on species native to the region of interest. We are starting to see advances in the integration of high-throughput screening, omics techniques and bioinformatics together with more traditional indicator endpoints that are relevant to regulators. Integration of such approaches with high-throughput testing of zebrafish embryos, leading to the discovery of adverse outcome pathways, promises to make a major contribution to ensuring the safety of chemicals in the environment.

Methylene Blue Facilitates Memory Retention in Zebrafish in a Dose-Dependent Manner.

PubMed

Echevarria, David J; Caramillo, Erika M; Gonzalez-Lima, Francisco

2016-12-01

Methylene blue (MB) is an FDA-grandfathered drug with memory-enhancing effects at low doses, but opposite effects at high doses. We investigated the effects of four MB doses (0.1, 0.5, 5.0, or 10.0 μM) on zebrafish memory retention in the T-maze task. After training fish to swim into a certain arm of the T-maze, the fish were placed into a tank containing one of the four MB doses or a control tank containing blue food dye. Subsequently, fish were placed into the T-maze for memory retention testing. Results indicated that MB produced hormetic dose-response effects on memory. Fish that received the 0.5 μM dose performed significantly better at the T-maze than those that received higher doses. Fish who received 5.0 μM did not exhibit a significant difference in performance from control fish, and the fish that received the 10.0 μM dose performed significantly worse than lower doses. These findings support the utility of zebrafish in comparative research and their potential value for testing of MB and other neuropsychopharmacological treatments in animal models of memory disorders.

OpenSource lab-on-a-chip physiometer for accelerated zebrafish embryo biotests.

PubMed

Akagi, Jin; Hall, Chris J; Crosier, Kathryn E; Cooper, Jonathan M; Crosier, Philip S; Wlodkowic, Donald

2014-01-02

Zebrafish (Danio rerio) embryo assays have recently come into the spotlight as convenient experimental models in both biomedicine and ecotoxicology. As a small aquatic model organism, zebrafish embryo assays allow for rapid physiological, embryo-, and genotoxic tests of drugs and environmental toxins that can be simply dissolved in water. This protocol describes prototyping and application of an innovative, miniaturized, and polymeric chip-based device capable of immobilizing a large number of living fish embryos for real-time and/or time-lapse microscopic examination. The device provides a physical address designation to each embryo during analysis, continuous perfusion of medium, and post-analysis specimen recovery. Miniaturized embryo array is a new concept of immobilization and real-time drug perfusion of multiple individual and developing zebrafish embryos inside the mesofluidic device. The OpenSource device presented in this protocol is particularly suitable to perform accelerated fish embryo biotests in ecotoxicology and phenotype-based pharmaceutical screening. Copyright © 2014 John Wiley & Sons, Inc.

Danio rerio: Small Fish Making a Big Splash in Leukemia.

PubMed

Squiban, Barbara; Frazer, J Kimble

2014-06-01

Zebrafish ( Danio rerio ) are widely used for developmental biology studies. In the past decade, D. rerio have become an important oncology model as well. Leukemia is one type of cancer where zebrafish are particularly valuable. As vertebrates, fish have great anatomic and biologic similarity to humans, including their hematopoietic and immune systems. As an experimental platform, D. rerio offer many advantages that mammalian models lack. These include their ease of genetic manipulation, capacity for imaging, and suitability for large-scale phenotypic and drug screens. In this review, we present examples of these strategies and others to illustrate how zebrafish have been and can be used to study leukemia. Besides appraising the techniques researchers apply and introducing the leukemia models they have created, we also highlight recent and exciting discoveries made using D. rerio with an eye to where the field is likely headed.

Measuring zebrafish turning rate.

PubMed

Mwaffo, Violet; Butail, Sachit; di Bernardo, Mario; Porfiri, Maurizio

2015-06-01

Zebrafish is becoming a popular animal model in preclinical research, and zebrafish turning rate has been proposed for the analysis of activity in several domains. The turning rate is often estimated from the trajectory of the fish centroid that is output by commercial or custom-made target tracking software run on overhead videos of fish swimming. However, the accuracy of such indirect methods with respect to the turning rate associated with changes in heading during zebrafish locomotion is largely untested. Here, we compare two indirect methods for the turning rate estimation using the centroid velocity or position data, with full shape tracking for three different video sampling rates. We use tracking data from the overhead video recorded at 60, 30, and 15 frames per second of zebrafish swimming in a shallow water tank. Statistical comparisons of absolute turning rate across methods and sampling rates indicate that, while indirect methods are indistinguishable from full shape tracking, the video sampling rate significantly influences the turning rate measurement. The results of this study can aid in the selection of the video capture frame rate, an experimental design parameter in zebrafish behavioral experiments where activity is an important measure.

Swimming behavior of zebrafish is accurately classified by direct modeling and behavioral space analysis

NASA Astrophysics Data System (ADS)

Feng, Ruopei; Chemla, Yann; Gruebele, Martin

Larval zebrafish is a popular organism in the search for the correlation between locomotion behavior and neural pathways because of their highly stereotyped and temporally episodic swimming motion. This correlation is usually investigated using electrophysiological recordings of neural activities in partially immobilized fish. Seeking for a way to study animal behavior without constraints or intruding electrodes, which can in turn modify their behavior, our lab has introduced a parameter-free approach which allows automated classification of the locomotion behaviors of freely swimming fish. We looked into several types of swimming bouts including free swimming and two modes of escape responses and established a new classification of these behaviors. Combined with a neurokinematic model, our analysis showed the capability to probe intrinsic properties of the underlying neural pathways of freely swimming larval zebrafish by inspecting swimming movies only.

Fluorescent transgenic zebrafish as a biosensor for growth-related effects of methyl parathion.

PubMed

Almeida, Daniela Volcan; Vaz, Bernardo; Azevedo Figueiredo, Márcio; Junior, Antonio Sergio Varela; Marins, Luis Fernando

2014-07-01

Transgenic fish models are potential alternative subjects in toxicological studies, since they can provide in vivo information on the deleterious effects of different substances. Here, we used a transgenic zebrafish (Danio rerio) lineage, which expresses a destabilized fluorescent protein (DsRED) driven by the myosin light chain promoter (Mylz2), in order to propose a new research tool for environmental biomonitoring. For validating the MYO-RED lineage, we exposed fish to the organophosphorated pesticide methyl parathion (MP). The effect of MP on fish growth was assessed by evaluating weight, length, condition factor and muscle fiber diameter. All factors suffered reduction at both tested concentrations (0.13μM and 13μM of MP). Similarly, fluorescence intensity decreased in a concentration-dependent manner, suggesting muscle protein catabolism. However, DsRED gene expression lowered only at the higher MP concentration. Results indicate that the MYO-RED transgenic zebrafish is an interesting model for detecting the growth-related effects of pollutants. Destabilized proteins such as reporter genes are apparently sensitive biomarkers, since effects were observed even at the lower, environmentally acceptable concentration. Therefore, this transgenic fish is a promising candidate model for sensitive, fast, and easy environmental monitoring. Copyright © 2014 Elsevier B.V. All rights reserved.

From the Cover: Zebrafish Larvae Are Insensitive to Stimulation by Cocaine: Importance of Exposure Route and Toxicokinetics.

PubMed

Kirla, Krishna Tulasi; Groh, Ksenia J; Steuer, Andrea E; Poetzsch, Michael; Banote, Rakesh Kumar; Stadnicka-Michalak, Julita; Eggen, Rik I L; Schirmer, Kristin; Kraemer, Thomas

2016-11-01

Zebrafish (Danio rerio) larvae have been suggested as vertebrate model to complement or even replace mammals for rapidly assessing behavioral effects of psychoactive drugs. Yet, divergent responses have been reported in mammals and fish despite the conservation of many drug targets. Cocaine, eg, acts as stimulant in mammals but no such response has been documented for zebrafish larvae. We hypothesized that differences in exposure routes (inhalation or injection in mammals vs waterborne in fish) may be a reason for differences in behavioral responses. We characterized cocaine toxicokinetics by liquid chromatography-mass spectrometry and found its rapid uptake into larvae. We used Matrix-assisted laser desorption ionization-mass spectrometry imaging for the first time to characterize internal distribution of cocaine in zebrafish larvae. Surprisingly, eyes accumulated the highest amount of cocaine and retained most of it even after 48 h depuration. We attribute this to trapping by pigment melanin, a thus far little explored mechanism that may also be relevant for other basic drugs. Cocaine also reached the brain but with levels similar to those in trunk indicating simple passive diffusion as means of distribution which was supported by toxicokinetic models. Although brain levels covered those known to cause hyperactivity in mammals, only hypoactivity (decreased locomotion) was recorded in zebrafish larvae. Our results therefore point to cocaine's anesthetic properties as the dominant mechanism of interaction in the fish: upon entry through the fish skin and gills, it first acts on peripheral nerves rapidly overriding any potential stimulatory response in the brain. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Zebrafish as a useful model for zoonotic Vibrio parahaemolyticus pathogenicity in fish and human.

PubMed

Zhang, Qinghua; Dong, Xuehong; Chen, Biao; Zhang, Yonghua; Zu, Yao; Li, Weiming

2016-02-01

Vibrio parahaemolyticus is an important aquatic zoonotic pathogen worldwide that causes vibriosis in many marine fish, and sepsis, gastroenteritis and wound infection in humans. However, the pathogenesis of different sources of V. parahaemolyticus is not fully understood. Here, we examined the pathogenicity and histopathology of fish (V. parahaemolyticus 1.2164) and human (V. parahaemolyticus 17) strains in a zebrafish (Danio rerio). We found that different infection routes resulted in different mortality in zebrafish. Moreover, death due to V. parahaemolyticus 1.2164 infection occurred quicker than that caused by V. parahaemolyticus 17 infection. Hematoxylin-eosin staining of liver, kidney and intestine sections showed histological lesions in all three organs after infection with either strain. V. parahaemolyticus 1.2164 caused more severe damage than V. parahaemolyticus 17. In particular, V. parahaemolyticus 1.2164 treatment induced more serious hydropic degeneration and venous sinus necrosis in the liver than V. parahaemolyticus 17 treatment. The expression levels of three proinflammatory cytokines, interleukin 1β (il1β), interferon phi 1 (ifnϕ1) and tumor necrosis factor α (tnfα), as determined by quantitative real-time PCR, were upregulated in all examined tissues of infected fish. Notably, the peak levels of tnfα were significantly higher than those of il1β and ifnϕ1, suggesting, together with pathological results, that tnfα and il1β play an important role in acute sepsis. High amounts of tnfα may be related to acute liver necrosis, while ifnϕ1 may respond to V. parahaemolyticus and play an antibacterial role for chronically infected adult zebrafish. Taken together, our results suggest that the zebrafish model of V. parahaemolyticus infection is useful for studying strain differences in V. parahaemolyticus pathogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Developmental effects of simulated microgravity on zebrafish, (Danio rerio)

NASA Astrophysics Data System (ADS)

Stoyek, Matthew; Edsall, Sara; Franz-Odendaal, Tamara; Smith, Frank; Croll, Roger

Zebrafish are widely used model vertebrates in research and recently this species has been used to study the effects of microgravity on fundamental biological processes. In this study we used a NASA-designed rotating wall vessel (RWV) to investigate the effects of simulated microgravity (SMG) on zebrafish development up to 14 days post fertilization (dpf). At developmental stages beyond the 3-4 somite stage we found SMG-exposed embryos reached key developmental stag-ing points more rapidly than fish raised within a non-rotating vessel. By the 21 somite stage, both groups were again synchronized in their developmental staging. However, SMG-exposed embryos eventually exhibited a delay in hatching time compared to controls. Otolith and to-tal body size were observed to be greater in larvae raised in SMG. In addition, pigmentation patterns in SMG exposed fish differed, with larger and differentially aggregated melanocytes . Heart development was slowed in SMG exposed fish, but no change in nervous system de-velopment was detected. Ongoing research will focus on differences in heart and respiration rates. Finally, by developing a method to extend the duration of SMG exposure, we found the swimming behaviour of SMG-exposed animals was altered with time in the RWV. Initially SMG-exposed animals swam in the direction of RWV rotation (5-9dpf) but older (9+dpf) fish swam against rotation and demonstrated righting behaviour with each rotation. These results suggest that vestibular reflexes may develop normally and be maintained in animals exposed to SMG. Together, our data provide insights into how zebrafish may develop when flown in space, permitting better formulation of experiments to test mechanisms by which microgravity may affect ontogeny of this model organism. Keywords: microgravity, zebrafish, growth, development

Inhibited Carnitine Synthesis Causes Systemic Alteration of Nutrient Metabolism in Zebrafish

PubMed Central

Li, Jia-Min; Li, Ling-Yu; Qin, Xuan; Degrace, Pascal; Demizieux, Laurent; Limbu, Samwel M.; Wang, Xin; Zhang, Mei-Ling; Li, Dong-Liang; Du, Zhen-Yu

2018-01-01

Impaired mitochondrial fatty acid β-oxidation has been correlated with many metabolic syndromes, and the metabolic characteristics of the mammalian models of mitochondrial dysfunction have also been intensively studied. However, the effects of the impaired mitochondrial fatty acid β-oxidation on systemic metabolism in teleost have never been investigated. In the present study, we established a low-carnitine zebrafish model by feeding fish with mildronate as a specific carnitine synthesis inhibitor [0.05% body weight (BW)/d] for 7 weeks, and the systemically changed nutrient metabolism, including carnitine and triglyceride (TG) concentrations, fatty acid (FA) β-oxidation capability, and other molecular and biochemical assays of lipid, glucose, and protein metabolism, were measured. The results indicated that mildronate markedly decreased hepatic carnitine concentrations while it had no effect in muscle. Liver TG concentrations increased by more than 50% in mildronate-treated fish. Mildronate decreased the efficiency of liver mitochondrial β-oxidation, increased the hepatic mRNA expression of genes related to FA β-oxidation and lipolysis, and decreased the expression of lipogenesis genes. Mildronate decreased whole body glycogen content, increased glucose metabolism rate, and upregulated the expression of glucose uptake and glycolysis genes. Mildronate also increased whole body protein content and hepatic mRNA expression of mechanistic target of rapamycin (mtor), and decreased the expression of a protein catabolism-related gene. Liver, rather than muscle, was the primary organ targeted by mildronate. In short, mildronate-induced hepatic inhibited carnitine synthesis in zebrafish caused decreased mitochondrial FA β-oxidation efficiency, greater lipid accumulation, and altered glucose and protein metabolism. This reveals the key roles of mitochondrial fatty acid β-oxidation in nutrient metabolism in fish, and this low-carnitine zebrafish model could also be used as a novel fish model for future metabolism studies. PMID:29867554

Inhibited Carnitine Synthesis Causes Systemic Alteration of Nutrient Metabolism in Zebrafish.

PubMed

Li, Jia-Min; Li, Ling-Yu; Qin, Xuan; Degrace, Pascal; Demizieux, Laurent; Limbu, Samwel M; Wang, Xin; Zhang, Mei-Ling; Li, Dong-Liang; Du, Zhen-Yu

2018-01-01

Impaired mitochondrial fatty acid β-oxidation has been correlated with many metabolic syndromes, and the metabolic characteristics of the mammalian models of mitochondrial dysfunction have also been intensively studied. However, the effects of the impaired mitochondrial fatty acid β-oxidation on systemic metabolism in teleost have never been investigated. In the present study, we established a low-carnitine zebrafish model by feeding fish with mildronate as a specific carnitine synthesis inhibitor [0.05% body weight (BW)/d] for 7 weeks, and the systemically changed nutrient metabolism, including carnitine and triglyceride (TG) concentrations, fatty acid (FA) β-oxidation capability, and other molecular and biochemical assays of lipid, glucose, and protein metabolism, were measured. The results indicated that mildronate markedly decreased hepatic carnitine concentrations while it had no effect in muscle. Liver TG concentrations increased by more than 50% in mildronate-treated fish. Mildronate decreased the efficiency of liver mitochondrial β-oxidation, increased the hepatic mRNA expression of genes related to FA β-oxidation and lipolysis, and decreased the expression of lipogenesis genes. Mildronate decreased whole body glycogen content, increased glucose metabolism rate, and upregulated the expression of glucose uptake and glycolysis genes. Mildronate also increased whole body protein content and hepatic mRNA expression of mechanistic target of rapamycin ( mtor ), and decreased the expression of a protein catabolism-related gene. Liver, rather than muscle, was the primary organ targeted by mildronate. In short, mildronate-induced hepatic inhibited carnitine synthesis in zebrafish caused decreased mitochondrial FA β-oxidation efficiency, greater lipid accumulation, and altered glucose and protein metabolism. This reveals the key roles of mitochondrial fatty acid β-oxidation in nutrient metabolism in fish, and this low-carnitine zebrafish model could also be used as a novel fish model for future metabolism studies.

Zebrafish and Streptococcal Infections.

PubMed

Saralahti, A; Rämet, M

2015-09-01

Streptococcal bacteria are a versatile group of gram-positive bacteria capable of infecting several host organisms, including humans and fish. Streptococcal species are common colonizers of the human respiratory and gastrointestinal tract, but they also cause some of the most common life-threatening, invasive infections in humans and aquaculture. With its unique characteristics and efficient tools for genetic and imaging applications, the zebrafish (Danio rerio) has emerged as a powerful vertebrate model for infectious diseases. Several zebrafish models introduced so far have shown that zebrafish are suitable models for both zoonotic and human-specific infections. Recently, several zebrafish models mimicking human streptococcal infections have also been developed. These models show great potential in providing novel information about the pathogenic mechanisms and host responses associated with human streptococcal infections. Here, we review the zebrafish infection models for the most relevant streptococcal species: the human-specific Streptococcus pneumoniae and Streptococcus pyogenes, and the zoonotic Streptococcus iniae and Streptococcus agalactiae. The recent success and the future potential of these models for the study of host-pathogen interactions in streptococcal infections are also discussed. © 2015 The Foundation for the Scandinavian Journal of Immunology.

Preparing for a Semiannual IACUC Inspection of a Satellite Zebrafish (Danio rerio) Facility

PubMed Central

Koerber, Amy S; Kalishman, Jennifer

2009-01-01

Institutions worldwide have experienced a rapid growth in the use of zebrafish as a research model for a variety of molecular and genetic studies of vertebrate development. This expansion in zebrafish research essentially has outpaced the establishment of specific recommendations for the care and use of fish in research. In some cases, this situation has created a dilemma where an Institutional Animal Care and Use Committee, which is responsible for oversight of vertebrate animal research, is not fully prepared to undertake this role for a decentralized zebrafish facility. IACUC inspectors will be more equipped to ask pertinent questions by understanding the basic principles of zebrafish health and facility management. Concurrently, zebrafish facility managers can contribute to the progress of a semiannual facility inspection by maintaining fully accessible operating records. In the context of presenting a well-established and useful model of zebrafish management and recordkeeping to the zebrafish facility operator, the information we present here also prepares a potential IACUC inspector to conduct a constructive and positive inspection. PMID:19245754

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

PubMed Central

Lawrence, Christian; Best, Jason; Cockington, Jason; Henry, Eric C.; Hurley, Shane; James, Althea; Lapointe, Christopher; Maloney, Kara; Sanders, Erik

2016-01-01

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an "on-demand", nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding. PMID:26863035

Influence of robotic shoal size, configuration, and activity on zebrafish behavior in a free-swimming environment.

PubMed

Butail, Sachit; Polverino, Giovanni; Phamduy, Paul; Del Sette, Fausto; Porfiri, Maurizio

2014-12-15

In animal studies, robots have been recently used as a valid tool for testing a wide spectrum of hypotheses. These robots often exploit visual or auditory cues to modulate animal behavior. The propensity of zebrafish, a model organism in biological studies, toward fish with similar color patterns and shape has been leveraged to design biologically inspired robots that successfully attract zebrafish in preference tests. With an aim of extending the application of such robots to field studies, here, we investigate the response of zebrafish to multiple robotic fish swimming at different speeds and in varying arrangements. A soft real-time multi-target tracking and control system remotely steers the robots in circular trajectories during the experimental trials. Our findings indicate a complex behavioral response of zebrafish to biologically inspired robots. More robots produce a significant change in salient measures of stress, with a fast robot swimming alone causing more freezing and erratic activity than two robots swimming slowly together. In addition, fish spend more time in the proximity of a robot when they swim far apart than when the robots swim close to each other. Increase in the number of robots also significantly alters the degree of alignment of fish motion with a robot. Results from this study are expected to advance our understanding of robot perception by live animals and aid in hypothesis-driven studies in unconstrained free-swimming environments. Copyright © 2014 Elsevier B.V. All rights reserved.

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish.

PubMed

Lawrence, Christian; Best, Jason; Cockington, Jason; Henry, Eric C; Hurley, Shane; James, Althea; Lapointe, Christopher; Maloney, Kara; Sanders, Erik

2016-01-17

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an "on-demand", nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding.

Auditory sensitivity of larval zebrafish (Danio rerio) measured using a behavioral prepulse inhibition assay

PubMed Central

Bhandiwad, Ashwin A.; Zeddies, David G.; Raible, David W.; Rubel, Edwin W.; Sisneros, Joseph A.

2013-01-01

SUMMARY Zebrafish (Danio rerio) have become a valuable model for investigating the molecular genetics and development of the inner ear in vertebrates. In this study, we employed a prepulse inhibition (PPI) paradigm to assess hearing in larval wild-type (AB) zebrafish during early development at 5–6 days post-fertilization (d.p.f.). We measured the PPI of the acoustic startle response in zebrafish using a 1-dimensional shaker that simulated the particle motion component of sound along the fish's dorsoventral axis. The thresholds to startle-inducing stimuli were determined in 5–6 d.p.f. zebrafish, and their hearing sensitivity was then characterized using the thresholds of prepulse tone stimuli (90–1200 Hz) that inhibited the acoustic startle response to a reliable startle stimulus (820 Hz at 20 dB re. 1 m s−2). Hearing thresholds were defined as the minimum prepulse tone level required to significantly reduce the startle response probability compared with the baseline (no-prepulse) condition. Larval zebrafish showed greatest auditory sensitivity from 90 to 310 Hz with corresponding mean thresholds of −19 to −10 dB re. 1 m s−2, respectively. Hearing thresholds of prepulse tones were considerably lower than previously predicted by startle response assays. The PPI assay was also used to investigate the relative contribution of the lateral line to the detection of acoustic stimuli. After aminoglycoside-induced neuromast hair-cell ablation, we found no difference in PPI thresholds between treated and control fish. We propose that this PPI assay can be used to screen for novel zebrafish hearing mutants and to investigate the ontogeny of hearing in zebrafish and other fishes. PMID:23966590

Diving deeper into Zebrafish development of social behavior: analyzing high resolution data.

PubMed

Buske, Christine; Gerlai, Robert

2014-08-30

Vertebrate model organisms have been utilized in high throughput screening but only with substantial cost and human capital investment. The zebrafish is a vertebrate model species that is a promising and cost effective candidate for efficient high throughput screening. Larval zebrafish have already been successfully employed in this regard (Lessman, 2011), but adult zebrafish also show great promise. High throughput screening requires the use of a large number of subjects and collection of substantial amount of data. Collection of data is only one of the demanding aspects of screening. However, in most screening approaches that involve behavioral data the main bottleneck that slows throughput is the time consuming aspect of analysis of the collected data. Some automated analytical tools do exist, but often they only work for one subject at a time, eliminating the possibility of fully utilizing zebrafish as a screening tool. This is a particularly important limitation for such complex phenotypes as social behavior. Testing multiple fish at a time can reveal complex social interactions but it may also allow the identification of outliers from a group of mutagenized or pharmacologically treated fish. Here, we describe a novel method using a custom software tool developed within our laboratory, which enables tracking multiple fish, in combination with a sophisticated analytical approach for summarizing and analyzing high resolution behavioral data. This paper focuses on the latter, the analytic tool, which we have developed using the R programming language and environment for statistical computing. We argue that combining sophisticated data collection methods with appropriate analytical tools will propel zebrafish into the future of neurobehavioral genetic research. Copyright © 2014. Published by Elsevier B.V.

Development of mandibular, hyoid and hypobranchial muscles in the zebrafish: homologies and evolution of these muscles within bony fishes and tetrapods

PubMed Central

Diogo, Rui; Hinits, Yaniv; Hughes, Simon M

2008-01-01

Background During vertebrate head evolution, muscle changes accompanied radical modification of the skeleton. Recent studies have suggested that muscles and their innervation evolve less rapidly than cartilage. The freshwater teleostean zebrafish (Danio rerio) is the most studied actinopterygian model organism, and is sometimes taken to represent osteichthyans as a whole, which include bony fishes and tetrapods. Most work concerning zebrafish cranial muscles has focused on larval stages. We set out to describe the later development of zebrafish head muscles and compare muscle homologies across the Osteichthyes. Results We describe one new muscle and show that the number of mandibular, hyoid and hypobranchial muscles found in four day-old zebrafish larvae is similar to that found in the adult. However, the overall configuration and/or the number of divisions of these muscles change during development. For example, the undivided adductor mandibulae of early larvae gives rise to the adductor mandibulae sections A0, A1-OST, A2 and Aω, and the protractor hyoideus becomes divided into dorsal and ventral portions in adults. There is not always a correspondence between the ontogeny of these muscles in the zebrafish and their evolution within the Osteichthyes. All of the 13 mandibular, hyoid and hypobranchial muscles present in the adult zebrafish are found in at least some other living teleosts, and all except the protractor hyoideus are found in at least some extant non-teleost actinopterygians. Of these muscles, about a quarter (intermandibularis anterior, adductor mandibulae, sternohyoideus) are found in at least some living tetrapods, and a further quarter (levator arcus palatini, adductor arcus palatini, adductor operculi) in at least some extant sarcopterygian fish. Conclusion Although the zebrafish occupies a rather derived phylogenetic position within actinopterygians and even within teleosts, with respect to the mandibular, hyoid and hypobranchial muscles it seems justified to consider it an appropriate representative of these two groups. Among these muscles, the three with clear homologues in tetrapods and the further three identified in sarcopterygian fish are particularly appropriate for comparisons of results between the actinopterygian zebrafish and the sarcopterygians. PMID:18307809

The influence of exercise on anxiety-like behavior in zebrafish (Danio rerio).

PubMed

DePasquale, C; Leri, J

2018-04-12

In non-human mammals, exercise has been shown to decrease anxiety-like behavior. Conversely, a number of studies have reported no effect or even an increase in anxiety-like behavior after exercise, however, inconsistent training regimes and behavioral paradigms across studies may be confounding the results. Zebrafish (Danio rerio) are a well-established animal model in neurobehavioral research, and have the potential to shed new insight into the effects of exercise on anxiety-like behavior where previous research has been limited, due to the ability to precisely control intensity and duration of exercise, and the validation of tests for measuring different aspects of anxiety-like behaviors. In the current study, fish were split between two treatment groups; Exercised and Control. Fish in the exercised condition were aerobically challenged (max water velocity: 0.5 m/s) using a swim tunnel one hour a day, five days a week, for six weeks. Control fish spent an equal amount of time in the swim tunnel but were not aerobically challenged (max water velocity: 0.05 m/s). After six weeks, all fish were tested individually in two standard complimentary anxiety tests for zebrafish: the novel tank test and the light-dark test. Exercised fish exhibited reduced anxiety-like behaviors in the novel tank test; they spent more time in the top and were quicker to enter the top of a novel tank compared to Control fish. In addition, Exercised fish spent more time in the light compartment of the light-dark test compared to Control fish. Our results demonstrate the beneficial effect of exercise on anxiety-like behavior in zebrafish. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Current Status of Sperm Cryopreservation in Biomedical Research Fish Models: Zebrafish, Medaka, and Xiphophorus*

PubMed Central

Yang, Huiping; Tiersch, Terrence R.

2009-01-01

Aquarium fishes are becoming increasingly important because of their value in biomedical research and the ornamental fish trade, and because many have become threatened or endangered in the wild. This review summarizes the current status of sperm cryopreservation in three fishes widely used in biomedical research: zebrafish, medaka, and live-bearing fishes of the genus Xiphophorus, and will focus on the needs and opportunities for future research and application of cryopreservation in aquarium fish. First, we summarize the basic biological characteristics regarding natural habitat, testis structure, spermatogenesis, sperm morphology, and sperm physiology. Second, we compare protocol development of sperm cryopreservation. Third, we emphasize the importance of artificial fertilization in sperm cryopreservation to evaluate the viability of thawed sperm. We conclude with a look to future research directions for sperm cryopreservation and the application of this technique in aquarium species. PMID:18691673

Fish Oncology: Diseases, Diagnostics, and Therapeutics.

PubMed

Vergneau-Grosset, Claire; Nadeau, Marie-Eve; Groff, Joseph M

2017-01-01

The scientific literature contains a wealth of information concerning spontaneous fish neoplasms, although ornamental fish oncology is still in its infancy. The occurrence of fish neoplasms has often been associated with oncogenic viruses and environmental insults, making them useful markers for environmental contaminants. The use of fish, including zebrafish, as models of human carcinogenesis has been developed and knowledge gained from these models may also be applied to ornamental fish, although more studies are required. This review summarizes information available about fish oncology pertaining to veterinary clinicians. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantification of birefringence readily measures the level of muscle damage in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berger, Joachim, E-mail: Joachim.Berger@Monash.edu; Sztal, Tamar; Currie, Peter D.

2012-07-13

Highlights: Black-Right-Pointing-Pointer Report of an unbiased quantification of the birefringence of muscle of fish larvae. Black-Right-Pointing-Pointer Quantification method readily identifies level of overall muscle damage. Black-Right-Pointing-Pointer Compare zebrafish muscle mutants for level of phenotype severity. Black-Right-Pointing-Pointer Proposed tool to survey treatments that aim to ameliorate muscular dystrophy. -- Abstract: Muscular dystrophies are a group of genetic disorders that progressively weaken and degenerate muscle. Many zebrafish models for human muscular dystrophies have been generated and analysed, including dystrophin-deficient zebrafish mutants dmd that model Duchenne Muscular Dystrophy. Under polarised light the zebrafish muscle can be detected as a bright area in anmore » otherwise dark background. This light effect, called birefringence, results from the diffraction of polarised light through the pseudo-crystalline array of the muscle sarcomeres. Muscle damage, as seen in zebrafish models for muscular dystrophies, can readily be detected by a reduction in the birefringence. Therefore, birefringence is a very sensitive indicator of overall muscle integrity within larval zebrafish. Unbiased documentation of the birefringence followed by densitometric measurement enables the quantification of the birefringence of zebrafish larvae. Thereby, the overall level of muscle integrity can be detected, allowing the identification and categorisation of zebrafish muscle mutants. In addition, we propose that the establish protocol can be used to analyse treatments aimed at ameliorating dystrophic zebrafish models.« less

An emerging role for gasotransmitters in the control of breathing and ionic regulation in fish.

PubMed

Perry, Steve; Kumai, Y; Porteus, C S; Tzaneva, V; Kwong, R W M

2016-02-01

Three gases comprising nitric oxide, carbon monoxide and hydrogen sulphide, collectively are termed gasotransmitters. The gasotransmitters control several physiological functions in fish by acting as intracellular signaling molecules. Hydrogen sulphide, first implicated in vasomotor control in fish, plays a critical role in oxygen chemoreception owing to its production and downstream effects within the oxygen chemosensory cells, the neuroepithelial cells. Indeed, there is emerging evidence that hydrogen sulphide may contribute to oxygen sensing in both fish and mammals by promoting membrane depolarization of the chemosensory cells. Unlike hydrogen sulphide which stimulates breathing in zebrafish, carbon monoxide inhibits ventilation in goldfish and zebrafish whereas nitric oxide stimulates breathing in zebrafish larvae while inhibiting breathing in adults. Gasotransmitters also modulate ionic uptake in zebrafish. Though nothing is known about the role of CO, reduced activities of branchial Na(+)/K(+)-ATPase and H(+)-ATPase activities in the presence of NO donors suggest an inhibitory role of NO in fish osmoregulation. Hydrogen sulphide inhibits Na(+) uptake in zebrafish larvae and contributes to lowering Na(+) uptake capacity in fish acclimated to Na(+)-enriched water whereas it stimulates Ca(2+) uptake in larvae exposed to Ca(2+)-poor water.

Zebrafish: swimming towards a role for fanconi genes in DNA repair.

PubMed

Scata, Kimberly A; El-Deiry, Wafik S

2004-06-01

The zebrafish, Danio rerio, has become a favorite model organism for geneticists and developmental biologists. Recently cancer biologists have turned to this tiny fish to help them unravel the mysteries of conserved pathways such as the Fanconi Anemia (FA) pathway. Although a relatively rare disease, the genes involved in FA are part of a large network of DNA damage response/repair genes. Liu and colleagues have recapitulated some of the clinical manifestations of human FA by knocking down the zebrafish FANC-D2 gene thereby providing a new model for probing the underlying causes of these phenotypes.

Chlorella diet alters mitochondrial cardiolipin contents differentially in organs of Danio rerio analyzed by a lipidomics approach

PubMed Central

Chao, Yu-Jen; Wu, Wen-Hsin; Balazova, Maria; Wu, Ting-Yuan; Lin, Jamie; Liu, Yi-Wen

2018-01-01

The zebrafish (Danio rerio) is an important and widely used vertebrate model organism for the study of human diseases which include disorders caused by dysfunctional mitochondria. Mitochondria play an essential role in both energy metabolism and apoptosis, which are mediated through a mitochondrial phospholipid cardiolipin (CL). In order to examine the cardiolipin profile in the zebrafish model, we developed a CL analysis platform by using liquid chromatography-mass spectrometry (LC-MS). Meanwhile, we tested whether chlorella diet would alter the CL profile in the larval fish, and in various organs of the adult fish. The results showed that chlorella diet increased the chain length of CL in larval fish. In the adult zebrafish, the distribution patterns of CL species were similar between the adult brain and eye tissues, and between the heart and muscles. Interestingly, monolyso-cardiolipin (MLCL) was not detected in brain and eyes but found in other examined tissues, indicating a different remodeling mechanism to maintain the CL integrity. While the adult zebrafish were fed with chlorella for four weeks, the CL distribution showed an increase of the species of saturated acyl chains in the brain and eyes, but a decrease in the other organs. Moreover, chlorella diet led to a decrease of MLCL percentage in organs except the non-MLCL-containing brain and eyes. The CL analysis in the zebrafish provides an important tool for studying the mechanism of mitochondria diseases, and may also be useful for testing medical regimens targeting against the Barth Syndrome. PMID:29494608

Effectiveness of Rapid Cooling as a Method of Euthanasia for Young Zebrafish (Danio rerio).

PubMed

Wallace, Chelsea K; Bright, Lauren A; Marx, James O; Andersen, Robert P; Mullins, Mary C; Carty, Anthony J

2018-01-01

Despite increased use of zebrafish (Danio rerio) in biomedical research, consistent information regarding appropriate euthanasia methods, particularly for embryos, is sparse. Current literature indicates that rapid cooling is an effective method of euthanasia for adult zebrafish, yet consistent guidelines regarding zebrafish younger than 6 mo are unavailable. This study was performed to distinguish the age at which rapid cooling is an effective method of euthanasia for zebrafish and the exposure times necessary to reliably euthanize zebrafish using this method. Zebrafish at 3, 4, 7, 14, 16, 19, 21, 28, 60, and 90 d postfertilization (dpf) were placed into an ice water bath for 5, 10, 30, 45, or 60 min (n = 12 to 40 per group). In addition, zebrafish were placed in ice water for 12 h (age ≤14 dpf) or 30 s (age ≥14 dpf). After rapid cooling, fish were transferred to a recovery tank and the number of fish alive at 1, 4, and 12-24 h after removal from ice water was documented. Euthanasia was defined as a failure when evidence of recovery was observed at any point after removal from ice water. Results showed that younger fish required prolonged exposure to rapid cooling for effective euthanasia, with the required exposure time decreasing as fish age. Although younger fish required long exposure times, animals became immobilized immediately upon exposure to the cold water, and behavioral indicators of pain or distress rarely occurred. We conclude that zebrafish 14 dpf and younger require as long as 12 h, those 16 to 28 dpf of age require 5 min, and those older than 28 dpf require 30 s minimal exposure to rapid cooling for reliable euthanasia.

Differential gene expression associated with dietary methylmercury (MeHg) exposure in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio)

PubMed Central

Liu, Qing; Basu, Niladri; Goetz, Giles; Jiang, Nan; Hutz, Reinhold J.; Tonellato, Peter J.; Carvan, Michael J.

2013-01-01

The objective of this study was to identify and evaluate conserved biomarkers that could be used in most species of teleost fish at most life-stages. We investigated the effects of sublethal methylmercury (MeHg) exposure on developing rainbow trout and zebrafish. Juvenile rainbow trout and young adult zebrafish were fed food with MeHg added at 0, 0.5, 5 and 50 ppm. Atomic absorption spectrometry was applied to measure whole body total Hg levels, and pathologic analysis was performed to identify MeHg-induced toxicity. Fish at six weeks were sampled from each group for microarray analysis using RNA from whole fish. MeHg-exposed trout and zebrafish did not show overt signs of toxicity or pathology, nor were significant differences seen in mortality, length, mass, or condition factor. The accumulation of MeHg in trout and zebrafish exhibited dose- and time-dependent patterns during six weeks, and zebrafish exhibited greater assimilation of total Hg than rainbow trout. The dysregulated genes in MeHg-treated fish have multiple functional annotations, such as iron ion homeostasis, glutathione transferase activity, regulation of muscle contraction, troponin I binding and calcium-dependent protein binding. Genes were selected as biomarker candidates based on their microarray data and their expression was evaluated by QPCR. Unfortunately, these genes are not good consistent biomarkers for both rainbow trout and zebrafish from QPCR evaluation using individual fish. Our conclusion is that biomarker analysis for aquatic toxicant assessment using fish needs to be based on tissue-, sex- and species-specific consideration. PMID:23529582

Behaviour of telomere and telomerase during aging and regeneration in zebrafish.

PubMed

Anchelin, Monique; Murcia, Laura; Alcaraz-Pérez, Francisca; García-Navarro, Esther M; Cayuela, María L

2011-02-09

Telomere length and telomerase activity are important factors in the pathobiology of human diseases. Age-related diseases and premature aging syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase. The zebrafish (Danio rerio) offers multiple experimental manipulation advantages over other vertebrate models and, therefore, it has been recently considered as a potential model for aging, cancer, and regeneration studies. However, it has only partially been exploited to shed light on these fundamental biological processes. The aim of this study was, therefore, to investigate telomere length and telomerase expression and activity in different strains of zebrafish obtained from different stock centres to determine whether they undergo any changes during aging and regeneration. We found that although both telomerase expression and telomere length increased from embryo to adulthood stages, they drastically declined in aged fish despite telomerase activity was detected in different tissues of old fish. In addition, we observed a weaker upregulation of telomerase expression in regenerating fins of old fish, which well correlates with their impaired regeneration capacity. Strikingly, telomeres were elongated or maintained during the fin regeneration process at all ages and after repeated amputations, likely to support high cell proliferation rates. We conclude that the expression of telomerase and telomere length are closely related during the entire life cycle of the fish and that these two parameters can be used as biomarkers of aging in zebrafish. Our results also reveal a direct relationship between the expression of telomerase, telomere length and the efficiency of tissue regeneration.

Induction of Social Behavior in Zebrafish: Live Versus Computer Animated Fish as Stimuli

PubMed Central

Qin, Meiying; Wong, Albert; Seguin, Diane

2014-01-01

Abstract The zebrafish offers an excellent compromise between system complexity and practical simplicity and has been suggested as a translational research tool for the analysis of human brain disorders associated with abnormalities of social behavior. Unlike laboratory rodents zebrafish are diurnal, thus visual cues may be easily utilized in the analysis of their behavior and brain function. Visual cues, including the sight of conspecifics, have been employed to induce social behavior in zebrafish. However, the method of presentation of these cues and the question of whether computer animated images versus live stimulus fish have differential effects have not been systematically analyzed. Here, we compare the effects of five stimulus presentation types: live conspecifics in the experimental tank or outside the tank, playback of video-recorded live conspecifics, computer animated images of conspecifics presented by two software applications, the previously employed General Fish Animator, and a new application Zebrafish Presenter. We report that all stimuli were equally effective and induced a robust social response (shoaling) manifesting as reduced distance between stimulus and experimental fish. We conclude that presentation of live stimulus fish, or 3D images, is not required and 2D computer animated images are sufficient to induce robust and consistent social behavioral responses in zebrafish. PMID:24575942

Induction of social behavior in zebrafish: live versus computer animated fish as stimuli.

PubMed

Qin, Meiying; Wong, Albert; Seguin, Diane; Gerlai, Robert

2014-06-01

The zebrafish offers an excellent compromise between system complexity and practical simplicity and has been suggested as a translational research tool for the analysis of human brain disorders associated with abnormalities of social behavior. Unlike laboratory rodents zebrafish are diurnal, thus visual cues may be easily utilized in the analysis of their behavior and brain function. Visual cues, including the sight of conspecifics, have been employed to induce social behavior in zebrafish. However, the method of presentation of these cues and the question of whether computer animated images versus live stimulus fish have differential effects have not been systematically analyzed. Here, we compare the effects of five stimulus presentation types: live conspecifics in the experimental tank or outside the tank, playback of video-recorded live conspecifics, computer animated images of conspecifics presented by two software applications, the previously employed General Fish Animator, and a new application Zebrafish Presenter. We report that all stimuli were equally effective and induced a robust social response (shoaling) manifesting as reduced distance between stimulus and experimental fish. We conclude that presentation of live stimulus fish, or 3D images, is not required and 2D computer animated images are sufficient to induce robust and consistent social behavioral responses in zebrafish.

Evaluating the effectiveness of common disinfectants at preventing the propagation of Mycobacterium spp. isolated from zebrafish

PubMed Central

Chang, Carolyn T.; Colicino, Erica G.; DiPaola, Elizabeth J.; Al-Hasnawi, Hadi Jabbar; Whipps, Christopher M.

2016-01-01

Mycobacteriosis is a bacterial disease that is common in captive, wild and research fish. There is no one causative agent of mycobacteriosis, as several strains and species of Mycobacterium have been identified in zebrafish. With increased usage and investment in wild-type and mutant zebrafish strains, considerable value is placed on preserving zebrafish health. One control measure used to prevent mycobacterial spread within and between zebrafish facilities is egg disinfection. Here we investigate the effectiveness of three disinfectants [chlorine bleach, hydrogen peroxide, and povidone iodine (PVPI)] commonly included in egg disinfection protocols for laboratory fish as well as aquaculture fish and compare the knockdown effect of these treatments on Mycobacterium spp. in vitro. Despite current usage, comparison of these disinfection regimes’ abilities to prevent mycobacterial growth has not been tested. We found that the germicidal effect of different disinfectants vary by Mycobacterium spp.. Hydrogen peroxide was the least effective disinfectant, followed by unbuffered chlorine bleach, which is commonly used to disinfect embryos in zebrafish facilities. Disinfection with 25 ppm PVPI for 5 min was very effective, and may be an improved alternative to chlorine bleach for embryo disinfection. Results from this study can be utilized by laboratory fish facilities in order to prevent the spread of mycobacteriosis in research fish. PMID:26423444

The hydrodynamics of predator-prey interactions in zebrafish

NASA Astrophysics Data System (ADS)

McHenry, Matthew; Soto, Alberto; Carrillo, Andres; Byron, Margaret

2017-11-01

Hydrodynamics govern the behavior of fishes when they operate as predators or prey. In addition to the role of fluid forces in propulsion, fishes relay on flow stimuli to sense a predatory threat and to localize palatable prey. We have performed a series of experiments on zebrafish (Danio rerio) that aim to resolve the major factors that determine whether prey survive an encounter with a predator. Zebrafish serve as a model system in this pursuit because the adults prey on larvae of the same species and the larvae are often successful in evading the attacks of the adults. We use a combination of theoretical and experimental approaches to resolve the behavioral algorithms and kinematics that determined the outcome of these interactions. In this context, the hydrodynamics of intermediate Reynolds numbers largely determines the range of flow stimuli and the limits to locomotor performance at dictate prey survival. These principles have the potential to apply to a broad diversity of fishes and other aquatic animals. ONR: N00014-15-1-2249.

The Presence or Absence of Intestinal Microbiota Affects Lipid Deposition and Related Genes Expression in Zebrafish (Danio rerio).

PubMed

Sheng, Yi; Ren, Hui; Limbu, Samwel M; Sun, Yuhong; Qiao, Fang; Zhai, Wanying; Du, Zhen-Yu; Zhang, Meiling

2018-01-01

Understanding how intestinal microbiota alters energy homeostasis and lipid metabolism is a critical process in energy balance and health. However, the exact role of intestinal microbiota in the regulation of lipid metabolism in fish remains unclear. Here, we used two zebrafish models (germ-free and antibiotics-treated zebrafish) to identify the role of intestinal microbiota in lipid metabolism. Conventional and germ-free zebrafish larvae were fed with egg yolk. Transmission electron microscopy was used to detect the presence of lipid droplets in the intestinal epithelium. The results showed that, microbiota increased lipid accumulation in the intestinal epithelium. The mRNA sequencing technology was used to assess genes expression level. We found majority of the differentially expressed genes were related to lipid metabolism. Due to the limitation of germ-free zebrafish larvae, antibiotics-treated zebrafish were also used to identify the relationship between the gut microbiota and the host lipid metabolism. Oil-red staining showed antibiotics-treated zebrafish had less intestinal lipid accumulation than control group. The mRNA expression of genes related to lipid metabolism in liver and intestine was also quantified by using real-time PCR. The results indicated that apoa4 , hsl , cox15 , slc2a1a , and lss were more related to intestinal bacteria in fish, while the influence of intestinal microbiota on the activity of fabp6 , acsl5 , cd36 , and gpat2 was different between the liver and intestine. This study identified several genes regulated by intestinal microbiota. Furthermore, the advantages and disadvantages of each model have been discussed. This study provides valuable information for exploring host-microbiota interactions in zebrafish in future.

Progress Towards the Development of a Fathead Minnow Embryo Test and Comparison to the Zebrafish Embryo Test for Assessing Acute Fish Toxicity

EPA Science Inventory

The Zebrafish Embryo Test (ZFET) for acute fish toxicity is a well developed method nearing adoption as an OECD Test Guideline. Early drafts of the test guideline (TG) envisioned a suite of potential test species to be covered including zebrafish, fathead minnow, Japanese Medaka...

Advancing Toxicology Research Using In Vivo High Throughput Toxicology with Small Fish Models

PubMed Central

Planchart, Antonio; Mattingly, Carolyn J.; Allen, David; Ceger, Patricia; Casey, Warren; Hinton, David; Kanungo, Jyotshna; Kullman, Seth W.; Tal, Tamara; Bondesson, Maria; Burgess, Shawn M.; Sullivan, Con; Kim, Carol; Behl, Mamta; Padilla, Stephanie; Reif, David M.; Tanguay, Robert L.; Hamm, Jon

2017-01-01

Summary Small freshwater fish models, especially zebrafish, offer advantages over traditional rodent models, including low maintenance and husbandry costs, high fecundity, genetic diversity, physiology similar to that of traditional biomedical models, and reduced animal welfare concerns. The Collaborative Workshop on Aquatic Models and 21st Century Toxicology was held at North Carolina State University on May 5-6, 2014, in Raleigh, North Carolina, USA. Participants discussed the ways in which small fish are being used as models to screen toxicants and understand mechanisms of toxicity. Workshop participants agreed that the lack of standardized protocols is an impediment to broader acceptance of these models, whereas development of standardized protocols, validation, and subsequent regulatory acceptance would facilitate greater usage. Given the advantages and increasing application of small fish models, there was widespread interest in follow-up workshops to review and discuss developments in their use. In this article, we summarize the recommendations formulated by workshop participants to enhance the utility of small fish species in toxicology studies, as well as many of the advances in the field of toxicology that resulted from using small fish species, including advances in developmental toxicology, cardiovascular toxicology, neurotoxicology, and immunotoxicology. We also review many emerging issues that will benefit from using small fish species, especially zebrafish, and new technologies that will enable using these organisms to yield results unprecedented in their information content to better understand how toxicants affect development and health. PMID:27328013

Physical exercise improves learning in zebrafish, Danio rerio.

PubMed

Luchiari, Ana Carolina; Chacon, Diana Marques Martins

2013-11-01

Zebrafish is an ideal vertebrate model for neuroscience studies focusing on learning and memory. Although genetic manipulation of zebrafish is available, behavioral protocols are often lacking. In this study we tested whether physical activity can facilitate zebrafish's learning process in an associative conditioning task. Learning was inferred by the approach of the feeding area just after the conditioned stimulus (light). Unexercised zebrafish showed conditioning response from the 5th testing day while fish previously submitted to swim against the water current showed learning by the 3rd day of testing. It seems that physical activity may accelerate associative learning response in zebrafish, indicating the benefits of exercise for cognitive processes. We suggest that this preliminary work could be useful for high throughput screening. Copyright © 2013 Elsevier B.V. All rights reserved.

Embryonic senescence and laminopathies in a progeroid zebrafish model.

PubMed

Koshimizu, Eriko; Imamura, Shintaro; Qi, Jie; Toure, Jamal; Valdez, Delgado M; Carr, Christopher E; Hanai, Jun-ichi; Kishi, Shuji

2011-03-30

Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist. We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with relatively mild phenotypes only, but had shortened lifespan with obvious distortion of body shape. We generated new zebrafish models for a human premature aging disorder, and further demonstrated the utility for studying laminopathies. Premature aging could also be modeled in zebrafish embryos. This genetic model may thus provide a new platform for future drug screening as well as genetic analyses aimed at identifying modifier genes that influence not only progeria and laminopathies but also other age-associated human diseases common in vertebrates.

Transient Overexpression of adh8a Increases Allyl Alcohol Toxicity in Zebrafish Embryos

PubMed Central

Klüver, Nils; Ortmann, Julia; Paschke, Heidrun; Renner, Patrick; Ritter, Axel P.; Scholz, Stefan

2014-01-01

Fish embryos are widely used as an alternative model to study toxicity in vertebrates. Due to their complexity, embryos are believed to more resemble an adult organism than in vitro cellular models. However, concerns have been raised with respect to the embryo's metabolic capacity. We recently identified allyl alcohol, an industrial chemical, to be several orders of magnitude less toxic to zebrafish embryo than to adult zebrafish (embryo LC50 = 478 mg/L vs. fish LC50 = 0.28 mg/L). Reports on mammals have indicated that allyl alcohol requires activation by alcohol dehydrogenases (Adh) to form the highly reactive and toxic metabolite acrolein, which shows similar toxicity in zebrafish embryos and adults. To identify if a limited metabolic capacity of embryos indeed can explain the low allyl alcohol sensitivity of zebrafish embryos, we compared the mRNA expression levels of Adh isoenzymes (adh5, adh8a, adh8b and adhfe1) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for adh8a and adh8b expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of adh8a, but not adh8b mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC50 = 0.42 mg/L in adh8a mRNA-injected embryos). Furthermore, GC/MS analysis of adh8a-injected embryos indicated a significant decline of internal allyl alcohol concentrations from 0.23-58 ng/embryo to levels below the limit of detection (< 4.6 µg/L). Injection of neither adh8b nor gfp mRNA had an impact on internal allyl alcohol levels supporting that the increased allyl alcohol toxicity was mediated by an increase in its metabolization. These results underline the necessity to critically consider metabolic activation in the zebrafish embryo. As demonstrated here, mRNA injection is one useful approach to study the role of candidate enzymes involved in metabolization. PMID:24594943

A New Model Army: Emerging fish models to study the genomics of vertebrate Evo-Devo

PubMed Central

Braasch, Ingo; Peterson, Samuel M.; Desvignes, Thomas; McCluskey, Braedan M.; Batzel, Peter; Postlethwait, John H.

2014-01-01

Many fields of biology – including vertebrate Evo-Devo research – are facing an explosion of genomic and transcriptomic sequence information and a multitude of fish species are now swimming in this ‘genomic tsunami’. Here, we first give an overview of recent developments in sequencing fish genomes and transcriptomes that identify properties of fish genomes requiring particular attention and propose strategies to overcome common challenges in fish genomics. We suggest that the generation of chromosome-level genome assemblies - for which we introduce the term ‘chromonome’ – should be a key component of genomic investigations in fish because they enable large-scale conserved synteny analyses that inform orthology detection, a process critical for connectivity of genomes. Orthology calls in vertebrates, especially in teleost fish, are complicated by divergent evolution of gene repertoires and functions following two rounds of genome duplication in the ancestor of vertebrates and a third round at the base of teleost fish. Second, using examples of spotted gar, basal teleosts, zebrafish-related cyprinids, cavefish, livebearers, icefish, and lobefin fish, we illustrate how next generation sequencing technologies liberate emerging fish systems from genomic ignorance and transform them into a new model army to answer longstanding questions on the genomic and developmental basis of their biodiversity. Finally, we discuss recent progress in the genetic toolbox for the major fish models for functional analysis, zebrafish and medaka, that can be transferred to many other fish species to study in vivo the functional effect of evolutionary genomic change as Evo-Devo research enters the postgenomic era. PMID:25111899

Effect of social isolation on anxiety-related behaviors, cortisol, and monoamines in adult zebrafish.

PubMed

Shams, Soaleha; Seguin, Diane; Facciol, Amanda; Chatterjee, Diptendu; Gerlai, Robert

2017-12-01

Social isolation can be used to study behavioral, neural, and hormonal mechanisms that regulate interactions in social animals. Although isolation effects have been reported in social mammals and various fish species, systematic studies with isolated zebrafish are rare. Here, the authors examined behavior (social and nonsocial), physiological stress (whole-body cortisol levels), and neurochemicals (serotonin, dopamine, and their metabolites), following acute and chronic social isolation in adult zebrafish. To observe how isolated fish respond behaviorally to social stimuli, they exposed zebrafish to live conspecifics or animated images after acute (24 hr) or chronic (6 months) social isolation. The authors observed that isolation did not affect locomotor activity, but acute isolation had weak nonsignificant anxiogenic effects in adult zebrafish. They also found that all isolated fish responded to both live and animated social stimuli, and the stress hormone, cortisol was lower in chronically isolated fish. Finally, neurochemical analyses showed that serotonin levels increased when fish were exposed to social stimulus after acute isolation, but its metabolite 5HIAA decreased in response to social stimulus following both acute and chronic isolation. Levels of both dopamine and its metabolite DOPAC were also reduced in fish exposed to social stimulus after acute and chronic isolation. Overall, these results show that isolation in zebrafish is an effective tool to study fundamental mechanisms controlling social interaction at behavioral and physiological levels. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

LITTLE FISH, BIG DATA: ZEBRAFISH AS A MODEL FOR CARDIOVASCULAR AND METABOLIC DISEASE.

PubMed

Gut, Philipp; Reischauer, Sven; Stainier, Didier Y R; Arnaout, Rima

2017-07-01

The burden of cardiovascular and metabolic diseases worldwide is staggering. The emergence of systems approaches in biology promises new therapies, faster and cheaper diagnostics, and personalized medicine. However, a profound understanding of pathogenic mechanisms at the cellular and molecular levels remains a fundamental requirement for discovery and therapeutics. Animal models of human disease are cornerstones of drug discovery as they allow identification of novel pharmacological targets by linking gene function with pathogenesis. The zebrafish model has been used for decades to study development and pathophysiology. More than ever, the specific strengths of the zebrafish model make it a prime partner in an age of discovery transformed by big-data approaches to genomics and disease. Zebrafish share a largely conserved physiology and anatomy with mammals. They allow a wide range of genetic manipulations, including the latest genome engineering approaches. They can be bred and studied with remarkable speed, enabling a range of large-scale phenotypic screens. Finally, zebrafish demonstrate an impressive regenerative capacity scientists hope to unlock in humans. Here, we provide a comprehensive guide on applications of zebrafish to investigate cardiovascular and metabolic diseases. We delineate advantages and limitations of zebrafish models of human disease and summarize their most significant contributions to understanding disease progression to date. Copyright © 2017 the American Physiological Society.

Zebrafish tracking using convolutional neural networks.

PubMed

Xu, Zhiping; Cheng, Xi En

2017-02-17

Keeping identity for a long term after occlusion is still an open problem in the video tracking of zebrafish-like model animals, and accurate animal trajectories are the foundation of behaviour analysis. We utilize the highly accurate object recognition capability of a convolutional neural network (CNN) to distinguish fish of the same congener, even though these animals are indistinguishable to the human eye. We used data augmentation and an iterative CNN training method to optimize the accuracy for our classification task, achieving surprisingly accurate trajectories of zebrafish of different size and age zebrafish groups over different time spans. This work will make further behaviour analysis more reliable.

Zebrafish tracking using convolutional neural networks

NASA Astrophysics Data System (ADS)

Xu, Zhiping; Cheng, Xi En

2017-02-01

Keeping identity for a long term after occlusion is still an open problem in the video tracking of zebrafish-like model animals, and accurate animal trajectories are the foundation of behaviour analysis. We utilize the highly accurate object recognition capability of a convolutional neural network (CNN) to distinguish fish of the same congener, even though these animals are indistinguishable to the human eye. We used data augmentation and an iterative CNN training method to optimize the accuracy for our classification task, achieving surprisingly accurate trajectories of zebrafish of different size and age zebrafish groups over different time spans. This work will make further behaviour analysis more reliable.

The Vital Relationship Between Nutrition and Health in Zebrafish.

PubMed

Watts, Stephen A; Lawrence, Christian; Powell, Mickie; D'Abramo, Louis R

2016-07-01

In the relatively short span of four decades, the zebrafish (Danio rerio) has emerged as an increasingly important model organism for biomedicine and other scientific disciplines. As the scale and sophistication of zebrafish research expands, so too does the need to develop standards that promote the production and maintenance of healthy animals for experiments. A major, but long overlooked, contributor to fish health is nutrition. Historically, feeding practices for laboratory zebrafish have been designed to promote growth and reproductive function. However, as the field matures, it is becoming increasingly clear that the nutritional goals for these animals should evolve beyond basic production to the maintenance of clinically healthy research subjects. This review outlines weaknesses and limitations of current approaches and provides a justification for the development of defined standardized diets that will strengthen and facilitate the continued growth of the zebrafish model system.

Larval rearing of zebrafish at suboptimal temperatures.

PubMed

Delomas, Thomas A; Dabrowski, Konrad

2018-05-01

Temperature-sensitive mutants have been widely utilized in single-cell and invertebrate model systems, particularly to study the function of essential genes. Few temperature-sensitive mutants have been identified in zebrafish, likely due to the difficulty of raising zebrafish at low temperatures. We describe a novel rearing protocol that allows rapid growth of larval and juvenile zebrafish at 23 °C compared to previous data in the literature. Embryos collected from four breeding pairs were maintained at 28.5 ± 0.5 °C until 5 days post-fertilization (dpf) - the onset of exogenous feeding. Larvae were then divided to six tanks and three tanks were cooled to 23 ± 0.2 °C. Fish were fed a live diet (marine rotifers Brachionus plicatilis and Artemia nauplii) and maintained under a set of environmental parameters shown to increase growth rate: continuous light, low salinity (3ppt), and algal turbidity. Mean total length and weight of fish at 21dpf were 12.7 ± 0.3 mm and 20.5 ± 1.5 mg for the 23 °C treatment and 18.5 ± 0.4 mm and 67.3 ± 3.4 mg for the 28.5 °C control. By 35 dpf, the fish raised at 23 °C had reached a mean length and weight of 18.9 ± 0.7 mm and 76.4 ± 6.7 mg, approximately the size control fish reached at 21 dpf. At 35 dpf, water temperature was raised to 28 °C and fish were reared to maturity (75 dpf) under standard conditions (freshwater, 13 L:11D photoperiod, dry diet, no added algal turbidity). Sex ratio and fertility were assessed and compared between temperature groups. There were no significant differences in sex ratio, fertilization rate, embryo viability at 1 dpf, clutch size, or relative fecundity. This rearing protocol will allow for efficient utilization of temperature-sensitive mutations in the zebrafish model system. Copyright © 2018 Elsevier Ltd. All rights reserved.

The French press: a repeatable and high-throughput approach to exercising zebrafish (Danio rerio).

PubMed

Usui, Takuji; Noble, Daniel W A; O'Dea, Rose E; Fangmeier, Melissa L; Lagisz, Malgorzata; Hesselson, Daniel; Nakagawa, Shinichi

2018-01-01

Zebrafish are increasingly used as a vertebrate model organism for various traits including swimming performance, obesity and metabolism, necessitating high-throughput protocols to generate standardized phenotypic information. Here, we propose a novel and cost-effective method for exercising zebrafish, using a coffee plunger and magnetic stirrer. To demonstrate the use of this method, we conducted a pilot experiment to show that this simple system provides repeatable estimates of maximal swim performance (intra-class correlation [ICC] = 0.34-0.41) and observe that exercise training of zebrafish on this system significantly increases their maximum swimming speed. We propose this high-throughput and reproducible system as an alternative to traditional linear chamber systems for exercising zebrafish and similarly sized fishes.

The French press: a repeatable and high-throughput approach to exercising zebrafish (Danio rerio)

PubMed Central

Usui, Takuji; Noble, Daniel W.A.; O’Dea, Rose E.; Fangmeier, Melissa L.; Lagisz, Malgorzata; Hesselson, Daniel

2018-01-01

Zebrafish are increasingly used as a vertebrate model organism for various traits including swimming performance, obesity and metabolism, necessitating high-throughput protocols to generate standardized phenotypic information. Here, we propose a novel and cost-effective method for exercising zebrafish, using a coffee plunger and magnetic stirrer. To demonstrate the use of this method, we conducted a pilot experiment to show that this simple system provides repeatable estimates of maximal swim performance (intra-class correlation [ICC] = 0.34–0.41) and observe that exercise training of zebrafish on this system significantly increases their maximum swimming speed. We propose this high-throughput and reproducible system as an alternative to traditional linear chamber systems for exercising zebrafish and similarly sized fishes. PMID:29372124

Fishing on chips: up-and-coming technological advances in analysis of zebrafish and Xenopus embryos.

PubMed

Zhu, Feng; Skommer, Joanna; Huang, Yushi; Akagi, Jin; Adams, Dany; Levin, Michael; Hall, Chris J; Crosier, Philip S; Wlodkowic, Donald

2014-11-01

Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole-organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high-throughput cell-based in vitro assays and low-throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor-intensive. Recently, a new generation of miniaturized chip-based devices have been developed for zebrafish and Xenopus embryo on-chip culture and experimentation. In this work, we review the critical developments in the field of Lab-on-a-Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

Comparative Analyses of Zebrafish Anxiety-Like Behavior Using Conflict-Based Novelty Tests.

PubMed

Kysil, Elana V; Meshalkina, Darya A; Frick, Erin E; Echevarria, David J; Rosemberg, Denis B; Maximino, Caio; Lima, Monica Gomes; Abreu, Murilo S; Giacomini, Ana C; Barcellos, Leonardo J G; Song, Cai; Kalueff, Allan V

2017-06-01

Modeling of stress and anxiety in adult zebrafish (Danio rerio) is increasingly utilized in neuroscience research and central nervous system (CNS) drug discovery. Representing the most commonly used zebrafish anxiety models, the novel tank test (NTT) focuses on zebrafish diving in response to potentially threatening stimuli, whereas the light-dark test (LDT) is based on fish scototaxis (innate preference for dark vs. bright areas). Here, we systematically evaluate the utility of these two tests, combining meta-analyses of published literature with comparative in vivo behavioral and whole-body endocrine (cortisol) testing. Overall, the NTT and LDT behaviors demonstrate a generally good cross-test correlation in vivo, whereas meta-analyses of published literature show that both tests have similar sensitivity to zebrafish anxiety-like states. Finally, NTT evokes higher levels of cortisol, likely representing a more stressful procedure than LDT. Collectively, our study reappraises NTT and LDT for studying anxiety-like states in zebrafish, and emphasizes their developing utility for neurobehavioral research. These findings can help optimize drug screening procedures by choosing more appropriate models for testing anxiolytic or anxiogenic drugs.

Zebrafish ( Danio rerio) as a model for investigating the safety of GM feed ingredients (soya and maize); performance, stress response and uptake of dietary DNA sequences.

PubMed

Sissener, Nini H; Johannessen, Lene E; Hevrøy, Ernst M; Wiik-Nielsen, Christer R; Berdal, Knut G; Nordgreen, Andreas; Hemre, Gro-Ingunn

2010-01-01

A 20-d zebrafish (Danio rerio) feeding trial, in which a near doubling of fish weight was achieved, was conducted with GM feed ingredients to evaluate feed intake, growth, stress response and uptake of dietary DNA. A partial aim of the study was to assess zebrafish as a model organism in GM safety assessments. Roundup Ready soya (RRS), YieldGard Bt maize (MON810) and their non-modified, maternal, near-isogenic lines were used in a 2 x 2 factorial design. Soya variety and maize variety were the main factors, both with two levels; non-GM and GM. Compared with fish fed non-GM maize, those fed GM maize exhibited significantly better growth, had lower mRNA transcription levels of superoxide dismutase (SOD)-1 and a tendency (non-significant) towards lower transcription of heat shock protein 70 in liver. Sex of the fish and soya variety had significant interaction effects on total RNA yield from the whole liver and transcription of SOD-1, suggesting that some diet component affecting males and females differently was present in different levels in the GM and the non-GM soya used in the present study. Dietary DNA sequences were detected in all of the organs analysed, but not all of the samples. Soya and maize rubisco (non-transgenic, multicopy genes) were most frequently detected, while MON810 transgenic DNA fragments were detected in some samples and RRS fragments were not detected. In conclusion, zebrafish shows promise as a model for this application.

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish

PubMed Central

Nagao, Yusuke; Takada, Hiroyuki; Miyadai, Motohiro; Adachi, Tomoko; Kamei, Yasuhiro; Hara, Ikuyo; Naruse, Kiyoshi; Hibi, Masahiko

2018-01-01

Mechanisms generating diverse cell types from multipotent progenitors are fundamental for normal development. Pigment cells are derived from multipotent neural crest cells and their diversity in teleosts provides an excellent model for studying mechanisms controlling fate specification of distinct cell types. Zebrafish have three types of pigment cells (melanocytes, iridophores and xanthophores) while medaka have four (three shared with zebrafish, plus leucophores), raising questions about how conserved mechanisms of fate specification of each pigment cell type are in these fish. We have previously shown that the Sry-related transcription factor Sox10 is crucial for fate specification of pigment cells in zebrafish, and that Sox5 promotes xanthophores and represses leucophores in a shared xanthophore/leucophore progenitor in medaka. Employing TILLING, TALEN and CRISPR/Cas9 technologies, we generated medaka and zebrafish sox5 and sox10 mutants and conducted comparative analyses of their compound mutant phenotypes. We show that specification of all pigment cells, except leucophores, is dependent on Sox10. Loss of Sox5 in Sox10-defective fish partially rescued the formation of all pigment cells in zebrafish, and melanocytes and iridophores in medaka, suggesting that Sox5 represses Sox10-dependent formation of these pigment cells, similar to their interaction in mammalian melanocyte specification. In contrast, in medaka, loss of Sox10 acts cooperatively with Sox5, enhancing both xanthophore reduction and leucophore increase in sox5 mutants. Misexpression of Sox5 in the xanthophore/leucophore progenitors increased xanthophores and reduced leucophores in medaka. Thus, the mode of Sox5 function in xanthophore specification differs between medaka (promoting) and zebrafish (repressing), which is also the case in adult fish. Our findings reveal surprising diversity in even the mode of the interactions between Sox5 and Sox10 governing specification of pigment cell types in medaka and zebrafish, and suggest that this is related to the evolution of a fourth pigment cell type. PMID:29621239

Discrimination reversal and attentional sets in zebrafish (Danio rerio)

PubMed Central

Parker, Matthew O.; Gaviria, Jessica; Haigh, Alastair; Millington, Mollie E.; Brown, Verity J.; Combe, Fraser J.; Brennan, Caroline H.

2014-01-01

The potential of zebrafish as a comparative model in behavioural neuroscience is currently hampered only by the lack of reliable and validated behavioural assays available to researchers. In the present experiment, we describe the performance of zebrafish in a test of attentional set formation. The fish were initially trained on a two-choice colour discrimination. Upon reaching acquisition criterion, the reinforced alternative was switched to the previously unreinforced alternative. Again, upon reaching criterion, the cues were replaced with a novel pair of colours (intra-dimensional shift) and reversed again on reaching criteria. We found that zebrafish show a steady decrease in trials-to-criteria over the four phases of the experiment, suggesting that they are forming and maintaining an attentional set, as has previously been demonstrated with mammals. Reversal learning deficits have been implicated in a variety of human psychological disorders (e.g., disorders of impulse control) and as such, we propose that performance of zebrafish in this procedure may represent a useful comparative model to complement existing rodent models. PMID:22561034

Stab wound injury of the zebrafish adult telencephalon: a method to investigate vertebrate brain neurogenesis and regeneration.

PubMed

Schmidt, Rebecca; Beil, Tanja; Strähle, Uwe; Rastegar, Sepand

2014-08-04

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury. In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

Efficient mutation identification in zebrafish by microarray capturing and next generation sequencing.

PubMed

Bontems, Franck; Baerlocher, Loic; Mehenni, Sabrina; Bahechar, Ilham; Farinelli, Laurent; Dosch, Roland

2011-02-18

Fish models like medaka, stickleback or zebrafish provide a valuable resource to study vertebrate genes. However, finding genetic variants e.g. mutations in the genome is still arduous. Here we used a combination of microarray capturing and next generation sequencing to identify the affected gene in the mozartkugelp11cv (mzlp11cv) mutant zebrafish. We discovered a 31-bp deletion in macf1 demonstrating the potential of this technique to efficiently isolate mutations in a vertebrate genome. Copyright © 2011 Elsevier Inc. All rights reserved.

ScreenCube: A 3D Printed System for Rapid and Cost-Effective Chemical Screening in Adult Zebrafish.

PubMed

Monstad-Rios, Adrian T; Watson, Claire J; Kwon, Ronald Y

2018-02-01

Phenotype-based small molecule screens in zebrafish embryos and larvae have been successful in accelerating pathway and therapeutic discovery for diverse biological processes. Yet, the application of chemical screens to adult physiologies has been relatively limited due to additional demands on cost, space, and labor associated with screens in adult animals. In this study, we present a 3D printed system and methods for intermittent drug dosing that enable rapid and cost-effective chemical administration in adult zebrafish. Using prefilled screening plates, the system enables dosing of 96 fish in ∼3 min, with a 10-fold reduction in drug quantity compared to that used in previous chemical screens in adult zebrafish. We characterize water quality kinetics during immersion in the system and use these kinetics to rationally design intermittent dosing regimens that result in 100% fish survival. As a demonstration of system fidelity, we show the potential to identify two known chemical inhibitors of adult tail fin regeneration, cyclopamine and dorsomorphin. By developing methods for rapid and cost-effective chemical administration in adult zebrafish, this study expands the potential for small molecule discovery in postembryonic models of development, disease, and regeneration.

Operant models of relapse in zebrafish (Danio rerio): Resurgence, renewal, and reinstatement.

PubMed

Kuroda, Toshikazu; Mizutani, Yuto; Cançado, Carlos R X; Podlesnik, Christopher A

2017-09-29

Zebrafish are a widely used animal model in biomedical research, as an alternative to mammals, for having features such as a fully sequenced genome, high fecundity, and low-cost maintenance, but behavioral research with these fish remains scarce. The present study investigated whether zebrafish could be a new animal model for studies on the relapse of behavior (e.g., addiction and overeating) after the behavior has been extinguished. Specifically, we examined whether zebrafish would show three different types of relapse commonly studied with other species: resurgence, renewal, and reinstatement. For resurgence, a target response (i.e., approaching a sensor) was established by presenting a reinforcer (i.e., shrimp eggs) contingent upon the response in Phase 1; the target response was extinguished while introducing reinforcement for an alternative response in Phase 2; neither response produced the reinforcer in Phase 3. For renewal, a target response was established under Context A in Phase 1 and was extinguished under Context B in Phase 2; the fish were placed back in Context A in Phase 3, where extinction remained in effect. For reinstatement, a target response was established in Phase 1 and was extinguished in Phase 2; the reinforcer was presented independently of responding in Phase 3. Each type of relapse occurred in Phase 3. These results replicate and extend previous findings on relapse to a new species and suggest that zebrafish can be a useful animal model for studying the interactions of biological and environmental factors that lead to relapse. Copyright © 2017 Elsevier B.V. All rights reserved.

Haploinsufficiency of the genes encoding the tumor suppressor Pten predisposes zebrafish to hemangiosarcoma.

PubMed

Choorapoikayil, Suma; Kuiper, Raoul V; de Bruin, Alain; den Hertog, Jeroen

2012-03-01

PTEN is an essential tumor suppressor that antagonizes Akt/PKB signaling. The zebrafish genome encodes two Pten genes, ptena and ptenb. Here, we report that zebrafish mutants that retain a single wild-type copy of ptena or ptenb (ptena(+/-)ptenb(-/-) or ptena(-/-)ptenb(+/-)) are viable and fertile. ptena(+/-)ptenb(-/-) fish develop tumors at a relatively high incidence (10.2%) and most tumors developed close to the eye (26/30). Histopathologically, the tumor masses were associated with the retrobulbar vascular network and diagnosed as hemangiosarcomas. A single tumor was identified in 42 ptena(-/-)ptenb(+/-) fish and was also diagnosed as hemangiosarcoma. Immunohistochemistry indicated that the tumor cells in ptena(+/-)ptenb(-/-) and ptena(-/-)ptenb(+/-) fish proliferated rapidly and were of endothelial origin. Akt/PKB signaling was activated in the tumors, whereas Ptena was still detected in tumor tissue from ptena(+/-)ptenb(-/-) zebrafish. We conclude that haploinsufficiency of the genes encoding Pten predisposes to hemangiosarcoma in zebrafish.

Differential sensitivities to dioxin-like compounds PCB 126 and PeCDF between Tg(cyp1a:gfp) transgenic medaka and zebrafish larvae.

PubMed

Xu, Hongyan; Li, Caixia; Suklai, Pacharaporn; Zeng, Qinghua; Chong, Raymond; Gong, Zhiyuan

2018-02-01

It has been intensively documented that there are species-differences in the sensitivity to dioxin-like compounds (DLCs) in mammalian and avian. However, this issue is still unclear in fish. This study aimed at evaluating the differential sensitivities to DLCs in fish larvae. Here, larvae of Tg(cyp1a:gfp) medaka and Tg(cyp1a:gfp) zebrafish were tested with 2,3,7,8-Tetrachlorodibenzodioxin (TCDD), polychlorinated biphenyl 126 (PCB 126) and 2,3,4,7,8,-Pentachlorodibenzofuran (PeCDF). Comparative analyses were performed on induction of GFP fluorescence, expression of endogenous cyp1a mRNAs and EROD activity between the two species after exposure to these chemicals. We found that PCB 126 and PeCDF exposure at high concentrations induced strong GFP expression in multiple organs (liver, head kidney and gut) in both medaka and zebrafish larvae. Moreover, the expression of endogenous cyp1a mRNA was significantly elevated in the zebrafish larvae exposed to TCDD, PCB 126 and PeCDF at different concentrations. Likewise, almost all the exposure conditions could cause prominent elevation of EROD activity in the zebrafish larvae, while the EROD activities were just slightly elevated in the medaka larvae exposed to 1 nM and 0.5 nM of TCDD as well as to 1.5 nM and 15 nM of PeCDF, but not in the medaka larvae exposed to PCB 126. Taken together, zebrafish was proved to be more sensitive than medaka to PCB 126 and to PeCDF in this study. The findings suggested species-specific sensitivity to DLCs in fish and will facilitate choosing a sensitive and reliable fish model or tool to evaluate the risk of dioxins and DLCs exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of the Cultivable Gut Microflora in Wild-Caught 
Mediterranean Fish Species.

PubMed

Jammal, Ahmad; Bariche, Michel; Zu Dohna, Heinrich; Kambris, Zakaria

2017-05-01

Microflora of the gastrointestinal tract plays important roles in food digestion, nutrient absorption and in host defense against ingested pathogens. Several studies have focused on the microflora of farmed fishes, but the gut flora of wild fishes remains poorly characterized. The aim of this work was to provide an overview of the bacteria colonizing the gut of wild-caught fishes and to determine whether some bacterial species can be pathogenic. We isolated cultivable bacteria from fifteen wild-caught Mediterranean fish species corresponding to different habitat, diet and origin. Bacterial species identity was determined by 16s rRNA gene sequencing for the 61 isolates. The potential pathogenicity of isolated bacteria was investigated using fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio) as model organisms. Two bacterial strains (Serratia sp. and Aeromonas salmonicida) were lethal when microinjected to Drosophila, while zebrafish did not develop any disease when exposed to any of 34 isolated bacterial strains. However, it was interesting to note that two bacterial strains (Shewanella and Arthrobacter) isolated from marine fishes were able to colonize the guts of freshwater zebrafish. The results of this study give an overview of the bacterial species found in the guts of wild fishes living off Beirut seashore. It shows that some parameters believed to be limiting factors to host-gut colonization by bacteria can be overcome by some species. This pilot study could be extended by sampling a larger number of fish species with several specimens per fish species, and by identifying uncultivable bacteria that reside in the fish guts. Our results may have implications for the utilization of certain bacterial species in fish farming or their use as bio-indicators for water and/or food quality.

Learning to swim, again: Axon regeneration in fish.

PubMed

Rasmussen, Jeffrey P; Sagasti, Alvaro

2017-01-01

Damage to the central nervous system (CNS) of fish can often be repaired to restore function, but in mammals recovery from CNS injuries usually fails due to a lack of axon regeneration. The relatively growth-permissive environment of the fish CNS may reflect both the absence of axon inhibitors found in the mammalian CNS and the presence of pro-regenerative environmental factors. Despite their different capacities for axon regeneration, many of the physiological processes, intrinsic molecular pathways, and cellular behaviors that control an axon's ability to regrow are conserved between fish and mammals. Fish models have thus been useful both for identifying factors differing between mammals and fish that may account for differences in CNS regeneration and for characterizing conserved intrinsic pathways that regulate axon regeneration in all vertebrates. The majority of adult axon regeneration studies have focused on the optic nerve or spinal axons of the teleosts goldfish and zebrafish, which have been productive models for identifying genes associated with axon regeneration, cellular mechanisms of circuit reestablishment, and the basis of functional recovery. Lampreys, which are jawless fish lacking myelin, have provided an opportunity to study regeneration of well defined spinal cord circuits. Newer larval zebrafish models offer numerous genetic tools and the ability to monitor the dynamic behaviors of extrinsic cell types regulating axon regeneration in live animals. Recent advances in imaging and gene editing methods are making fish models yet more powerful for investigating the cellular and molecular underpinnings of axon regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

Can zebrafish be used as animal model to study Alzheimer's disease?

PubMed Central

Santana, Soraya; Rico, Eduardo P; Burgos, Javier S

2012-01-01

Zebrafish is rapidly emerging as a promising model organism to study various central nervous system (CNS) disorders, including Alzheimer’s disease (AD). AD is the main cause of dementia in the human population and there is an urgency to understand the causes of this neurodegenerative disease. In this respect, the development of new animal models to study the underlying neurodegenerative mechanisms of AD is an urgent need. In this review we analyze the current situation in the use of zebrafish as a model for AD, discussing the reasons to use this experimental paradigm in CNS investigation and analyzing the several strategies adopted to induce an AD-like pathology in zebrafish. We discuss the strategies of performing interventions to cause damage in the zebrafish brain by altering the major neurotransmitter systems (such as cholinergic, glutamatergic or GABAergic circuits). We also analyze the several transgenic zebrafish constructed for the AD study, discussing both the familial-AD models based on APP processing pathway (APP and presenilins) and in the TAU hyperphosphorylation, together with the genes involved in sporadic-AD, as apolipoprotein E. We conclude that zebrafish is in a preliminary stage of development in the AD field, and that the transgenic animals must be improved to use this fish as an optimal model for AD research. Furthermore, a deeper knowledge of the zebrafish brain and a better characterization of the injury caused by alterations in the major neurotransmitter systems are needed. PMID:23383380

Heat-induced masculinization in domesticated zebrafish is family-specific and yields a set of different gonadal transcriptomes.

PubMed

Ribas, Laia; Liew, Woei Chang; Díaz, Noèlia; Sreenivasan, Rajini; Orbán, László; Piferrer, Francesc

2017-02-07

Understanding environmental influences on sex ratios is important for the study of the evolution of sex-determining mechanisms and for evaluating the effects of global warming and chemical pollution. Fishes exhibit sexual plasticity, but the underlying mechanisms of environmental effects on their reproduction are unclear even in the well-established teleost research model, the zebrafish. Here we established the conditions to study the effects of elevated temperature on zebrafish sex. We showed that sex ratio response to elevated temperature is family-specific and typically leads to masculinization (female-to-male sex reversal), resulting in neomales. These results uncovered genotype-by-environment interactions that support a polygenic sex determination system in domesticated (laboratory) zebrafish. We found that some heat-treated fish had gene expression profiles similar to untreated controls of the same sex, indicating that they were resistant to thermal effects. Further, most neomales had gonadal transcriptomes similar to that of regular males. Strikingly, we discovered heat-treated females that displayed a normal ovarian phenotype but with a "male-like" gonadal transcriptome. Such major transcriptomic reprogramming with preserved organ structure has never been reported. Juveniles were also found to have a male-like transcriptome shortly after exposure to heat. These findings were validated by analyzing the expression of genes and signaling pathways associated with sex differentiation. Our results revealed a lasting thermal effect on zebrafish gonads, suggesting new avenues for detection of functional consequences of elevated temperature in natural fish populations in a global warming scenario.

Miniaturized Embryo Array for Automated Trapping, Immobilization and Microperfusion of Zebrafish Embryos

PubMed Central

Akagi, Jin; Khoshmanesh, Khashayar; Evans, Barbara; Hall, Chris J.; Crosier, Kathryn E.; Cooper, Jonathan M.; Crosier, Philip S.; Wlodkowic, Donald

2012-01-01

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale. PMID:22606275

Zebrafish: an animal model for research in veterinary medicine.

PubMed

Nowik, N; Podlasz, P; Jakimiuk, A; Kasica, N; Sienkiewicz, W; Kaleczyc, J

2015-01-01

The zebrafish (Danio rerio) has become known as an excellent model organism for studies of vertebrate biology, vertebrate genetics, embryonal development, diseases and drug screening. Nevertheless, there is still lack of detailed reports about usage of the zebrafish as a model in veterinary medicine. Comparing to other vertebrates, they can lay hundreds of eggs at weekly intervals, externally fertilized zebrafish embryos are accessible to observation and manipulation at all stages of their development, which makes possible to simplify the research techniques such as fate mapping, fluorescent tracer time-lapse lineage analysis and single cell transplantation. Although zebrafish are only 2.5 cm long, they are easy to maintain. Intraperitoneal and intracerebroventricular injections, blood sampling and measurement of food intake are possible to be carry out in adult zebrafish. Danio rerio is a useful animal model for neurobiology, developmental biology, drug research, virology, microbiology and genetics. A lot of diseases, for which the zebrafish is a perfect model organism, affect aquatic animals. For a part of them, like those caused by Mycobacterium marinum or Pseudoloma neutrophila, Danio rerio is a natural host, but the zebrafish is also susceptible to the most of fish diseases including Itch, Spring viraemia of carp and Infectious spleen and kidney necrosis. The zebrafish is commonly used in research of bacterial virulence. The zebrafish embryo allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host. Plenty of common pathogens can be examined using zebrafish model: Streptococcus iniae, Vibrio anguillarum or Listeria monocytogenes. The steps are taken to use the zebrafish also in fungal research, especially that dealing with Candida albicans and Cryptococcus neoformans. Although, the zebrafish is used commonly as an animal model to study diseases caused by external agents, it is also useful in studies of metabolic disorders including fatty liver disease and diabetes. The zebrafish is also a valuable tool as a model in behavioral studies connected with feeding, predator evasion, habituation and memory or lateralized control of behavior. The aim of the present article is to familiarize the reader with the possibilities of Danio rerio as an experimental model for veterinary medicine.

New frontiers for zebrafish management.

PubMed

Lawrence, C

2016-01-01

The zebrafish (Danio rerio) is a preeminent model organism with a wide and expanding utility for numerous scientific disciplines. The same features that once endeared this small freshwater minnow to developmental biologists combined with its relatively high genetic similarity to mammals and the advent of new, more efficient methods for genome editing are now helping to spur expanded growth in its usage in various fields, including toxicology, drug discovery, transplant biology, disease modeling, and even aquaculture. Continued maturation and adoption of the zebrafish model system in these and other fields of science will require that methods and approaches for husbandry and management of these fish in controlled settings be refined and improved to the extent that, ultimately, zebrafish research becomes more reproducible, defined, cost-effective, and accessible to the masses. Knowledge and technology transfer from laboratory animal science and commercial aquaculture will be a necessary part of this development. Copyright © 2016 Elsevier Inc. All rights reserved.

Zebrafish knockout of Down syndrome gene, DYRK1A, shows social impairments relevant to autism.

PubMed

Kim, Oc-Hee; Cho, Hyun-Ju; Han, Enna; Hong, Ted Inpyo; Ariyasiri, Krishan; Choi, Jung-Hwa; Hwang, Kyu-Seok; Jeong, Yun-Mi; Yang, Se-Yeol; Yu, Kweon; Park, Doo-Sang; Oh, Hyun-Woo; Davis, Erica E; Schwartz, Charles E; Lee, Jeong-Soo; Kim, Hyung-Goo; Kim, Cheol-Hee

2017-01-01

DYRK1A maps to the Down syndrome critical region at 21q22. Mutations in this kinase-encoding gene have been reported to cause microcephaly associated with either intellectual disability or autism in humans. Intellectual disability accompanied by microcephaly was recapitulated in a murine model by overexpressing Dyrk1a which mimicked Down syndrome phenotypes. However, given embryonic lethality in homozygous knockout (KO) mice, no murine model studies could present sufficient evidence to link Dyrk1a dysfunction with autism. To understand the molecular mechanisms underlying microcephaly and autism spectrum disorders (ASD), we established an in vivo dyrk1aa KO model using zebrafish. We identified a patient with a mutation in the DYRK1A gene using microarray analysis. Circumventing the barrier of murine model studies, we generated a dyrk1aa KO zebrafish using transcription activator-like effector nuclease (TALEN)-mediated genome editing. For social behavioral tests, we have established a social interaction test, shoaling assay, and group behavior assay. For molecular analysis, we examined the neuronal activity in specific brain regions of dyrk1aa KO zebrafish through in situ hybridization with various probes including c-fos and crh which are the molecular markers for stress response. Microarray detected an intragenic microdeletion of DYRK1A in an individual with microcephaly and autism. From behavioral tests of social interaction and group behavior, dyrk1aa KO zebrafish exhibited social impairments that reproduce human phenotypes of autism in a vertebrate animal model. Social impairment in dyrk1aa KO zebrafish was further confirmed by molecular analysis of c-fos and crh expression. Transcriptional expression of c-fos and crh was lower than that of wild type fish in specific hypothalamic regions, suggesting that KO fish brains are less activated by social context. In this study, we established a zebrafish model to validate a candidate gene for autism in a vertebrate animal. These results illustrate the functional deficiency of DYRK1A as an underlying disease mechanism for autism. We also propose simple social behavioral assays as a tool for the broader study of autism candidate genes.

Transgenic expression of omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection.

PubMed

Cheng, Chih-Lun; Huang, Shin-Jie; Wu, Chih-Lu; Gong, Hong-Yi; Ken, Chuian-Fu; Hu, Shao-Yang; Wu, Jen-Leih

2015-11-17

Highly desaturated n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are synthesized by desaturases and elongase. They exert hepatoprotective effects to prevent alcoholic fatty liver syndrome or cholestatic liver injury. However, it is unclear how n-3 PUFAs improve immune function in liver. Vibrio vulnificus, a gram-negative bacterial pathogen, causes high mortality of aquaculture fishes upon infection. Humans can become infected with V. vulnificus through open wounds or by eating raw seafood, and such infections may result in systemic septicemia. Moreover, patients with liver diseases are vulnerable to infection, and are more likely than healthy persons to present with liver inflammation following infection. This study quantified n-3 PUFAs and their anti-bacterial effects in Fadsd6 and Elvol5a transgenic zebrafish. Two transgenic zebrafish strains with strong liver specific expression of Fadsd6 and Elvol5a (driven by the zebrafish Fabp10 promoter) were established using the Tol2 system. Synthesis of n-3 PUFAs in these strains were increased by 2.5-fold as compared to wild type (Wt) fish. The survival rate in 24 h following challenge with V. vulnificus was 20 % in Wt, but 70 % in the transgenic strains. In addition, the bacteria counts in transgenic fish strains were significantly decreased. The expression levels of pro-inflammatory genes, such as TNF-α, IL-1β, and NF-κB, were suppressed between 9 and 12 h after challenge. This study confirms the anti-bacterial function of n-3 PUFAs in a transgenic zebrafish model. Fadsd6 and Elvol5a transgenic zebrafish are more resistant to V. vulnificus infection, and enhance survival by diminishing the attendant inflammatory response.

Pax2.1 is required for the development of thyroid follicles in zebrafish.

PubMed

Wendl, Thomas; Lun, Klaus; Mione, Marina; Favor, Jack; Brand, Michael; Wilson, Stephen W; Rohr, Klaus B

2002-08-01

The thyroid gland is an organ primarily composed of endoderm-derived follicular cells. Although disturbed embryonic development of the thyroid gland leads to congenital hypothyroidism in humans and mammals, the underlying principles of thyroid organogenesis are largely unknown. In this study, we introduce zebrafish as a model to investigate the molecular and genetic mechanisms that control thyroid development. Marker gene expression suggests that the molecular pathways of early thyroid development are essentially conserved between fish and mammals. However during larval stages, we find both conserved and divergent features of development compared with mammals. A major difference is that in fish, we find evidence for hormone production not only in thyroid follicular cells, but also in an anterior non-follicular group of cells. We show that pax2.1 and pax8, members of the zebrafish pax2/5/8 paralogue group, are expressed in the thyroid primordium. Whereas in mice, only Pax8 has a function during thyroid development, analysis of the zebrafish pax2.1 mutant no isthmus (noi(-/-)) demonstrates that pax2.1 has a role comparable with mouse Pax8 in differentiation of the thyroid follicular cells. Early steps of thyroid development are normal in noi(-/-), but later expression of molecular markers is lost and the formation of follicles fails. Interestingly, the anterior non-follicular site of thyroid hormone production is not affected in noi(-/-). Thus, in zebrafish, some remaining thyroid hormone synthesis takes place independent of the pathway leading to thyroid follicle formation. We suggest that the noi(-/-) mutant serves as a new zebrafish model for hypothyroidism.

Glutathione S-Transferase Protein Expression in Different Life Stages of Zebrafish (Danio rerio)

PubMed Central

Tierbach, Alena; Groh, Ksenia J; Schönenberger, René; Schirmer, Kristin

2018-01-01

Abstract Zebrafish is a widely used animal model in biomedical sciences and toxicology. Although evidence for the presence of phases I and II xenobiotic defense mechanisms in zebrafish exists on the transcriptional and enzyme activity level, little is known about the protein expression of xenobiotic metabolizing enzymes. Given the important role of glutathione S-transferases (GSTs) in phase II biotransformation, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The established multiple reaction monitoring-based assays enable the measurement of the relative abundance of specific GST isoenzymes and GST classes in zebrafish through a combination of proteotypic peptides and peptides shared within the same class. GSTs of the classes alpha, mu, pi and rho are expressed in zebrafish embryo as early as 4 h postfertilization (hpf). The majority of GST enzymes are present at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ dependent, with most of the GST classes showing the highest expression in the liver. The expression of a wide range of cytosolic GST isoenzymes and classes in zebrafish early life stages and adulthood supports the use of zebrafish as a model organism in chemical-related investigations. PMID:29361160

Characterization of behavioral and endocrine effects of LSD on zebrafish.

PubMed

Grossman, Leah; Utterback, Eli; Stewart, Adam; Gaikwad, Siddharth; Chung, Kyung Min; Suciu, Christopher; Wong, Keith; Elegante, Marco; Elkhayat, Salem; Tan, Julia; Gilder, Thomas; Wu, Nadine; Dileo, John; Cachat, Jonathan; Kalueff, Allan V

2010-12-25

Lysergic acid diethylamide (LSD) is a potent hallucinogenic drug that strongly affects animal and human behavior. Although adult zebrafish (Danio rerio) are emerging as a promising neurobehavioral model, the effects of LSD on zebrafish have not been investigated previously. Several behavioral paradigms (the novel tank, observation cylinder, light-dark box, open field, T-maze, social preference and shoaling tests), as well as modern video-tracking tools and whole-body cortisol assay were used to characterize the effects of acute LSD in zebrafish. While lower doses (5-100 microg/L) did not affect zebrafish behavior, 250 microg/L LSD increased top dwelling and reduced freezing in the novel tank and observation cylinder tests, also affecting spatiotemporal patterns of activity (as assessed by 3D reconstruction of zebrafish traces and ethograms). LSD evoked mild thigmotaxis in the open field test, increased light behavior in the light-dark test, reduced the number of arm entries and freezing in the T-maze and social preference test, without affecting social preference. In contrast, LSD affected zebrafish shoaling (increasing the inter-fish distance in a group), and elevated whole-body cortisol levels. Overall, our findings show sensitivity of zebrafish to LSD action, and support the use of zebrafish models to study hallucinogenic drugs of abuse. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Analyzing Cold Tolerance Mechanism in Transgenic Zebrafish (Danio rerio)

PubMed Central

Wang, Qian; Tan, Xungang; Jiao, Shuang; You, Feng; Zhang, Pei-Jun

2014-01-01

Low temperatures may cause severe growth inhibition and mortality in fish. In order to understand the mechanism of cold tolerance, a transgenic zebrafish Tg (smyd1:m3ck) model was established to study the effect of energy homeostasis during cold stress. The muscle-specific promoter Smyd1 was used to express the carp muscle form III of creatine kinase (M3-CK), which maintained enzymatic activity at a relatively low temperature, in zebrafish skeletal muscle. In situ hybridization showed that M3-CK was expressed strongly in the skeletal muscle. When exposed to 13°C, Tg (smyd1:m3ck) fish maintained their swimming behavior, while the wild-type could not. Energy measurements showed that the concentration of ATP increased in Tg (smyd1:m3ck) versus wild-type fish at 28°C. After 2 h at 13°C, ATP concentrations were 2.16-fold higher in Tg (smyd1:m3ck) than in wild-type (P<0.05). At 13°C, the ATP concentration in Tg (smyd1:m3ck) fish and wild-type fish was 63.3% and 20.0%, respectively, of that in wild-type fish at 28°C. Microarray analysis revealed differential expression of 1249 transcripts in Tg (smyd1:m3ck) versus wild-type fish under cold stress. Biological processes that were significantly overrepresented in this group included circadian rhythm, energy metabolism, lipid transport, and metabolism. These results are clues to understanding the mechanisms underlying temperature acclimation in fish. PMID:25058652

The synthetic substance hypoxanthine 3-N-oxide elicits alarm reactions in zebrafish (Danio rerio)

PubMed Central

Parra, Kevin V.; Adrian, James C.; Gerlai, Robert

2009-01-01

Zebrafish, one of the preferred study species of geneticists, is gaining increasing popularity in behavioral neuroscience. This small and prolific species may be an excellent tool with which the biological mechanisms of vertebrate brain function and behavior are investigated. Zebrafish has been proposed as a model organism in the analysis of fear responses and human anxiety disorders. Species-specific cues signaling the presence of predators have been successfully utilized in such research. Zebrafish has been shown to respond to its natural alarm substance with species-typical fear reactions. However, the extraction of this alarm substance and ascertaining its consistent dosing has been problematic. A synthetic substance with a known chemical identity and molecular weight would allow precise dosing and experimental control. Previously, the chemical component, hypoxanthine 3-N-oxide, common to several fish alarm substances has been identified and has been shown to elicit alarm reactions in fish species belonging to the Osteriophysan superorder. In the current study we investigate the effect of hypoxanthine 3-N-oxide by exposing zebrafish to three different concentrations of this synthetic substance. Our results show that the substance efficaciously induces species-typical fear reactions increasing the number of erratic movement episodes and jumps in zebrafish. We discuss the translational relevance of our findings and conclude that hypoxanthine 3-N-oxide will have utility to elicit fear responses in the laboratory in a precisely controlled manner in zebrafish. PMID:19583985

In Vivo Determination of Body Composition in Zebrafish (Danio rerio) by Quantitative Magnetic Resonance

PubMed Central

Fowler, L. Adele; Dennis, Lacey N.; Barry, R. Jeff; Powell, Mickie L.; Watts, Stephen A.

2016-01-01

Abstract Zebrafish (Danio rerio) as a model research organism continues to expand its relevance and role in multiple research disciplines, with recent work directed toward models of metabolism, nutrition, and energetics. Multiple technologies exist to assess body composition in animal research models at various levels of detail (tissues/organs, body regions, and whole organism). The development and/or validation of body composition assessment tools can open new areas of research questions for a given organism. Using fish from a comparative nutrition study, quantitative magnetic resonance (QMR) assessment of whole body fat and fat-free mass (FFM) in live fish was performed. QMR measures from two cohorts (n = 26 and n = 27) were compared with chemical carcass analysis (CCA) of FM and FFM. QMR was significantly correlated with chemical carcass values (fat, p < 0.001; lean, p = 0.002), although QMR significantly overestimated fat mass (FM) (0.011 g; p < 0.0001) and underestimated FFM (−0.024 g; p < 0.0001) relative to CCA. In a separate cross-validation group of fish, prediction equations corrected carcass values for FM (p = 0.121) and FFM (p = 0.753). These results support the utilization of QMR—a nonlethal nondestructive method—for cross-sectional or longitudinal body composition assessment outcomes in zebrafish. PMID:26974510

Use of TSHβ:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Cheng; Graduate University of Chinese Academy of Sciences, Beijing; Jin, Xia

Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit β (TSHβ) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSHβ in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSHβ promoter, the similar responsive patterns between the expression levels of TSHβ:EGFP and endogenous TSHβ mRNA in themore » pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSHβ:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system. Highlights: ► The promoter of zebrafish TSHβ gene has been identified. ► The stable TSHβ:EGFP transgenic zebrafish reporter germline has been generated. ► The EGFP in the transgenic fish recapitulated the pattern of pituitary TSHβ mRNA. ► The transgenic zebrafish may be an in vivo model for EDC assessment.« less

Report of Workshop on Euthanasia for Zebrafish-A Matter of Welfare and Science.

PubMed

Köhler, Almut; Collymore, Chereen; Finger-Baier, Karin; Geisler, Robert; Kaufmann, Larissa; Pounder, Kieran C; Schulte-Merker, Stefan; Valentim, Ana; Varga, Zoltan M; Weiss, Jürgen; Strähle, Uwe

2017-12-01

The increasing importance of zebrafish as a biomedical model organism is reflected by the steadily growing number of publications and laboratories working with this species. Regulatory recommendations for euthanasia as issued in Directive 2010/63/EU are, however, based on experience with fish species used for food production and do not take the small size and specific physiology of zebrafish into account. Consequently, the currently recommended methods of euthanasia in the Directive 2010/63/EU are either not applicable or may interfere with research goals. An international workshop was held in Karlsruhe, Germany, March 9, 2017, to discuss and propose alternative methods for euthanasia of zebrafish. The aim was to identify methods that adequately address the physiology of zebrafish and its use as a biomedical research model, follow the principles of the 3Rs (Replacement, Reduction, and Refinement) in animal experimentation and consider animal welfare during anesthesia and euthanasia. The results of the workshop are summarized here in the form of a white paper.

Fish from Head to Tail: The 9th European Zebrafish Meeting in Oslo.

PubMed

Griffiths, Gareth; Müller, Ferenc; Ledin, Johan; Patton, E Elizabeth; Gjøen, Tor; Lobert, Viola Hélène; Winther-Larsen, Hanne Cecilie; Mullins, Mary; Joly, Jean-Stephane; Weltzien, Finn-Arne; Press, Charles McLean; Aleström, Peter

2016-04-01

The 9th European Zebrafish Meeting took place recently in Oslo (June 28-July 2, 2015). A total of 650 participants came to hear the latest research news focused on the zebrafish, Danio rerio, and to its distant evolutionary relative medaka, Oryzias latipes. The packed program included keynote and plenary talks, short oral presentations and poster sessions, workshops, and strategic discussions. The meeting was a great success and revealed dramatically how important the zebrafish in particular has become as a model system for topics, such as developmental biology, functional genomics, biomedicine, toxicology, and drug development. A new emphasis was given to its potential as a model for aquaculture, a topic of great economic interest to the host country Norway and for the future global food supply in general. Zebrafish husbandry as well as its use in teaching were also covered in separate workshops. As has become a tradition in these meetings, there was a well-attended Wellcome Trust Sanger Institute and ZFIN workshop focused on Zebrafish Genome Resources on the first day. The full EZM 2015 program with abstracts can be read and downloaded from the EZM 2015 Web site zebrafish2015.org .

A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12

PubMed Central

Burger, Alexa; Vasilyev, Aleksandr; Tomar, Ritu; Selig, Martin K.; Nielsen, G. Petur; Peterson, Randall T.; Drummond, Iain A.; Haber, Daniel A.

2014-01-01

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer. PMID:24311731

Whole plant based treatment of hypercholesterolemia with Crataegus laevigata in a zebrafish model.

PubMed

Littleton, Robert M; Miller, Matthew; Hove, Jay R

2012-07-23

Consumers are increasingly turning to plant-based complementary and alternative medicines to treat hypercholesterolemia. Many of these treatments are untested and their efficacy is unknown. This multitude of potential remedies necessitates a model system amenable to testing large numbers of organisms that maintains similarity to humans in both mode of drug administration and overall physiology. Here we develop the larval zebrafish (4-30 days post fertilization) as a vertebrate model of dietary plant-based treatment of hypercholesterolemia and test the effects of Crataegus laevigata in this model. Larval zebrafish were fed high cholesterol diets infused with fluorescent sterols and phytomedicines. Plants were ground with mortar and pestle into a fine powder before addition to food. Fluorescent sterols were utilized to optically quantify relative difference in intravascular cholesterol levels between groups of fish. We utilized the Zeiss 7-Live Duo high-speed confocal platform in order to both quantify intravascular sterol fluorescence and to capture video of the heart beat for determination of cardiac output. In this investigation we developed and utilized a larval zebrafish model to investigate dietary plant-based intervention of the pathophysiology of hypercholesterolemia. We found BODIPY-cholesterol effectively labels diet-introduced intravascular cholesterol levels (P < 0.05, Student's t-test). We also established that zebrafish cardiac output declines as cholesterol dose increases (difference between 0.1% and 8% (w/w) high cholesterol diet-treated cardiac output significant at P < 0.05, 1-way ANOVA). Using this model, we found hawthorn leaves and flowers significantly reduce intravascular cholesterol levels (P < 0.05, 1-way ANOVA) and interact with cholesterol to impact cardiac output in hypercholesterolemic fish (2-way ANOVA, P < 0.05 for interaction effect). The results of this study demonstrate that the larval zebrafish has the potential to become a powerful model to test plant based dietary intervention of hypercholesterolemia. Using this model we have shown that hawthorn leaves and flowers have the potential to affect cardiac output as well as intravascular cholesterol levels. Further, our observation that hawthorn leaves and flowers interact with cholesterol to impact cardiac output indicates that the physiological effects of hawthorn may depend on diet.

It's time to swim! Zebrafish and the circadian clock.

PubMed

Vatine, Gad; Vallone, Daniela; Gothilf, Yoav; Foulkes, Nicholas S

2011-05-20

The zebrafish represents a fascinating model for studying key aspects of the vertebrate circadian timing system. Easy access to early embryonic development has made this species ideal for investigating how the clock is first established during embryogenesis. In particular, the molecular basis for the functional development of the zebrafish pineal gland has received much attention. In addition to this dedicated clock and photoreceptor organ, and unlike the situation in mammals, the clocks in zebrafish peripheral tissues and even cell lines are entrainable by direct exposure to light thus providing unique insight into the function and evolution of the light input pathway. Finally, the small size, low maintenance costs and high fecundity of this fish together with the availability of genetic tools make this an attractive model for forward genetic analysis of the circadian clock. Here, we review the work that has established the zebrafish as a valuable clock model organism and highlight the key questions that will shape the future direction of research. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Development of a transgenic zebrafish model expressing GFP in the notochord, somite and liver directed by the hfe2 gene promoter.

PubMed

Bian, Yue-Hong; Xu, Cheng; Li, Junling; Xu, Jin; Zhang, Hongwei; Du, Shao Jun

2011-08-01

Hemojuvelin, also known as RGMc, is encoded by hfe2 gene that plays an important role in iron homeostasis. hfe2 is specifically expressed in the notochord, developing somite and skeletal muscles during development. The molecular regulation of hfe2 expression is, however, not clear. We reported here the characterization of hfe2 gene expression and the regulation of its tissue-specific expression in zebrafish embryos. We demonstrated that the 6 kb 5'-flanking sequence upstream of the ATG start codon in the zebrafish hfe2 gene could direct GFP specific expression in the notochord, somites, and skeletal muscle of zebrafish embryos, recapitulating the expression pattern of the endogenous gene. However, the Tg(hfe2:gfp) transgene is also expressed in the liver of fish embryos, which did not mimic the expression of the endogenous hfe2 at the early stage. Nevertheless, the Tg(hfe2:gfp) transgenic zebrafish provides a useful model to study liver development. Treating Tg(hfe2:gfp) transgenic zebrafish embryos with valproic acid, a liver development inhibitor, significantly inhibited GFP expression in zebrafish. Together, these data indicate that the tissue specific expression of hfe2 in the notochord, somites and muscles is regulated by regulatory elements within the 6 kb 5'-flanking sequence of the hfe2 gene. Moreover, the Tg(hfe2:gfp) transgenic zebrafish line provides a useful model system for analyzing liver development in zebrafish.

Effect of the dietary inclusion of soybean components on the innate immune system in zebrafish.

PubMed

Fuentes-Appelgren, Pamela; Opazo, Rafael; Barros, Luis; Feijoó, Carmen G; Urzúa, Victoria; Romero, Jaime

2014-02-01

Some components of plant-based meals, such as saponins and vegetal proteins, have been proposed as inducers of intestinal inflammation in some fish. However, the molecular and cellular bases for this phenomenon have not been reported. In this work, zebrafish were used as a model to evaluate the effects of individual soybean meal components, such as saponins and soy proteins. Zebrafish larvae fed a fish meal feed containing soy components were assessed according to low and high inclusion levels. The granulocytes associated with the digestive tract and the induction of genes related to the immune system were quantitated as markers of the effects of the dietary components. A significant increase in the number of granulocytes was observed after feeding fish diets containing high saponin or soy protein contents. These dietary components also induced the expression of genes related to the innate immune system, including myeloid-specific peroxidase, as well as the complement protein and cytokines. These results reveal the influence of dietary components on the stimulation of the immune system. These observations could be significant to understanding the contributions of saponin and soy protein to the onset of enteritis in aqua-cultured fish, and this knowledge may aid in defining the role of the innate immune system in other inflammatory diseases involving dietary components in mammals.

Mature osteoblasts dedifferentiate in response to traumatic bone injury in the zebrafish fin and skull.

PubMed

Geurtzen, Karina; Knopf, Franziska; Wehner, Daniel; Huitema, Leonie F A; Schulte-Merker, Stefan; Weidinger, Gilbert

2014-06-01

Zebrafish have an unlimited capacity to regenerate bone after fin amputation. In this process, mature osteoblasts dedifferentiate to osteogenic precursor cells and thus represent an important source of newly forming bone. By contrast, differentiated osteoblasts do not appear to contribute to repair of bone injuries in mammals; rather, osteoblasts form anew from mesenchymal stem cells. This raises the question whether osteoblast dedifferentiation is specific to appendage regeneration, a special feature of the lepidotrichia bone of the fish fin, or a process found more generally in fish bone. Here, we show that dedifferentiation of mature osteoblasts is not restricted to fin regeneration after amputation, but also occurs during repair of zebrafish fin fractures and skull injuries. In both models, mature osteoblasts surrounding the injury downregulate the expression of differentiation markers, upregulate markers of the pre-osteoblast state and become proliferative. Making use of photoconvertible Kaede protein as well as Cre-driven genetic fate mapping, we show that osteoblasts migrate to the site of injury to replace damaged tissue. Our findings suggest a fundamental role for osteoblast dedifferentiation in reparative bone formation in fish and indicate that adult fish osteoblasts display elevated cellular plasticity compared with mammalian bone-forming cells. © 2014. Published by The Company of Biologists Ltd.

Heat-induced masculinization in domesticated zebrafish is family-specific and yields a set of different gonadal transcriptomes

PubMed Central

2017-01-01

Understanding environmental influences on sex ratios is important for the study of the evolution of sex-determining mechanisms and for evaluating the effects of global warming and chemical pollution. Fishes exhibit sexual plasticity, but the underlying mechanisms of environmental effects on their reproduction are unclear even in the well-established teleost research model, the zebrafish. Here we established the conditions to study the effects of elevated temperature on zebrafish sex. We showed that sex ratio response to elevated temperature is family-specific and typically leads to masculinization (female-to-male sex reversal), resulting in neomales. These results uncovered genotype-by-environment interactions that support a polygenic sex determination system in domesticated (laboratory) zebrafish. We found that some heat-treated fish had gene expression profiles similar to untreated controls of the same sex, indicating that they were resistant to thermal effects. Further, most neomales had gonadal transcriptomes similar to that of regular males. Strikingly, we discovered heat-treated females that displayed a normal ovarian phenotype but with a “male-like” gonadal transcriptome. Such major transcriptomic reprogramming with preserved organ structure has never been reported. Juveniles were also found to have a male-like transcriptome shortly after exposure to heat. These findings were validated by analyzing the expression of genes and signaling pathways associated with sex differentiation. Our results revealed a lasting thermal effect on zebrafish gonads, suggesting new avenues for detection of functional consequences of elevated temperature in natural fish populations in a global warming scenario. PMID:28115725

In vivo cell tracking and quantification method in adult zebrafish

NASA Astrophysics Data System (ADS)

Zhang, Li; Alt, Clemens; Li, Pulin; White, Richard M.; Zon, Leonard I.; Wei, Xunbin; Lin, Charles P.

2012-03-01

Zebrafish have become a powerful vertebrate model organism for drug discovery, cancer and stem cell research. A recently developed transparent adult zebrafish using double pigmentation mutant, called casper, provide unparalleled imaging power in in vivo longitudinal analysis of biological processes at an anatomic resolution not readily achievable in murine or other systems. In this paper we introduce an optical method for simultaneous visualization and cell quantification, which combines the laser scanning confocal microscopy (LSCM) and the in vivo flow cytometry (IVFC). The system is designed specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish casper, under physiological conditions in the same fish over time. The confocal imaging part in this system serves the dual purposes of imaging fish tissue microstructure and a 3D navigation tool to locate a suitable vessel for circulating cell counting. The multi-color, multi-channel instrument allows the detection of multiple cell populations or different tissues or organs simultaneously. We demonstrate initial testing of this novel instrument by imaging vasculature and tracking circulating cells in CD41: GFP/Gata1: DsRed transgenic casper fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. Circulating fluorescent cell incidents were recorded and counted repeatedly over time and in different types of vessels. Great application opportunities in cancer and stem cell researches are discussed.

Longitudinal Effects of Embryonic Exposure to Cocaine on Morphology, Cardiovascular Physiology, and Behavior in Zebrafish.

PubMed

Mersereau, Eric J; Boyle, Cody A; Poitra, Shelby; Espinoza, Ana; Seiler, Joclyn; Longie, Robert; Delvo, Lisa; Szarkowski, Megan; Maliske, Joshua; Chalmers, Sarah; Darland, Diane C; Darland, Tristan

2016-05-31

A sizeable portion of the societal drain from cocaine abuse results from the complications of in utero drug exposure. Because of challenges in using humans and mammalian model organisms as test subjects, much debate remains about the impact of in utero cocaine exposure. Zebrafish offer a number of advantages as a model in longitudinal toxicology studies and are quite sensitive physiologically and behaviorally to cocaine. In this study, we have used zebrafish to model the effects of embryonic pre-exposure to cocaine on development and on subsequent cardiovascular physiology and cocaine-induced conditioned place preference (CPP) in longitudinal adults. Larval fish showed a progressive decrease in telencephalic size with increased doses of cocaine. These treated larvae also showed a dose dependent response in heart rate that persisted 24 h after drug cessation. Embryonic cocaine exposure had little effect on overall health of longitudinal adults, but subtle changes in cardiovascular physiology were seen including decreased sensitivity to isoproterenol and increased sensitivity to cocaine. These longitudinal adult fish also showed an embryonic dose-dependent change in CPP behavior, suggesting an increased sensitivity. These studies clearly show that pre-exposure during embryonic development affects subsequent cocaine sensitivity in longitudinal adults.

The Nordic Countries Meeting on the Zebrafish as a Model for Development and Disease 2012

PubMed Central

Zetterberg, Henrik

2013-01-01

Abstract The first Nordic Countries Meeting on the Zebrafish as a Model for Development and Disease took place at Karolinska Institutet in Stockholm, November 21–23, 2012. The meeting gathered 130 scientists, students, and company representatives from Iceland, Finland, Norway, Denmark, and Sweden, as well as invited guests and keynote speakers from England, Scotland, Germany, Poland, The Netherlands, Singapore, Japan, and the United States. Presentations covered a wide range of topics, including developmental biology, genetics, evolutionary biology, toxicology, behavioral studies, and disease mechanisms. The need for formal guidance and training in zebrafish housing, husbandry, and health monitoring was recognized, and the meeting expressed its support for the joint working group of the FELASA/COST action BM0804 EuFishBioMed. The decision was made to turn the Nordic meeting into an annual event and create a Nordic network of zebrafish researchers. PMID:23590403

The Nordic countries meeting on the zebrafish as a model for development and disease 2012.

PubMed

Andersson Lendahl, Monika; Zetterberg, Henrik

2013-03-01

The first Nordic Countries Meeting on the Zebrafish as a Model for Development and Disease took place at Karolinska Institutet in Stockholm, November 21-23, 2012. The meeting gathered 130 scientists, students, and company representatives from Iceland, Finland, Norway, Denmark, and Sweden, as well as invited guests and keynote speakers from England, Scotland, Germany, Poland, The Netherlands, Singapore, Japan, and the United States. Presentations covered a wide range of topics, including developmental biology, genetics, evolutionary biology, toxicology, behavioral studies, and disease mechanisms. The need for formal guidance and training in zebrafish housing, husbandry, and health monitoring was recognized, and the meeting expressed its support for the joint working group of the FELASA/COST action BM0804 EuFishBioMed. The decision was made to turn the Nordic meeting into an annual event and create a Nordic network of zebrafish researchers.

Learning to Fish with Genetics: A Primer on the Vertebrate Model Danio rerio

PubMed Central

Holtzman, Nathalia G.; Iovine, M. Kathryn; Liang, Jennifer O.; Morris, Jacqueline

2016-01-01

In the last 30 years, the zebrafish has become a widely used model organism for research on vertebrate development and disease. Through a powerful combination of genetics and experimental embryology, significant inroads have been made into the regulation of embryonic axis formation, organogenesis, and the development of neural networks. Research with this model has also expanded into other areas, including the genetic regulation of aging, regeneration, and animal behavior. Zebrafish are a popular model because of the ease with which they can be maintained, their small size and low cost, the ability to obtain hundreds of embryos on a daily basis, and the accessibility, translucency, and rapidity of early developmental stages. This primer describes the swift progress of genetic approaches in zebrafish and highlights recent advances that have led to new insights into vertebrate biology. PMID:27384027

The use of fish models to study human neurological disorders.

PubMed

Matsui, Hideaki

2017-07-01

Small teleost fish including zebrafish and medaka have been used as animal models in basic science research due to the relative ease of handling and transparency during embryogenesis. Current advances in genetic engineering and progress in disease genetics allowed utilization of these fish to study neurological diseases and psychiatric disorders. This review summarizes the advantages and disadvantages of using fish for neuropsychiatric research using primarily our own studies as examples. We discuss how fish belong to a class of vertebrates, are feasible for imaging, and include diverse species with multiple research possibilities yet to be discovered. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

Fishing for causes and cures of motor neuron disorders

PubMed Central

Patten, Shunmoogum A.; Armstrong, Gary A. B.; Lissouba, Alexandra; Kabashi, Edor; Parker, J. Alex; Drapeau, Pierre

2014-01-01

Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount. The zebrafish (Danio rerio) is increasingly becoming a valuable model for studying human diseases and in screening for potential therapeutics. In this Review, we highlight recent progress in using zebrafish to study the pathology of the most common MNDs: spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). These studies indicate the power of zebrafish as a model to study the consequences of disease-related genes, because zebrafish homologues of human genes have conserved functions with respect to the aetiology of MNDs. Zebrafish also complement other animal models for the study of pathological mechanisms of MNDs and are particularly advantageous for the screening of compounds with therapeutic potential. We present an overview of their potential usefulness in MND drug discovery, which is just beginning and holds much promise for future therapeutic development. PMID:24973750

Natural Variation in Fish Transcriptomes: Comparative Analysis of the Fathead Minnow (Pimephales promelas) and Zebrafish (Danio rerio)

EPA Science Inventory

Fathead minnow and zebrafish are among the most intensively studied fish species in environmental toxicogenomics. To aid the assessment and interpretation of subtle transcriptomic effects from treatment conditions of interest, there needs to be a better characterization and unde...

Sensory hair cell death and regeneration in fishes

PubMed Central

Monroe, Jerry D.; Rajadinakaran, Gopinath; Smith, Michael E.

2015-01-01

Sensory hair cells are specialized mechanotransductive receptors required for hearing and vestibular function. Loss of hair cells in humans and other mammals is permanent and causes reduced hearing and balance. In the early 1980’s, it was shown that hair cells continue to be added to the inner ear sensory epithelia in cartilaginous and bony fishes. Soon thereafter, hair cell regeneration was documented in the chick cochlea following acoustic trauma. Since then, research using chick and other avian models has led to great insights into hair cell death and regeneration. However, with the rise of the zebrafish as a model organism for studying disease and developmental processes, there has been an increased interest in studying sensory hair cell death and regeneration in its lateral line and inner ears. Advances derived from studies in zebrafish and other fish species include understanding the effect of ototoxins on hair cells and finding otoprotectants to mitigate ototoxin damage, the role of cellular proliferation vs. direct transdifferentiation during hair cell regeneration, and elucidating cellular pathways involved in the regeneration process. This review will summarize research on hair cell death and regeneration using fish models, indicate the potential strengths and weaknesses of these models, and discuss several emerging areas of future studies. PMID:25954154

Vision in two cyprinid fish: implications for collective behavior

PubMed Central

Moore, Bret A.; Tyrrell, Luke P.; Fernández-Juricic, Esteban

2015-01-01

Many species of fish rely on their visual systems to interact with conspecifics and these interactions can lead to collective behavior. Individual-based models have been used to predict collective interactions; however, these models generally make simplistic assumptions about the sensory systems that are applied without proper empirical testing to different species. This could limit our ability to predict (and test empirically) collective behavior in species with very different sensory requirements. In this study, we characterized components of the visual system in two species of cyprinid fish known to engage in visually dependent collective interactions (zebrafish Danio rerio and golden shiner Notemigonus crysoleucas) and derived quantitative predictions about the positioning of individuals within schools. We found that both species had relatively narrow binocular and blind fields and wide visual coverage. However, golden shiners had more visual coverage in the vertical plane (binocular field extending behind the head) and higher visual acuity than zebrafish. The centers of acute vision (areae) of both species projected in the fronto-dorsal region of the visual field, but those of the zebrafish projected more dorsally than those of the golden shiner. Based on this visual sensory information, we predicted that: (a) predator detection time could be increased by >1,000% in zebrafish and >100% in golden shiners with an increase in nearest neighbor distance, (b) zebrafish schools would have a higher roughness value (surface area/volume ratio) than those of golden shiners, (c) and that nearest neighbor distance would vary from 8 to 20 cm to visually resolve conspecific striping patterns in both species. Overall, considering between-species differences in the sensory system of species exhibiting collective behavior could change the predictions about the positioning of individuals in the group as well as the shape of the school, which can have implications for group cohesion. We suggest that more effort should be invested in assessing the role of the sensory system in shaping local interactions driving collective behavior. PMID:26290783

Fish can show emotional fever: stress-induced hyperthermia in zebrafish.

PubMed

Rey, Sonia; Huntingford, Felicity A; Boltaña, Sebastian; Vargas, Reynaldo; Knowles, Toby G; Mackenzie, Simon

2015-11-22

Whether fishes are sentient beings remains an unresolved and controversial question. Among characteristics thought to reflect a low level of sentience in fishes is an inability to show stress-induced hyperthermia (SIH), a transient rise in body temperature shown in response to a variety of stressors. This is a real fever response, so is often referred to as 'emotional fever'. It has been suggested that the capacity for emotional fever evolved only in amniotes (mammals, birds and reptiles), in association with the evolution of consciousness in these groups. According to this view, lack of emotional fever in fishes reflects a lack of consciousness. We report here on a study in which six zebrafish groups with access to a temperature gradient were either left as undisturbed controls or subjected to a short period of confinement. The results were striking: compared to controls, stressed zebrafish spent significantly more time at higher temperatures, achieving an estimated rise in body temperature of about 2-4°C. Thus, zebrafish clearly have the capacity to show emotional fever. While the link between emotion and consciousness is still debated, this finding removes a key argument for lack of consciousness in fishes. © 2015 The Authors.

Fish can show emotional fever: stress-induced hyperthermia in zebrafish

PubMed Central

Huntingford, Felicity A.; Boltaña, Sebastian; Vargas, Reynaldo; Knowles, Toby G.; Mackenzie, Simon

2015-01-01

Whether fishes are sentient beings remains an unresolved and controversial question. Among characteristics thought to reflect a low level of sentience in fishes is an inability to show stress-induced hyperthermia (SIH), a transient rise in body temperature shown in response to a variety of stressors. This is a real fever response, so is often referred to as ‘emotional fever’. It has been suggested that the capacity for emotional fever evolved only in amniotes (mammals, birds and reptiles), in association with the evolution of consciousness in these groups. According to this view, lack of emotional fever in fishes reflects a lack of consciousness. We report here on a study in which six zebrafish groups with access to a temperature gradient were either left as undisturbed controls or subjected to a short period of confinement. The results were striking: compared to controls, stressed zebrafish spent significantly more time at higher temperatures, achieving an estimated rise in body temperature of about 2–4°C. Thus, zebrafish clearly have the capacity to show emotional fever. While the link between emotion and consciousness is still debated, this finding removes a key argument for lack of consciousness in fishes. PMID:26609087

Mortality Caused by Bath Exposure of Zebrafish (Danio rerio) Larvae to Nervous Necrosis Virus Is Limited to the Fourth Day Postfertilization

PubMed Central

Morick, Danny; Faigenbaum, Or; Smirnov, Margarita; Fellig, Yakov; Inbal, Adi

2015-01-01

Nervous necrosis virus (NNV) is a member of the Betanodavirus genus that causes fatal diseases in over 40 species of fish worldwide. Mortality among NNV-infected fish larvae is almost 100%. In order to elucidate the mechanisms responsible for the susceptibility of fish larvae to NNV, we exposed zebrafish larvae to NNV by bath immersion at 2, 4, 6, and 8 days postfertilization (dpf). Here, we demonstrate that developing zebrafish embryos are resistant to NNV at 2 dpf due to the protection afforded by the egg chorion and, to a lesser extent, by the perivitelline fluid. The zebrafish larvae succumbed to NNV infection during a narrow time window around the 4th dpf, while 6- and 8-day-old larvae were much less sensitive, with mortalities of 24% and 28%, respectively. PMID:25746990

Mortality Caused by Bath Exposure of Zebrafish (Danio rerio) Larvae to Nervous Necrosis Virus Is Limited to the Fourth Day Postfertilization.

PubMed

Morick, Danny; Faigenbaum, Or; Smirnov, Margarita; Fellig, Yakov; Inbal, Adi; Kotler, Moshe

2015-05-15

Nervous necrosis virus (NNV) is a member of the Betanodavirus genus that causes fatal diseases in over 40 species of fish worldwide. Mortality among NNV-infected fish larvae is almost 100%. In order to elucidate the mechanisms responsible for the susceptibility of fish larvae to NNV, we exposed zebrafish larvae to NNV by bath immersion at 2, 4, 6, and 8 days postfertilization (dpf). Here, we demonstrate that developing zebrafish embryos are resistant to NNV at 2 dpf due to the protection afforded by the egg chorion and, to a lesser extent, by the perivitelline fluid. The zebrafish larvae succumbed to NNV infection during a narrow time window around the 4th dpf, while 6- and 8-day-old larvae were much less sensitive, with mortalities of 24% and 28%, respectively. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Non-invasive imaging of zebrafish with spinal deformities using optical coherence tomography: a preliminary study

NASA Astrophysics Data System (ADS)

Bernstein, Liane; Beaudette, Kathy; Patten, Kessen; Beaulieu-Ouellet, Émilie; Strupler, Mathias; Moldovan, Florina; Boudoux, Caroline

2013-03-01

A zebrafish model has recently been introduced to study various genetic mutations that could lead to spinal deformities such as scoliosis. However, current imaging techniques make it difficult to perform longitudinal studies of this condition in zebrafish, especially in the early stages of development. The goal of this project is to determine whether optical coherence tomography (OCT) is a viable non-invasive method to image zebrafish exhibiting spinal deformities. Images of both live and fixed malformed zebrafish (5 to 21 days postfertilization) as well as wild-type fish (5 to 29 days postfertilization) were acquired non-invasively using a commercial SD-OCT system, with a laser source centered at 930nm (λ=100nm), permitting axial and lateral resolutions of 7 and 8μm respectively. Using two-dimensional images and three-dimensional reconstructions, it was possible to identify the malformed notochord as well as deformities in other major organs at different stages of formation. Visualization of the notochord was facilitated with the development of a segmentation algorithm. OCT images were compared to HE histological sections and images obtained by calcein staining. Because of the possibility of performing longitudinal studies on a same fish and reducing image processing time as compared with staining techniques and histology, the use of OCT could facilitate phenotypic characterization in studying genetic factors leading to spinal deformities in zebrafish and could eventually contribute to the identification of the genetic causes of spinal deformities such as scoliosis.

Evaluation of rapid cooling and tricaine methanesulfonate (MS222) as methods of euthanasia in zebrafish (Danio rerio).

PubMed

Wilson, Jolaine M; Bunte, Ralph M; Carty, Anthony J

2009-11-01

Despite the progressively increasing use of zebrafish (Danio rerio) in research, the most humane method of euthanasia for these fish has not been determined. Contemporary guidance documents state that hypothermia is an unacceptable method of euthanasia. The goal of this study was to compare rapid cooling and tricaine methanesulfonate (MS222) for zebrafish euthanasia. Zebrafish (n = 46) were euthanized by immersion in either an ice-water (4 degrees C or less) bath or unbuffered MS222 solution (250 mg/L; 25 to 30 degrees C). Another cohort (n = 10) was exposed to buffered MS222 to determine whether the acidity of the water alone caused distress. The times from exposure until the animals became unable to swim, right themselves, and death were measured, and signs of distress were recorded. Fish then were placed in a 'recovery tank' of system water to verify that recovery did not occur. Tissues were examined histologically. The mean time for euthanasia was longer and the number of fish exhibiting signs of distress was greater for fish exposed to MS222 than those exposed to chilled water. In addition, 4 of the 23 fish exposed to MS222 regained consciousness in the recovery tank, whereas none of 23 fish exposed to chilled water recovered. No histopathologic changes or evidence of ice crystal formation were seen in either group. In light of the faster time to death and fewer signs of distress in zebrafish euthanized by rapid cooling, we advocate this method as a humane veterinary practice.

A Rotifer-Based Technique to Rear Zebrafish Larvae in Small Academic Settings.

PubMed

Allen, Raymond L; Wallace, Robert L; Sisson, Barbara E

2016-08-01

Raising zebrafish from larvae to juveniles can be laborious, requiring frequent water exchanges and continuous culturing of live feed. This task becomes even more difficult for small institutions that do not have access to the necessary funding, equipment, or personnel to maintain large-scale systems usually employed in zebrafish husbandry. To open this opportunity to smaller institutions, a cost-efficient protocol was developed to culture Nannochloropsis to feed the halophilic, planktonic rotifer Brachionus plicatilis; the rotifers were then used to raise larval zebrafish to juveniles. By using these methods, small institutions can easily raise zebrafish embryos in a cost-efficient manner without the need to establish an extensive fish-raising facility. In addition, culturing rotifers provides a micrometazoan that serves as a model organism for teaching and undergraduate research studies for a variety of topics, including aging, toxicology, and predator-prey dynamics.

Zebrafish Embryo as an In Vivo Model for Behavioral and Pharmacological Characterization of Methylxanthine Drugs.

PubMed

Basnet, Ram Manohar; Guarienti, Michela; Memo, Maurizio

2017-03-09

Zebrafish embryo is emerging as an important tool for behavior analysis as well as toxicity testing. In this study, we compared the effect of nine different methylxanthine drugs using zebrafish embryo as a model. We performed behavioral analysis, biochemical assay and Fish Embryo Toxicity (FET) test in zebrafish embryos after treatment with methylxanthines. Each drug appeared to behave in different ways and showed a distinct pattern of results. Embryos treated with seven out of nine methylxanthines exhibited epileptic-like pattern of movements, the severity of which varied with drugs and doses used. Cyclic AMP measurement showed that, despite of a significant increase in cAMP with some compounds, it was unrelated to the observed movement behavior changes. FET test showed a different pattern of toxicity with different methylxanthines. Each drug could be distinguished from the other based on its effect on mortality, morphological defects and teratogenic effects. In addition, there was a strong positive correlation between the toxic doses (TC 50 ) calculated in zebrafish embryos and lethal doses (LD 50 ) in rodents obtained from TOXNET database. Taken together, all these findings elucidate the potentiality of zebrafish embryos as an in vivo model for behavioral and toxicity testing of methylxanthines and other related compounds.

Haploinsufficiency of the genes encoding the tumor suppressor Pten predisposes zebrafish to hemangiosarcoma

PubMed Central

Choorapoikayil, Suma; Kuiper, Raoul V.; de Bruin, Alain; den Hertog, Jeroen

2012-01-01

SUMMARY PTEN is an essential tumor suppressor that antagonizes Akt/PKB signaling. The zebrafish genome encodes two Pten genes, ptena and ptenb. Here, we report that zebrafish mutants that retain a single wild-type copy of ptena or ptenb (ptena+/−ptenb−/− or ptena−/−ptenb+/−) are viable and fertile. ptena+/−ptenb−/− fish develop tumors at a relatively high incidence (10.2%) and most tumors developed close to the eye (26/30). Histopathologically, the tumor masses were associated with the retrobulbar vascular network and diagnosed as hemangiosarcomas. A single tumor was identified in 42 ptena−/−ptenb+/− fish and was also diagnosed as hemangiosarcoma. Immunohistochemistry indicated that the tumor cells in ptena+/−ptenb−/− and ptena−/−ptenb+/− fish proliferated rapidly and were of endothelial origin. Akt/PKB signaling was activated in the tumors, whereas Ptena was still detected in tumor tissue from ptena+/−ptenb−/− zebrafish. We conclude that haploinsufficiency of the genes encoding Pten predisposes to hemangiosarcoma in zebrafish. PMID:22071262

Effects of ZnSO4-induced peripheral anosmia on zebrafish behavior and physiology.

PubMed

Abreu, Murilo S; Giacomini, Ana C V V; Rodriguez, Rubens; Kalueff, Allan V; Barcellos, Leonardo J G

2017-03-01

Olfaction plays a key role in modulating behavioral and physiological responses of various animal species, including fishes. Olfactory deficits can be induced in fish experimentally, and utilized to examine the role of olfaction in their normal and pathological behaviors. Here, we examine whether experimental anosmia, evoked by ZnSO 4 in adult zebrafish can be associated with behavioral and/or physiological responses. We show that experimental ZnSO 4 -induced anosmia caused acute, but not prolonged, anxiogenic-like effects on zebrafish behavior tested in the novel tank test. The procedure also elevated whole-body cortisol levels in zebrafish. Moreover, ZnSO4 treatment, but not sham, produced damage to olfactory epithelium, inducing overt basal cell vacuolization and intercellular edema. The loss of olfaction, assessed by the fish food preference behavior in the aquatic Y-maze, was present 1h, but not 24h, after the treatment. Collectively, this suggests that transient experimental anosmia by ZnSO 4 modulates zebrafish behavior and olfaction, which can be used to evoke and assess their stress-related anxiety-like states. Copyright © 2016 Elsevier B.V. All rights reserved.

Halogenated Bisphenol-A Analogs Act as Obesogens in Zebrafish Larvae (Danio rerio)

PubMed Central

Pinto, Caroline L.; Grimaldi, Marina; Hillenweck, Anne; Perdu, Elisabeth; Zalko, Daniel; Bernard, Laure; Laudet, Vincent; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Ake

2014-01-01

Obesity has increased dramatically over the past decades, reaching epidemic proportions. The reasons are likely multifactorial. One of the suggested causes is the accelerated exposure to obesity-inducing chemicals (obesogens). However, out of the tens of thousands of industrial chemicals humans are exposed to, very few have been tested for their obesogenic potential, mostly due to the limited availability of appropriate in vivo screening models. In this study, we investigated whether two commonly used flame retardants, the halogenated bisphenol-A (BPA) analogs tetrabromobisphenol-A (TBBPA) and tetrachlorobisphenol-A (TCBPA), could act as obesogens using zebrafish larvae as an in vivo animal model. The effect of embryonic exposure to these chemicals on lipid accumulation was analyzed by Oil Red-O staining, and correlated to their capacity to activate human and zebrafish peroxisome proliferator-activated receptor gamma (PPARγ) in zebrafish and in reporter cell lines. Then, the metabolic fate of TBBPA and TCBPA in zebrafish larvae was analyzed by high-performance liquid chromatography (HPLC) . TBBPA and TCBPA were readily taken up by the fish embryo and both compounds were biotransformed to sulfate-conjugated metabolites. Both halogenated-BPAs, as well as TBBPA-sulfate induced lipid accumulation in zebrafish larvae. TBBPA and TCBPA also induced late-onset weight gain in juvenile zebrafish. These effects correlated to their capacity to act as zebrafish PPARγ agonists. Screening of chemicals for inherent obesogenic capacities through the zebrafish lipid accumulation model could facilitate prioritizing chemicals for further investigations in rodents, and ultimately, help protect humans from exposure to environmental obesogens. PMID:24591153

Inhibition of memory consolidation by antibodies against cell adhesion molecules after active avoidance conditioning in zebrafish.

PubMed

Pradel, G; Schachner, M; Schmidt, R

1999-05-01

Cell adhesion molecules are expected to play an important role in long-term storage of information in the central nervous system. Several of these glycoproteins, such as NCAM, L1, and the ependymins, express the HNK-1 carbohydrate structure, which is known to be involved in cell-cell and cell-matrix interactions. To investigate the contribution of the HNK-1 epitope and the secretory glycoproteins ependymins to memory formation in zebrafish (Brachydanio rerio), we developed an active avoidance conditioning paradigm. Zebrafish were trained in a shuttle-box to cross a hurdle, to avoid mild electric shocks following a conditioning light signal. One hour after acquisition of the task, zebrafish were injected intracerebroventricularly with monoclonal antibodies against the HNK-1 epitope or polyclonal antibodies against ependymins. Control fish received immunoglobulins G (IgGs) from nonimmune rat serum or the monoclonal antibody C183 against an unrelated cell-surface protein of the cyprinid brain. Two days later, injected zebrafish were tested for recall, and for quantitative evaluation a retention score (RS), ranging from 1.0 for immediate recall to 0.0, indicating no saving, was calculated. The average RS of anti-HNK-1-injected fish (RS = 0.30) and anti-ependymin-injected fish (0.24) were significantly different from the RS of uninjected fish (0.77), of controls injected with nonimmune serum IgGs (0.68), of C183-injected controls (0.78), and of overtrained fish injected with anti-HNK-1 antibodies (0.81). Anti-HNK-1 and anti-ependymin antibodies were characterized by Western blot analyses of subcellular brain fractions and immunohistochemical staining of the zebrafish optic tectum. Our data suggest that the antibodies influence cell recognition events at synaptic membranes and/or associated intracellular signaling cascades, and thereby block memory consolidation.

Fish-robot interactions in a free-swimming environment: Effects of speed and configuration of robots on live fish

NASA Astrophysics Data System (ADS)

Butail, Sachit; Polverino, Giovanni; Phamduy, Paul; Del Sette, Fausto; Porfiri, Maurizio

2014-03-01

We explore fish-robot interactions in a comprehensive set of experiments designed to highlight the effects of speed and configuration of bioinspired robots on live zebrafish. The robot design and movement is inspired by salient features of attraction in zebrafish and includes enhanced coloration, aspect ratio of a fertile female, and carangiform/subcarangiformlocomotion. The robots are autonomously controlled to swim in circular trajectories in the presence of live fish. Our results indicate that robot configuration significantly affects both the fish distance to the robots and the time spent near them.

A Novel Method for Rearing Zebrafish by Using Freshwater Rotifers (Brachionus calyciflorus)

PubMed Central

Aoyama, Yuta; Moriya, Natsumi; Tanaka, Shingo; Taniguchi, Tomoko; Hosokawa, Hiroshi

2015-01-01

Abstract The zebrafish (Danio rerio) has become a powerful model organism for studying developmental processes and genetic diseases. However, there remain several problems in previous rearing methods. In this study, we demonstrate a novel method for rearing zebrafish larvae by using a new first food, freshwater rotifers (Brachionus calyciflorus). Feeding experiments indicated that freshwater rotifers are suitable as the first food for newly hatched larval fish. In addition, we revisited and improved a feeding schedule from 5 to 40 days postfertilization. Our feeding method using freshwater rotifers accelerated larval growth. At 49 dpf, one pair out of 10 pairs successfully produced six fertilized eggs. At 56, 63, and 71 dpf, 6 out of the 10 pairs constantly produced normal embryos. Our method will improve the husbandry of the zebrafish. PMID:25938499

Fishing for Nature's Hits: Establishment of the Zebrafish as a Model for Screening Antidiabetic Natural Products.

PubMed

Tabassum, Nadia; Tai, Hongmei; Jung, Da-Woon; Williams, Darren R

2015-01-01

Diabetes mellitus affects millions of people worldwide and significantly impacts their quality of life. Moreover, life threatening diseases, such as myocardial infarction, blindness, and renal disorders, increase the morbidity rate associated with diabetes. Various natural products from medicinal plants have shown potential as antidiabetes agents in cell-based screening systems. However, many of these potential "hits" fail in mammalian tests, due to issues such as poor pharmacokinetics and/or toxic side effects. To address this problem, the zebrafish (Danio rerio) model has been developed as a "bridge" to provide an experimentally convenient animal-based screening system to identify drug candidates that are active in vivo. In this review, we discuss the application of zebrafish to drug screening technologies for diabetes research. Specifically, the discovery of natural product-based antidiabetes compounds using zebrafish will be described. For example, it has recently been demonstrated that antidiabetic natural compounds can be identified in zebrafish using activity guided fractionation of crude plant extracts. Moreover, the development of fluorescent-tagged glucose bioprobes has allowed the screening of natural product-based modulators of glucose homeostasis in zebrafish. We hope that the discussion of these advances will illustrate the value and simplicity of establishing zebrafish-based assays for antidiabetic compounds in natural products-based laboratories.

The effects of dietary Myrtle (Myrtus communis) on skin mucus immune parameters and mRNA levels of growth, antioxidant and immune related genes in zebrafish (Danio rerio).

PubMed

Safari, Roghieh; Hoseinifar, Seyed Hossein; Van Doan, Hien; Dadar, Maryam

2017-07-01

Myrtle (Myrtus communis L., Myrtaceae) is a significant plant which naturally distributed around the globe. Although numerous studies have demonstrated the benefits of myrtle in different species, studies using the oral route are rare in the literature. In the present study, we evaluated the effect of myrtle intake on the antioxidant, immune, appetite and growth related genes as well as mucosal immune responses in zebrafish (Danio rerio) model. Zebrafish were fed control or myrtle (5, 10 and 20 g kg -1 myrtle) supplemented diets for sixty days. The results showed that, oral administration of Myrtle significantly improved mucosal immune responses (the activity of lysozyme, total Ig and protease). Furthermore, fish fed 20 g kg -1 showed remarkably higher antioxidant (sod and cat) enzymes gene expression compared other treatment. There were significant difference between myrtle fed fish and control group regarding tnf-alpha and lyz expression. Also, evaluation of growth (gh and igf1) related genes revealed remarkable upregulation in 20 g kg -1 myrtle treatment compared other myrtle treatments and control group. Similar results was observed regarding the mRNA levels of appetite related genes (ghrl) in zebrafish fed 20 g kg -1 myrtle. The present results indicated that dietary administration of myrtle improved mucosal immune parameters and altered mRNA levels of selected genes. These results on zebrafish model also highlights the potential use of Myrtle supplements as additive in human diets. Copyright © 2017 Elsevier Ltd. All rights reserved.

The smell of "anxiety": Behavioral modulation by experimental anosmia in zebrafish.

PubMed

Abreu, Murilo S; Giacomini, Ana C V V; Kalueff, Allan V; Barcellos, Leonardo J G

2016-04-01

Olfaction is strongly involved in the regulation of fish behavior, including reproductive, defensive, social and migration behaviors. In fish, anosmia (the lack of olfaction) can be induced experimentally, impairing their ability to respond to various olfactory stimuli. Here, we examine the effects of experimental lidocaine-induced anosmia on anxiety-like behavior and whole-body cortisol levels in adult zebrafish (Danio rerio). We show that experimentally-induced anosmia reduces anxiolytic-like behavioral effects of fluoxetine and seems to interact with anxiogenic effect of stress also paralleling cortisol responses in zebrafish. These findings provide first experimental evidence that temporary anosmia modulates anxiety-like behaviors and physiology in adult zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

PubMed Central

Blackburn, Jessica S; Liu, Sali; Raimondi, Aubrey R; Ignatius, Myron S; Salthouse, Christopher D; Langenau, David M

2011-01-01

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis. PMID:21293462

A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12.

PubMed

Burger, Alexa; Vasilyev, Aleksandr; Tomar, Ritu; Selig, Martin K; Nielsen, G Petur; Peterson, Randall T; Drummond, Iain A; Haber, Daniel A

2014-07-01

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer. © 2014. Published by The Company of Biologists Ltd.

Fish is Fish: the use of experimental model species to reveal causes of skeletal diversity in evolution and disease

PubMed Central

Harris, M. P.; Henke, K.; Hawkins, M. B.; Witten, P. E.

2014-01-01

Summary Fishes are wonderfully diverse. This variety is a result of the ability of ray-finned fishes to adapt to a wide range of environments, and has made them more specious than the rest of vertebrates combined. With such diversity it is easy to dismiss comparisons between distantly related fishes in efforts to understand the biology of a particular fish species. However, shared ancestry and the conservation of developmental mechanisms, morphological features and physiology provide the ability to use comparative analyses between different organisms to understand mechanisms of development and physiology. The use of species that are amenable to experimental investigation provides tools to approach questions that would not be feasible in other ‘non-model’ organisms. For example, the use of small teleost fishes such as zebrafish and medaka has been powerful for analysis of gene function and mechanisms of disease in humans, including skeletal diseases. However, use of these fish to aid in understanding variation and disease in other fishes has been largely unexplored. This is especially evident in aquaculture research. Here we highlight the utility of these small laboratory fishes to study genetic and developmental factors that underlie skeletal malformations that occur under farming conditions. We highlight several areas in which model species can serve as a resource for identifying the causes of variation in economically important fish species as well as to assess strategies to alleviate the expression of the variant phenotypes in farmed fish. We focus on genetic causes of skeletal deformities in the zebrafish and medaka that closely resemble phenotypes observed both in farmed as well as natural populations of fishes. PMID:25221374

A novel model for development, organization, and function of gonadotropes in fish pituitary.

PubMed

Golan, Matan; Biran, Jakob; Levavi-Sivan, Berta

2014-01-01

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are key regulators of the reproductive axis in vertebrates. Despite the high popularity of zebrafish as a model organism for studying reproductive functions, to date no transgenic zebrafish with labeled gonadotropes have been introduced. Using gonadotropin regulatory elements from tilapia, we generated two transgenic zebrafish lines with labeled gonadotropes. The tilapia and zebrafish regulatory sequences were highly divergent but several conserved elements allowed the tilapia promoters to correctly drive the transgenes in zebrafish pituitaries. FSH cells reacted to stimulation with gonadotropin releasing hormone by proliferating and showing increased transgene fluorescence, whereas estrogen exposure caused a decrease in cell number and transgene fluorescence. Transgene fluorescence reflected the expression pattern of the endogenous fshb gene. Ontogenetic expression of the transgenes followed typical patterns, with FSH cells appearing early in development, and LH cells appearing later and increasing dramatically in number with the onset of puberty. Our transgenic lines provide a powerful tool for investigating the development, anatomy, and function of the reproductive axis in lower vertebrates.

Repairing quite swimmingly: advances in regenerative medicine using zebrafish.

PubMed

Goessling, Wolfram; North, Trista E

2014-07-01

Regenerative medicine has the promise to alleviate morbidity and mortality caused by organ dysfunction, longstanding injury and trauma. Although regenerative approaches for a few diseases have been highly successful, some organs either do not regenerate well or have no current treatment approach to harness their intrinsic regenerative potential. In this Review, we describe the modeling of human disease and tissue repair in zebrafish, through the discovery of disease-causing genes using classical forward-genetic screens and by modulating clinically relevant phenotypes through chemical genetic screening approaches. Furthermore, we present an overview of those organ systems that regenerate well in zebrafish in contrast to mammalian tissue, as well as those organs in which the regenerative potential is conserved from fish to mammals, enabling drug discovery in preclinical disease-relevant models. We provide two examples from our own work in which the clinical translation of zebrafish findings is either imminent or has already proven successful. The promising results in multiple organs suggest that further insight into regenerative mechanisms and novel clinically relevant therapeutic approaches will emerge from zebrafish research in the future. © 2014. Published by The Company of Biologists Ltd.

Recording the adult zebrafish cerebral field potential during pentylenetetrazole seizures

PubMed Central

Pineda, Ricardo; Beattie, Christine E.; Hall, Charles W.

2017-01-01

Although the zebrafish is increasingly used as a model organism to study epilepsy, no standard electrophysiological technique for recording electrographic seizures in adult fish exists. The purpose of this paper is to introduce a readily implementable technique for recording pentylenetetrazole seizures in the adult zebrafish. We find that we can consistently record a high quality field potential over the zebrafish cerebrum using an amplification of 5000 V/V and bandpass filtering at corner frequencies of 1.6 and 16 Hz. The cerebral field potential recordings show consistent features in the baseline, pre-seizure, seizure and post-seizure time periods that can be easily recognized by visual inspection as is the case with human and rodent electroencephalogram. Furthermore, numerical analysis of the field potential at the time of seizure onset reveals an increase in the total power, bandwidth and peak frequency in the power spectrum, as is also the case with human and rodent electroencephalogram. The techniques presented herein stand to advance the utility of the adult zebrafish in the study of epilepsy by affording an equivalent to the electroencephalogram used in mammalian models and human patients. PMID:21689682

Fishing for Fetal Alcohol Spectrum Disorders: Zebrafish as a Model for Ethanol Teratogenesis.

PubMed

Lovely, Charles Ben; Fernandes, Yohaan; Eberhart, Johann K

2016-10-01

Fetal Alcohol Spectrum Disorders (FASD) describes a wide array of ethanol-induced developmental defects, including craniofacial dysmorphology and cognitive impairments. It affects ∼1 in 100 children born in the United States each year. Due to the pleiotropic effects of ethanol, animal models have proven critical in characterizing the mechanisms of ethanol teratogenesis. In this review, we focus on the utility of zebrafish in characterizing ethanol-induced developmental defects. A growing number of laboratories have focused on using zebrafish to examine ethanol-induced defects in craniofacial, cardiac, ocular, and neural development, as well as cognitive and behavioral impairments. Growing evidence supports that genetic predisposition plays a role in these ethanol-induced defects, yet little is understood about these gene-ethanol interactions. With a high degree of genetic amenability, zebrafish is at the forefront of identifying and characterizing the gene-ethanol interactions that underlie FASD. Because of the conservation of gene function between zebrafish and humans, these studies will directly translate to studies of candidate genes in human populations and allow for better diagnosis and treatment of FASD.

Assessment of diclofenac LC50 reference values in juvenile and embryonic stages of the zebrafish (Danio rerio).

PubMed

Praskova, E; Voslarova, E; Siroka, Z; Plhalova, L; Macova, S; Marsalek, P; Pistekova, V; Svobodova, Z

2011-01-01

The aim of the study was to compare the acute toxicity of diclofenac to juvenile and embryonic stages of the zebrafish (Danio rerio). Acute toxicity tests were performed on the aquarium fish Danio rerio, which is one of the model organisms most commonly used in toxicity testing. The tests were performed using a semi-static method according to OECD guideline No. 203 (Fish, acute toxicity test). Embryo toxicity tests were performed in zebrafish embryos (Danio rerio) in compliance with OECD No. 212 methodology (Fish, short-term toxicity test on embryo and sac-fry stages). The results were subjected to a probit analysis using the EKO-TOX 5.2 programme to determine 96hLC50 and 144hLC50 (median lethal concentration, 50% mortality after a 96 h or 144 h interval, respectively) values of diclofenac. The statistical significance of the difference between LC50 values in juvenile and embryonic stages of Danio rerio was tested using the Mann-Whitney non-parametric test implemented in the Unistat 5.1 programme. The LC50 mean value of diclofenac was 166.6 +/- 9.8 mg/L in juvenile Danio rerio, and 6.11 +/- 2.48 mg/L in embryonic stages of Danio rerio. The study demonstrated a statistically higher sensitivity to diclofenac (P < 0.05) in embryonic stages compared to the juvenile fish.

Understanding the regulation of vertebrate hematopoiesis and blood disorders: big lessons from a small fish

PubMed Central

Robertson, Anne L.; Avagyan, Serine; Gansner, John M.; Zon, Leonard I.

2017-01-01

Hematopoietic stem cells (HSCs) give rise to all differentiated blood cells. Understanding the mechanisms that regulate self-renewal and lineage specification of HSCs is key for developing treatments for many human diseases. Zebrafish have emerged as an excellent model for studying vertebrate hematopoiesis. This review will highlight the unique strengths of zebrafish and important findings that have emerged from studies of blood development and disorders using this system. We discuss recent advances in our understanding of hematopoiesis, including the origin of HSCs, molecular control of their development, and key signaling pathways involved in their regulation. We highlight significant findings from zebrafish models of blood disorders, and discuss their application for investigating stem cell dysfunction in disease and for developing new therapeutics. PMID:27616157

Treatment of Francisella infections via PLGA- and lipid-based nanoparticle delivery of antibiotics in a zebrafish model.

PubMed

Ulanova, Lilia S; Pinheiro, Marina; Vibe, Carina; Nunes, Claudia; Misaghian, Dorna; Wilson, Steven; Zhu, Kaizheng; Fenaroli, Federico; Winther-Larsen, Hanne C; Reis, Salette; Griffiths, Gareth

2017-06-19

We tested the efficiency of 2 different antibiotics, rifampicin and oxolinic acid, against an established infection caused by fish pathogen Francisella noatunensis ssp. orientalis (F.n.o.) in zebrafish. The drugs were tested in the free form as well as encapsulated into biodegradable nanoparticles, either polylactic-co-glycolic acid (PLGA) nanoparticles or nanostructured lipid carriers. The most promising therapies were PLGA-rifampicin nanoparticles and free oxolinic acid; the PLGA nanoparticles significantly delayed embryo mortality while free oxolinic acid prevented it. Encapsulation of rifampicin in both PLGA and nanostructured lipid carriers enhanced its efficiency against F.n.o. infection relative to the free drug. We propose that the zebrafish model is a robust, rapid system for initial testing of different treatments of bacterial diseases important for aquaculture.

Understanding spatio-temporal strategies of adult zebrafish exploration in the open field test.

PubMed

Stewart, Adam Michael; Gaikwad, Siddharth; Kyzar, Evan; Kalueff, Allan V

2012-04-27

Zebrafish (Danio rerio) are emerging as a useful model organism for neuroscience research. Mounting evidence suggests that various traditional rodent paradigms may be adapted for testing zebrafish behavior. The open field test is a popular rodent test of novelty exploration, recently applied to zebrafish research. To better understand fish novelty behavior, we exposed adult zebrafish to two different open field arenas for 30 min, assessing the amount and temporal patterning of their exploration. While (similar to rodents) zebrafish scale their locomotory activity depending on the size of the tank, the temporal patterning of their activity was independent of arena size. These observations strikingly parallel similar rodent behaviors, suggesting that spatio-temporal strategies of animal exploration may be evolutionarily conserved across vertebrate species. In addition, we found interesting oscillations in zebrafish exploration, with the per-minute distribution of their horizontal activity demonstrating sinusoidal-like patterns. While such patterning is not reported for rodents and other higher vertebrates, a nonlinear regression analysis confirmed the oscillation patterning of all assessed zebrafish behavioral endpoints in both open field arenas, revealing a potentially important aspect of novelty exploration in lower vertebrates. Copyright © 2012 Elsevier B.V. All rights reserved.

BAC Recombineering of the Agouti Loci from Spotted Gar and Zebrafish Reveals the Evolutionary Ancestry of Dorsal-Ventral Pigment Asymmetry in Fish.

PubMed

Cal, Laura; MegÍas, Manuel; Cerdá-Reverter, José Miguel; Postlethwait, John H; Braasch, Ingo; Rotllant, Josep

2017-11-01

Dorsoventral pigment patterning, characterized by a light ventrum and a dark dorsum, is one of the most widespread chromatic adaptations in vertebrate body coloration. In mammals, this countershading depends on differential expression of agouti-signaling protein (ASIP), which drives a switch of synthesis of one type of melanin to another within melanocytes. Teleost fish share countershading, but the pattern results from a differential distribution of multiple types of chromatophores, with black-brown melanophores most abundant in the dorsal body and reflective iridophores most abundant in the ventral body. We previously showed that Asip1 (a fish ortholog of mammalian ASIP) plays a role in patterning melanophores. This observation leads to the surprising hypothesis that agouti may control an evolutionarily conserved pigment pattern by regulating different mechanisms in mammals and fish. To test this hypothesis, we compared two ray-finned fishes: the teleost zebrafish and the nonteleost spotted gar (Lepisosteus oculatus). By examining the endogenous pattern of asip1 expression in gar, we demonstrate a dorsoventral-graded distribution of asip1 expression that is highest ventrally, similar to teleosts. Additionally, in the first reported experiments to generate zebrafish transgenic lines carrying a bacterial artificial chromosome (BAC) from spotted gar, we show that both transgenic zebrafish lines embryos replicate the endogenous asip1 expression pattern in adult zebrafish, showing that BAC transgenes from both species contain all of the regulatory elements required for regular asip1 expression within adult ray-finned fishes. These experiments provide evidence that the mechanism leading to an environmentally important pigment pattern was likely in place before the origin of teleosts. © 2017 Wiley Periodicals, Inc.

Whole plant based treatment of hypercholesterolemia with Crataegus laevigata in a zebrafish model

PubMed Central

2012-01-01

Background Consumers are increasingly turning to plant-based complementary and alternative medicines to treat hypercholesterolemia. Many of these treatments are untested and their efficacy is unknown. This multitude of potential remedies necessitates a model system amenable to testing large numbers of organisms that maintains similarity to humans in both mode of drug administration and overall physiology. Here we develop the larval zebrafish (4–30 days post fertilization) as a vertebrate model of dietary plant-based treatment of hypercholesterolemia and test the effects of Crataegus laevigata in this model. Methods Larval zebrafish were fed high cholesterol diets infused with fluorescent sterols and phytomedicines. Plants were ground with mortar and pestle into a fine powder before addition to food. Fluorescent sterols were utilized to optically quantify relative difference in intravascular cholesterol levels between groups of fish. We utilized the Zeiss 7-Live Duo high-speed confocal platform in order to both quantify intravascular sterol fluorescence and to capture video of the heart beat for determination of cardiac output. Results In this investigation we developed and utilized a larval zebrafish model to investigate dietary plant-based intervention of the pathophysiology of hypercholesterolemia. We found BODIPY-cholesterol effectively labels diet-introduced intravascular cholesterol levels (P < 0.05, Student’s t-test). We also established that zebrafish cardiac output declines as cholesterol dose increases (difference between 0.1% and 8% (w/w) high cholesterol diet-treated cardiac output significant at P < 0.05, 1-way ANOVA). Using this model, we found hawthorn leaves and flowers significantly reduce intravascular cholesterol levels (P < 0.05, 1-way ANOVA) and interact with cholesterol to impact cardiac output in hypercholesterolemic fish (2-way ANOVA, P < 0.05 for interaction effect). Conclusions The results of this study demonstrate that the larval zebrafish has the potential to become a powerful model to test plant based dietary intervention of hypercholesterolemia. Using this model we have shown that hawthorn leaves and flowers have the potential to affect cardiac output as well as intravascular cholesterol levels. Further, our observation that hawthorn leaves and flowers interact with cholesterol to impact cardiac output indicates that the physiological effects of hawthorn may depend on diet. PMID:22824306

From inflation to flotation: contribution of the swimbladder to whole-body density and swimming depth during development of the zebrafish (Danio rerio).

PubMed

Lindsey, Benjamin W; Smith, Frank M; Croll, Roger P

2010-03-01

Teleost fishes have body tissues that are denser than water, causing them to sink. Many teleosts therefore possess a gas-filled swimbladder that provides lift, allowing fish to attain neutral buoyancy. The importance of the swimbladder as a buoyancy aid during changing body sizes over ontogeny and its role in determining the swimming depth of fish remain unclear. In this study, we have used the zebrafish (Danio rerio) to investigate changes in the size and shape of the swimbladder during development and examine whether these changes affect the hydrostatic contribution of the swimbladder during swimming. Our results showed that swim-up behavior is critical for larvae to first inflate their swimbladder, decrease body density, and attain neutral buoyancy. Following inflation, we found a strong linear correlation between fish volume and swimbladder volume over ontogeny. This trend was supported by measures of the density of zebrafish, which was conserved within a narrow range between 1.00 +/- 0.001 and 0.996 +/- 0.001 g/cm(3) despite an increase in the swimming depth of zebrafish, which occurred upon transition to a double-chambered organ. Finally, we demonstrated that the contribution of the swimbladder keeps the fish within 1.7% of neutral buoyancy throughout larval development.

Long-Term Hyperphagia and Caloric Restriction Caused by Low- or High-Density Husbandry Have Differential Effects on Zebrafish Postembryonic Development, Somatic Growth, Fat Accumulation and Reproduction

PubMed Central

Leibold, Sandra; Hammerschmidt, Matthias

2015-01-01

In recent years, the zebrafish (Danio rerio) has emerged as an alternative vertebrate model for energy homeostasis and metabolic diseases, including obesity and anorexia. It has been shown that diet-induced obesity (DIO) in zebrafish shares multiple pathophysiological features with obesity in mammals. However, a systematic and comprehensive analysis of the different pathways of energy expenditure in obese and starved fish had been missing thus far. Here, we carry out long-term ad libitum feeding (hyperphagia) and caloric restriction studies induced by low- or high-density husbandry, respectively, to investigate the impact of caloric intake on the timing of scale formation, a crucial step of postembryonic development and metamorphosis, and on somatic growth, body weight, fat storage and female reproduction. We show that all of them are positively affected by increased caloric intake, that middle-aged fish develop severe DIO, and that the body mass index (BMI) displays a strict linear correlation with whole-body triglyceride levels in adult zebrafish. Interestingly, juvenile fish are largely resistant to DIO, while BMI and triglyceride values drop in aged fish, pointing to aging-associated anorexic effects. Histological analyses further indicate that increased fat storage in white adipose tissue involves both hyperplasia and hypertrophy of adipocytes. Furthermore, in ovaries, caloric intake primarily affects the rate of oocyte growth, rather than total oocyte numbers. Finally, comparing the different pathways of energy expenditure with each other, we demonstrate that they are differentially affected by caloric restriction / high-density husbandry. In juvenile fish, scale formation is prioritized over somatic growth, while in sexually mature adults, female reproduction is prioritized over somatic growth, and somatic growth over fat storage. Our data will serve as a template for future functional studies to dissect the neuroendocrine regulators of energy homeostasis mediating differential energy allocation. PMID:25799180

Long-term hyperphagia and caloric restriction caused by low- or high-density husbandry have differential effects on zebrafish postembryonic development, somatic growth, fat accumulation and reproduction.

PubMed

Leibold, Sandra; Hammerschmidt, Matthias

2015-01-01

In recent years, the zebrafish (Danio rerio) has emerged as an alternative vertebrate model for energy homeostasis and metabolic diseases, including obesity and anorexia. It has been shown that diet-induced obesity (DIO) in zebrafish shares multiple pathophysiological features with obesity in mammals. However, a systematic and comprehensive analysis of the different pathways of energy expenditure in obese and starved fish had been missing thus far. Here, we carry out long-term ad libitum feeding (hyperphagia) and caloric restriction studies induced by low- or high-density husbandry, respectively, to investigate the impact of caloric intake on the timing of scale formation, a crucial step of postembryonic development and metamorphosis, and on somatic growth, body weight, fat storage and female reproduction. We show that all of them are positively affected by increased caloric intake, that middle-aged fish develop severe DIO, and that the body mass index (BMI) displays a strict linear correlation with whole-body triglyceride levels in adult zebrafish. Interestingly, juvenile fish are largely resistant to DIO, while BMI and triglyceride values drop in aged fish, pointing to aging-associated anorexic effects. Histological analyses further indicate that increased fat storage in white adipose tissue involves both hyperplasia and hypertrophy of adipocytes. Furthermore, in ovaries, caloric intake primarily affects the rate of oocyte growth, rather than total oocyte numbers. Finally, comparing the different pathways of energy expenditure with each other, we demonstrate that they are differentially affected by caloric restriction / high-density husbandry. In juvenile fish, scale formation is prioritized over somatic growth, while in sexually mature adults, female reproduction is prioritized over somatic growth, and somatic growth over fat storage. Our data will serve as a template for future functional studies to dissect the neuroendocrine regulators of energy homeostasis mediating differential energy allocation.

Fishing for causes and cures of motor neuron disorders.

PubMed

Patten, Shunmoogum A; Armstrong, Gary A B; Lissouba, Alexandra; Kabashi, Edor; Parker, J Alex; Drapeau, Pierre

2014-07-01

Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount. The zebrafish (Danio rerio) is increasingly becoming a valuable model for studying human diseases and in screening for potential therapeutics. In this Review, we highlight recent progress in using zebrafish to study the pathology of the most common MNDs: spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). These studies indicate the power of zebrafish as a model to study the consequences of disease-related genes, because zebrafish homologues of human genes have conserved functions with respect to the aetiology of MNDs. Zebrafish also complement other animal models for the study of pathological mechanisms of MNDs and are particularly advantageous for the screening of compounds with therapeutic potential. We present an overview of their potential usefulness in MND drug discovery, which is just beginning and holds much promise for future therapeutic development. © 2014. Published by The Company of Biologists Ltd.

Production of Zebrafish Offspring from Cultured Female Germline Stem Cells

PubMed Central

Wong, Ten-Tsao; Tesfamichael, Abraham; Collodi, Paul

2013-01-01

Zebrafish female germline stem cell (FGSC) cultures were generated from a transgenic line of fish that expresses Neo and DsRed under the control of the germ cell specific promoter, ziwi [Tg(ziwi:neo);Tg(ziwi:DsRed)]. Homogeneous FGSC cultures were established by G418 selection and continued to express ziwi for more than 6 weeks along with the germ cell markers nanos3, dnd, dazl and vasa. A key component of the cell culture system was the use of a feeder cell line that was initiated from ovaries of a transgenic line of fish [Tg(gsdf:neo)] that expresses Neo controlled by the zebrafish gonadal soma derived factor (gsdf) promoter. The feeder cell line was selected in G418 and engineered to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and glial-cell-line derived neurotrophic factor (Gdnf). These factors were shown to significantly enhance FGSC growth, survival and germline competency in culture. Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes. Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months. The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome. PMID:23671620

A MULTIFACETED, MEDIUM-THROUGHPUT APPROACH FOR DETECTING AND CHARACTERIZING DEVELOPMENTAL NEUROTOXICITY USING ZEBRAFISH.

EPA Science Inventory

To address the EPA's need to prioritize hundreds to thousands of chemicals for testing, we are developing a rapid, cost-effective in vivo screen for developmental neurotoxicity using zebrafish (Danio rerio), a small freshwater fish with external fertilization. Zebrafish embryos d...

Lidocaine Hydrochloride Compared with MS222 for the Euthanasia of Zebrafish (Danio rerio).

PubMed

Collymore, Chereen; Banks, E Kate; Turner, Patricia V

2016-11-01

Despite several shortcomings, MS222 is the most commonly used chemical agent for euthanasia of zebrafish. Although lidocaine hydrochloride has some advantages over MS222, its effectiveness as a euthanasia agent for zebrafish is unknown. Larvae at 9 to 16 d postfertilization were exposed to 250 mg/L MS222 or 400, 500, 600, 700, 800, 900, or 1000 mg/L lidocaine and observed for cessation of heartbeat. Adult zebrafish were exposed to 250 mg/L MS222 or 400, 500, or 600 mg/L lidocaine; times to loss of righting reflex, cessation of opercular movement, and complete recovery; body length; aversive behavior; and gross and microscopic evidence of acute toxicity were evaluated. The heartbeat was not lost from any larvae in any group, regardless of drug or dosage. For adults, time to loss of righting reflex was greatest in the 500-mg/L lidocaine group. Opercular movement ceased earlier in all lidocaine groups compared with the MS222 group. Fish in the 500-mg/L lidocaine group were smaller than those in other groups. Fewer fish in the lidocaine groups displayed aversive behavior (erratic swimming and piping) compared with the MS222 group. No fish in the lidocaine hydrochloride groups (n = 30) recovered from euthanasia, whereas one fish in the MS222 group did (n = 10). Neither the MS222 nor lidocaine groups showed any gross or histologic changes suggestive of acute toxicity. Our results suggest that lidocaine hydrochloride may be an effective alternative chemical euthanasia agent for adult zebrafish but should not be used in larval fish.

Lidocaine Hydrochloride Compared with MS222 for the Euthanasia of Zebrafish (Danio rerio)

PubMed Central

Collymore, Chereen; Banks, E Kate; Turner, Patricia V

2016-01-01

Despite several shortcomings, MS222 is the most commonly used chemical agent for euthanasia of zebrafish. Although lidocaine hydrochloride has some advantages over MS222, its effectiveness as a euthanasia agent for zebrafish is unknown. Larvae at 9 to 16 d postfertilization were exposed to 250 mg/L MS222 or 400, 500, 600, 700, 800, 900, or 1000 mg/L lidocaine and observed for cessation of heartbeat. Adult zebrafish were exposed to 250 mg/L MS222 or 400, 500, or 600 mg/L lidocaine; times to loss of righting reflex, cessation of opercular movement, and complete recovery; body length; aversive behavior; and gross and microscopic evidence of acute toxicity were evaluated. The heartbeat was not lost from any larvae in any group, regardless of drug or dosage. For adults, time to loss of righting reflex was greatest in the 500-mg/L lidocaine group. Opercular movement ceased earlier in all lidocaine groups compared with the MS222 group. Fish in the 500-mg/L lidocaine group were smaller than those in other groups. Fewer fish in the lidocaine groups displayed aversive behavior (erratic swimming and piping) compared with the MS222 group. No fish in the lidocaine hydrochloride groups (n = 30) recovered from euthanasia, whereas one fish in the MS222 group did (n = 10). Neither the MS222 nor lidocaine groups showed any gross or histologic changes suggestive of acute toxicity. Our results suggest that lidocaine hydrochloride may be an effective alternative chemical euthanasia agent for adult zebrafish but should not be used in larval fish. PMID:27931323

A novel method for rearing first-feeding larval zebrafish: polyculture with Type L saltwater rotifers (Brachionus plicatilis).

PubMed

Best, Jason; Adatto, Isaac; Cockington, Jason; James, Althea; Lawrence, Christian

2010-09-01

Promoting high rates of growth and survival can be a major challenge in zebrafish culture, especially during the first-feeding stage. Here we describe a new rearing technique in which zebrafish larvae are polycultured in static tanks with Type "L" saltwater rotifers (Brachionus plicatilis) for the first 5 days of feeding (days 5-9 postfertilization). To demonstrate the effectiveness of this technique, we conducted rearing trials using fish from two different strains: AB and nacre. Growth, survival, water quality, and rotifer density were assayed daily through the polyculture phase (days 5-9), and during the transition to standard rearing conditions (days 10-12). After that point, once the fish were fully integrated onto recirculating systems, parameters were measured once per week out to day 30. In all trials, the fish displayed high rates of growth and survival throughout the three phases (polyculture, transition, and recirculating flow), indicating that this method may be employed during the critical first-feeding stage to help improve rearing performance in zebrafish facilities. Additionally, water quality parameters observed during the polyculture phase of the trials reveal that early zebrafish larvae are much more tolerant of elevated levels of ammonia and salinity than previously believed.

A Sketch of the Taiwan Zebrafish Core Facility.

PubMed

You, May-Su; Jiang, Yun-Jin; Yuh, Chiou-Hwa; Wang, Chien-Ming; Tang, Chih-Hao; Chuang, Yung-Jen; Lin, Bo-Hung; Wu, Jen-Leih; Hwang, Sheng-Ping L

2016-07-01

In the past three decades, the number of zebrafish laboratories has significantly increased in Taiwan. The Taiwan Zebrafish Core Facility (TZCF), a government-funded core facility, was launched to serve this growing community. The Core Facility was built on two sites, one located at the National Health Research Institutes (NHRI, called Taiwan Zebrafish Core Facility at NHRI or TZeNH) and the other is located at the Academia Sinica (Taiwan Zebrafish Core Facility at AS a.k.a. TZCAS). The total surface area of the TZCF is about 180 m(2) encompassing 2880 fish tanks. Each site has a separate quarantine room and centralized water recirculating systems, monitoring key water parameters. To prevent diseases, three main strategies have been implemented: (1) imported fish must be quarantined; (2) only bleached embryos are introduced into the main facilities; and (3) working practices were implemented to minimize pathogen transfer between stocks and facilities. Currently, there is no health program in place; however, a fourth measure for the health program, specific regular pathogen tests, is being planned. In March 2015, the TZCF at NHRI has been AAALAC accredited. It is our goal to ensure that we provide "disease-free" fish and embryos to the Taiwanese research community.

Modeling consequences of prolonged strong unpredictable stress in zebrafish: Complex effects on behavior and physiology.

PubMed

Song, Cai; Liu, Bai-Ping; Zhang, Yong-Ping; Peng, Zhilan; Wang, JiaJia; Collier, Adam D; Echevarria, David J; Savelieva, Katerina V; Lawrence, Robert F; Rex, Christopher S; Meshalkina, Darya A; Kalueff, Allan V

2018-02-02

Chronic stress is the major pathogenetic factor of human anxiety and depression. Zebrafish (Danio rerio) have become a novel popular model species for neuroscience research and CNS drug discovery. The utility of zebrafish for mimicking human affective disorders is also rapidly growing. Here, we present a new zebrafish model of clinically relevant, prolonged unpredictable strong chronic stress (PUCS). The 5-week PUCS induced overt anxiety-like and motor retardation-like behaviors in adult zebrafish, also elevating whole-body cortisol and proinflammatory cytokines - interleukins IL-1β and IL-6. PUCS also elevated whole-body levels of the anti-inflammatory cytokine IL-10 and increased the density of dendritic spines in zebrafish telencephalic neurons. Chronic treatment of fish with an antidepressant fluoxetine (0.1mg/L for 8days) normalized their behavioral and endocrine phenotypes, as well as corrected stress-elevated IL-1β and IL-6 levels, similar to clinical and rodent data. The CNS expression of the bdnf gene, the two genes of its receptors (trkB, p75), and the gfap gene of glia biomarker, the glial fibrillary acidic protein, was unaltered in all three groups. However, PUCS elevated whole-body BDNF levels and the telencephalic dendritic spine density (which were corrected by fluoxetine), thereby somewhat differing from the effects of chronic stress in rodents. Together, these findings support zebrafish as a useful in-vivo model of chronic stress, also calling for further cross-species studies of both shared/overlapping and distinct neurobiological responses to chronic stress. Copyright © 2017 Elsevier Inc. All rights reserved.

Reversal learning and resurgence of operant behavior in zebrafish (Danio rerio).

PubMed

Kuroda, Toshikazu; Mizutani, Yuto; Cançado, Carlos R X; Podlesnik, Christopher A

2017-09-01

Zebrafish are used extensively as vertebrate animal models in biomedical research for having such features as a fully sequenced genome and transparent embryo. Yet, operant-conditioning studies with this species are scarce. The present study investigated reversal learning and resurgence of operant behavior in zebrafish. A target response (approaching a sensor) was reinforced in Phase 1. In Phase 2, the target response was extinguished while reinforcing an alternative response (approaching a different sensor). In Phase 3, extinction was in effect for the target and alternative responses. Reversal learning was demonstrated when responding tracked contingency changes between Phases 1 and 2. Moreover, resurgence occurred in 10 of 13 fish in Phase 3: Target response rates increased transiently and exceeded rates of an unreinforced control response. The present study provides the first evidence with zebrafish supporting reversal learning between discrete operant responses and a laboratory model of relapse. These findings open the possibility to assessing genetic influences of operant behavior generally and in models of relapse (e.g., resurgence, renewal, reinstatement). Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of chronic perfluorooctanoic acid (PFOA) at low concentration on morphometrics, gene expression, and fecundity in zebrafish (Danio rerio).

PubMed

Jantzen, Carrie E; Toor, Fatima; Annunziato, Kate A; Cooper, Keith R

2017-04-01

Perfluorooctanoic acid (PFOA) is a persistent, toxic, anthropogenic chemical recalcitrant to biodegradation. Based on previous studies in lower and higher vertebrates, it was hypothesized that chronic, sub-lethal, embryonic exposure to PFOA in zebrafish (Danio rerio) would adversely impact fish development, survival, and fecundity. Zebrafish embryo/sac-fry were water exposed to 2.0 or 0nM PFOA from 3 to 120hpf, and juvenile to adult cohorts were fed spiked food (8 pM) until 6 months. After chronic exposure, PFOA exposed fish were significantly smaller in total weight and length. Gene expression analysis found a significant decrease of transporters slco2b1, slco4a1, slco3a1 and tgfb1a, and a significant increase of slco1d1 expression. PFOA exposed fish produced significantly fewer eggs with reduced viability and developmental stage delay in F 1 . Chronic, low-dose exposure of zebrafish to PFOA significantly altered normal development, survival and fecundity and would likely impact wild fish population fitness in watersheds chronically exposed to PFOA. Copyright © 2017 Elsevier Inc. All rights reserved.

Whole-body cortisol response of zebrafish to acute net handling stress

USGS Publications Warehouse

Ramsay, J.M.; Feist, G.W.; Varga, Z.M.; Westerfield, M.; Kent, M.L.; Schreck, C.B.

2009-01-01

Zebrafish, Danio rerio, are frequently handled during husbandry and experimental procedures in the laboratory, yet little is known about the physiological responses to such stressors. We measured the whole-body cortisol levels of adult zebrafish subjected to net stress and air exposure at intervals over a 24 h period; cortisol recovered to near control levels by about 1 h post-net-stress (PNS). We then measured cortisol at frequent intervals over a 1 h period. Cortisol levels were more than 2-fold higher in net stressed fish at 3 min PNS and continued to increase peaking at 15 min PNS, when cortisol levels were 6-fold greater than the control cortisol. Mean cortisol declined from 15 to 60 min PNS, and at 60 min, net-stressed cortisol was similar to control cortisol. Because the age of fish differed between studies, we examined resting cortisol levels of fish of different ages (3, 7, 13, and 19 months). The resting cortisol values among tanks with the same age fish differed significantly but there was no clear effect of age. Our study is the first to report the response and recovery of cortisol after net handling for laboratory-reared zebrafish. ?? 2009 Elsevier B.V.

Early life exposure to PCB126 results in delayed mortality and growth impairment in the zebrafish larvae.

PubMed

Di Paolo, Carolina; Groh, Ksenia J; Zennegg, Markus; Vermeirssen, Etiënne L M; Murk, Albertinka J; Eggen, Rik I L; Hollert, Henner; Werner, Inge; Schirmer, Kristin

2015-12-01

The occurrence of chronic or delayed toxicity resulting from the exposure to sublethal chemical concentrations is an increasing concern in environmental risk assessment. The Fish Embryo Toxicity (FET) test with zebrafish provides a reliable prediction of acute toxicity in adult fish, but it cannot yet be applied to predict the occurrence of chronic or delayed toxicity. Identification of sublethal FET endpoints that can assist in predicting the occurrence of chronic or delayed toxicity would be advantageous. The present study characterized the occurrence of delayed toxicity in zebrafish larvae following early exposure to PCB126, previously described to cause delayed effects in the common sole. The first aim was to investigate the occurrence and temporal profiles of delayed toxicity during zebrafish larval development and compare them to those previously described for sole to evaluate the suitability of zebrafish as a model fish species for delayed toxicity assessment. The second aim was to examine the correlation between the sublethal endpoints assessed during embryonal and early larval development and the delayed effects observed during later larval development. After exposure to PCB126 (3-3000ng/L) until 5 days post fertilization (dpf), larvae were reared in clean water until 14 or 28 dpf. Mortality and sublethal morphological and behavioural endpoints were recorded daily, and growth was assessed at 28 dpf. Early life exposure to PCB126 caused delayed mortality (300 ng/L and 3000 ng/L) as well as growth impairment and delayed development (100 ng/L) during the clean water period. Effects on swim bladder inflation and cartilaginous tissues within 5 dpf were the most promising for predicting delayed mortality and sublethal effects, such as decreased standard length, delayed metamorphosis, reduced inflation of swim bladder and column malformations. The EC50 value for swim bladder inflation at 5 dpf (169 ng/L) was similar to the LC50 value at 8 dpf (188 and 202 ng/L in two experiments). Interestingly, the patterns of delayed mortality and delayed effects on growth and development were similar between sole and zebrafish. This indicates the comparability of critical developmental stages across divergent fish species such as a cold water marine flatfish and a tropical freshwater cyprinid. Additionally, sublethal effects in early embryo-larval stages were found promising for predicting delayed lethal and sublethal effects of PCB126. Therefore, the proposed method with zebrafish is expected to provide valuable information on delayed mortality and delayed sublethal effects of chemicals and environmental samples that may be extrapolated to other species. Copyright © 2015 Elsevier B.V. All rights reserved.

Contextual Fear Conditioning in Zebrafish

ERIC Educational Resources Information Center

Kenney, Justin W.; Scott, Ian C.; Josselyn, Sheena A.; Frankland, Paul W.

2017-01-01

Zebrafish are a genetically tractable vertebrate that hold considerable promise for elucidating the molecular basis of behavior. Although numerous recent advances have been made in the ability to precisely manipulate the zebrafish genome, much less is known about many aspects of learning and memory in adult fish. Here, we describe the development…

A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

PubMed

Radev, Zlatko; Hermel, Jean-Michel; Elipot, Yannick; Bretaud, Sandrine; Arnould, Sylvain; Duchateau, Philippe; Ruggiero, Florence; Joly, Jean-Stéphane; Sohm, Frédéric

2015-01-01

Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish

PubMed Central

Elipot, Yannick; Bretaud, Sandrine; Arnould, Sylvain; Duchateau, Philippe; Ruggiero, Florence; Joly, Jean-Stéphane; Sohm, Frédéric

2015-01-01

Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders. PMID:26221953

Rapid, Accurate, and Non-Invasive Measurement of Zebrafish Axial Length and Other Eye Dimensions Using SD-OCT Allows Longitudinal Analysis of Myopia and Emmetropization

PubMed Central

Collery, Ross F.; Veth, Kerry N.; Dubis, Adam M.; Carroll, Joseph; Link, Brian A.

2014-01-01

Refractive errors in vision can be caused by aberrant axial length of the eye, irregular corneal shape, or lens abnormalities. Causes of eye length overgrowth include multiple genetic loci, and visual parameters. We evaluate zebrafish as a potential animal model for studies of the genetic, cellular, and signaling basis of emmetropization and myopia. Axial length and other eye dimensions of zebrafish were measured using spectral domain-optical coherence tomography (SD-OCT). We used ocular lens and body metrics to normalize and compare eye size and relative refractive error (difference between observed retinal radial length and controls) in wild-type and lrp2 zebrafish. Zebrafish were dark-reared to assess effects of visual deprivation on eye size. Two relative measurements, ocular axial length to body length and axial length to lens diameter, were found to accurately normalize comparisons of eye sizes between different sized fish (R2 = 0.9548, R2 = 0.9921). Ray-traced focal lengths of wild-type zebrafish lenses were equal to their retinal radii, while lrp2 eyes had longer retinal radii than focal lengths. Both genetic mutation (lrp2) and environmental manipulation (dark-rearing) caused elongated eye axes. lrp2 mutants had relative refractive errors of −0.327 compared to wild-types, and dark-reared wild-type fish had relative refractive errors of −0.132 compared to light-reared siblings. Therefore, zebrafish eye anatomy (axial length, lens radius, retinal radius) can be rapidly and accurately measured by SD-OCT, facilitating longitudinal studies of regulated eye growth and emmetropization. Specifically, genes homologous to human myopia candidates may be modified, inactivated or overexpressed in zebrafish, and myopia-sensitizing conditions used to probe gene-environment interactions. Our studies provide foundation for such investigations into genetic contributions that control eye size and impact refractive errors. PMID:25334040

Effects of Clove Oil as a Euthanasia Agent on Blood Collection Efficiency and Serum Cortisol Levels in Danio rerio

PubMed Central

Davis, Daniel J; Klug, Jenna; Hankins, Miriam; Doerr, Holly M; Monticelli, Stephanie R; Song, Ava; Gillespie, Catherine H; Bryda, Elizabeth C

2015-01-01

Zebrafish are an important laboratory animal model for biomedical research and are increasingly being used for behavioral neuroscience. Tricaine methanesulfonate (MS222) is the standard agent used for euthanasia of zebrafish. However, recent studies of zebrafish behavior suggest that MS222 may be aversive, and clove oil might be a possible alternative. In this study, we compared the effects of MS222 or clove oil as a euthanasia agent in zebrafish on the volume of blood collected and on serum levels of cortisol. Greater amounts of serum could be collected and lower serum levels of cortisol were present in fish euthanized with clove oil compared with equipotent dose of MS222. Euthanasia with clove oil did not blunt the expected elevation of serum cortisol levels elicited by an acute premortem stress. According to our findings, clove oil is a fast-acting agent that minimizes the cortisol response to euthanasia in zebrafish and allows the collection of large volumes of blood postmortem. These results represent a significant refinement in euthanasia methods for zebrafish. PMID:26424256

Effects of Clove Oil as a Euthanasia Agent on Blood Collection Efficiency and Serum Cortisol Levels in Danio rerio.

PubMed

Davis, Daniel J; Klug, Jenna; Hankins, Miriam; Doerr, Holly M; Monticelli, Stephanie R; Song, Ava; Gillespie, Catherine H; Bryda, Elizabeth C

2015-09-01

Zebrafish are an important laboratory animal model for biomedical research and are increasingly being used for behavioral neuroscience. Tricaine methanesulfonate (MS222) is the standard agent used for euthanasia of zebrafish. However, recent studies of zebrafish behavior suggest that MS222 may be aversive, and clove oil might be a possible alternative. In this study, we compared the effects of MS222 or clove oil as a euthanasia agent in zebrafish on the volume of blood collected and on serum levels of cortisol. Greater amounts of serum could be collected and lower serum levels of cortisol were present in fish euthanized with clove oil compared with equipotent dose of MS222. Euthanasia with clove oil did not blunt the expected elevation of serum cortisol levels elicited by an acute premortem stress. According to our findings, clove oil is a fast-acting agent that minimizes the cortisol response to euthanasia in zebrafish and allows the collection of large volumes of blood postmortem. These results represent a significant refinement in euthanasia methods for zebrafish.

A faster escape does not enhance survival in zebrafish larvae

PubMed Central

Nair, Arjun; Nguyen, Christy

2017-01-01

An escape response is a rapid manoeuvre used by prey to evade predators. Performing this manoeuvre at greater speed, in a favourable direction, or from a longer distance have been hypothesized to enhance the survival of prey, but these ideas are difficult to test experimentally. We examined how prey survival depends on escape kinematics through a novel combination of experimentation and mathematical modelling. This approach focused on zebrafish (Danio rerio) larvae under predation by adults and juveniles of the same species. High-speed three-dimensional kinematics were used to track the body position of prey and predator and to determine the probability of behavioural actions by both fish. These measurements provided the basis for an agent-based probabilistic model that simulated the trajectories of the animals. Predictions of survivorship by this model were found by Monte Carlo simulations to agree with our observations and we examined how these predictions varied by changing individual model parameters. Contrary to expectation, we found that survival may not be improved by increasing the speed or altering the direction of the escape. Rather, zebrafish larvae operate with sufficiently high locomotor performance due to the relatively slow approach and limited range of suction feeding by fish predators. We did find that survival was enhanced when prey responded from a greater distance. This is an ability that depends on the capacity of the visual and lateral line systems to detect a looming threat. Therefore, performance in sensing, and not locomotion, is decisive for improving the survival of larval fish prey. These results offer a framework for understanding the evolution of predator–prey strategy that may inform prey survival in a broad diversity of animals. PMID:28404783

Kcnh1 Voltage-gated Potassium Channels Are Essential for Early Zebrafish Development*

PubMed Central

Stengel, Rayk; Rivera-Milla, Eric; Sahoo, Nirakar; Ebert, Christina; Bollig, Frank; Heinemann, Stefan H.; Schönherr, Roland; Englert, Christoph

2012-01-01

The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning. PMID:22927438

Tetraodon nigroviridis as a nonlethal model of infectious spleen and kidney necrosis virus (ISKNV) infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu Xiaopeng; Huang Lichao; Weng Shaoping

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus, family Iridoviridae. We have previously established a high mortality ISKNV infection model of zebrafish (Danio rerio). In this study, a nonlethal Tetraodon nigroviridis model of ISKNV infection was established. ISKNV infection did not cause lethal disease in Tetraodon but could infect almost all the organs of this species. Electron microscopy showed ISKNV particles were present in infected tissues. Immunofluorescence and quantitative real-time PCR analysis showed that nearly all the virions and infected cells were cleared at 14 d postinfection. The expression profiles of interferon-{gamma} andmore » tumor necrosis factor-{alpha} gene in response to ISKNV infection were significantly different in Tetraodon and zebrafish. The establishment of the nonlethal Tetraodon model of ISKNV infection can offer a valuable tool complementary to the zebrafish infection model for studying megalocytivirus disease, fish immune systems, and viral tropism.« less

Mind the fish: zebrafish as a model in cognitive social neuroscience

PubMed Central

Oliveira, Rui F.

2013-01-01

Understanding how the brain implements social behavior on one hand, and how social processes feedback on the brain to promote fine-tuning of behavioral output according to changes in the social environment is a major challenge in contemporary neuroscience. A critical step to take this challenge successfully is finding the appropriate level of analysis when relating social to biological phenomena. Given the enormous complexity of both the neural networks of the brain and social systems, the use of a cognitive level of analysis (in an information processing perspective) is proposed here as an explanatory interface between brain and behavior. A conceptual framework for a cognitive approach to comparative social neuroscience is proposed, consisting of the following steps to be taken across different species with varying social systems: (1) identification of the functional building blocks of social skills; (2) identification of the cognitive mechanisms underlying the previously identified social skills; and (3) mapping these information processing mechanisms onto the brain. Teleost fish are presented here as a group of choice to develop this approach, given the diversity of social systems present in closely related species that allows for planned phylogenetic comparisons, and the availability of neurogenetic tools that allows the visualization and manipulation of selected neural circuits in model species such as the zebrafish. Finally, the state-of-the art of zebrafish social cognition and of the tools available to map social cognitive abilities to neural circuits in zebrafish are reviewed. PMID:23964204

Mind the fish: zebrafish as a model in cognitive social neuroscience.

PubMed

Oliveira, Rui F

2013-01-01

Understanding how the brain implements social behavior on one hand, and how social processes feedback on the brain to promote fine-tuning of behavioral output according to changes in the social environment is a major challenge in contemporary neuroscience. A critical step to take this challenge successfully is finding the appropriate level of analysis when relating social to biological phenomena. Given the enormous complexity of both the neural networks of the brain and social systems, the use of a cognitive level of analysis (in an information processing perspective) is proposed here as an explanatory interface between brain and behavior. A conceptual framework for a cognitive approach to comparative social neuroscience is proposed, consisting of the following steps to be taken across different species with varying social systems: (1) identification of the functional building blocks of social skills; (2) identification of the cognitive mechanisms underlying the previously identified social skills; and (3) mapping these information processing mechanisms onto the brain. Teleost fish are presented here as a group of choice to develop this approach, given the diversity of social systems present in closely related species that allows for planned phylogenetic comparisons, and the availability of neurogenetic tools that allows the visualization and manipulation of selected neural circuits in model species such as the zebrafish. Finally, the state-of-the art of zebrafish social cognition and of the tools available to map social cognitive abilities to neural circuits in zebrafish are reviewed.

Effects of two strobilurins (azoxystrobin and picoxystrobin) on embryonic development and enzyme activities in juveniles and adult fish livers of zebrafish (Danio rerio).

PubMed

Jia, Wei; Mao, Liangang; Zhang, Lan; Zhang, Yanning; Jiang, Hongyun

2018-09-01

Azoxystrobin and picoxystrobin are two primary strobilurin fungicides used worldwide. This study was conducted to test their effects on embryonic development and the activity of several enzyme in the zebrafish (Danio rerio). After fish eggs were separately exposed to azoxystrobin and picoxystrobin from 24 to 144 h post fertilization (hpf), the mortality, hatching, and teratogenetic rates were measured. Additionally, effects of azoxystrobin and picoxystrobin on activities of three important antioxidant enzymes [catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD)] and two primary detoxification enzymes [carboxylesterase (CarE) and glutathione S-transferase (GST)] and malondialdehyde (MDA) content in zebrafish larvae (96 h) and livers of adult zebrafish of both sexes were also assessed for potential toxicity mechanisms. Based on the embryonic development test results, the mortality, hatching, and teratogenetic rates of eggs treated with azoxystrobin and picoxystrobin all showed significant dose- and time-dependent effects, and the 144-h LC 50 values of azoxystrobin and picoxystrobin were 1174.9 and 213.8 μg L -1 , respectively. In the larval zebrafish (96 h) test, activities of CAT, POD, CarE, and GST and MDA content in azoxystrobin and picoxystrobin-treated zebrafish larvae increased significantly with concentrations of the pesticides compared with those in the control. We further revealed that azoxystrobin and picoxystrobin exposure both caused significant oxidative stress in adult fish livers and the changes differed between the sexes. Our results indicated that picoxystrobin led to higher embryonic development toxicity and oxidative stress than azoxystrobin in zebrafish and the male zebrafish liver had stronger ability to detoxify than that of the females. Copyright © 2018 Elsevier Ltd. All rights reserved.

DarT: The embryo test with the Zebrafish Danio rerio--a general model in ecotoxicology and toxicology.

PubMed

Nagel, Roland

2002-01-01

The acute fish test is an animal test whose ecotoxicological relevance is worthy of discussion. The primary aim of protection in ecotoxicology is the population and not the individual. Furthermore the concentration of pollutants in the environment is normally not in the lethal range. Therefore the acute fish test covers solely the situation after chemical spills. Nevertheless, acute fish toxicity data still belong to the base set used for the assessment of chemicals. The embryo test with the zebrafish Danio rerio (DarT) is recommended as a substitute for the acute fish test. For validation an international laboratory comparison test was carried out. A summary of the results is presented in this paper. Based on the promising results of testing chemicals and waste water the test design was validated by the DIN-working group "7.6 Fischei-Test". A normed test guideline for testing waste water with fish is available. The test duration is short (48 h) and within the test different toxicological endpoints can be examined. Endpoints from the embryo test are suitable for QSAR-studies. Besides the use in ecotoxicology the introduction as a toxicological model was investigated. Disturbance of pigmentation and effects on the frequency of heart-beat were examined. A further important application is testing of teratogenic chemicals. Based on the results DarT could be a screening test within preclinical studies.

Effectiveness of recommended euthanasia methods in larval zebrafish (Danio rerio).

PubMed

Strykowski, Jennifer L; Schech, Joseph M

2015-01-01

The popularity of zebrafish and its use as a model organism in biomedical research including genetics, development, and toxicology, has increased over the past 20 y and continues to grow. However, guidelines for euthanasia remain vague, and the responsibility of creating appropriate euthanasia protocols essentially falls on individual facilities. To reduce variation in experimental results among labs, a standard method of euthanasia for zebrafish would be useful. Although various euthanasia methods have been compared, few studies focus on the effectiveness of euthanasia methods for larval zebrafish. In this study, we exposed larval zebrafish to each of 3 euthanasia agents (MS222, eugenol, and hypothermic shock) and assessed the recovery rate. Hypothermic shock appeared to be the most effective method for euthanizing zebrafish at 14 d after fertilization; however, this method may not be considered an efficient method for large numbers of larval zebrafish. Exposure to chemicals, such as MS222 and eugenol, were ineffective methods for euthanasia at this stage of development. When these agents are used, secondary measures should be taken to ensure death. Choosing a euthanasia method that is effective, efficient, and humane can be challenging. Determining a method of euthanasia that is suitable for fish of all stages will bring the zebrafish community closer to meeting this challenge.

Effectiveness of Recommended Euthanasia Methods in Larval Zebrafish (Danio rerio)

PubMed Central

Strykowski, Jennifer L; Schech, Joseph M

2015-01-01

The popularity of zebrafish and its use as a model organism in biomedical research including genetics, development, and toxicology, has increased over the past 20 y and continues to grow. However, guidelines for euthanasia remain vague, and the responsibility of creating appropriate euthanasia protocols essentially falls on individual facilities. To reduce variation in experimental results among labs, a standard method of euthanasia for zebrafish would be useful. Although various euthanasia methods have been compared, few studies focus on the effectiveness of euthanasia methods for larval zebrafish. In this study, we exposed larval zebrafish to each of 3 euthanasia agents (MS222, eugenol, and hypothermic shock) and assessed the recovery rate. Hypothermic shock appeared to be the most effective method for euthanizing zebrafish at 14 d after fertilization; however, this method may not be considered an efficient method for large numbers of larval zebrafish. Exposure to chemicals, such as MS222 and eugenol, were ineffective methods for euthanasia at this stage of development. When these agents are used, secondary measures should be taken to ensure death. Choosing a euthanasia method that is effective, efficient, and humane can be challenging. Determining a method of euthanasia that is suitable for fish of all stages will bring the zebrafish community closer to meeting this challenge. PMID:25651096

Advancements in zebrafish applications for 21st century toxicology.

PubMed

Garcia, Gloria R; Noyes, Pamela D; Tanguay, Robert L

2016-05-01

The zebrafish model is the only available high-throughput vertebrate assessment system, and it is uniquely suited for studies of in vivo cell biology. A sequenced and annotated genome has revealed a large degree of evolutionary conservation in comparison to the human genome. Due to our shared evolutionary history, the anatomical and physiological features of fish are highly homologous to humans, which facilitates studies relevant to human health. In addition, zebrafish provide a very unique vertebrate data stream that allows researchers to anchor hypotheses at the biochemical, genetic, and cellular levels to observations at the structural, functional, and behavioral level in a high-throughput format. In this review, we will draw heavily from toxicological studies to highlight advances in zebrafish high-throughput systems. Breakthroughs in transgenic/reporter lines and methods for genetic manipulation, such as the CRISPR-Cas9 system, will be comprised of reports across diverse disciplines. Copyright © 2016 Elsevier Inc. All rights reserved.

Advancements in zebrafish applications for 21st century toxicology

PubMed Central

Garcia, Gloria R.; Noyes, Pamela D.; Tanguay, Robert L.

2016-01-01

The zebrafish model is the only available high-throughput vertebrate assessment system, and it is uniquely suited for studies of in vivo cell biology. A sequenced and annotated genome has revealed a large degree of evolutionary conservation in comparison to the human genome. Due to our shared evolutionary history, the anatomical and physiological features of fish are highly homologous to humans, which facilitates studies relevant to human health. In addition, zebrafish provide a very unique vertebrate data stream that allows researchers to anchor hypotheses at the biochemical, genetic, and cellular levels to observations at the structural, functional, and behavioral level in a high-throughput format. In this review, we will draw heavily from toxicological studies to highlight advances in zebrafish high-throughput systems. Breakthroughs in transgenic/reporter lines and methods for genetic manipulation, such as the CRISPR-Cas9 system, will be comprised of reports across diverse disciplines. PMID:27016469

Stable multilineage xenogeneic replacement of definitive hematopoiesis in adult zebrafish.

PubMed

Hess, Isabell; Boehm, Thomas

2016-01-18

Bony fishes are the most numerous and phenotypically diverse group of vertebrates inhabiting our planet, making them an ideal target for identifying general principles of tissue development and function. However, lack of suitable experimental platforms prevents the exploitation of this rich source of natural phenotypic variation. Here, we use a zebrafish strain lacking definitive hematopoiesis for interspecific analysis of hematopoietic cell development. Without conditioning prior to transplantation, hematopoietic progenitor cells from goldfish stably engraft in adult zebrafish homozygous for the c-myb(I181N) mutation. However, in competitive repopulation experiments, zebrafish hematopoietic cells exhibit an advantage over their goldfish counterparts, possibly owing to subtle species-specific functional differences in hematopoietic microenvironments resulting from over 100 million years of independent evolution. Thus, our unique animal model provides an unprecedented opportunity to genetically and functionally disentangle universal and species-specific contributions of the microenvironment to hematopoietic progenitor cell maintenance and development.

Zebrafish housing systems: a review of basic operating principles and considerations for design and functionality.

PubMed

Lawrence, Christian; Mason, Timothy

2012-01-01

The strategies for housing zebrafish used in biomedical research have evolved considerably over the past three decades. To keep pace with the rapid expansion and development of the zebrafish model system, the field has generally moved from keeping fish at the level of aquarium hobbyist to that of industrialized, recirculating aquaculture. Numerous commercial system vendors now offer increasingly sophisticated housing systems based on design principles that maximize the number of animals that can be housed in a given space footprint, and they are thus able to support large and diverse research programs. This review is designed to provide managers, lab animal veterinarians, investigators, and other parties responsible for care and use of these animals with a comprehensive overview of the basic operating and design principles of zebrafish housing systems. This information can be used to help plan the construction of new facilities and/or the upgrade and maintenance of existing operations.

Anxiolytic-like effects of noribogaine in zebrafish.

PubMed

Kalueff, Allan V; Kaluyeva, Aleksandra; Maillet, Emeline L

2017-07-14

Noribogaine is the main psychoactive metabolite of the hallucinogenic drug ibogaine, and is a particularly interesting compound potentially useful to treat dependence and various psychiatric disorders. Here, we report the effects of noribogaine on anxiety and locomotion in zebrafish (Danio rerio), a new promising model organism in neurobehavioral and psychopharmacological research. Adult zebrafish were subjected to the 5min novel tank test (NTT) following an acute, 20-min drug immersion in 1, 5 and 10mg/L noribogaine. Overall, noribogaine produced robust anxiolytic-like behavior in zebrafish (increasing the time spent and transitions to the top half compartment and reducing freezing bouts) without overt effects on fish locomotion. Taken together, these results indicate that noribogaine modulates the components of the acute stress response related to emotionality and anxiety behaviors, implicating this drug as a potentially useful non-sedative anxiolytic agent. Copyright © 2017 Elsevier B.V. All rights reserved.

Successive and discrete spaced conditioning in active avoidance learning in young and aged zebrafish.

PubMed

Yang, Peng; Kajiwara, Riki; Tonoki, Ayako; Itoh, Motoyuki

2018-05-01

We designed an automated device to study active avoidance learning abilities of zebrafish. Open source tools were used for the device control, statistical computing, and graphic outputs of data. Using the system, we developed active avoidance tests to examine the effects of trial spacing and aging on learning. Seven-month-old fish showed stronger avoidance behavior as measured by color preference index with discrete spaced training as compared to successive spaced training. Fifteen-month-old fish showed a similar trend, but with reduced cognitive abilities compared with 7-month-old fish. Further, in 7-month-old fish, an increase in learning ability during trials was observed with discrete, but not successive, spaced training. In contrast, 15-month-old fish did not show increase in learning ability during trials. Therefore, these data suggest that discrete spacing is more effective for learning than successive spacing, with the zebrafish active avoidance paradigm, and that the time course analysis of active avoidance using discrete spaced training is useful to detect age-related learning impairment. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

The serotonergic system in fish.

PubMed

Lillesaar, Christina

2011-07-01

Neurons using serotonin (5-HT) as neurotransmitter and/or modulator have been identified in the central nervous system in representatives from all vertebrate clades, including jawless, cartilaginous and ray-finned fishes. The aim of this review is to summarize our current knowledge about the anatomical organization of the central serotonergic system in fishes. Furthermore, selected key functions of 5-HT will be described. The main focus will be the adult brain of teleosts, in particular zebrafish, which is increasingly used as a model organism. It is used to answer not only genetic and developmental biology questions, but also issues concerning physiology, behavior and the underlying neuronal networks. The many evolutionary conserved features of zebrafish combined with the ever increasing number of genetic tools and its practical advantages promise great possibilities to increase our understanding of the serotonergic system. Further, comparative studies including several vertebrate species will provide us with interesting insights into the evolution of this important neurotransmitter system. Copyright © 2011 Elsevier B.V. All rights reserved.

Comparative studies on the influence of "simulated weigthlessness" on fish otolith growth

NASA Astrophysics Data System (ADS)

Brungs, Sonja; Hendrik Anken, Ralf; Li, Xiao-Yan; Hauslage, Jens; Wang, Gaohong; Liu, Yongding; Hilbig, Reinhard; Hemmersbach, Ruth

Stimulus dependence is a general feature of all developing sensory systems. Concerning the vestibular organ of fish, it has been shown earlier that the growth of inner ear otoliths of developing Cichlid fish (Oreochromis mossambicus) and Zebrafish (Danio rerio) is slowed down by increased gravity (hypergravity) as an adaptation. Several studies proposed that otolith growth actively is adjusted via a feedback mechanism to produce a test mass of the appropriate physical capacity. Applying diminished gravity such as microgravity during spaceflight yielded an opposite effect, i.e., larger than normal otoliths in swordtails Xiphophorus helleri. Since there are no data on spaceflown early larval stages of the Cichlid fish and the Zebrafish available, these model organisms were subjected to simulated weightlessness using a submersed clinostat with one axis of rotation (O. mossambicus) and rotating-wall vessels (RWVs; O. mossambicus was maintained within a submersed RWV, which was recently developed at DLR, whereas D. rerio was kept within a modified RWV, developed by NASA). Developmental stages were subjected to clinorotation (60 rpm) and wall vessel rotation (Cichlid fish: 44 rpm; Zebrafish: 12.5 rpm; at these speeds, the larvae did neither sediment nor were they centrifuged away from the center of the RWVs) at a point of time when inner ear otolith mineralisation began. The experimental runs were discontinued when the animals hatched (O. mossambicus, stage 12, reached after 2-3 days at 22° C) or when they began to actively move within the devices (D. rerio, after 6 days at 28° C). After clinostat exposure, both utricular and saccular otoliths (Lapilli and Sagittae, respectively) of the Cichlids were significantly larger as compared to otoliths from the 1g controls. A similar result was obtained after wall vessel rotation for 3 and 6 days of the Zebrafish. These results support the idea that a feedback mechanism correlates the gravity level with the physical capacity of an otolithic test mass during early development and after calcification of otoliths has begun. Interestingly, wall vessel rotation had no effects on Cichlid fish otolith growth. In contrast to D. rerio, O. mossambicus is a mouth-breeding species: the mother animal turns around the larvae in her mouth for supply with fresh (aerated) water. Possibly, wall vessel rotation (rather than clinorotation) mimics this passive, natural movement, which likely will not be perceived as "weightlessness" by the offspring. In the course of further studies, Zebrafish should be subjected to clinorotation and experiments using further devices to simulate weightlessness (e.g., Levitron) should be carried out.

Toxicity of analytically cleaned pentabromodiphenylether after prolonged exposure in estuarine European flounder (Platichthys flesus), and partial life-cycle exposure in fresh water zebrafish (Danio rerio).

PubMed

Kuiper, Raoul V; Vethaak, A D; Cantón, Roćio F; Anselmo, Henrique; Dubbeldam, Marco; van den Brandhof, Evert-Jan; Leonards, Pim E G; Wester, Piet W; van den Berg, Martin

2008-09-01

Residues of polybrominated diphenylethers (PBDEs), extensively applied as flame retardants, are widely spread in the aquatic environment and biota. The present study investigates effects of the environmentally relevant lower brominated diphenylethers in two fish species in vivo under controlled laboratory conditions. Euryhaline flounder (Platichthys flesus) and freshwater zebrafish (Danio rerio) were exposed to a range of concentrations of a commercial pentabromodiphenylether mixture, DE-71. Chemical analysis of exposed fish showed a pattern of PBDE congeners that was very similar to that in wild fish. The resulting range included environmentally relevant, as well as higher levels. Animals were investigated histopathologically with emphasis on endocrine and reproductive organs. In zebrafish, hatching of embryos and larval development were assessed. Biochemical parameters were investigated in flounder as markers for suggested dioxin-like activity (ethoxyresorufin-O-deethylase=EROD), and activation of endogenous estrogen synthesis (gonad aromatase activity). Thyroid hormones were analyzed in plasma in both species. Benchmark analysis using internal PBDE concentrations showed a mild dose-dependent decrease of hepatic EROD and ovarian aromatase activities, and plasma thyroxin levels in flounder, and an increase of plasma thyroid hormone levels in zebrafish. These trends did not result in statistically significant differences from control fish, and major histopathological changes were not observed. Reproduction in zebrafish appeared to be the most sensitive parameter with statistically significantly reduced larval survival and non-significant indications for decreased egg production at internal levels that were more than 55 times the highest environmental recordings. The present results indicate limited risk for endocrine or reproductive effects of current environmental PBDE contamination in fish.

Evaluating the Zebrafish Embryo Toxicity Test for Pesticide ...

EPA Pesticide Factsheets

Given the numerous chemicals used in society, it is critical to develop tools for accurate and efficient evaluation of potential risks to human and ecological receptors. Fish embryo acute toxicity tests are 1 tool that has been shown to be highly predictive of standard, more resource-intensive, juvenile fish acute toxicity tests. However, there is also evidence that fish embryos are less sensitive than juvenile fish for certain types of chemicals, including neurotoxicants. The utility of fish embryos for pesticide hazard assessment was investigated by comparing published zebrafish embryo toxicity data from pesticides with median lethal concentration 50% (LC50) data for juveniles of 3 commonly tested fish species: rainbow trout, bluegill sunfish, and sheepshead minnow. A poor, albeit significant, relationship (r2 = 0.28; p < 0.05) was found between zebrafish embryo and juvenile fish toxicity when pesticides were considered as a single group, but a much better relationship (r2 = 0.64; p < 0.05) when pesticide mode of action was factored into an analysis of covariance. This discrepancy is partly explained by the large number of neurotoxic pesticides in the dataset, supporting previous findings that commonly used fish embryo toxicity test endpoints are particularly insensitive to neurotoxicants. These results indicate that it is still premature to replace juvenile fish toxicity tests with embryo-based tests such as the Organisation for Economic Co-op

The Effect of Water Hardness on Mortality of Zebrafish (Danio rerio) During Exposure to Oxytetracycline.

PubMed

Hundt, Matthias; Schreiber, Benjamin; Eckmann, Reiner; Lunestad, Bjørn Tore; Wünneman, Hannah; Schulz, Ralf

2016-02-01

Marking of fish otoliths with oxytetracycline and tetracycline is a widely used method to evaluate the effectiveness of stocking operations. Available protocols for the labeling of fish specify a number of factors influencing mark quality and potential risk for fish during marking. This study investigates the influence of water hardness on mortality of freshwater fish during marking with OTC. In order to pursue this question complexation of OTC with Mg(2+) and Ca(2+) cations was measured spectrophotometrically. Furthermore, zebrafish (Danio rerio) were immersed in OTC solutions (1200 mg/L; 48 h immersion) combined with varying levels of water hardness (5.5, 15.5, 25.5, 32.5°dH). The amount of OTC-Mg-Ca-complexes was positively correlated to water hardness. Moreover, it could be demonstrated that mortality of zebrafish during marking varied as a factor of water hardness. Highest mortalities occurred at the lowest (5.5°dH) and the highest (32.5°dH) tested levels during marking with OTC.

FishFace: interactive atlas of zebrafish craniofacial development at cellular resolution

PubMed Central

2013-01-01

Background The vertebrate craniofacial skeleton may exhibit anatomical complexity and diversity, but its genesis and evolution can be understood through careful dissection of developmental programs at cellular resolution. Resources are lacking that include introductory overviews of skeletal anatomy coupled with descriptions of craniofacial development at cellular resolution. In addition to providing analytical guidelines for other studies, such an atlas would suggest cellular mechanisms underlying development. Description We present the Fish Face Atlas, an online, 3D-interactive atlas of craniofacial development in the zebrafish Danio rerio. Alizarin red-stained skulls scanned by fluorescent optical projection tomography and segmented into individual elements provide a resource for understanding the 3D structure of the zebrafish craniofacial skeleton. These data provide the user an anatomical entry point to confocal images of Alizarin red-stained zebrafish with transgenically-labelled pharyngeal arch ectomesenchyme, chondrocytes, and osteoblasts, which illustrate the appearance, morphogenesis, and growth of the mandibular and hyoid cartilages and bones, as viewed in live, anesthetized zebrafish during embryonic and larval development. Confocal image stacks at high magnification during the same stages provide cellular detail and suggest developmental and evolutionary hypotheses. Conclusion The FishFace Atlas is a novel learning tool for understanding craniofacial skeletal development, and can serve as a reference for a variety of studies, including comparative and mutational analyses. PMID:23714426

A simple automated system for appetitive conditioning of zebrafish in their home tanks.

PubMed

Doyle, Jillian M; Merovitch, Neil; Wyeth, Russell C; Stoyek, Matthew R; Schmidt, Michael; Wilfart, Florentin; Fine, Alan; Croll, Roger P

2017-01-15

We describe here an automated apparatus that permits rapid conditioning paradigms for zebrafish. Arduino microprocessors were used to control the delivery of auditory or visual stimuli to groups of adult or juvenile zebrafish in their home tanks in a conventional zebrafish facility. An automatic feeder dispensed precise amounts of food immediately after the conditioned stimuli, or at variable delays for controls. Responses were recorded using inexpensive cameras, with the video sequences analysed with ImageJ or Matlab. Fish showed significant conditioned responses in as few as 5 trials, learning that the conditioned stimulus was a predictor of food presentation at the water surface and at the end of the tank where the food was dispensed. Memories of these conditioned associations persisted for at least 2days after training when fish were tested either as groups or as individuals. Control fish, for which the auditory or visual stimuli were specifically unpaired with food, showed no comparable responses. This simple, low-cost, automated system permits scalable conditioning of zebrafish with minimal human intervention, greatly reducing both variability and labour-intensiveness. It will be useful for studies of the neural basis of learning and memory, and for high-throughput screening of compounds modifying those processes. Copyright © 2016 Elsevier B.V. All rights reserved.

Fibroblast growth factor (Fgf) signaling pathway regulates liver homeostasis in zebrafish.

PubMed

Tsai, Su-Mei; Liu, Da-Wei; Wang, Wen-Pin

2013-04-01

In mammals, fibroblast growth factor (FGF) signaling controls liver specification and regulates the metabolism of lipids, cholesterol, and bile acids. FGF signaling also promotes hepatocyte proliferation, and helps detoxify hepatotoxin during liver regeneration after partial hepatectomy. However, the function of Fgf in zebrafish liver is not yet well understood, specifically for postnatal homeostasis. The current study analyzed the expression of fgf receptors (fgfrs) in the liver of zebrafish. We then investigated the function of Fgf signaling in the zebrafish liver by expressing a dominant-negative Fgf receptor in hepatocytes (lfabp:dnfgfr1-egfp, lf:dnfr). Histological analysis showed that our genetic intervention resulted in a small liver size with defected medial expansion of developing livers in transgenic (Tg) larvae. Morphologically, the liver lobe of lf:dnfr adult fish was shorter than that of control. Ballooning degeneration of hepatocytes was observed in fish as young as 3 months. Further examination revealed the development of hepatic steatosis and cholestasis. In adult Tg fish, we unexpectedly observed increased liver-to-body-weight ratios, with higher percentages of proliferating hepatocytes. Considering all these findings, we concluded that as in mammals, in adult zebrafish the metabolism of lipid and bile acids in the liver are regulated by Fgf signaling. Disruption of the Fgf signal-mediated metabolism might indirectly affect hepatocyte proliferation.

Place versus response learning in fish: a comparison between species.

PubMed

McAroe, Claire L; Craig, Cathy M; Holland, Richard A

2016-01-01

Place learning is thought to be an adaptive and flexible facet of navigation. Due to the flexibility of this learning, it is thought to be more complex than the simpler strategies such as learning a particular route or navigating through the use of cues. Place learning is crucial in a familiar environment as it allows an individual to successfully navigate to the same endpoint, regardless of where in the environment the journey begins. Much of the research to date focusing on different strategies employed for navigation has used human subjects or other mammals such as rodents. In this series of experiments, the spatial memory of four different species of fish (goldfish, killifish, zebrafish and Siamese fighting fish) was analysed using a plus maze set-up. Results suggest that three of the species showed a significant preference for the adoption of a place strategy during this task, whereas zebrafish showed no significant preference. Furthermore, zebrafish took significantly longer to learn the task than the other species. Finally, results suggest that zebrafish took the least amount of time (seconds) to complete trials both during training and probe.

Potential of zebrafish as a model for exploring the role of the amygdala in emotional memory and motivational behavior.

PubMed

Perathoner, Simon; Cordero-Maldonado, Maria Lorena; Crawford, Alexander D

2016-06-01

Emotion is a key aspect of behavior, enabling humans and animals to assign either positive or negative values to sensory inputs and thereby to make appropriate decisions. Classical experiments in mammalian models, mainly in primates and rodents, have shown that the amygdala is essential for appetitive and aversive associative processing and that dysfunction of this brain region leads to various psychiatric conditions, including depression, generalized anxiety disorder, panic disorder, phobias, autism, and posttraumatic stress disorder. In the past 2 decades, the zebrafish (Danio rerio; Cyprinidae) has emerged as a versatile, reliable vertebrate model organism for the in vivo study of development, gene function, and numerous aspects of human pathologies. Small size, high fecundity, rapid external development, transparency, genetic tractability, and high genetic and physiologic homology with humans are among the factors that have contributed to the success with this small fish in different biomedical research areas. Recent findings indicate that, despite the anatomical differences in the brain structure of teleosts and tetrapods, fish possess a structure homologous to the mammalian amygdala, a hypothesis that is supported by the expression of molecular markers, analyses of neuronal projections in different brain areas, and behavioral studies. This Review summarizes this evidence and highlights a number of relevant bioassays in zebrafish to study emotional memory and motivational behavior. © 2016 Wiley Periodicals, Inc.

SELDI PROTEINCHIP-BASED LIVER BIOMARKERS IN FUNGICIDE EXPOSED ZEBRAFISH

EPA Science Inventory

The research presented here is part of a three-phased small fish computational toxicology project using a combination of 1) whole organism endpoints, 2) genomic, proteomic, and metabolomic approaches, and 3) computational modeling to (a) identify new molecular biomarkers of expos...

Effect of acute ethanol administration on zebrafish tail-beat motion.

PubMed

Bartolini, Tiziana; Mwaffo, Violet; Butail, Sachit; Porfiri, Maurizio

2015-11-01

Zebrafish is becoming a species of choice in neurobiological and behavioral studies of alcohol-related disorders. In these efforts, the activity of adult zebrafish is typically quantified using indirect activity measures that are either scored manually or identified automatically from the fish trajectory. The analysis of such activity measures has produced important insight into the effect of acute ethanol exposure on individual and social behavior of this vertebrate species. Here, we leverage a recently developed tracking algorithm that reconstructs fish body shape to investigate the effect of acute ethanol administration on zebrafish tail-beat motion in terms of amplitude and frequency. Our results demonstrate a significant effect of ethanol on the tail-beat amplitude as well as the tail-beat frequency, both of which were found to robustly decrease for high ethanol concentrations. Such a direct measurement of zebrafish motor functions is in agreement with evidence based on indirect activity measures, offering a complementary perspective in behavioral screening. Copyright © 2015 Elsevier Inc. All rights reserved.

From fish bowl to bedside: The power of zebrafish to unravel melanoma pathogenesis and discover new therapeutics.

PubMed

van Rooijen, Ellen; Fazio, Maurizio; Zon, Leonard I

2017-07-01

Melanoma is the most aggressive and deadliest form of skin cancer. A detailed knowledge of the cellular, molecular, and genetic events underlying melanoma progression is highly relevant to diagnosis, prognosis and risk stratification, and the development of new therapies. In the last decade, zebrafish have emerged as a valuable model system for the study of melanoma. Pathway conservation, coupled with the availability of robust genetic, transgenic, and chemical tools, has made the zebrafish a powerful model for identifying novel disease genes, visualizing cancer initiation, interrogating tumor-microenvironment interactions, and discovering new therapeutics that regulate melanocyte and melanoma development. In this review, we will give an overview of these studies, and highlight recent advancements that will help unravel melanoma pathogenesis and impact human disease. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mycobacteriosis in zebrafish colonies.

PubMed

Whipps, Christopher M; Lieggi, Christine; Wagner, Robert

2012-01-01

Mycobacteriosis, a chronic bacterial infection, has been associated with severe losses in some zebrafish facilities and low-level mortalities and unknown impacts in others. The occurrence of at least six different described species (Mycobacterium abscessus, M. chelonae, M. fortuitum, M. haemophilum, M. marinum, M. peregrinum) from zebrafish complicates diagnosis and control because each species is unique. As a generalization, mycobacteria are often considered opportunists, but M. haemophilum and M. marinum appear to be more virulent. Background genetics of zebrafish and environmental conditions influence the susceptibility of fish and progression of disease, emphasizing the importance of regular monitoring and good husbandry practices. A combined approach to diagnostics is ultimately the most informative, with histology as a first-level screen, polymerase chain reaction for rapid detection and species identification, and culture for strain differentiation. Occurrence of identical strains of Mycobacterium in both fish and biofilms in zebrafish systems suggests transmission can occur when fish feed on infected tissues or tank detritus containing mycobacteria. Within a facility, good husbandry practices and sentinel programs are essential for minimizing the impacts of mycobacteria. In addition, quarantine and screening of animals coming into a facility is important for eliminating the introduction of the more severe pathogens. Elimination of mycobacteria from an aquatic system is likely not feasible because these species readily establish biofilms on surfaces even in extremely low nutrient conditions. Risks associated with each commonly encountered species need to be identified and informed management plans developed. Basic research on the growth characteristics, disinfection, and pathogenesis of zebrafish mycobacteria is critical moving forward.

Multimodal LA-ICP-MS and nanoSIMS imaging enables copper mapping within photoreceptor megamitochondria in a zebrafish model of Menkes disease.

PubMed

Ackerman, Cheri M; Weber, Peter K; Xiao, Tong; Thai, Bao; Kuo, Tiffani J; Zhang, Emily; Pett-Ridge, Jennifer; Chang, Christopher J

2018-03-01

Copper is essential for eukaryotic life, and animals must acquire this nutrient through the diet and distribute it to cells and organelles for proper function of biological targets. Indeed, mutations in the central copper exporter ATP7A contribute to a spectrum of diseases, including Menkes disease, with symptoms ranging from neurodegeneration to lax connective tissue. As such, a better understanding of the fundamental impacts of ATP7A mutations on in vivo copper distributions is of relevance to those affected by these diseases. Here we combine metal imaging and optical imaging techniques at a variety of spatial resolutions to identify tissues and structures with altered copper levels in the Calamity gw71 zebrafish model of Menkes disease. Rapid profiling of tissue slices with LA-ICP-MS identified reduced copper levels in the brain, neuroretina, and liver of Menkes fish compared to control specimens. High resolution nanoSIMS imaging of the neuroretina, combined with electron and confocal microscopies, identified the megamitochondria of photoreceptors as loci of copper accumulation in wildtype fish, with lower levels of megamitochondrial copper observed in Calamity gw71 zebrafish. Interestingly, this localized copper decrease does not result in impaired photoreceptor development or altered megamitochondrial morphology, suggesting the prioritization of copper at sufficient levels for maintaining essential mitochondrial functions. Together, these data establish the Calamity gw71 zebrafish as an optically transparent in vivo model for the study of neural copper misregulation, illuminate a role for the ATP7A copper exporter in trafficking copper to the neuroretina, and highlight the utility of combining multiple imaging techniques for studying metals in whole organism settings with spatial resolution.

anyFish 2.0: An open-source software platform to generate and share animated fish models to study behavior

NASA Astrophysics Data System (ADS)

Ingley, Spencer J.; Rahmani Asl, Mohammad; Wu, Chengde; Cui, Rongfeng; Gadelhak, Mahmoud; Li, Wen; Zhang, Ji; Simpson, Jon; Hash, Chelsea; Butkowski, Trisha; Veen, Thor; Johnson, Jerald B.; Yan, Wei; Rosenthal, Gil G.

2015-12-01

Experimental approaches to studying behaviors based on visual signals are ubiquitous, yet these studies are limited by the difficulty of combining realistic models with the manipulation of signals in isolation. Computer animations are a promising way to break this trade-off. However, animations are often prohibitively expensive and difficult to program, thus limiting their utility in behavioral research. We present anyFish 2.0, a user-friendly platform for creating realistic animated 3D fish. anyFish 2.0 dramatically expands anyFish's utility by allowing users to create animations of members of several groups of fish from model systems in ecology and evolution (e.g., sticklebacks, Poeciliids, and zebrafish). The visual appearance and behaviors of the model can easily be modified. We have added several features that facilitate more rapid creation of realistic behavioral sequences. anyFish 2.0 provides a powerful tool that will be of broad use in animal behavior and evolution and serves as a model for transparency, repeatability, and collaboration.

Chronic Unpredictable Stress (CUS)-Induced Anxiety and Related Mood Disorders in a Zebrafish Model: Altered Brain Proteome Profile Implicates Mitochondrial Dysfunction

PubMed Central

Chakravarty, Sumana; Reddy, Bommana R.; Sudhakar, Sreesha R.; Saxena, Sandeep; Das, Tapatee; Meghah, Vuppalapaty; Brahmendra Swamy, Cherukuvada V.; Kumar, Arvind; Idris, Mohammed M.

2013-01-01

Anxiety and depression are major chronic mood disorders, and the etiopathology for each appears to be repeated exposure to diverse unpredictable stress factors. Most of the studies on anxiety and related mood disorders are performed in rodents, and a good model is chronic unpredictable stress (CUS). In this study, we have attempted to understand the molecular basis of the neuroglial and behavioral changes underlying CUS-induced mood disorders in the simplest vertebrate model, the zebrafish, Danio rerio. Zebrafish were subjected to a CUS paradigm in which two different stressors were used daily for 15 days, and thorough behavioral analyses were performed to assess anxiety and related mood disorder phenotypes using the novel tank test, shoal cohesion and scototaxis. Fifteen days of exposure to chronic stressors appears to induce an anxiety and related mood disorder phenotype. Decreased neurogenesis, another hallmark of anxiety and related disorders in rodents, was also observed in this zebrafish model. The common molecular markers of rodent anxiety and related disorders, corticotropin-releasing factor (CRF), calcineurin (ppp3r1a) and phospho cyclic AMP response element binding protein (pCREB), were also replicated in the fish model. Finally, using 2DE FTMS/ITMSMS proteomics analyses, 18 proteins were found to be deregulated in zebrafish anxiety and related disorders. The most affected process was mitochondrial function, 4 of the 18 differentially regulated proteins were mitochondrial proteins: PHB2, SLC25A5, VDAC3 and IDH2, as reported in rodent and clinical samples. Thus, the zebrafish CUS model and proteomics can facilitate not only uncovering new molecular targets of anxiety and related mood disorders but also the routine screening of compounds for drug development. PMID:23691016

Thyroid hormone regulates muscle function during cold acclimation in zebrafish (Danio rerio).

PubMed

Little, Alexander G; Seebacher, Frank

2013-09-15

Thyroid hormone (TH) is a universal regulator of growth, development and metabolism during cold exposure in mammals. In zebrafish (Danio rerio), TH regulates locomotor performance and metabolism during cold acclimation. The influence of TH on locomotor performance may be via its effect on metabolism or, as has been shown in mammals, by modulating muscle phenotypes. Our aim was to determine whether TH influences muscle phenotypes in zebrafish, and whether this could explain changes in swimming capacity in response to thermal acclimation. We used propylthiouracil and iopanoic acid to induce hypothyroidism in zebrafish over a 3-week acclimation period to either 18 or 28°C. To verify that physiological changes following hypothyroid treatment were in fact due to the action of TH, we supplemented hypothyroid fish with 3,5-diiodothryronine (T2) or 3,5,3'-triiodothyronine (T3). Cold-acclimated fish had significantly greater sustained swimming performance (Ucrit) but not burst speed. Greater Ucrit was accompanied by increased tail beat frequency, but there was no change in tail beat amplitude. Hypothyroidism significantly decreased Ucrit and burst performance, as well as tail beat frequency and SERCA activity in cold-acclimated fish. However, myofibrillar ATPase activity increased in cold-acclimated hypothyroid fish. Hypothyroid treatment also decreased mRNA concentrations of myosin heavy chain fast isoforms and SERCA 1 isoform in cold-acclimated fish. SERCA 1 mRNA increased in warm-acclimated hypothyroid fish, and SERCA 3 mRNA decreased in both cold- and warm-acclimated hypothyroid fish. Supplementation with either T2 or T3 restored Ucrit, burst speed, tail beat frequency, SERCA activity and myosin heavy chain and SERCA 1 and 3 mRNA levels of hypothyroid fish back to control levels. We show that in addition to regulating development and metabolism in vertebrates, TH also regulates muscle physiology in ways that affect locomotor performance in fish. We suggest that the role of TH in modulating SERCA1 expression during cold exposure may have predisposed it to regulate endothermic thermogenesis.

Culturable Gut Microbiota Diversity in Zebrafish

PubMed Central

Sørby, Jan Roger Torp; Aleström, Peter; Sørum, Henning

2012-01-01

Abstract The zebrafish (Danio rerio) is an increasingly used laboratory animal model in basic biology and biomedicine, novel drug development, and toxicology. The wide use has increased the demand for optimized husbandry protocols to ensure animal health care and welfare. The knowledge about the correlation between culturable zebrafish intestinal microbiota and health in relation to environmental factors and management procedures is very limited. A semi-quantitative level of growth of individual types of bacteria was determined and associated with sampling points. A total of 72 TAB line zebrafish from four laboratories (Labs A–D) in the Zebrafish Network Norway were used. Diagnostic was based on traditional bacterial culture methods and biochemical characterization using commercial kits, followed by 16S rDNA gene sequencing from pure subcultures. Also selected Gram-negative isolates were analyzed for antibiotic susceptibility to 8 different antibiotics. A total of 13 morphologically different bacterial species were the most prevalent: Aeromonas hydrophila, Aeromonas sobria, Vibrio parahaemolyticus, Photobacterium damselae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas luteola, Comamonas testosteroni, Ochrobactrum anthropi, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus warneri. Only Lab B had significantly higher levels of total bacterial growth (OR=2.03), whereas numbers from Lab C (OR=1.01) and Lab D (OR=1.12) were found to be similar to the baseline Lab A. Sexually immature individuals had a significantly higher level of harvested total bacterial growth than mature fish (OR=0.82), no statistically significant differences were found between male and female fish (OR=1.01), and the posterior intestinal segment demonstrated a higher degree of culturable bacteria than the anterior segment (OR=4.1). Multiple antibiotic (>3) resistance was observed in 17% of the strains. We propose that a rapid conventional diagnostic bacteriological assay on the culturable microbiota profiles can be designed and used as quality measure of the husbandry routines of a zebrafish facility to ensure a bacterial standard safeguarding the zebrafish health and welfare. PMID:22428747

Culturable gut microbiota diversity in zebrafish.

PubMed

Cantas, Leon; Sørby, Jan Roger Torp; Aleström, Peter; Sørum, Henning

2012-03-01

The zebrafish (Danio rerio) is an increasingly used laboratory animal model in basic biology and biomedicine, novel drug development, and toxicology. The wide use has increased the demand for optimized husbandry protocols to ensure animal health care and welfare. The knowledge about the correlation between culturable zebrafish intestinal microbiota and health in relation to environmental factors and management procedures is very limited. A semi-quantitative level of growth of individual types of bacteria was determined and associated with sampling points. A total of 72 TAB line zebrafish from four laboratories (Labs A-D) in the Zebrafish Network Norway were used. Diagnostic was based on traditional bacterial culture methods and biochemical characterization using commercial kits, followed by 16S rDNA gene sequencing from pure subcultures. Also selected Gram-negative isolates were analyzed for antibiotic susceptibility to 8 different antibiotics. A total of 13 morphologically different bacterial species were the most prevalent: Aeromonas hydrophila, Aeromonas sobria, Vibrio parahaemolyticus, Photobacterium damselae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas luteola, Comamonas testosteroni, Ochrobactrum anthropi, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus warneri. Only Lab B had significantly higher levels of total bacterial growth (OR=2.03), whereas numbers from Lab C (OR=1.01) and Lab D (OR=1.12) were found to be similar to the baseline Lab A. Sexually immature individuals had a significantly higher level of harvested total bacterial growth than mature fish (OR=0.82), no statistically significant differences were found between male and female fish (OR=1.01), and the posterior intestinal segment demonstrated a higher degree of culturable bacteria than the anterior segment (OR=4.1). Multiple antibiotic (>3) resistance was observed in 17% of the strains. We propose that a rapid conventional diagnostic bacteriological assay on the culturable microbiota profiles can be designed and used as quality measure of the husbandry routines of a zebrafish facility to ensure a bacterial standard safeguarding the zebrafish health and welfare.

An automated device for appetitive conditioning in zebrafish (Danio rerio).

PubMed

Manabe, Kazuchika; Dooling, R J; Takaku, Shinichi

2013-12-01

An automated device and a procedure for the operant conditioning individual zebrafish were developed. The key feature of this procedure was the construction of a simple, inexpensive feeder that can deliver extremely small amounts of food, thus preventing rapid satiation. This allows the experimenter to run multiple trails in a single test session and multiple sessions in one day. In addition, small response keys made from acryl rods and fiber sensors were developed that were sufficiently sensitive to detect fish contact. To illustrate the efficiency and utility of the device for traditional learning paradigms, we trained zebrafish in a fixed ratio schedule where subjects were reinforced with food after 10 responses. Zebrafish reliably responded on the response key for sessions that lasted as long 80-reinforcements. They also showed the traditional "break and run" response pattern that has been found in many species. These results show that this system will be valuable for behavioral studies with zebrafish, especially for experiments that need many repeated trials using food reinforcer in a session. The present system can be used for sensory and learning investigations, as well applications in behavioral pharmacology, behavioral genetics, and toxicology where the zebrafish is becoming the vertebrate model of choice.

Boldness predicts social status in zebrafish (Danio rerio).

PubMed

Dahlbom, S Josefin; Lagman, David; Lundstedt-Enkel, Katrin; Sundström, L Fredrik; Winberg, Svante

2011-01-01

This study explored if boldness could be used to predict social status. First, boldness was assessed by monitoring individual zebrafish behaviour in (1) an unfamiliar barren environment with no shelter (open field), (2) the same environment when a roof was introduced as a shelter, and (3) when the roof was removed and an unfamiliar object (Lego® brick) was introduced. Next, after a resting period of minimum one week, social status of the fish was determined in a dyadic contest and dominant/subordinate individuals were determined as the winner/loser of two consecutive contests. Multivariate data analyses showed that males were bolder than females and that the behaviours expressed by the fish during the boldness tests could be used to predict which fish would later become dominant and subordinate in the ensuing dyadic contest. We conclude that bold behaviour is positively correlated to dominance in zebrafish and that boldness is not solely a consequence of social dominance.

Boldness Predicts Social Status in Zebrafish (Danio rerio)

PubMed Central

Dahlbom, S. Josefin; Lagman, David; Lundstedt-Enkel, Katrin; Sundström, L. Fredrik; Winberg, Svante

2011-01-01

This study explored if boldness could be used to predict social status. First, boldness was assessed by monitoring individual zebrafish behaviour in (1) an unfamiliar barren environment with no shelter (open field), (2) the same environment when a roof was introduced as a shelter, and (3) when the roof was removed and an unfamiliar object (Lego® brick) was introduced. Next, after a resting period of minimum one week, social status of the fish was determined in a dyadic contest and dominant/subordinate individuals were determined as the winner/loser of two consecutive contests. Multivariate data analyses showed that males were bolder than females and that the behaviours expressed by the fish during the boldness tests could be used to predict which fish would later become dominant and subordinate in the ensuing dyadic contest. We conclude that bold behaviour is positively correlated to dominance in zebrafish and that boldness is not solely a consequence of social dominance. PMID:21858168

The role of taurine on anxiety-like behaviors in zebrafish: A comparative study using the novel tank and the light-dark tasks.

PubMed

Mezzomo, Nathana J; Silveira, Ariane; Giuliani, Giulie S; Quadros, Vanessa A; Rosemberg, Denis B

2016-02-02

Taurine (TAU) is an amino sulfonic acid with several functions in central nervous system. Mounting evidence suggests that it acts in osmoregulation, neuromodulation and also as an inhibitory neurotransmitter. However, the effects of TAU on behavioral functions, especially on anxiety-related parameters, are limited. The adult zebrafish is a suitable model organism to examine anxiety-like behaviors since it presents neurotransmitter systems and behavioral functions evolutionary conserved. Anxiety in zebrafish can be measured by different tasks, analyzing the habituation to novelty, as well as the response to brightly lit environments. The aim of this study was to investigate whether acute TAU treatment alters anxiety-like behavior in zebrafish using the novel tank and the light-dark tests. Fish were individually treated with TAU (42, 150, and 400mg/L) for 1h and the behaviors were further analyzed for 6min in the novel tank or in the light-dark test. Control fish were handled in a similar manner, but kept only in home tank water. Although TAU did not alter locomotor and vertical activities, all concentrations significantly increased shuttling and time spent in lit compartment. Moreover, TAU 150 group showed a significant decrease in the number of risk assessment episodes. Overall, these data suggest that TAU exerts an anxiolytic-like effect in zebrafish and the comparative analysis of behavior using different tasks is an interesting strategy for neuropsychiatric studies related to anxiety in this species. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Exercise quantity-dependent muscle hypertrophy in adult zebrafish (Danio rerio).

PubMed

Hasumura, Takahiro; Meguro, Shinichi

2016-07-01

Exercise is very important for maintaining and increasing skeletal muscle mass, and is particularly important to prevent and care for sarcopenia and muscle disuse atrophy. However, the dose-response relationship between exercise quantity, duration/day, and overall duration and muscle mass is poorly understood. Therefore, we investigated the effect of exercise duration on skeletal muscle to reveal the relationship between exercise quantity and muscle hypertrophy in zebrafish forced to exercise. Adult male zebrafish were exercised 6 h/day for 4 weeks, 6 h/day for 2 weeks, or 3 h/day for 2 weeks. Flow velocity was adjusted to maximum velocity during continual swimming (initial 43 cm/s). High-speed consecutive photographs revealed that zebrafish mainly drove the caudal part. Additionally, X-ray micro computed tomography measurements indicated muscle hypertrophy of the mid-caudal half compared with the mid-cranial half part. The cross-sectional analysis of the mid-caudal half muscle revealed that skeletal muscle (red, white, or total) mass increased with increasing exercise quantity, whereas that of white muscle and total muscle increased only under the maximum exercise load condition of 6 h/day for 4 weeks. Additionally, the muscle fiver size distributions of exercised fish were larger than those from non-exercised fish. We revealed that exercise quantity, duration/day, and overall duration were correlated with skeletal muscle hypertrophy. The forced exercise model enabled us to investigate the relationship between exercise quantity and skeletal muscle mass. These results open up the possibility for further investigations on the effects of exercise on skeletal muscle in adult zebrafish.

Simple, economical heat-shock devices for zebrafish housing racks.

PubMed

Duszynski, Robert J; Topczewski, Jacek; LeClair, Elizabeth E

2011-12-01

One reason for the popularity of the zebrafish (Danio rerio) as a model vertebrate is the ability to manipulate gene expression in this organism. A common method is to induce gene expression transiently under control of a heat-shock promoter (e.g., hsp70l). By making simple mechanical adjustments to small aquarium heaters (25-50W), we were able to produce consistent and reliable heat-shock conditions within a conventional zebrafish housing system. Up to two heat-shock intervals per day (>37°C) could be maintained under conditions of continuous flow (5-25 mL/min). Temperature logging every 30 s indicated rapid warm up times, consistent heat-shock lengths, and accurate and precise peak water temperatures (mean±SD=38°C±0.2°C). The biological effects of these heat-shock treatments were confirmed by observing inducible expression of enhanced green fluorescent protein (EGFP) and inhibition of caudal fin regeneration in a transgenic fish line expressing a dominant negative fibroblast growth factor receptor (Tg(hsp70l:dnfgfr1-EGFP)(pd1)). These devices are inexpensive, easily modified, and can be calibrated to accommodate a variety of experimental designs. After setup on a programmable timer, the heaters require no intervention to produce consistent daily heat shocks, and all other standard care protocols can be followed in the fish facility. The simplicity and stability of these devices make them suitable for long-term heat shocks at any stage of the zebrafish lifecycle (>7 days postfertilization), and useful for both laboratory and classroom experiments on transgenic zebrafish.

10th European Zebrafish Meeting 2017, Budapest: Husbandry Workshop Summary.

PubMed

Oltová, Jana; Barton, Carrie; Certal, Ana Catarina; Argenton, Francesco; Varga, Zoltán M

2018-01-02

A husbandry workshop on July 3, 2017, at the 10th European Zebrafish Meeting in Budapest, Hungary (July 3-July 7, 2017), focused on the standardization, optimization, and streamlining of fish facility procedures. Standardization can be achieved for example by developing novel software and hardware tools, such as a fish facility database for husbandry and environmental facility management (Zebrabase, Oltova), or a hand-held, air-pressurized fish feeder for consistent food distribution (Blowfish, Argenton). Streamlining is achieved when work hours are reduced, as with the standardized fish feeder, or by limiting the number and types of fish diets and observing the effect on animal welfare and performance (Barton). Testing the characteristics of new fish diets and observing whether they produce better experimental outcomes (Certal) optimizes diets and improves fish productivity. Collectively, the workshop presentations emphasized how consistency and harmonization of husbandry procedures within and across aquatic facilities yield reproducible scientific outcomes.

Zebrafish AID is capable of deaminating methylated deoxycytidines

PubMed Central

Abdouni, Hala; King, Justin J.; Suliman, Mussa; Quinlan, Matthew; Fifield, Heather; Larijani, Mani

2013-01-01

Activation-induced cytidine deaminase (AID) deaminates deoxycytidine (dC) to deoxyuracil (dU) at immunoglobulin loci in B lymphocytes to mediate secondary antibody diversification. Recently, AID has been proposed to also mediate epigenetic reprogramming by demethylating methylated cytidines (mC) possibly through deamination. AID overexpression in zebrafish embryos was shown to promote genome demethylation through G:T lesions, implicating a deamination-dependent mechanism. We and others have previously shown that mC is a poor substrate for human AID. Here, we examined the ability of bony fish AID to deaminate mC. We report that zebrafish AID was unique among all orthologs in that it efficiently deaminates mC. Analysis of domain-swapped and mutant AID revealed that mC specificity is independent of the overall high-catalytic efficiency of zebrafish AID. Structural modeling with or without bound DNA suggests that efficient deamination of mC by zebrafish AID is likely not due to a larger catalytic pocket allowing for better fit of mC, but rather because of subtle differences in the flexibility of its structure. PMID:23585279

Urban stormwater runoff negatively impacts lateral line development in larval zebrafish and salmon embryos.

PubMed

Young, Alexander; Kochenkov, Valentin; McIntyre, Jenifer K; Stark, John D; Coffin, Allison B

2018-02-12

After a storm, water often runs off of impervious urban surfaces directly into aquatic ecosystems. This stormwater runoff is a cocktail of toxicants that have serious effects on the ecological integrity of aquatic habitats. Zebrafish that develop in stormwater runoff suffer from cardiovascular toxicity and impaired growth, but the effects of stormwater on fish sensory systems are not understood. Our study investigated the effect of stormwater on hair cells of the lateral line in larval zebrafish and coho salmon. Our results showed that although toxicants in stormwater did not kill zebrafish hair cells, these cells did experience damage. Zebrafish developing in stormwater also experienced impaired growth, fewer neuromasts in the lateral line, and fewer hair cells per neuromast. A similar reduction in neuromast number was observed in coho salmon reared in stormwater. Bioretention treatment, intended to filter out harmful constituents of stormwater, rescued the lateral line defects in zebrafish but not in coho salmon, suggesting that not all of the harmful constituents were removed by the filtration media and that salmonids are particularly sensitive to aquatic toxicants. Collectively, these data demonstrate that sub-lethal exposure to stormwater runoff negatively impacts a fish sensory system, which may have consequences for organismal fitness.

Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis.

PubMed

Knöbel, Melanie; Busser, Frans J M; Rico-Rico, Angeles; Kramer, Nynke I; Hermens, Joop L M; Hafner, Christoph; Tanneberger, Katrin; Schirmer, Kristin; Scholz, Stefan

2012-09-04

The zebrafish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, which is required by various regulations for environmental risk assessment of chemicals. We investigated the reliability of the embryo test by probing organic industrial chemicals with a wide range of physicochemical properties, toxicities, and modes of toxic action. Moreover, the relevance of using measured versus nominal (intended) exposure concentrations, inclusion of sublethal endpoints, and different exposure durations for the comparability with reported fish acute toxicity was explored. Our results confirm a very strong correlation of zebrafish embryo to fish acute toxicity. When toxicity values were calculated based on measured exposure concentrations, the slope of the type II regression line was 1 and nearly passed through the origin (1 to 1 correlation). Measured concentrations also explained several apparent outliers. Neither prolonged exposure (up to 120 h) nor consideration of sublethal effects led to a reduced number of outliers. Yet, two types of compounds were less lethal to embryos than to adult fish: a neurotoxic compound acting via sodium channels (permethrin) and a compound requiring metabolic activation (allyl alcohol).

Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults

PubMed Central

Le Fol, Vincent; Brion, François; Hillenweck, Anne; Perdu, Elisabeth; Bruel, Sandrine; Aït-Aïssa, Selim; Cravedi, Jean-Pierre; Zalko, Daniel

2017-01-01

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using 3H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances. PMID:28346357

Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults.

PubMed

Le Fol, Vincent; Brion, François; Hillenweck, Anne; Perdu, Elisabeth; Bruel, Sandrine; Aït-Aïssa, Selim; Cravedi, Jean-Pierre; Zalko, Daniel

2017-03-25

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using ³H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances.

Generation and Characterization of Neurogeninl-GFP Transgenic Medaka for High Throughput Developmental Neurotoxicity Screening

EPA Science Inventory

Fish models such as zebrafish and medaka are increasingly used as alternatives to rodents in developmental and toxicological studies. These developmental and toxicological studies can be facilitated by the use of transgenic reporters that permit the real-time, noninvasive observa...

Imaging Subcellular Structures in the Living Zebrafish Embryo.

PubMed

Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

2016-04-02

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Impact of stress, fear and anxiety on the nociceptive responses of larval zebrafish.

PubMed

Lopez-Luna, Javier; Al-Jubouri, Qussay; Al-Nuaimy, Waleed; Sneddon, Lynne U

2017-01-01

Both adult and larval zebrafish have been demonstrated to show behavioural responses to noxious stimulation but also to potentially stress- and fear or anxiety- eliciting situations. The pain or nociceptive response can be altered and modulated by these situations in adult fish through a mechanism called stress-induced analgesia. However, this phenomenon has not been described in larval fish yet. Therefore, this study explores the behavioural changes in larval zebrafish after noxious stimulation and exposure to challenges that can trigger a stress, fear or anxiety reaction. Five-day post fertilization zebrafish were exposed to either a stressor (air emersion), a predatory fear cue (alarm substance) or an anxiogenic (caffeine) alone or prior to immersion in acetic acid 0.1%. Pre- and post-stimulation behaviour (swimming velocity and time spent active) was recorded using a novel tracking software in 25 fish at once. Results show that larvae reduced both velocity and activity after exposure to the air emersion and alarm substance challenges and that these changes were attenuated using etomidate and diazepam, respectively. Exposure to acetic acid decreased velocity and activity as well, whereas air emersion and alarm substance inhibited these responses, showing no differences between pre- and post-stimulation. Therefore, we hypothesize that an antinociceptive mechanism, activated by stress and/or fear, occur in 5dpf zebrafish, which could have prevented the larvae to display the characteristic responses to pain.

Evolution of the duplicated intracellular lipid-binding protein genes of teleost fishes.

PubMed

Venkatachalam, Ananda B; Parmar, Manoj B; Wright, Jonathan M

2017-08-01

Increasing organismal complexity during the evolution of life has been attributed to the duplication of genes and entire genomes. More recently, theoretical models have been proposed that postulate the fate of duplicated genes, among them the duplication-degeneration-complementation (DDC) model. In the DDC model, the common fate of a duplicated gene is lost from the genome owing to nonfunctionalization. Duplicated genes are retained in the genome either by subfunctionalization, where the functions of the ancestral gene are sub-divided between the sister duplicate genes, or by neofunctionalization, where one of the duplicate genes acquires a new function. Both processes occur either by loss or gain of regulatory elements in the promoters of duplicated genes. Here, we review the genomic organization, evolution, and transcriptional regulation of the multigene family of intracellular lipid-binding protein (iLBP) genes from teleost fishes. Teleost fishes possess many copies of iLBP genes owing to a whole genome duplication (WGD) early in the teleost fish radiation. Moreover, the retention of duplicated iLBP genes is substantially higher than the retention of all other genes duplicated in the teleost genome. The fatty acid-binding protein genes, a subfamily of the iLBP multigene family in zebrafish, are differentially regulated by peroxisome proliferator-activated receptor (PPAR) isoforms, which may account for the retention of iLBP genes in the zebrafish genome by the process of subfunctionalization of cis-acting regulatory elements in iLBP gene promoters.

Comprehensive transcriptome analysis reveals accelerated genic evolution in a Tibet fish, Gymnodiptychus pachycheilus.

PubMed

Yang, Liandong; Wang, Ying; Zhang, Zhaolei; He, Shunping

2014-12-26

Elucidating the genetic mechanisms of organismal adaptation to the Tibetan Plateau at a genomic scale can provide insights into the process of adaptive evolution. Many highland species have been investigated and various candidate genes that may be responsible for highland adaptation have been identified. However, we know little about the genomic basis of adaptation to Tibet in fishes. Here, we performed transcriptome sequencing of a schizothoracine fish (Gymnodiptychus pachycheilus) and used it to identify potential genetic mechanisms of highland adaptation. We obtained totally 66,105 assembled unigenes, of which 7,232 were assigned as putative one-to-one orthologs in zebrafish. Comparative gene annotations from several species indicated that at least 350 genes lost and 41 gained since the divergence between G. pachycheilus and zebrafish. An analysis of 6,324 orthologs among zebrafish, fugu, medaka, and spotted gar identified consistent evidence for genome-wide accelerated evolution in G. pachycheilus and only the terminal branch of G. pachycheilus had an elevated Ka/Ks ratio than the ancestral branch. Many functional categories related to hypoxia and energy metabolism exhibited rapid evolution in G. pachycheilus relative to zebrafish. Genes showing signature of rapid evolution and positive selection in the G. pachycheilus lineage were also enriched in functions associated with energy metabolism and hypoxia. The first genomic resources for fish in the Tibetan Plateau and evolutionary analyses provided some novel insights into highland adaptation in fishes and served as a foundation for future studies aiming to identify candidate genes underlying the genetic bases of adaptation to Tibet in fishes. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Fluoride caused thyroid endocrine disruption in male zebrafish (Danio rerio).

PubMed

Jianjie, Chen; Wenjuan, Xue; Jinling, Cao; Jie, Song; Ruhui, Jia; Meiyan, Li

2016-02-01

Excessive fluoride in natural water ecosystem has the potential to detrimentally affect thyroid endocrine system, but little is known of such effects or underlying mechanisms in fish. In the present study, we evaluated the effects of fluoride on growth performance, thyroid histopathology, thyroid hormone levels, and gene expressions in the HPT axis in male zebrafish (Danio rerio) exposed to different determined concentrations of 0.1, 0.9, 2.0 and 4.1 M of fluoride to investigate the effects of fluoride on thyroid endocrine system and the potential toxic mechanisms caused by fluoride. The results indicated that the growth of the male zebrafish used in the experiments was significantly inhibited, the thyroid microtrastructure was changed, and the levels of T3 and T4 were disturbed in fluoride-exposed male fish. In addition, the expressional profiles of genes in HPT axis displayed alteration. The expressions of all studied genes were significantly increased in all fluoride-exposed male fish after exposure for 45 days. The transcriptional levels of corticotrophin-releasing hormone (CRH), thyroid-stimulating hormone (TSH), thyroglobulin (TG), sodium iodide symporter (NIS), iodothyronine I (DIO1), and thyroid hormone receptor alpha (TRα) were also elevated in all fluoride-exposed male fish after 90 days of exposure, while the inconsistent expressions were found in the mRNA of iodothyronineⅡ (DIO2), UDP glucuronosyltransferase 1 family a, b (UGT1ab), transthyretin (TTR), and thyroid hormone receptor beta (TRβ). These results demonstrated that fluoride could notably inhibit the growth of zebrafish, and significantly affect thyroid endocrine system by changing the microtrastructure of thyroid, altering thyroid hormone levels and endocrine-related gene expressions in male zebrafish. All above indicated that fluoride could pose a great threat to thyroid endocrine system, thus detrimentally affected the normal function of thyroid of male zebrafish. Copyright © 2015. Published by Elsevier B.V.

Contextual fear conditioning in zebrafish.

PubMed

Kenney, Justin W; Scott, Ian C; Josselyn, Sheena A; Frankland, Paul W

2017-10-01

Zebrafish are a genetically tractable vertebrate that hold considerable promise for elucidating the molecular basis of behavior. Although numerous recent advances have been made in the ability to precisely manipulate the zebrafish genome, much less is known about many aspects of learning and memory in adult fish. Here, we describe the development of a contextual fear conditioning paradigm using an electric shock as the aversive stimulus. We find that contextual fear conditioning is modulated by shock intensity, prevented by an established amnestic agent (MK-801), lasts at least 14 d, and exhibits extinction. Furthermore, fish of various background strains (AB, Tu, and TL) are able to acquire fear conditioning, but differ in fear extinction rates. Taken together, we find that contextual fear conditioning in zebrafish shares many similarities with the widely used contextual fear conditioning paradigm in rodents. Combined with the amenability of genetic manipulation in zebrafish, we anticipate that our paradigm will prove to be a useful complementary system in which to examine the molecular basis of vertebrate learning and memory. © 2017 Kenney et al.; Published by Cold Spring Harbor Laboratory Press.

Pseudoloma neurophilia infections in zebrafish Danio rerio: effects of stress on survival, growth, and reproduction.

USGS Publications Warehouse

Ramsay, J.M.; Watral, Virginia G.; Schreck, C.B.; Kent, M.L.

2009-01-01

Pseudoloma neurophilia (Microsporidia) is a common disease of zebrafish Danio rerio, including those used as research models. We conducted a study comprised of 4 separate experiments to determine the effects of husbandry stress on preexisting and experimental P. neurophilia infections and the subsequent effects on survival, infection onset and intensity, fish growth, and reproduction. In fish (AB strain) with preexisting infections, stress or feeding cortisol significantly increased mortality over 7 wk compared to no stress or cortisol treatment. In contrast, no mortality was observed in fish (TL strain) experimentally exposed to P. neurophilia over 10 wk. A third experiment involved experimental exposure of AB fish to P. neurophilia and exposure to crowding and handling stressors. No mortality was associated with P. neurophilia regardless of stress treatment over a period of 20 wk. However, the onset of infection occurred sooner in stress-treated fish. Stress significantly increased the mean intensity of infection (described as xenoma area/spinal cord area in histological sections) at Week 20 post-exposure (PE). In fish with preexisting infections, myositis was significantly greater in stressed and cortisol-treated fish than those not stressed. With experimental exposure of AB fish, stressed and infected groups weighed significantly less than the control group at Week 20 PE. Regarding fecundity, the number of larvae hatched at 5 d post fertilization was negatively associated with mean infection intensity among P. neurophilia-infected and stressed AB fish. These experiments are the first to show empirically that P. neurophilia can be associated with reduced weight and fecundity, and that stress can exacerbate the severity of the infection.

MEETING IN PORTUGAL: LINKAGE OF EXPOSURE AND EFFECTS USING GENOMICS, PROTEOMICS AND METABOLOMICS IN SMALL FISH MODELS

EPA Science Inventory

With an interdisciplinary team of scientists from U.S. Government Agencies and Universities, we are utilizing zebrafish and fathead minnow to develop techniques for extrapolation of chemical stressor impacts across species, chemicals and endpoints. The linkage of responses acros...

Zebrafish Housing Systems: A Review of Basic Operating Principles and Considerations for Design and Functionality

PubMed Central

Lawrence, Christian; Mason, Timothy

2015-01-01

The strategies for housing zebrafish used in biomedical research have evolved considerably over the past three decades. To keep pace with the rapid expansion and development of the zebrafish model system, the field has generally moved from keeping fish at the level of aquarium hobbyist to that of industrialized, recirculating aquaculture. Numerous commercial system vendors now offer increasingly sophisticated housing systems based on design principles that maximize the number of animals that can be housed in a given space footprint, and they are thus able to support large and diverse research programs. This review is designed to provide managers, lab animal veterinarians, investigators, and other parties responsible for care and use of these animals with a comprehensive overview of the basic operating and design principles of zebrafish housing systems. This information can be used to help plan the construction of new facilities and/or the upgrade and maintenance of existing operations. PMID:23382349

Development and automation of a test of impulse control in zebrafish

PubMed Central

Parker, Matthew O.; Ife, Dennis; Ma, Jun; Pancholi, Mahesh; Smeraldi, Fabrizio; Straw, Chris; Brennan, Caroline H.

2013-01-01

Deficits in impulse control (difficulties in inhibition of a pre-potent response) are fundamental to a number of psychiatric disorders, but the molecular and cellular basis is poorly understood. Zebrafish offer a very useful model for exploring these mechanisms, but there is currently a lack of validated procedures for measuring impulsivity in fish. In mammals, impulsivity can be measured by examining rates of anticipatory responding in the 5-choice serial reaction time task (5-CSRTT), a continuous performance task where the subject is reinforced upon accurate detection of a briefly presented light in one of five distinct spatial locations. This paper describes the development of a fully-integrated automated system for testing impulsivity in adult zebrafish. We outline the development of our image analysis software and its integration with National Instruments drivers and actuators to produce the system. We also describe an initial validation of the system through a one-generation screen of chemically mutagenized zebrafish, where the testing parameters were optimized. PMID:24133417

Effects of piracetam on behavior and memory in adult zebrafish.

PubMed

Grossman, Leah; Stewart, Adam; Gaikwad, Siddharth; Utterback, Eli; Wu, Nadine; Dileo, John; Frank, Kevin; Hart, Peter; Howard, Harry; Kalueff, Allan V

2011-04-25

Piracetam, a derivative of γ-aminobutyric acid, exerts memory-enhancing and mild anxiolytic effects in human and rodent studies. To examine the drug's behavioral profile further, we assessed its effects on behavioral and endocrine (cortisol) responses of adult zebrafish (Danio rerio)--a novel model species rapidly gaining popularity in neurobehavioral research. Overall, acute piracetam did not affect zebrafish novel tank and light-dark box behavior at mild doses (25-400mg/L), but produced nonspecific behavioral inhibition at 700mg/L. No effects on cortisol levels or inter-/intra-session habituation in the novel tank test were observed for acute or chronic mild non-sedative dose of 200mg/L. In contrast, fish exposed to chronic piracetam at this dose performed significantly better in the cued learning plus-maze test. This observation parallels clinical and rodent literature on the behavioral profile of piracetam, supporting the utility of zebrafish paradigms for testing nootropic agents. Copyright © 2011 Elsevier Inc. All rights reserved.

Developmental Toxicity of Louisiana Crude Oil-Spiked Sediment to Zebrafish

EPA Science Inventory

Embryonic exposures to the components of petroleum, including polycyclic aromatic hydrocarbons (PAHs), cause a characteristic suite of developmental defects and cardiotoxicity in a variety of fish species. We exposed zebrafish embryos to reference sediment mixed with laboratory w...

Developmental Toxicity of Louisiana Crude Oiled Sediment to Zebrafish

EPA Science Inventory

Embryonic exposures to polycyclic aromatic hydrocarbons (PAHs) and petroleum products cause a characteristic suite of developmental defects in a variety of fish species. We exposed zebrafish embryos to sediment mixed with laboratory weathered South Louisiana crude oil. Oiled sedi...

Detecting Developmental Neurotoxicants Using Zebrafish Embryos

EPA Science Inventory

As part of EPA’s program on the screening and prioritization of chemicals for developmental neurotoxicity, a rapid, cost-effective in vivo vertebrate screen is being developed using an alternative species approach. Zebrafish (Danio rerio), a small freshwater fish with external f...

Sleep–wake regulation and hypocretin–melatonin interaction in zebrafish

PubMed Central

Appelbaum, Lior; Wang, Gordon X.; Maro, Geraldine S.; Mori, Rotem; Tovin, Adi; Marin, Wilfredo; Yokogawa, Tohei; Kawakami, Koichi; Smith, Stephen J.; Gothilf, Yoav; Mignot, Emmanuel; Mourrain, Philippe

2009-01-01

In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep–wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant (hcrtr−/−). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr−/− pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr−/− fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr−/− fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation. PMID:19966231

Neural defects and cardiac arrhythmia in fish larvae following embryonic exposure to 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47).

PubMed

Lema, Sean C; Schultz, Irvin R; Scholz, Nathaniel L; Incardona, John P; Swanson, Penny

2007-05-31

Polybrominated diphenyl ethers (PBDEs) are added to plastics, polyurethane foam, and textiles as a flame retardant. While PBDEs play a key role in reducing loss of human life and property from fires, these flame retardants have become pervasive organic contaminants in the environment and in the tissues of fish, birds, marine mammals, and humans. Levels of PBDEs in wildlife and humans continue to rise, raising concerns about potential ecological and health risks associated with exposure to these chemicals. Nevertheless, there is little currently known about the toxicological effects of PBDE exposure. Here, we examined the developmental toxicity of the PBDE congener 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47) using the zebrafish (Danio rerio) as an ontogenetic model. Zebrafish embryos were exposed continuously to dissolved phase PBDE 47 (100-5000 microg/l) beginning 3-5 h post-fertilization (hpf). Fish treated with the highest concentrations of PBDE 47 delayed hatching, had reduced growth post-hatching, and displayed an abnormal dorsal curvature of the body with flexion at the hindbrain. By 96h post-fertilization larvae exposed to PBDE 47 had significant tachycardia, which progressed into atrioventricular block arrhythmias. Microinjection of fluorescent dye into the hindbrain ventricle revealed that cerebrospinal fluid in the neural tube and brain ventricles flowed more slowly in fish larvae exposed to PBDE 47, a likely etiology for the dorsal curvature. Similar, though much less pronounced, developmental toxicity also occurred in larvae exposed to PBDE 47 only for a 20h period during early embryogenesis (3-23 hpf), suggesting that PBDEs incorporated in lipid of the egg are bioavailable and cause toxicity later in life. Taken together, this work indicates that exposure to PBDE 47 can cause morphological abnormalities, impair cardiovascular function and cerebrospinal fluid flow, and provides a tractable starting point for using the zebrafish model to explore molecular mechanisms of PBDE toxicity.

Biosecurity and Health Monitoring at the Zebrafish International Resource Center

PubMed Central

Varga, Zoltán M.; Kent, Michael L.

2016-01-01

Abstract The Zebrafish International Resource Center (ZIRC) is a repository and distribution center for mutant, transgenic, and wild-type zebrafish. In recent years annual imports of new zebrafish lines to ZIRC have increased tremendously. In addition, after 15 years of research, we have identified some of the most virulent pathogens affecting zebrafish that should be avoided in large production facilities, such as ZIRC. Therefore, while importing a high volume of new lines we prioritize safeguarding the health of our in-house fish colony. Here, we describe the biosecurity and health-monitoring program implemented at ZIRC. This strategy was designed to prevent introduction of new zebrafish pathogens, minimize pathogens already present in the facility, and ensure a healthy zebrafish colony for in-house uses and shipment to customers. PMID:27031282

Dichlorvos Exposure Results in Large Scale Disruption of Energy Metabolism in the Liver of the Zebra Fish, Danio Rerio

DTIC Science & Technology

2015-10-24

zebrafish reference genome sequence and its relationship to the human genome . Nature. 2013;496(7446):498–503. 21. Linney E, Upchurch L, Donerly S. Zebrafish...To obtain a broader understanding of the effects of dichlorvos on liver metabolism, we per- formed a genome -wide analysis of gene expression in the ...condition) for whole genome transcript ana- lysis, and fixed another set of fish for histological evaluation (n = 5/condition). We determined the target

Gene expression analysis in zebrafish embryos: a potential approach to predict effect concentrations in the fish early life stage test.

PubMed

Weil, Mirco; Scholz, Stefan; Zimmer, Michaela; Sacher, Frank; Duis, Karen

2009-09-01

Based on the hypothesis that analysis of gene expression could be used to predict chronic fish toxicity, the zebrafish (Danio rerio) embryo test (DarT), developed as a replacement method for the acute fish test, was expanded to a gene expression D. rerio embryo test (Gene-DarT). The effects of 14 substances on lethal and sublethal endpoints of the DarT and on expression of potential marker genes were investigated: the aryl hydrocarbon receptor 2, cytochrome P450 1A (cypla), heat shock protein 70, fizzy-related protein 1, the transcription factors v-maf musculoaponeurotic fibrosarcoma oncogene family protein g (avian) 1 and NF-E2-p45-related factor, and heme oxygenase 1 (hmox1). After exposure of zebrafish embryos for 48 h, differential gene expression was evaluated using reverse transcriptase-polymerase chain reaction, gel electrophoresis, and densitometric analysis of the gels. All tested compounds significantly affected the expression of at least one potential marker gene, with cyp1a and hmox1 being most sensitive. Lowest-observed-effect concentrations (LOECs) for gene expression were below concentrations resulting in 10% lethal effects in the DarT. For 10 (3,4- and 3,5-dichloroaniline, 1,4-dichlorobenzene, 2,4-dinitrophenol, atrazine, parathion-ethyl, chlorotoluron, genistein, 4-nitroquinoline-1-oxide, and cadmium) out of the 14 tested substances, LOEC values derived with the Gene-DarT differ by a factor of less than 10 from LOEC values of fish early life stage tests with zebrafish. For pentachloroaniline and pentachlorobenzene, the Gene-DarT showed a 23- and 153-fold higher sensitivity, respectively, while for lindane, it showed a 13-fold lower sensitivity. For ivermectin, the Gene-DarT was by a factor of more than 1,000 less sensitive than the acute fish test. The results of the present study indicate that gene expression analysis in zebrafish embryos could principally be used to predict effect concentrations in the fish early life stage test.

Zebrafish in the sea of mineral (iron, zinc, and copper) metabolism

PubMed Central

Zhao, Lu; Xia, Zhidan; Wang, Fudi

2014-01-01

Iron, copper, zinc, and eight other minerals are classified as essential trace elements because they present in minute in vivo quantities and are essential for life. Because either excess or insufficient levels of trace elements can be detrimental to life (causing human diseases such as iron-deficiency anemia, hemochromatosis, Menkes syndrome and Wilson's disease), the endogenous levels of trace minerals must be tightly regulated. Many studies have demonstrated the existence of systems that maintain trace element homeostasis, and these systems are highly conserved in multiple species ranging from yeast to mice. As a model for studying trace mineral metabolism, the zebrafish is indispensable to researchers. Several large-scale mutagenesis screens have been performed in zebrafish, and these screens led to the identification of a series of metal transporters and the generation of several mutagenesis lines, providing an in-depth functional analysis at the system level. Moreover, because of their developmental advantages, zebrafish have also been used in mineral metabolism-related chemical screens and toxicology studies. Here, we systematically review the major findings of trace element homeostasis studies using the zebrafish model, with a focus on iron, zinc, copper, selenium, manganese, and iodine. We also provide a homology analysis of trace mineral transporters in fish, mice and humans. Finally, we discuss the evidence that zebrafish is an ideal experimental tool for uncovering novel mechanisms of trace mineral metabolism and for improving approaches to treat mineral imbalance-related diseases. PMID:24639652

An automatic method to calculate heart rate from zebrafish larval cardiac videos.

PubMed

Kang, Chia-Pin; Tu, Hung-Chi; Fu, Tzu-Fun; Wu, Jhe-Ming; Chu, Po-Hsun; Chang, Darby Tien-Hao

2018-05-09

Zebrafish is a widely used model organism for studying heart development and cardiac-related pathogenesis. With the ability of surviving without a functional circulation at larval stages, strong genetic similarity between zebrafish and mammals, prolific reproduction and optically transparent embryos, zebrafish is powerful in modeling mammalian cardiac physiology and pathology as well as in large-scale high throughput screening. However, an economical and convenient tool for rapid evaluation of fish cardiac function is still in need. There have been several image analysis methods to assess cardiac functions in zebrafish embryos/larvae, but they are still improvable to reduce manual intervention in the entire process. This work developed a fully automatic method to calculate heart rate, an important parameter to analyze cardiac function, from videos. It contains several filters to identify the heart region, to reduce video noise and to calculate heart rates. The proposed method was evaluated with 32 zebrafish larval cardiac videos that were recording at three-day post-fertilization. The heart rate measured by the proposed method was comparable to that determined by manual counting. The experimental results show that the proposed method does not lose accuracy while largely reducing the labor cost and uncertainty of manual counting. With the proposed method, researchers do not have to manually select a region of interest before analyzing videos. Moreover, filters designed to reduce video noise can alleviate background fluctuations during the video recording stage (e.g. shifting), which makes recorders generate usable videos easily and therefore reduce manual efforts while recording.

Immunostaining of dissected zebrafish embryonic heart.

PubMed

Yang, Jingchun; Xu, Xiaolei

2012-01-10

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects. The expression of any gene can be manipulated via morpholino technology or RNA injection. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis. Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals. Copyright © 2012 Journal of Visualized Experiments

Conspecific alarm substance differently alters group behavior of zebrafish populations: Putative involvement of cholinergic and purinergic signaling in anxiety- and fear-like responses.

PubMed

Canzian, Julia; Fontana, Barbara D; Quadros, Vanessa A; Rosemberg, Denis B

2017-03-01

The zebrafish (Danio rerio) is an emergent model organism for assessing fear and anxiety-like phenotypes. The short fin wild type (WT), and leopard (leo) are two zebrafish populations that present several behavioral differences, in which leo displays pronounced defensive responses. Mounting evidence suggests a modulatory role for cholinergic and purinergic signaling in fear and anxiety, but the involvement of these neurotransmitter systems in the behavioral profile of zebrafish is obscure. Here we tested whether the acute exposure to conspecific alarm substance (AS), an experimental protocol that induces fear, alters shoaling behavior, diving response, acetylcholinesterase (AChE) activity, and nucleotide hydrolysis in brain tissue of WT and leo. When four fish were concomitantly exposed to AS extracted from a donor fish of similar phenotype, both populations presented a significant increase of erratic movements without changes in freezing bouts. An increased shoal cohesion and a decreased vertical distribution were observed only in WT exposed to AS. The respective population also revealed a significant increase in AChE and ecto-5'-nucleotidase activities after the exposure period. The comparison of basal endpoints between populations showed that leo displays a higher social cohesion, few vertical transitions and enhanced AChE and ecto-5'-nucleotidase activities. In conclusion, we suggest that the effects of AS on defensive behaviors depend on the population, indicating the existence of distinct neurochemical mechanisms involved. Furthermore, this report shows the first evidence of a potential role of cholinergic and purinergic systems in fear- and anxiety-like responses of zebrafish populations. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitative GFP fluorescence as an indicator of arsenite developmental toxicity in mosaic heat shock protein 70 transgenic zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seok, Seung-Hyeok; Baek, Min-Won; Lee, Hui-Young

2007-12-01

In transgenic zebrafish (Danio rerio), green fluorescent protein (GFP) is a promising marker for environmental pollutants. In using GFP, one of the obstacles which we faced was how to compare toxicity among different toxicants or among a specific toxicant in different model species with the intensity of GFP expression. Using a fluorescence detection method, we first validated our method for estimating the amount of GFP fluorescence present in transgenic fish, which we used as an indicator of developmental toxicity caused by the well-known toxicant, arsenite. To this end, we developed mosaic transgenic zebrafish with the human heat shock response elementmore » (HSE) fused to the enhanced GFP (EGFP) reporter gene to indicate exposure to arsenite. We confirmed that EGFP expression sites correlate with gross morphological disruption caused by arsenite exposure. Arsenite (300.0 {mu}M) caused stronger EGFP fluorescence intensity and quantity than 50.0 {mu}M and 10.0 {mu}M arsenite in our transgenic zebrafish. Furthermore, arsenite-induced apoptosis was demonstrated by TUNEL assay. Apoptosis was inhibited by the antioxidant, N-acetyl-cystein (NAC) in this transgenic zebrafish. The distribution of TUNEL-positive cells in embryonic tissues was correlated with the sites of arsenite toxicity and EGFP expression. The EGFP values quantified using the standard curve equation from the known GFP quantity were consistent with the arsenite-induced EGFP expression pattern and arsenite concentration, indicating that this technique can be a reliable and applicable measurement. In conclusion, we propose that fluorescence-based EGFP quantification in transgenic fish containing the hsp70 promoter-EGFP reporter-gene construct is a useful indicator of development toxicity caused by arsenite.« less

Brief Embryonic Strychnine Exposure in Zebrafish Causes Long-Term Adult Behavioral Impairment with Indications of Embryonic Synaptic Changes

PubMed Central

Roy, Nicole M.; Arpie, Brianna; Lugo, Joseph; Linney, Elwood; Levin, Edward D.; Cerutti, Daniel

2015-01-01

Zebrafish provide a powerful model of the impacts of embryonic toxicant exposure on neural development that may result in long-term behavioral dysfunction. In this study, zebrafish embryos were treated with 1.5 mM strychnine for short embryonic time windows to induce transient changes in inhibitory neural signaling, and were subsequently raised in untreated water until adulthood. PCR analysis showed indications that strychnine exposure altered expression of some genes related to glycinergic, GABAergic and glutamatergic neuronal synapses during embryonic development. In adulthood, treated fish showed significant changes in swimming speed and tank diving behavior compared to controls. Taken together, these data show that a short embryonic exposure to a neurotoxicant can alter development of neural synapses and lead to changes in adult behavior. PMID:23022260

Brief embryonic strychnine exposure in zebrafish causes long-term adult behavioral impairment with indications of embryonic synaptic changes.

PubMed

Roy, Nicole M; Arpie, Brianna; Lugo, Joseph; Linney, Elwood; Levin, Edward D; Cerutti, Daniel

2012-01-01

Zebrafish provide a powerful model of the impacts of embryonic toxicant exposure on neural development that may result in long-term behavioral dysfunction. In this study, zebrafish embryos were treated with 1.5mM strychnine for short embryonic time windows to induce transient changes in inhibitory neural signaling, and were subsequently raised in untreated water until adulthood. PCR analysis showed indications that strychnine exposure altered expression of some genes related to glycinergic, GABAergic and glutamatergic neuronal synapses during embryonic development. In adulthood, treated fish showed significant changes in swimming speed and tank diving behavior compared to controls. Taken together, these data show that a short embryonic exposure to a neurotoxicant can alter development of neural synapses and lead to changes in adult behavior. Copyright © 2012 Elsevier Inc. All rights reserved.

Rosmarinic acid plays a protective role in the embryogenesis of zebrafish exposed to food colours through its influence on aurora kinase A level.

PubMed

Swarnalatha, Y; Jerrine Joseph, I S; Jayakrishna, Tippabathani

2017-05-01

To evaluate the protective nature of the rosmarinic acid from Sphaeranthus amaranthoides during zebra fish embryogenesis. Rosmarinic acid was isolated from the S. amaranthoides. An accurate, sensitive and simple LC-MS analysis was performed to determine the rosmarinic acid from S. amaranthoides. In the present study, zebrafish embryos were exposed to crimson red and sunset yellow at a concentration of 0.1 and 0.5mg/l and the effect of these food colours on the levels of aurora kinase A was studied individually. Aurora kinase A levels are crucial for embryogenesis in zebrafish which is used as model in this study. The decrease of aurora kinase A levels in food colour treated embryos influences the embryogenesis, resulting in short and bent trunk leading to cell death and growth retardation. Elevated levels of aurora kinase A in rosmarinic acid treated groups can be attributed to the restoration of normal growth in zebra fish embryos with well developed brain and eyes. Further insilico docking studies were carried out and target was identified as rosmarinic acid. From the docking studies the docking poses and binding energy confirms that aurora kinase A is the target for rosmarinic acid. Rosmarinic acid was found to play a protective role in the embryogenesis of zebra fish exposed to food colours (crimson red and sunset yellow) through its influence on aurora kinase A levels. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Phototoxicity of TiO2 nanoparticles to zebrafish (Danio rerio) is dependent on life stage.

PubMed

Ma, Hongbo; Diamond, Stephen A

2013-09-01

Zebrafish embryos have been used increasingly to evaluate nanomaterial toxicity. The present study compared phototoxicity of TiO2 nanoparticles with zebrafish at 4 life stages (embryos, yolk-sac larvae, free-swimming larvae, and juvenile) under simulated sunlight using the 96-h standard toxicity assay. Yolk-sac larvae were found to be the most sensitive to TiO2 phototoxicity, suggesting that the widely used zebrafish embryo test may not fully or accurately predict hazard and risk of these nanoparticles to small fish. Copyright © 2013 SETAC.

Lxr regulates lipid metabolic and visual perception pathways during zebrafish development.

PubMed

Pinto, Caroline Lucia; Kalasekar, Sharanya Maanasi; McCollum, Catherine W; Riu, Anne; Jonsson, Philip; Lopez, Justin; Swindell, Eric C; Bouhlatouf, Abdel; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Åke

2016-01-05

The Liver X Receptors (LXRs) play important roles in multiple metabolic pathways, including fatty acid, cholesterol, carbohydrate and energy metabolism. To expand the knowledge of the functions of LXR signaling during embryonic development, we performed a whole-genome microarray analysis of Lxr target genes in zebrafish larvae treated with either one of the synthetic LXR ligands T0901317 or GW3965. Assessment of the biological processes enriched by differentially expressed genes revealed a prime role for Lxr in regulating lipid metabolic processes, similarly to the function of LXR in mammals. In addition, exposure to the Lxr ligands induced changes in expression of genes in the neural retina and lens of the zebrafish eye, including the photoreceptor guanylate cyclase activators and lens gamma crystallins, suggesting a potential novel role for Lxr in modulating the transcription of genes associated with visual function in zebrafish. The regulation of expression of metabolic genes was phenotypically reflected in an increased absorption of yolk in the zebrafish larvae, and changes in the expression of genes involved in visual perception were associated with morphological alterations in the retina and lens of the developing zebrafish eye. The regulation of expression of both lipid metabolic and eye specific genes was sustained in 1 month old fish. The transcriptional networks demonstrated several conserved effects of LXR activation between zebrafish and mammals, and also identified potential novel functions of Lxr, supporting zebrafish as a promising model for investigating the role of Lxr during development. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Lxr regulates lipid metabolic and visual perception pathways during zebrafish development

PubMed Central

Pinto, Caroline Lucia; Kalasekar, Sharanya Maanasi; McCollum, Catherine W.; Riu, Anne; Jonsson, Philip; Lopez, Justin; Swindell, Eric; Bouhlatouf, Abdel; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Åke

2015-01-01

The Liver X Receptors (LXRs) play important roles in multiple metabolic pathways, including fatty acid, cholesterol, carbohydrate and energy metabolism. To expand the knowledge of the functions of LXR signaling during embryonic development, we performed a whole-genome microarray analysis of Lxr target genes in zebrafish larvae treated with either one of the synthetic LXR ligands T0901317 or GW3965. Assessment of the biological processes enriched by differentially expressed genes revealed a prime role for Lxr in regulating lipid metabolic processes, similarly to the function of LXR in mammals. In addition, exposure to the Lxr ligands induced changes in expression of genes in the neural retina and lens of the zebrafish eye, including the photoreceptor guanylate cyclase activators and lens gamma crystallins, suggesting a potential novel role for Lxr in modulating the transcription of genes associated with visual function in zebrafish. The regulation of expression of metabolic genes was phenotypically reflected in an increased absorption of yolk in the zebrafish larvae, and changes in the expression of genes involved in visual perception were associated with morphological alterations in the retina and lens of the developing zebrafish eye. The regulation of expression of both lipid metabolic and eye specific genes was sustained in 1 month old fish. The transcriptional networks demonstrated several conserved effects of LXR activation between zebrafish and mammals, and also identified potential novel functions of Lxr, supporting zebrafish as a promising model for investigating the role of Lxr during development. PMID:26427652

Involvement of estradiol-17beta and its membrane receptor, G protein coupled receptor 30 (GPR30) in regulation of oocyte maturation in zebrafish, Danio rario.

PubMed

Pang, Yefei; Thomas, Peter

2009-03-01

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20beta-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17beta, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.

Differential Lectin Binding Patterns Identify Distinct Heart Regions in Giant Danio (Devario aequipinnatus) and Zebrafish (Danio rerio) Hearts

PubMed Central

Manalo, Trina; May, Adam; Quinn, Joshua; Lafontant, Dominique S.; Shifatu, Olubusola; He, Wei; Gonzalez-Rosa, Juan M.; Burns, Geoffrey C.; Burns, Caroline E.; Burns, Alan R.; Lafontant, Pascal J.

2016-01-01

Lectins are carbohydrate-binding proteins commonly used as biochemical and histochemical tools to study glycoconjugate (glycoproteins, glycolipids) expression patterns in cells, tissues, including mammalian hearts. However, lectins have received little attention in zebrafish (Danio rerio) and giant danio (Devario aequipinnatus) heart studies. Here, we sought to determine the binding patterns of six commonly used lectins—wheat germ agglutinin (WGA), Ulex europaeus agglutinin, Bandeiraea simplicifolia lectin (BS lectin), concanavalin A (Con A), Ricinus communis agglutinin I (RCA I), and Lycopersicon esculentum agglutinin (tomato lectin)—in these hearts. Con A showed broad staining in the myocardium. WGA stained cardiac myocyte borders, with binding markedly stronger in the compact heart and bulbus. BS lectin, which stained giant danio coronaries, was used to measure vascular reconstruction during regeneration. However, BS lectin reacted poorly in zebrafish. RCA I stained the compact heart of both fish. Tomato lectin stained the giant danio, and while low reactivity was seen in the zebrafish ventricle, staining was observed in their transitional cardiac myocytes. In addition, we observed unique staining patterns in the developing zebrafish heart. Lectins’ ability to reveal differential glycoconjugate expression in giant danio and zebrafish hearts suggests they can serve as simple but important tools in studies of developing, adult, and regenerating fish hearts. PMID:27680670

Behavioural responses to novelty or to a predator stimulus are not altered in adult zebrafish by early embryonic alcohol exposure

PubMed Central

Seguin, Diane; Shams, Soaleha; Gerlai, Robert

2016-01-01

Background Fetal Alcohol Spectrum Disorders (FASD) may vary in symptoms and severity. In the milder and more prevalent forms of the disease, behavioural abnormalities may include impaired social behaviour, e.g. difficulty interpreting social cues. FASD patients remain often undiagnosed due to lack of biomarkers, and treatment is unavailable because the mechanisms of the disease are not yet understood. Animal models have been proposed to facilitate addressing these problems. More recently, short exposure of the zebrafish embryo to low concentrations of alcohol was shown to lead to significant and lasting impairment of behaviour in response to social stimuli. The impairment may be the result of abnormal social behaviour or altered fear/anxiety. The goal of the current study was to investigate the latter. Methods Here, we employed the alcohol exposure regimen used previously (exposure of 24th hour post-fertilization embryos to 0.00, 0.25, 0.50, 0.75 or 1.00 vol/vol % alcohol for 2 hours), allowed the fish to reach adulthood, and measured the behavioural responses of these adults to a novel tank (anxiety related behaviours) as well as to an animated image of a sympatric predator of zebrafish (fear related behaviours). Results We found behavioural responses of embryonic alcohol exposed adult fish to remain statistically indistinguishable from those of controls, suggesting unaltered anxiety and fear in the embryonic alcohol treated fish. Conclusions Given that motor and perceptual function was previously shown to be also unaltered in the adults after embryonic alcohol exposure, our current results suggest that the impaired response of these fish to social stimuli may be the result of abnormal social behaviour. PMID:27790739

In silico genome wide mining of conserved and novel miRNAs in the brain and pineal gland of Danio rerio using small RNA sequencing data.

PubMed

Agarwal, Suyash; Nagpure, Naresh Sahebrao; Srivastava, Prachi; Kushwaha, Basdeo; Kumar, Ravindra; Pandey, Manmohan; Srivastava, Shreya

2016-03-01

MicroRNAs (miRNAs) are small, non-coding RNA molecules that bind to the mRNA of the target genes and regulate the expression of the gene at the post-transcriptional level. Zebrafish is an economically important freshwater fish species globally considered as a good predictive model for studying human diseases and development. The present study focused on uncovering known as well as novel miRNAs, target prediction of the novel miRNAs and the differential expression of the known miRNA using the small RNA sequencing data of the brain and pineal gland (dark and light treatments) obtained from NCBI SRA. A total of 165, 151 and 145 known zebrafish miRNAs were found in the brain, pineal gland (dark treatment) and pineal gland (light treatment), respectively. Chromosomes 4 and 5 of zebrafish reference assembly GRCz10 were found to contain maximum number of miR genes. The miR-181a and miR-182 were found to be highly expressed in terms of number of reads in the brain and pineal gland, respectively. Other ncRNAs, such as tRNA, rRNA and snoRNA, were curated against Rfam. Using GRCz10 as reference, the subsequent bioinformatic analyses identified 25, 19 and 9 novel miRNAs from the brain, pineal gland (dark treatment) and pineal gland (light treatment), respectively. Targets of the novel miRNAs were identified, based on sequence complementarity between miRNAs and mRNA, by searching for antisense hits in the 3'-UTR of reference RNA sequences of the zebrafish. The discovery of novel miRNAs and their targets in the zebrafish genome can be a valuable scientific resource for further functional studies not only in zebrafish but also in other economically important fishes.

"Young at heart": Regenerative potential linked to immature cardiac phenotypes.

PubMed

Gomes, Renata S M; Skroblin, Philipp; Munster, Alex B; Tomlins, Hannah; Langley, Sarah R; Zampetaki, Anna; Yin, Xiaoke; Wardle, Fiona C; Mayr, Manuel

2016-03-01

The adult human myocardium is incapable of regeneration; yet, the zebrafish (Danio rerio) can regenerate damaged myocardium. Similar to the zebrafish heart, hearts of neonatal, but not adult mice are capable of myocardial regeneration. We performed a proteomics analysis of adult zebrafish hearts and compared their protein expression profile to hearts from neonatal and adult mice. Using difference in-gel electrophoresis (DIGE), there was little overlap between the proteome from adult mouse (>8weeks old) and adult zebrafish (18months old) hearts. Similarly, there was a significant degree of mismatch between the protein expression in neonatal and adult mouse hearts. Enrichment analysis of the selected proteins revealed over-expression of DNA synthesis-related proteins in the cardiac proteome of the adult zebrafish heart similar to neonatal and 4days old mice, whereas in hearts of adult mice there was a mitochondria-related predominance in protein expression. Importantly, we noted pronounced differences in the myofilament composition: the adult zebrafish heart lacks many of the myofilament proteins of differentiated adult cardiomyocytes such as the ventricular isoforms of myosin light chains and nebulette. Instead, troponin I and myozenin 1 were expressed as skeletal isoforms rather than cardiac isoforms. The relative immaturity of the adult zebrafish heart was further supported by cardiac microRNA data. Our assessment of zebrafish and mammalian hearts challenges the assertions on the translational potential of cardiac regeneration in the zebrafish model. The immature myofilament composition of the fish heart may explain why adult mouse and human cardiomyocytes lack this endogenous repair mechanism. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Resveratrol ameliorates diet-induced dysregulation of lipid metabolism in zebrafish (Danio rerio)

PubMed Central

Li, Lin; Yan, Qiaoqiao; Yi, Weijie; Ying, Chenjiang; Wu, Hongmei

2017-01-01

Defective lipid metabolism is associated with increased risk of various chronic diseases, such as obesity, cardiovascular diseases, and diabetes. Resveratrol (RSV), a natural polyphenol, has been shown the potential of ameliorating disregulations of lipid metabolism. The objective of this study was to investigate the effects of feed intake and RSV on lipid metabolism in zebrafish (Danio rerio). The adult males were randomly allocated to 6 groups: control (Con, 8 mg cysts/fish/day), control with 20 μmol/L RSV (Con+RSV), calorie restriction (CR, 5 mg cysts/fish/day), calorie restriction with RSV (CR+RSV), overfeed (OF, 60 mg cysts/fish/day), and overfeed with RSV (OF+RSV) groups. The treatment period was 8 weeks. Results showed that CR reduced body length, body weight, and condition factor of zebrafish. CR reduced levels of plasma triglyceride (TG) and induced protein expression of phosphorylated AMP-activated protein kinase-α (pAMPKα), silent information regulator 2 homolog 1 (Sirt1), and peroxisome proliferator activated receptor gamma coactivator-1α (PGC1α). RSV attenuated CR-induced pAMPKα/AMPKαincreases. RSV increased levels of Sirt1 protein in the OF zebrafish, and decreased OF-induced increase in peroxisome proliferator-activated receptor-γ (PPARγ) protein level. Additionally, RSV down-regulated caveolin-1 and up-regulated microtubule-associated protein 1 light chain 3 -II (LC3-II) protein levels in OF zebrafish. In conclusion, these results suggest that 1) CR reduces plasma TG level through activation of the AMPKα-Sirt1- PGC1α pathway; 2) under different dietary stress conditions RSV might regulate AMPK phosphorylation bi-directionally; 3) RSV might regulate lipid metabolism through the AMPKα-Sirt1-PPARγ pathway in OF zebrafish. PMID:28686680

Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Xiaoxia; University of Chinese Academy of Sciences, Beijing; Chen, Xiaowen

The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were allmore » identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO{sub 4}, dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. - Highlights: • Identification of the promoter of zebrafish glycoprotein subunit α (gsuα) gene • Generation of stable transmission gsuα:EGFP transgenic zebrafish reporter • Demonstration of the recapitulation of the gsuα:EGFP and endogenous gsuα expression • Suggestion of the gsuα:EGFP transgenic zebrafish as a novel platform for EDC study.« less

Zebrafish embryos exposed to alcohol undergo abnormal development of motor neurons and muscle fibers.

PubMed

Sylvain, Nicole J; Brewster, Daniel L; Ali, Declan W

2010-01-01

Children exposed to alcohol in utero have significantly delayed gross and fine motor skills, as well as deficiencies in reflex development. The reasons that underlie the motor deficits caused by ethanol (EtOH) exposure remain to be fully elucidated. The present study was undertaken to investigate the effects of embryonic alcohol exposure (1.5%, 2% and 2.5% EtOH) on motor neuron and muscle fiber morphology in 3 days post fertilization (dpf) larval zebrafish. EtOH treated fish exhibited morphological deformities and fewer bouts of swimming in response to touch, compared with untreated fish. Immunolabelling with anti-acetylated tubulin indicated that fish exposed to 2.5% EtOH had significantly higher rates of motor neuron axon defects. Immunolabelling of primary and secondary motor neurons, using znp-1 and zn-8, revealed that fish exposed to 2% and 2.5% EtOH exhibited significantly higher rates of primary and secondary motor neuron axon defects compared to controls. Examination of red and white muscle fibers revealed that fish exposed to EtOH had significantly smaller fibers compared with controls. These findings indicate that motor neuron and muscle fiber morphology is affected by early alcohol exposure in zebrafish embryos, and that this may be related to deficits in locomotion. Copyright 2010 Elsevier Inc. All rights reserved.

Alternative methods for toxicity assessments in fish: comparison of the fish embryo toxicity and the larval growth and survival tests in zebrafish and fathead minnows.

PubMed

Jeffries, Marlo K Sellin; Stultz, Amy E; Smith, Austin W; Rawlings, Jane M; Belanger, Scott E; Oris, James T

2014-11-01

An increased demand for chemical toxicity evaluations has resulted in the need for alternative testing strategies that address animal welfare concerns. The fish embryo toxicity (FET) test developed for zebrafish (Danio rerio) is one such alternative, and the application of the FET test to other species such as the fathead minnow (Pimephales promelas) has been proposed. In the present study, the performances of the FET test and the larval growth and survival (LGS; a standard toxicity testing method) test in zebrafish and fathead minnows were evaluated. This required that testing methods for the fathead minnow FET and zebrafish LGS tests be harmonized with existing test methods and that the performance of these testing strategies be evaluated by comparing the median lethal concentrations of 2 reference toxicants, 3,4-dicholoraniline and ammonia, obtained via each of the test types. The results showed that procedures for the zebrafish FET test can be adapted and applied to the fathead minnow. Differences in test sensitivity were observed for 3,4-dicholoraniline but not ammonia; therefore, conclusions regarding which test types offer the least or most sensitivity could not be made. Overall, these results show that the fathead minnow FET test has potential as an alternative toxicity testing strategy and that further analysis with other toxicants is warranted in an effort to better characterize the sensitivity and feasibility of this testing strategy. © 2014 SETAC.

Evaluating the zebrafish embryo toxicity test for pesticide hazard screening.

PubMed

Glaberman, Scott; Padilla, Stephanie; Barron, Mace G

2017-05-01

Given the numerous chemicals used in society, it is critical to develop tools for accurate and efficient evaluation of potential risks to human and ecological receptors. Fish embryo acute toxicity tests are 1 tool that has been shown to be highly predictive of standard, more resource-intensive, juvenile fish acute toxicity tests. However, there is also evidence that fish embryos are less sensitive than juvenile fish for certain types of chemicals, including neurotoxicants. The utility of fish embryos for pesticide hazard assessment was investigated by comparing published zebrafish embryo toxicity data from pesticides with median lethal concentration 50% (LC50) data for juveniles of 3 commonly tested fish species: rainbow trout, bluegill sunfish, and sheepshead minnow. A poor, albeit significant, relationship (r 2  = 0.28; p < 0.05) was found between zebrafish embryo and juvenile fish toxicity when pesticides were considered as a single group, but a much better relationship (r 2  = 0.64; p < 0.05) when pesticide mode of action was factored into an analysis of covariance. This discrepancy is partly explained by the large number of neurotoxic pesticides in the dataset, supporting previous findings that commonly used fish embryo toxicity test endpoints are particularly insensitive to neurotoxicants. These results indicate that it is still premature to replace juvenile fish toxicity tests with embryo-based tests such as the Organisation for Economic Co-operation and Development Fish Embryo Acute Toxicity Test for routine pesticide hazard assessment, although embryo testing could be used with other screening tools for testing prioritization. Environ Toxicol Chem 2017;36:1221-1226. © 2016 SETAC. © 2016 SETAC.

Small Fish Species as Powerful Model Systems to Study Vertebrate Physiology in Space

NASA Astrophysics Data System (ADS)

Muller, M.; Aceto, J.; Dalcq, J.; Alestrom, P.; Nourizadeh-Lillabadi, R.; Goerlich, R.; Schiller, V.; Winkler, C.; Renn, J.; Eberius, M.; Slenzka, K.

2008-06-01

Small fish models, mainly zebrafish (Danio rerio) and medaka (Oryzias latipes), have been used for many years as powerful model systems for vertebrate developmental biology. Moreover, these species are increasingly recognized as valuable systems to study vertebrate physiology, pathology, pharmacology and toxicology, including in particular bone physiology. The biology of small fishes presents many advantages, such as transparency of the embryos, external and rapid development, small size and easy reproduction. Further characteristics are particularly useful for space research or for large scale screening approaches. Finally, many technologies for easily characterizing bones are available. Our objective is to investigate the changes induced by microgravity in small fish. By combining whole genome analysis (microarray, DNA methylation, chromatin modification) with live imaging of selected genes in transgenic animals, a comprehensive and integrated characterization of physiological changes in space could be gained, especially concerning bone physiology.

Edwardsiellosis Caused by Edwardsiella ictaluri in Laboratory Populations of Zebrafish Danio rerio

PubMed Central

Hawke, John P.; Kent, Michael; Rogge, Matt; Baumgartner, Wes; Wiles, Judy; Shelley, Johnny; Savolainen, L. Christine; Wagner, Robert; Murray, Katy; Peterson, Tracy S.

2014-01-01

We report the first cases of Edwardsiella ictaluri causing epizootics in laboratory populations of Zebrafish Danio rerio. Edwardsiella ictaluri is primarily recognized as a disease of catfish species and is known to cause an economically important bacterial disease of farm-raised catfish in the USA and abroad; however, it has been isolated on occasion from 10 other genera of nonictalurid fishes. We isolated E. ictaluri from moribund Zebrafish held in quarantine at two different universities in two states and from a research facility in a third state between February 23 and December 6, 2011. Edwardsiellosis in Zebrafish can be described as a severe systemic disease characterized by tissue necrosis and the presence of large numbers of extracellular and intracellular bacteria, often within macrophages. The kidneys (pronephros and mesonephros), spleen, nares, and forebrain were the most commonly and severely affected tissues. In outbreaks, mortality was acute and numerous fish died over a 1–2 week period. Mortality continued until the majority of the population was lost, at which time the remaining fish were euthanized. In addition to these cases, four cultures of bacteria isolated from Zebrafish by another diagnostic laboratory were submitted to the Louisiana Aquatic Diagnostic Laboratory for identification and were confirmed as E. ictaluri. In total, eight cultures of E. ictaluri from Zebrafish from Louisiana, Massachusetts, Pennsylvania, and Florida were identified. The isolates were confirmed as E. ictaluri by biochemical phenotype, API 20E (bioMérieux), and amplification and sequencing of a portion of the 16S rRNA gene. Edwardsiella ictaluri isolates from Zebrafish are believed to comprise a unique group and were differentiated from catfish isolates by exhibiting weaker motility, autoaggregation in broth, a different plasmid profile (two plasmids of 4.0 and 3.5 kb), a different API 20E code (4204000), and lack of lipopolysaccharide recognition with Mab Ed9. PMID:23865817

Behavioral and Molecular Analysis of Nicotine-Conditioned Place Preference in Zebrafish

PubMed Central

Kedikian, Ximena; Faillace, Maria Paula; Bernabeu, Ramón

2013-01-01

Studies using mice and rats have demonstrated that nicotine induces a conditioned place preference (CPP), with more effective results obtained by using biased procedures. Zebrafish have also been used as a model system to identify factors influencing nicotine-associated reward by using an unbiased design. Here, we report that zebrafish exhibited putative nicotine biased CPP to an initially aversive compartment (nicotine-paired group). A counterbalanced nicotine-exposed control group did not show a significant preference shift, providing evidence that the preference shift in the nicotine-paired group was not due to a reduction of aversion for this compartment. Zebrafish preference was corroborated by behavioral analysis of several indicators of drug preference, such as time spent in the drug-paired side, number of entries to the drug-paired side, and distance traveled. These results provided strong evidence that zebrafish may actually develop a preference for nicotine, although the drug was administrated in an aversive place for the fish, which was further supported by molecular studies. Reverse transcription-quantitative real-time PCR analysis depicted a significant increase in the expression of α7 and α6 but not α4 and β2 subunits of the nicotinic receptor in nicotine-paired zebrafish brains. In contrast, zebrafish brains from the counterbalanced nicotine group showed no significant changes. Moreover, CREB phosphorylation, an indicator of neural activity, accompanied the acquisition of nicotine-CPP. Our studies offered an incremental value to the drug addiction field, because they further describe behavioral features of CPP to nicotine in zebrafish. The results suggested that zebrafish exposed to nicotine in an unfriendly environment can develop a preference for that initially aversive place, which is likely due to the rewarding effect of nicotine. Therefore, this model can be used to screen exogenous and endogenous molecules involved in nicotine-associated reward in vertebrates. PMID:23894483

Facility Design and Health Management Program at the Sinnhuber Aquatic Research Laboratory

PubMed Central

Barton, Carrie L.; Johnson, Eric W.

2016-01-01

Abstract The number of researchers and institutions moving to the utilization of zebrafish for biomedical research continues to increase because of the recognized advantages of this model. Numerous factors should be considered before building a new or retooling an existing facility. Design decisions will directly impact the management and maintenance costs. We and others have advocated for more rigorous approaches to zebrafish health management to support and protect an increasingly diverse portfolio of important research. The Sinnhuber Aquatic Research Laboratory (SARL) is located ∼3 miles from the main Oregon State University campus in Corvallis, Oregon. This facility supports several research programs that depend heavily on the use of adult, larval, and embryonic zebrafish. The new zebrafish facility of the SARL began operation in 2007 with a commitment to build and manage an efficient facility that diligently protects human and fish health. An important goal was to ensure that the facility was free of Pseudoloma neurophilia (Microsporidia), which is very common in zebrafish research facilities. We recognize that there are certain limitations in space, resources, and financial support that are institution dependent, but in this article, we describe the steps taken to build and manage an efficient specific pathogen-free facility. PMID:26981844

Facility Design and Health Management Program at the Sinnhuber Aquatic Research Laboratory.

PubMed

Barton, Carrie L; Johnson, Eric W; Tanguay, Robert L

2016-07-01

The number of researchers and institutions moving to the utilization of zebrafish for biomedical research continues to increase because of the recognized advantages of this model. Numerous factors should be considered before building a new or retooling an existing facility. Design decisions will directly impact the management and maintenance costs. We and others have advocated for more rigorous approaches to zebrafish health management to support and protect an increasingly diverse portfolio of important research. The Sinnhuber Aquatic Research Laboratory (SARL) is located ∼3 miles from the main Oregon State University campus in Corvallis, Oregon. This facility supports several research programs that depend heavily on the use of adult, larval, and embryonic zebrafish. The new zebrafish facility of the SARL began operation in 2007 with a commitment to build and manage an efficient facility that diligently protects human and fish health. An important goal was to ensure that the facility was free of Pseudoloma neurophilia (Microsporidia), which is very common in zebrafish research facilities. We recognize that there are certain limitations in space, resources, and financial support that are institution dependent, but in this article, we describe the steps taken to build and manage an efficient specific pathogen-free facility.

Model of voluntary ethanol intake in zebrafish: Effect on behavior and hypothalamic orexigenic peptides

PubMed Central

Sterling, M.E.; Karatayev, O.; Chang, G.-Q.; Algava, D.B.; Leibowitz, S.F

2014-01-01

Recent studies in zebrafish have shown that exposure to ethanol in tank water affects various behaviors, including locomotion, anxiety and aggression, and produces changes in brain neurotransmitters, such as serotonin and dopamine. Building on these investigations, the present study had two goals: first, to develop a method for inducing voluntary ethanol intake in individual zebrafish, which can be used as a model in future studies to examine how this behavior is affected by various manipulations, and second, to characterize the effects of this ethanol intake on different behaviors and the expression of hypothalamic orexigenic peptides, galanin (GAL) and orexin (OX), which are known in rodents to stimulate consumption of ethanol and alter behaviors associated with alcohol abuse. Thus, we first developed a new model of voluntary intake of ethanol in fish by presenting this ethanol mixed with gelatin, which they readily consume. Using this model, we found that individual zebrafish can be trained in a short period of time to consume stable levels of 10% or 20% ethanol (v/v) mixed with gelatin and that their intake of this ethanol-gelatin mixture leads to pharmacologically-relevant blood ethanol concentrations which are strongly, positively correlated with the amount ingested. Intake of this ethanol-gelatin mixture increased locomotion, reduced anxiety, and stimulated aggressive behavior, while increasing expression of GAL and OX in specific hypothalamic areas. These findings, confirming results in rats, provide a method in zebrafish for investigating with forward genetics and pharmacological techniques the role of different brain mechanisms in controlling ethanol intake. PMID:25257106

Distribution of PEG-coated hollow polyelectrolyte microcapsules after introduction into the circulatory system and muscles of zebrafish

PubMed Central

2018-01-01

ABSTRACT The use of polyelectrolyte multilayer microcapsules as carriers for fluorescent molecular probes is a prospective technique for monitoring the physiological characteristics of animal vasculature and interstitial environment in vivo. Polyelectrolyte microcapsules have many features that favor their use as implantable carriers of optical sensors, but little information is available on their interactions with complex living tissues, distribution or residence time following different routes of administration in the body of vertebrates. Using the common fish model, the zebrafish Danio rerio, we studied in vivo the distribution of non-biodegradable microcapsules covered with polyethylene glycol (PEG) over time in the adults and evaluated potential side effects of their delivery into the fish bloodstream and muscles. Fluorescent microcapsules administered into the bloodstream and interstitially (in concentrations that were sufficient for visualization and spectral signal recording) both showed negligible acute toxicity to the fishes during three weeks of observation. The distribution pattern of microcapsules delivered into the bloodstream was stable for at least one week, with microcapsules prevalent in capillaries-rich organs. However, after intramuscular injection, the phagocytosis of the microcapsules by immune cells was manifested, indicating considerable immunogenicity of the microcapsules despite PEG coverage. The long-term negative effects of chronic inflammation were also investigated in fish muscles by histological analysis. PMID:29305467

Automated Lab-on-a-Chip Technology for Fish Embryo Toxicity Tests Performed under Continuous Microperfusion (μFET).

PubMed

Zhu, Feng; Wigh, Adriana; Friedrich, Timo; Devaux, Alain; Bony, Sylvie; Nugegoda, Dayanthi; Kaslin, Jan; Wlodkowic, Donald

2015-12-15

The fish embryo toxicity (FET) biotest has gained popularity as one of the alternative approaches to acute fish toxicity tests in chemical hazard and risk assessment. Despite the importance and common acceptance of FET, it is still performed in multiwell plates and requires laborious and time-consuming manual manipulation of specimens and solutions. This work describes the design and validation of a microfluidic Lab-on-a-Chip technology for automation of the zebrafish embryo toxicity test common in aquatic ecotoxicology. The innovative device supports rapid loading and immobilization of large numbers of zebrafish embryos suspended in a continuous microfluidic perfusion as a means of toxicant delivery. Furthermore, we also present development of a customized mechatronic automation interface that includes a high-resolution USB microscope, LED cold light illumination, and miniaturized 3D printed pumping manifolds that were integrated to enable time-resolved in situ analysis of developing fish embryos. To investigate the applicability of the microfluidic FET (μFET) in toxicity testing, copper sulfate, phenol, ethanol, caffeine, nicotine, and dimethyl sulfoxide were tested as model chemical stressors. Results obtained on a chip-based system were compared with static protocols performed in microtiter plates. This work provides evidence that FET analysis performed under microperfusion opens a brand new alternative for inexpensive automation in aquatic ecotoxicology.

Effects of dietary exposure to brominated flame retardant BDE-47 on thyroid condition, gonadal development and growth of zebrafish.

PubMed

Torres, Leticia; Orazio, Carl E; Peterman, Paul H; Patiño, Reynaldo

2013-10-01

Little is known about the effects of brominated flame retardants in teleosts and some of the information currently available is inconsistent. This study examined effects of dietary exposure to 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on thyroid condition, body mass and size, and gonadal development of zebrafish. Pubertal, 49-day-old (posthatch) fish were fed diets without BDE-47 (control) or with 1, 5 or 25 μg/g BDE-47/diet. Treatments were conducted in triplicate 30-L tanks each containing 50 zebrafish, and 15 fish per treatment (5 per tank) were sampled at days 40, 80 and 120 of exposure. Measurements were taken of body mass, standard length, head depth and head length. Sex (at 40-120 days of exposure), germ cell stage (at 40 days) and thyroid condition (at 120 days; follicular cell height, colloid depletion, angiogenesis) were histologically determined. Whole-body BDE-47 levels at study completion were within the high end of levels reported in environmentally exposed (wild) fishes. Analysis of variance was used to determine differences among treatments at each sampling time. No effects were observed on thyroid condition or germ cell stage in either sex. Reduced head length was observed in females exposed to BDE-47 at 80 days but not at 40 or 120 days. In males, no apparent effects of BDE-47 were observed at 40 and 80 days, but fish exposed to 25 μg/g had lower body mass at 120 days compared to control fish. These observations suggest that BDE-47 at environmentally relevant whole-body concentrations does not affect thyroid condition or pubertal development of zebrafish but does affect growth during the juvenile-to-adult transition, especially in males.

First Record of Transversotrema Witenberg, 1944 (Digenea) from the Americas, with Comments on the Taxonomy of Transversotrema patialense (Soparkar, 1924) Crusz and Sathananthan, 1960, and an Updated List of Its Hosts and Geographic Distribution.

PubMed

Womble, Matthew R; Cox-Gardiner, Stephanie J; Cribb, Thomas H; Bullard, Stephen A

2015-12-01

Specimens of Transversotrema patialense (sensu lato) ( Soparkar, 1924 ) Crusz and Sathananthan, 1960 (Digenea: Transversotrematidae) infected the skin (epidermal spaces beneath scales near pectoral fins) of 4 of 126 (prevalence 3%; mean intensity 1.8) zebrafish ( Danio rerio (Hamilton, 1822) [Cypriniformes: Cyprinidae]) purchased in 2009 and cultured by a California (USA) fish supplier. These fish were sold as "laboratory-reared" and "specific pathogen free," purportedly raised in a recirculating aquaculture system that included zebrafish only. We herein describe the morphological features of this transversotrematid using light and scanning electron microscopy, provide a comprehensive list of hosts (snails and fishes) and geographic locality records for specimens reported as T. patialense, which is perhaps a species complex, and provide a brief historical synopsis of the taxonomic and life history research that has been conducted on this fluke. No species of Transversotrema previously had been reported from the Americas; however, this discovery is not surprising given that: (1) a suitable intermediate host (red-rimmed melania, Melanoides tuberculata (Müller, 1774) [Cerithioidea: Thiaridae]) has been established in California and elsewhere in North America, (2) the zebrafish is a susceptible definitive host, and (3) T. patialense reportedly matures on a broad ecological and phylogenetic spectrum of freshwater fishes. To our knowledge, this is the northern-most geographic locality record for a species of this genus. We suspect this case study represents an example of a parasite that may now be established in North America by the fortuitous co-occurrence of a susceptible, exotic snail host (the red-rimmed melania) and a susceptible, widely distributed, exotic fish host (the zebrafish).

Genetic and environmental melanoma models in fish

PubMed Central

Patton, E Elizabeth; Mitchell, David L; Nairn, Rodney S

2010-01-01

Experimental animal models are extremely valuable for the study of human diseases, especially those with underlying genetic components. The exploitation of various animal models, from fruitflies to mice, has led to major advances in our understanding of the etiologies of many diseases, including cancer. Cutaneous malignant melanoma is a form of cancer for which both environmental insult (i.e., UV) and hereditary predisposition are major causative factors. Fish melanoma models have been used in studies of both spontaneous and induced melanoma formation. Genetic hybrids between platyfish and swordtails, different species of the genus Xiphophorus, have been studied since the 1920s to identify genetic determinants of pigmentation and melanoma formation. Recently, transgenesis has been used to develop zebrafish and medaka models for melanoma research. This review will provide a historical perspective on the use of fish models in melanoma research, and an updated summary of current and prospective studies using these unique experimental systems. PMID:20230482

Conditioned place avoidance of zebrafish (Danio rerio) to three chemicals used for euthanasia and anaesthesia.

PubMed

Wong, Devina; von Keyserlingk, Marina A G; Richards, Jeffrey G; Weary, Daniel M

2014-01-01

Zebrafish are becoming one of the most used vertebrates in developmental and biomedical research. Fish are commonly killed at the end of an experiment with an overdose of tricaine methanesulfonate (TMS, also known as MS-222), but to date little research has assessed if exposure to this or other agents qualifies as euthanasia (i.e. a "good death"). Alternative agents include metomidate hydrochloride and clove oil. We use a conditioned place avoidance paradigm to compare aversion to TMS, clove oil, and metomidate hydrochloride. Zebrafish (n = 51) were exposed to the different anaesthetics in the initially preferred side of a light/dark box. After exposure to TMS zebrafish spent less time in their previously preferred side; aversion was less pronounced following exposure to metomidate hydrochloride and clove oil. Nine of 17 fish exposed to TMS chose not to re-enter the previously preferred side, versus 2 of 18 and 3 of 16 refusals for metomidate hydrochloride and clove oil, respectively. We conclude that metomidate hydrochloride and clove oil are less aversive than TMS and that these agents be used as humane alternatives to TMS for killing zebrafish.

Functional and Structural Characterization of Zebrafish ASC.

PubMed

Li, Yajuan; Huang, Yi; Cao, Xiaocong; Yin, Xueying; Jin, Xiangyu; Liu, Sheng; Jiang, Jiansheng; Jiang, Wei; Xiao, Tsan Sam; Zhou, Rongbin; Cai, Gang; Hu, Bing; Jin, Tengchuan

2018-05-23

The zebrafish genome encodes homologs for most of the proteins involved in inflammatory pathways; however, the molecular components and activation mechanisms of fish inflammasomes are largely unknown. ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)) is the only adaptor involved in the formation of multiple types of inflammasomes. Here, we demonstrate that zASC is also involved in inflammasome activation in zebrafish. When overexpressed in vitro and in vivo in zebrafish, both the zASC and zASC pyrin domain (PYD) proteins form speck and filament structures. Importantly, the crystal structures of the N-terminal PYD and C-terminal CARD of zebrafish ASC were determined independently as two separate entities fused to maltose-binding protein (MBP). Structure-guided mutagenesis revealed the functional relevance of the PYD hydrophilic surface found in the crystal lattice. Finally, the fish caspase-1 homolog Caspy, but not the caspase-4/11 homolog Caspy2, interacts with zASC through homotypic PYD-PYD interactions, which differ from those in mammals. These observations establish the conserved and unique structural/functional features of the zASC-dependent inflammasome pathway. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Impaired tissue regeneration corresponds with altered expression of developmental genes that persists in the metabolic memory state of diabetic zebrafish

PubMed Central

Sarras, Michael P.; Leontovich, Alexey A.; Olsen, Ansgar S.; Intine, Robert V.

2013-01-01

As previously reported by our lab streptozocin-induced Diabetes mellitus (DM) in adult zebrafish results in an impairment of tissue regeneration as monitored by caudal fin regeneration. Following streptozocin withdrawal, a recovery phase occurs to re-establish euglycemia, via pancreatic beta-cell regeneration. However, DM-associated impaired fin regeneration continues indefinitely in the metabolic memory state (MM) allowing for subsequent molecular analysis of the underlying mechanisms of MM. This study focuses on elucidating the molecular basis that explains the DM-associated impaired fin regeneration and why it persists into the MM state with the aim of better understanding MM. Using a combination of microarray analysis and bioinformatics approaches our study found that of the 14,900 transcripts analyzed, aberrant expression of 71 genes relating to tissue developmental and regeneration processes were identified in DM fish and the altered expression of these 71 genes persisted in MM fish. Key regulatory genes of major development, and signal transduction pathways were identified among this group of 71. The aberrant expression of key regulatory genes in the DM state that persist into the MM state provides a plausible explanation for how hyperglycemia induced impaired fin regeneration in the adult zebrafish DM/MM model. PMID:23438205

Age matters: Developmental stage of Danio rerio larvae influences photomotor response thresholds to diazinion or diphenhydramine

PubMed Central

Kristofco, Lauren A.; Cruz, Luis Colon; Haddad, Samuel P.; Behra, Martine L; Chambliss, C. Kevin; Brooks, Bryan W.

2016-01-01

Because basic toxicological data is unavailable for the majority of industrial compounds, High Throughput Screening (HTS) assays using the embryonic and larval zebrafish provide promising approaches to define bioactivity profiles and identify potential adverse outcome pathways for previously understudied chemicals. Unfortunately, standardized approaches, including HTS experimental designs, for examining fish behavioral responses to contaminants are rarely available. In the present study, we examined movement behavior of larval zebrafish over 7 days (4–10 days post fertilization or dpf) during typical daylight workday hours to determine whether intrinsic activity differed with age and time of day. We then employed an early life stage approach using the Fish Embryo Test (FET) at multiple developmental ages to evaluate whether photomotor response (PMR) behavior differed with zebrafish age following exposure to diazinon (DZN), a well-studied orthophosphate insecticide, and diphenhydramine (DPH), an antihistamine that also targets serotonin reuptake transporters and the acetylcholine receptor. 72 h studies were conducted at 1–4, 4–7 and 7–10 dpf, followed by behavioral observations using a ViewPoint system at 4, 7 and 10 dpf. Distance traveled and swimming speeds were quantified; nominal treatment levels were analytically verified by isotope-dilution LC-MSMS. Larval zebrafish locomotion displayed significantly different (p < 0.05) activity profiles over the course of typical daylight and workday hours, and these time of day PMR activity profiles were similar across ages examined (4–10 dpf). 10 dpf zebrafish larvae were consistently more sensitive to DPH than either the 4 or 7 dpf larvae with an environmentally realistic lowest observed effect concentration of 200 ng/L. Though ELS and FET studies with zebrafish typically focus on mortality or teratogenicity in 0–4 dpf organisms, behavioral responses of slightly older fish were several orders of magnitude more sensitive to DPH. Our observations highlight the importance of understanding the influence of time of day on intrinsic locomotor activity, and the age-specific hazards of aquatic contaminants to fish behavior. PMID:26431593

Inducible Sterilization of Zebrafish by Disruption of Primordial Germ Cell Migration

PubMed Central

Wong, Ten-Tsao; Collodi, Paul

2013-01-01

During zebrafish development, a gradient of stromal-derived factor 1a (Sdf1a) provides the directional cue that guides the migration of the primordial germ cells (PGCs) to the gonadal tissue. Here we describe a method to produce large numbers of infertile fish by inducing ubiquitous expression of Sdf1a in zebrafish embryos resulting in disruption of the normal PGC migration pattern. A transgenic line of zebrafish, Tg(hsp70:sdf1a-nanos3, EGFP), was generated that expresses Sdf1a under the control of the heat-shock protein 70 (hsp70) promoter and nanos3 3?UTR. To better visualize the PGCs, the Tg(hsp70:sdf1a-nanos3, EGFP) fish were crossed with another transgenic line, Tg(kop:DsRed-nanos3), that expresses DsRed driven by the PGC-specific kop promoter. Heat treatment of the transgenic embryos caused an induction of Sdf1a expression throughout the embryo resulting in the disruption of their normal migration. Optimal embryo survival and disruption of PGC migration was achieved when transgenic embryos at the 4- to 8-cell stage were incubated at 34.5°C for 18 hours. Under these conditions, disruption of PGC migration was observed in 100% of the embryos. Sixty-four adult fish were developed from three separate batches of heat-treated embryos and all were found to be infertile males. When each male was paired with a wild-type female, only unfertilized eggs were produced and histological examination revealed that each of the adult male fish possessed severely under-developed gonads that lacked gametes. The results demonstrate that inducible Sdf1a expression is an efficient and reliable strategy to produce infertile fish. This approach makes it convenient to generate large numbers of infertile adult fish while also providing the capability to maintain a fertile brood stock. PMID:23826390

Gene Expression Profiling of Androgen Receptor Antagonists Flutamide and Vinclozolin in Zebrafish (Danio rerio) Gonads

EPA Science Inventory

The studies presented in this manuscript focus on characterization of genomic responses to anti-androgens in zebrafish (Danio rerio). Research of the effects of anti-androgens in fish has been characterized by a heavy reliance on apical endpoints, and molecular mechanisms of acti...

Transcriptomic changes in zebrafish embryos and larvae following benzo[a]pyrene exposure

USDA-ARS?s Scientific Manuscript database

Benzo[a]pyrene (BaP) is an environmentally relevant carcinogenic and endocrine disrupting compound that causes immediate, long-term, and multigenerational health deficits in mammals and fish. Previously, we found that BaP alters DNA methylation patterns in developing zebrafish, which may affect gene...

Estrogenic effect of the phytoestrogen biochanin A in zebrafish, Danio rerio, and brown trout, Salmo trutta.

PubMed

Holbech, Henrik; Schröder, Kristoffer D; Nielsen, Marie L; Brande-Lavridsen, Nanna; Holbech, Bente Frost; Bjerregaard, Poul

2013-11-15

Isoflavones with estrogenic activity produced in Fabaceae plants are known to leach from agricultural areas to freshwater systems, but the effect of waterborne isoflavones in fish has not been thoroughly characterized. Therefore, the estrogenic effect of waterborne biochanin A was investigated in zebrafish (Danio rerio) and juvenile brown trout (Salmo trutta). Exposure of juvenile brown trout to 10 μg biochanin AL(-1) or higher caused marked vitellogenin induction after 9-10 days of exposure and so did exposure to 186 μg biochanin AL(-1) for 6h. Following 8d of exposure, a NOEC for induction of vitellogenin production in male zebrafish was 70 and LOEC 114 μg biochanin AL(-1). Exposure to 209 μg biochanin AL(-1) from hatch to 60 days post hatch (dph) caused a skewing of the sex ratio toward more phenotypic female zebrafish, but did not cause induction of vitellogenin in male and undifferentiated fish. (1) biochanin A elicits estrogenic effects in trout at environmentally realistic concentrations, (2) brown trout plasma vitellogenin concentrations respond to lower biochanin A exposure concentrations than vitellogenin concentrations in zebrafish homogenates and (3) concerning vitellogenin induction, the hypothesis should be tested if short term tests with zebrafish may show a higher sensitivity than partial life cycle tests. Copyright © 2013 Elsevier B.V. All rights reserved.

Fluoride exposure abates pro-inflammatory response and induces in vivo apoptosis rendering zebrafish (Danio rerio) susceptible to bacterial infections.

PubMed

Singh, Rashmi; Khatri, Preeti; Srivastava, Nidhi; Jain, Shruti; Brahmachari, Vani; Mukhopadhyay, Asish; Mazumder, Shibnath

2017-04-01

The present study describes the immunotoxic effect of chronic fluoride exposure on adult zebrafish (Danio rerio). Zebrafish were exposed to fluoride (71.12 mg/L; 1/10 LC 50 ) for 30 d and the expression of selected genes studied. We observed significant elevation in the detoxification pathway gene cyp1a suggesting chronic exposure to non-lethal concentration of fluoride is indeed toxic to fish. Fluoride mediated pro-oxidative stress is implicated with the downregulation in superoxide dismutase 1 and 2 (sod1/2) genes. Fluoride affected DNA repair machinery by abrogating the expression of the DNA repair gene rad51 and growth arrest and DNA damage inducible beta a gene gadd45ba. The upregulated expression of casp3a coupled with altered Bcl-2 associated X protein/B-cell lymphoma 2 ratio (baxa/bcl2a) clearly suggested chronic fluoride exposure induced the apoptotic cascade in zebrafish. Fluoride-exposed zebrafish when challenged with non-lethal dose of fish pathogen A. hydrophila revealed gross histopathology in spleen, bacterial persistence and significant mortality. We report that fluoride interferes with system-level output of pro-inflammatory cytokines tumour necrosis factor-α, interleukin-1β and interferon-γ, as a consequence, bacteria replicate efficiently causing significant fish mortality. We conclude, chronic fluoride exposure impairs the redox balance, affects DNA repair machinery with pro-apoptotic implications and suppresses pro-inflammatory cytokines expression abrogating host immunity to bacterial infections. Copyright © 2017 Elsevier Ltd. All rights reserved.

Surface Defects on Plate-Shaped Silver Nanoparticles Contribute to Its Hazard Potential in a Fish Gill Cell Line and Zebrafish Embyos

PubMed Central

George, Saji; Lin, Sijie; Ji, Zhaoxia; Thomas, Courtney; Li, LinJiang; Mecklenburg, Mathew; Meng, Huan; Wang, Xiang; Zhang, Haiyuan; Xia, Tian; Lin, Shuo; Hohman, J. Nathan; Zink, Jeffrey I.; Weiss, Paul; Nel, André E.

2014-01-01

We investigated and compared nano-size Ag spheres, plates, and wires in a fish gill epithelial cell line (RT-W1) and in zebrafish embryos to understand the mechanism of toxicity of an engineered nanomaterial raising considerable environmental concern. While most of the Ag nanoparticles induced N-acetyl cysteine sensitive toxic oxidative stress effects in RT-W1, Ag nanoplates were considerably more toxic than other particle shapes. Interestingly, while Ag ion shedding and bioavailability failed to explain the high toxicity of the nanoplates, cellular injury required direct particle contact, resulting in cell membrane lysis in RT-W1 as well as red blood cells (RBC). Ag nanoplates were also considerably more toxic in zebrafish embryos in spite of their lesser ability to shed Ag into the exposure medium. In order to elucidate the “surface reactivity” of Ag nanoplates, high-resolution transmission electron microscopy was performed and demonstrated a high level of crystal defects (stacking faults and point defects) on the nanoplate surfaces. Surface coating with cysteine was used to passivate the surface defects and demonstrated a reduction of toxicity in RT-W1 cells, RBC, and zebrafish embryos. This study demonstrates the important role of crystal defects in contributing to Ag nanoparticle toxicity in addition to the established roles of Ag ion shed from spherical nanoparticles. The excellent correlation between the in vitro and in vivo toxicological assessment illustrates the utility of using a fish cell line in parallel with zebrafish embryos to perform a predictive environmental toxicological paradigm. PMID:22482460

Fish Stem Cell Cultures

PubMed Central

Hong, Ni; Li, Zhendong; Hong, Yunhan

2011-01-01

Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on “Fish Stem Cells and Nuclear Transfer”, we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer. PMID:21547056

Fish stem cell cultures.

PubMed

Hong, Ni; Li, Zhendong; Hong, Yunhan

2011-04-13

Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

A novel galanin receptor 1a gene in zebrafish: tissue distribution, developmental expression roles in nutrition regulation.

PubMed

Li, Linfang; Wei, Shulei; Huang, Qiaoyan; Feng, Dong; Zhang, Shicui; Liu, Zhenhui

2013-03-01

Galanin (Gal), a 29 (30 in human) amino acid neuropeptide, exerts its biological activities through three different G protein-coupled receptors, namely GalR1, GalR2 and GalR3. However, we previously found that only GalR1 and GalR2 exist in fish, and fish GalR1 has two genes (GalR1a and GalR1b), with GalR1a possibly representing the primitive gene form during fish evolution. To uncover the functions of GalR1a in fish, here the tissue distribution, developmental expression and the role in nutrition regulation of GalR1a were investigated in zebrafish (Danio rerio). Interestingly, the expression of GalR1a mRNA was restricted to the intestine and brain in adult zebrafish, while GalR1b mRNA was present in all tissues tested. During embryogenesis, GalR1a mRNA was abundant at 1hpf (hour past fertilization) and decreased gradually in abundance from 3 hpf to 10 hpf; then a significant increase in the amount of GalR1a transcripts was observed at 35 hpf, and this high level was maintained until 5 dpf (day past fertilization). In situ hybridization of embryos and larvae, expression pattern of GalR1a was mainly restricted to the intestine, pectoral fin, branchial arches and head, indicating a role of GalR1a during zebrafish embryogenesis. Quantitative real-time PCR assay suggested that fasting, high fat feeding or linoleic acid (LA) all could significantly induce up-regulation of GalR1a both in vitro and in vivo, suggesting roles of GalR1a in control of nutrition intake, especially to fat. In addition, a potential role of zebrafish GalR1a in accumulation of lipid droplets in cells was also demonstrated. Our study lays a foundation for further investigation of GalR1a function and evolution in fish. Copyright © 2012 Elsevier Inc. All rights reserved.

Impacts of 17beta-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish (Danio rerio).

PubMed

Brion, F; Tyler, C R; Palazzi, X; Laillet, B; Porcher, J M; Garric, J; Flammarion, P

2004-06-24

Zebrafish (Danio rerio) were exposed for 3 weeks to low concentrations of estradiol including environmentally relevant concentrations (5, 25 and 100 ng/l), encompassing either their embryo-larvae (from fertilization to 21 day post-fertilization (dpf)), juvenile (from 21 to 42 dpf) or adult life stages (>200 dpf) with a view to investigating the most sensitive life stage of the zebrafish to 17beta-estradiol (E2). At all sampling points, whole-body vitellogenin concentrations and gonadal development were analyzed in order to investigate the effects of estrogen exposure on these endpoint in the zebrafish. In the adult stage, additional endpoints were measured including secondary sexual characteristics (manifestation of the uro-genital papillae (UGP) in males), gonadal growth (the gonado-somatic index (GSI)) and sex ratio. For all the different life stage exposures, reproductive performance of the F0 generation was assessed (egg production) and survival and development of the F1 embryo-larvae. Exposure to low concentrations of E2 resulted in vitellogenin induction whatever the life stage exposed but these effects were reversible after depuration. The effective concentration for vitellogenin induction in zebrafish early life stages was 100 ng E2/l, and in adult male zebrafish the effective concentration for vitellogenin induction (between 5 and 25 ng/l) was lower than for the early life stage fish. Exposure to E2 prior to (from fertilization to 21 dpf) and during the time of sex differentiation (from 21 to 42 dpf) also caused disruptions in the process of sexual differentiation (resulting in formation of a retrogonadal cavity in presumptive male, germ cell development and leading to a significant change of the sex ratio towards the female sex at the dose of 100 ng E2/l for the fish exposure as embryo-larvae) and altered patterns of egg production in the subsequent adults. Exposure of adult fish to E2 resulted in a modification of the secondary sexual characteristic in males at 25 and 100 ng E2/l as well as a dose-dependent inhibition of egg production. The findings from this study show that the nature and intensity of the reproductive effects of E2 are dependent of the time and concentration of exposures of zebrafish to E2, some of these effects being permanent (effect on the sexual differentiation) while others being reversible (effect on the Vtg induction). This study demonstrated that early life stages of zebrafish are sensitive to low concentrations of E2 and provides relevant data that could be used for the adaptation of existing fish early life stage test for the in vivo testing of estrogenic compounds. The data presented raise further concerns about the effects of steroid estrogens in the environment on fish reproductive health.

Just Keep Swimming: Neuroendocrine, Metabolic, and Behavioral Changes After a Forced Swimming Test in Zebrafish.

PubMed

da Rosa, João Gabriel Santos; Barcellos, Heloísa Helena de Alcântara; Idalencio, Renan; Marqueze, Alessandra; Fagundes, Michele; Rossini, Mainara; Variani, Cristiane; Balbinoti, Francine; Tietböhl, Tássia Michele Huff; Rosemberg, Denis Broock; Barcellos, Leonardo José Gil

2017-02-01

In this study, we show that an adaptation of the spinning test can be used as a model to study the exercise-exhaustion-recovery paradigm in fish. This forced swimming test promotes a wide range of changes in the hypothalamus-pituitary-interrenal axis functioning, intermediary metabolism, as well in fish behavior at both exercise and recovery periods. Our results pointed that this adapted spinning test can be considered a valuable tool for evaluating drugs and contaminant effects on exercised fish. This can be a suitable protocol both to environmental-to evaluate contaminants that act in fish energy mobilization and recovery after stressors-and translational perspectives-effects of drugs on exercised or stressed humans.

Inhibitory Effects of Red Wine on Lipid Oxidation in Fish Oil Emulsion and Angiogenesis in Zebrafish Embryo.

PubMed

Sun, Haiyan; Zhang, Yulin; Shen, Yixiao; Zhu, Yongchao; Wang, Hua; Xu, Zhimin

2017-03-01

The capabilities of red wine against lipid oxidation and angiogenesis were evaluated by using a fish oil emulsion system and an in vivo zebrafish embryos model, respectively. The red wine contained 12 different antioxidant phenolics which levels were led by anthocyanins (140.46 mg/L), catechin (55.08 mg/L), and gallic acid (46.76 mg/L). The diversity of the phenolics in red wine was greater than the tea, coffee, or white wine selected as a peer control in this study. The total phenolics concentration of red wine was 305.53 mg/L, although the levels of tea, coffee, and white wine were 85.59, 76.85, and 26.57 mg/L, respectively. The activity of red wine in scavenging DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals was approximately 4 times higher than the tea and 8 times than the coffee or white wine. The red wine showed the highest capability in preventing long chain PUFA oxidation in the fish oil emulsion. Because of the outstanding antioxidant activity of red wine, the red wine dried extract was used to monitor its inhibitory effect against angiogenesis by using transgenic zebrafish embryos (Tg[fli1:egfp] y1 ) with fluorescent blood vessels. After incubated in 100 μg/mL of the extract solution for 26 h pf, each of the embryos had a lower number of intersegmental vessel than the control embryo. The inhibition rate of red wine extract against growing of angiogenic blood vessel reached 100%. © 2017 Institute of Food Technologists®.

GH indirectly enhances the regeneration of transgenic zebrafish fins through IGF2a and IGF2b.

PubMed

Nornberg, Bruna Félix; Almeida, Daniela Volcan; Figueiredo, Márcio Azevedo; Marins, Luis Fernando

2016-10-01

The somatotropic axis, composed essentially of the growth hormone (GH) and insulin-like growth factors (IGFs), is the main regulator of somatic growth in vertebrates. However, these protein hormones are also involved in various other major physiological processes. Although the importance of IGFs in mechanisms involving tissue regeneration has already been established, little is known regarding the direct effects of GH in these processes. In this study, we used a transgenic zebrafish (Danio rerio) model, which overexpresses GH from the beta-actin constitutive promoter. The regenerative ability of the caudal fin was assessed after repeated amputations, as well as the expression of genes related to the GH/IGF axis. The results revealed that GH overexpression increased the regenerated area of the caudal fin in transgenic fish after the second amputation. Transgenic fish also presented a decrease in gene expression of the GH receptor (ghrb), in opposition to the increased expression of the IGF1 receptors (igf1ra and igf1rb). These results suggest that transgenic fish have a higher sensitivity to IGFs than to GH during fin regeneration. With respect to the different IGFs produced locally, a decrease in igf1a expression and a significant increase in both igf2a and igf2b expression was observed, suggesting that igf1a is not directly involved in fin regeneration. Overall, the results revealed that excess GH enhances fin regeneration in zebrafish through igf2a and igf2b expression, acting indirectly on this major physiological process.

USING 1H-NMR METABOLOMICS TO CHARACTERIZE ALTERED METABOLIC PROFILES IN ZEBRAFISH (DANIO RERIO) EXPOSED TO THE MODEL EDCS 17 ALPHA-ETHINYLESTRADIOL (EE2) AND FADROZOLE

EPA Science Inventory

Elevated levels of endocrine-disrupting chemicals (EDCs) have been reported in waterways worldwide and have been shown to affect numerous aspects of development, behavior, reproduction, and survival in various fish species. We have examined the effects of the synthetic steroid 17...

Fish in a Dish: Drug Discovery for Hearing Habilitation.

PubMed

Esterberg, Robert; Coffin, Allison B; Ou, Henry; Simon, Julian A; Raible, David W; Rubel, Edwin W

2013-01-01

The majority of hearing loss is caused by the permanent loss of inner ear hair cells. The identification of drugs that modulate the susceptibility to hair cell loss or spur their regeneration is often hampered by the difficulties of assaying for such complex phenomena in mammalian models. The zebrafish has emerged as a powerful animal model for chemical screening in many contexts. Several characteristics of the zebrafish, such as its small size and external location of sensory hair cells, uniquely position it as an ideal model organism for the study of hair cell toxicity, protection, and regeneration. We have used this model to screen for drugs that affect each of these aspects of hair cell biology and have identified compounds that affect each of these processes. The identification of such drugs and drug-like compounds holds promise in the future ability to stem hearing loss in the human population.

Role of endogenous carbon monoxide in the control of breathing in zebrafish (Danio rerio).

PubMed

Tzaneva, Velislava; Perry, Steve F

2016-12-01

Carbon monoxide (CO) is a gaseous signaling molecule and is produced in vivo from the intracellular breakdown of heme via the heme oxygenase (HO) family of enzymes. In this study we investigated the role of the HO-1/CO system in the control of ventilation in zebrafish, Danio rerio Immunohistochemistry revealed the presence of HO-1 in the chemoreceptive neuroepithelial cells (NECs) of larvae (4 days postfertilization) and adults, indicating the potential for endogenous CO production in the NECs. Hypoxia (20 min, water Po 2 of 30 mmHg) caused a significant increase in HO-1 activity in whole larvae and in the gills of adult fish. Zebrafish with reduced HO-1 activity (via HO-1 knockdown in larvae or zinc protoporphyrin IX treatment in adults) exhibited increased ventilation frequency (V f ) under normoxic but not hypoxic conditions. The addition of exogenous CO restored resting V f in fish with diminished CO production, and in some cases (e.g., hypoxic sham larvae) CO modestly reduced V f below resting levels. Larval fish were treated with phenylhydrazine (PHZ) to eliminate the potential confounding effects of CO-hemoglobin interactions that might influence ventilation. PHZ treatment did not cause changes in V f of normoxic larvae, and the addition of CO to PHZ-exposed larvae resulted in a significant decrease in sham and HO-1-deficient fish under normoxic conditions. This study demonstrates for the first time that CO plays an inhibitory role in the control of breathing in larval and adult zebrafish. Copyright © 2016 the American Physiological Society.

Development of an opioid self-administration assay to study drug seeking in zebrafish.

PubMed

Bossé, Gabriel D; Peterson, Randall T

2017-09-29

The zebrafish (Danio rerio) has become an excellent tool to study mental health disorders, due to its physiological and genetic similarity to humans, ease of genetic manipulation, and feasibility of small molecule screening. Zebrafish have been shown to exhibit characteristics of addiction to drugs of abuse in non-contingent assays, including conditioned place preference, but contingent assays have been limited to a single assay for alcohol consumption. Using inexpensive electronic, mechanical, and optical components, we developed an automated opioid self-administration assay for zebrafish, enabling us to measure drug seeking and gain insight into the underlying biological pathways. Zebrafish trained in the assay for five days exhibited robust self-administration, which was dependent on the function of the μ-opioid receptor. In addition, a progressive ratio protocol was used to test conditioned animals for motivation. Furthermore, conditioned fish continued to seek the drug despite an adverse consequence and showed signs of stress and anxiety upon withdrawal of the drug. Finally, we validated our assay by confirming that self-administration in zebrafish is dependent on several of the same molecular pathways as in other animal models. Given the ease and throughput of this assay, it will enable identification of important biological pathways regulating drug seeking and could lead to the development of new therapeutic molecules to treat addiction. Copyright © 2017 Elsevier B.V. All rights reserved.

Transfer of benzo[a]pyrene from microplastics to Artemia nauplii and further to zebrafish via a trophic food web experiment: CYP1A induction and visual tracking of persistent organic pollutants.

PubMed

Batel, Annika; Linti, Frederic; Scherer, Martina; Erdinger, Lothar; Braunbeck, Thomas

2016-07-01

The uptake of microplastic particles and the transfer of potential harmful substances along with microplastics has been studied in a variety of organisms, especially invertebrates. However, the potential accumulation of very small microplastic particles along food webs ending with vertebrate models has not been investigated so far. Therefore, a simple artificial food chain with Artemia sp. nauplii and zebrafish (Danio rerio) was established to analyze the transfer of microplastic particles and associated persistent organic pollutants (POPs) between different trophic levels. Very small (1-20 μm) microplastic particles accumulated in Artemia nauplii and were subsequently transferred to fish. Virgin particles not loaded with POPs did not cause any observable physical harm in the intestinal tracts of zebrafish, although parts of the particles were retained within the mucus of intestinal villi and might even have been taken up by epithelial cells. The transfer of associated POPs was tested with the polycyclic aromatic hydrocarbon benzo[a]pyrene and an ethoxyresorufin-O-deethylase (EROD) assay for CYP1A induction in zebrafish liver as well as via fluorescence analyses. Whereas a significant induction in the EROD assay could not be shown, because of high individual variation and low sensitivity regarding substance concentration, the fluorescence tracking of benzo[a]pyrene indicates that food-borne microplastic-associated POPs may actually desorb in the intestine of fish and are thus transferred to the intestinal epithelium and liver. Environ Toxicol Chem 2016;35:1656-1666. © 2016 SETAC. © 2016 SETAC.

Earth-strength magnetic field affects the rheotactic threshold of zebrafish swimming in shoals.

PubMed

Cresci, Alessandro; De Rosa, Rosario; Putman, Nathan F; Agnisola, Claudio

2017-02-01

Rheotaxis, the unconditioned orienting response to water currents, is a main component of fish behavior. Rheotaxis is achieved using multiple sensory systems, including visual and tactile cues. Rheotactic orientation in open or low-visibility waters might also benefit from the stable frame of reference provided by the geomagnetic field, but this possibility has not been explored before. Zebrafish (Danio rerio) form shoals living in freshwater systems with low visibility, show a robust positive rheotaxis, and respond to geomagnetic fields. Here, we investigated whether a static magnetic field in the Earth-strength range influenced the rheotactic threshold of zebrafish in a swimming tunnel. The direction of the horizontal component of the magnetic field relative to water flow influenced the rheotactic threshold of fish as part of a shoal, but not of fish tested alone. Results obtained after disabling the lateral line of shoaling individuals with Co 2+ suggest that this organ system is involved in the observed magneto-rheotactic response. These findings constitute preliminary evidence that magnetic fields influence rheotaxis and suggest new avenues for further research. Copyright © 2016 Elsevier Inc. All rights reserved.

Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior.

PubMed

Ben-Moshe Livne, Zohar; Alon, Shahar; Vallone, Daniela; Bayleyen, Yared; Tovin, Adi; Shainer, Inbal; Nisembaum, Laura G; Aviram, Idit; Smadja-Storz, Sima; Fuentes, Michael; Falcón, Jack; Eisenberg, Eli; Klein, David C; Burgess, Harold A; Foulkes, Nicholas S; Gothilf, Yoav

2016-11-01

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior.

Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior

PubMed Central

Alon, Shahar; Vallone, Daniela; Tovin, Adi; Shainer, Inbal; Nisembaum, Laura G.; Aviram, Idit; Smadja-Storz, Sima; Fuentes, Michael; Falcón, Jack; Eisenberg, Eli; Klein, David C.; Burgess, Harold A.; Foulkes, Nicholas S.; Gothilf, Yoav

2016-01-01

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior. PMID:27870848

Fish as bioreactors: transgene expression of human coagulation factor VII in fish embryos.

PubMed

Hwang, Gyulin; Müller, Ferenc; Rahman, M Aziz; Williams, Darren W; Murdock, Paul J; Pasi, K John; Goldspink, Geoffrey; Farahmand, Hamid; Maclean, Norman

2004-01-01

A plasmid containing human coagulation factor VII (hFVII) complementary DNA regulated by a cytomegalovirus promoter was microinjected into fertilized eggs of zebrafish, African catfish, and tilapia. The active form of hFVll was detected in the fish embryos by various assays. This positive expression of human therapeutic protein in fish embryos demonstrates the possibility of exploitation of transgenic fish as bioreactors.

Zebrafish: a model animal for analyzing the impact of environmental pollutants on muscle and brain mitochondrial bioenergetics.

PubMed

Bourdineaud, Jean-Paul; Rossignol, R; Brèthes, D

2013-01-01

Mercury, anthropogenic release of uranium (U), and nanoparticles constitute hazardous environmental pollutants able to accumulate along the aquatic food chain with severe risk for animal and human health. The impact of such pollutants on living organisms has been up to now approached by classical toxicology in which huge doses of toxic compounds, environmentally irrelevant, are displayed through routes that never occur in the lifespan of organisms (for instance injecting a bolus of mercury to an animal although the main route is through prey and fish eating). We wanted to address the effect of such pollutants on the muscle and brain mitochondrial bioenergetics under realistic conditions, at unprecedented low doses, using an aquatic model animal, the zebrafish Danio rerio. We developed an original method to measure brain mitochondrial respiration: a single brain was put in 1.5 mL conical tube containing a respiratory buffer. Brains were gently homogenized by 13 strokes with a conical plastic pestle, and the homogenates were immediately used for respiration measurements. Skinned muscle fibers were prepared by saponin permeabilization. Zebrafish were contaminated with food containing 13 μg of methylmercury (MeHg)/g, an environmentally relevant dose. In permeabilized muscle fibers, we observed a strong inhibition of both state 3 mitochondrial respiration and cytochrome c oxidase activity after 49 days of MeHg exposure. We measured a dramatic decrease in the rate of ATP release by skinned muscle fibers. Contrarily to muscles, brain mitochondrial respiration was not modified by MeHg exposure although brain accumulated twice as much MeHg than muscles. When zebrafish were exposed to 30 μg/L of waterborne U, the basal mitochondrial respiratory control ratio was decreased in muscles after 28 days of exposure. This was due to an increase of the inner mitochondrial membrane permeability. The impact of a daily ration of food containing gold nanoparticles of two sizes (12 and 50 nm) was investigated at a very low dose for 60 days (40 ng gold/fish/day). Mitochondrial dysfunctions appeared in brain and muscle for both tested sizes. In conclusion, at low environmental doses, dietary or waterborne heavy metals impinged on zebrafish tissue mitochondrial respiration. Due to its incredible simplicity avoiding tedious and time-consuming mitochondria isolation, our one-pot method allowing brain respiratory analysis should give colleagues the incentive to use zebrafish brain as a model in bioenergetics. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy. Copyright © 2012 Elsevier Ltd. All rights reserved.

ZEBRAFISH AS AN IN VIVO MODEL FOR SUSTAINABLE CHEMICAL DESIGN.

PubMed

Noyes, Pamela D; Garcia, Gloria R; Tanguay, Robert L

2016-12-21

Heightened public awareness about the many thousands of chemicals in use and present as persistent contaminants in the environment has increased the demand for safer chemicals and more rigorous toxicity testing. There is a growing recognition that the use of traditional test models and empirical approaches is impractical for screening for toxicity the many thousands of chemicals in the environment and the hundreds of new chemistries introduced each year. These realities coupled with the green chemistry movement have prompted efforts to implement more predictive-based approaches to evaluate chemical toxicity early in product development. While used for many years in environmental toxicology and biomedicine, zebrafish use has accelerated more recently in genetic toxicology, high throughput screening (HTS), and behavioral testing. This review describes major advances in these testing methods that have positioned the zebrafish as a highly applicable model in chemical safety evaluations and sustainable chemistry efforts. Many toxic responses have been shown to be shared among fish and mammals owing to their generally well-conserved development, cellular networks, and organ systems. These shared responses have been observed for chemicals that impair endocrine functioning, development, and reproduction, as well as those that elicit cardiotoxicity and carcinogenicity, among other diseases. HTS technologies with zebrafish enable screening large chemical libraries for bioactivity that provide opportunities for testing early in product development. A compelling attribute of the zebrafish centers on being able to characterize toxicity mechanisms across multiple levels of biological organization from the genome to receptor interactions and cellular processes leading to phenotypic changes such as developmental malformations. Finally, there is a growing recognition of the links between human and wildlife health and the need for approaches that allow for assessment of real world multi-chemical exposures. The zebrafish is poised to be an important model in bridging these two conventionally separate areas of toxicology and characterizing the biological effects of chemical mixtures that could augment its role in sustainable chemistry.

Zebrafish: an exciting model for investigating the spatio-temporal pattern of enteric nervous system development.

PubMed

Doodnath, Reshma; Dervan, Adrian; Wride, Michael A; Puri, Prem

2010-12-01

Recently, the zebrafish (Danio rerio) has been shown to be an excellent model for human paediatric research. Advantages over other models include its small size, externally visually accessible development and ease of experimental manipulation. The enteric nervous system (ENS) consists of neurons and enteric glia. Glial cells permit cell bodies and processes of neurons to be arranged and maintained in a proper spatial arrangement, and are essential in the maintenance of basic physiological functions of neurons. Glial fibrillary acidic protein (GFAP) is expressed in astrocytes, but also expressed outside of the central nervous system. The aim of this study was to investigate the spatio-temporal pattern of GFAP expression in developing zebrafish ENS from 24 h post-fertilization (hpf), using transgenic fish that express green fluorescent protein (GFP). Zebrafish embryos were collected from transgenic GFP Tg(GFAP:GFP)(mi2001) adult zebrafish from 24 to 120 hpf, fixed and processed for whole mount immunohistochemistry. Antibodies to Phox2b were used to identify enteric neurons. Specimens were mounted on slides and imaging was performed using a fluorescent laser confocal microscope. GFAP:GFP labelling outside the spinal cord was identified in embryos from 48 hpf. The patterning was intracellular and consisted of elongated profiles that appeared to migrate away from the spinal cord into the periphery. At 72 and 96 hpf, GFAP:GFP was expressed dorsally and ventrally to the intestinal tract. At 120 hpf, GFAP:GFP was expressed throughout the intestinal wall, and clusters of enteric neurons were identified using Phox2b immunofluorescence along the pathway of GFAP:GFP positive processes, indicative of a migratory pathway of ENS precursors from the spinal cord into the intestine. The pattern of migration of GFAP:GFP expressing cells outside the spinal cord suggests an organized, early developing migratory pathway to the ENS. This shows for the first time that Tg(GFAP:GFP)(mi2001) zebrafish model is an ideal one to study spatio-temporal patterning of early ENS development.

ZEBRAFISH AS AN IN VIVO MODEL FOR SUSTAINABLE CHEMICAL DESIGN

PubMed Central

Noyes, Pamela D.; Garcia, Gloria R.; Tanguay, Robert L.

2016-01-01

Heightened public awareness about the many thousands of chemicals in use and present as persistent contaminants in the environment has increased the demand for safer chemicals and more rigorous toxicity testing. There is a growing recognition that the use of traditional test models and empirical approaches is impractical for screening for toxicity the many thousands of chemicals in the environment and the hundreds of new chemistries introduced each year. These realities coupled with the green chemistry movement have prompted efforts to implement more predictive-based approaches to evaluate chemical toxicity early in product development. While used for many years in environmental toxicology and biomedicine, zebrafish use has accelerated more recently in genetic toxicology, high throughput screening (HTS), and behavioral testing. This review describes major advances in these testing methods that have positioned the zebrafish as a highly applicable model in chemical safety evaluations and sustainable chemistry efforts. Many toxic responses have been shown to be shared among fish and mammals owing to their generally well-conserved development, cellular networks, and organ systems. These shared responses have been observed for chemicals that impair endocrine functioning, development, and reproduction, as well as those that elicit cardiotoxicity and carcinogenicity, among other diseases. HTS technologies with zebrafish enable screening large chemical libraries for bioactivity that provide opportunities for testing early in product development. A compelling attribute of the zebrafish centers on being able to characterize toxicity mechanisms across multiple levels of biological organization from the genome to receptor interactions and cellular processes leading to phenotypic changes such as developmental malformations. Finally, there is a growing recognition of the links between human and wildlife health and the need for approaches that allow for assessment of real world multi-chemical exposures. The zebrafish is poised to be an important model in bridging these two conventionally separate areas of toxicology and characterizing the biological effects of chemical mixtures that could augment its role in sustainable chemistry. PMID:28461781

Functional behavior and reproduction in androgenic sex reversed zebrafish (Danio rerio).

PubMed

Larsen, Mia G; Baatrup, Erik

2010-08-01

Endocrine-disrupting chemicals released into natural watercourses may cause biased sex ratios by sex reversal in fish populations. The present study investigated the androgenic sex reversal of zebrafish (Danio rerio) exposed to the androgenic compound 17beta-trenbolone (TB) and whether sex-changed females would revert to the female phenotype after cessation of TB exposure. 17beta-Trenbolone is a metabolite of trenbolone acetate, an anabolic steroid used as a growth promoter in beef cattle. 17beta-Trenbolone in runoff from cattle feedlots may reach concentrations that affect fish sexual development. Zebrafish were exposed to a concentration of 20 ng/L TB in a flow-through system for five months from egg until sexual maturity. This resulted in an all-male population. It was further found that all these phenotypic males displayed normal male courtship behavior and were able to reproduce successfully, implying that the sex reversal was complete and functional. None of the phenotypic males developed into females after six months in clean water, demonstrating that androgenic sex reversal of zebrafish is irreversible. Copyright 2010 SETAC

Safety Assessment of Bacillus thuringiensis Insecticidal Proteins Cry1C and Cry2A with a Zebrafish Embryotoxicity Test.

PubMed

Gao, Yan-Jie; Zhu, Hao-Jun; Chen, Yi; Li, Yun-He; Peng, Yu-Fa; Chen, Xiu-Ping

2018-05-02

As a result of the large-scale planting of transgenic Bacillus thuringiensis (Bt) crops, fish would be exposed to freely soluble Bt insecticidal protein(s) that are released from Bt crop tissues into adjacent bodies of water or by way of direct feeding on deposited plant material. To assess the safety of two Bt proteins Cry1C and Cry2A to fish, we used zebrafish as a representative species and exposed their embryos to 0.1, 1, and 10 mg/L of the two Cry proteins until 132 h post-fertilization and then several developmental, biochemical, and molecular parameters were evaluated. Chlorpyrifos (CPF), a known toxicant to aquatic organisms, was used as a positive control. Although CPF exposure resulted in significant developmental, biochemical, and molecular changes in the zebrafish embryos, there were almost no significant differences after Cry1C or Cry2A exposure. Thus, we conclude that zebrafish embryos are not sensitive to Cry1C and Cry2A insecticidal proteins at test concentrations.

Seeing the whole picture: A comprehensive imaging approach to functional mapping of circuits in behaving zebrafish.

PubMed

Feierstein, C E; Portugues, R; Orger, M B

2015-06-18

In recent years, the zebrafish has emerged as an appealing model system to tackle questions relating to the neural circuit basis of behavior. This can be attributed not just to the growing use of genetically tractable model organisms, but also in large part to the rapid advances in optical techniques for neuroscience, which are ideally suited for application to the small, transparent brain of the larval fish. Many characteristic features of vertebrate brains, from gross anatomy down to particular circuit motifs and cell-types, as well as conserved behaviors, can be found in zebrafish even just a few days post fertilization, and, at this early stage, the physical size of the brain makes it possible to analyze neural activity in a comprehensive fashion. In a recent study, we used a systematic and unbiased imaging method to record the pattern of activity dynamics throughout the whole brain of larval zebrafish during a simple visual behavior, the optokinetic response (OKR). This approach revealed the broadly distributed network of neurons that were active during the behavior and provided insights into the fine-scale functional architecture in the brain, inter-individual variability, and the spatial distribution of behaviorally relevant signals. Combined with mapping anatomical and functional connectivity, targeted electrophysiological recordings, and genetic labeling of specific populations, this comprehensive approach in zebrafish provides an unparalleled opportunity to study complete circuits in a behaving vertebrate animal. Copyright © 2014. Published by Elsevier Ltd.

Effects of Simulated Microgravity on Otolith Growth of Larval Zebrafish using a Rotating-Wall Vessel: Appropriate Rotation Speed and Fish Developmental Stage

NASA Astrophysics Data System (ADS)

Li, Xiaoyan; Anken, Ralf; Liu, Liyue; Wang, Gaohong; Liu, Yongding

2017-02-01

Stimulus dependence is a general feature of developing animal sensory systems. In this respect, it has extensively been shown earlier that fish inner ear otoliths can act as test masses as their growth is strongly affected by altered gravity such as hypergravity obtained using centrifuges, by (real) microgravity achieved during spaceflight or by simulated microgravity using a ground-based facility. Since flight opportunities are scarce, ground-based simulators of microgravity, using a wide variety of physical principles, have been developed to overcome this shortcoming. Not all of them, however, are equally well suited to provide functional weightlessness from the perspective of the biosystem under evaluation. Therefore, the range of applicability of a particular simulator has to be extensively tested. Earlier, we have shown that a Rotating-Wall Vessel (RWV) can be used to provide simulated microgravity for developing Zebrafish regarding the effect of rotation on otolith development. In the present study, we wanted to find the most effective speed of rotation and identify the appropriate developmental stage of Zebrafish, where effects are the largest, in order to provide a methodological basis for future in-depth analyses dedicated to the physiological processes underlying otolith growth at altered gravity. Last not least, we compared data on the effect of simulated microgravity on the size versus the weight of otoliths, since the size usually is measured in related studies due to convenience, but the weight more accurately approximates the physical capacity of an otolith. Maintaining embryos at 10 hours post fertilization for three days in the RWV, we found that 15 revolutions per minute (rpm) yielded the strongest effects on otolith growth. Maintenance of Zebrafish staged at 10 hpf, 1 day post fertilization (dpf), 4 dpf, 7 dpf and 14 dpf for three days at 15 rpm resulted in the most prominent effects in 7 dpf larvae. Weighing versus measuring the size of otoliths yielded basically similar results, but the data gained by weighing were more distinct. Overall, our results clearly support the concept that the environmental gravity vector regulates fish otolith growth in terms of the pendulum model of otolith test masses, and that wall vessel rotation is a valuable means to provide functional weightlessness from the perspective of developing Zebrafish. We recommend that Zebrafish embryos staged 7 dpf (or possibly slightly elder) are rotated at 15 rpm in a Rotating-Wall Vessel as used in the present study for further experiments designed to elucidate the mechanisms underlying (altered gravity affected) otolith growth.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish.

PubMed

Moore, Finola E; Garcia, Elaine G; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C; Garcia, Amaris J; Mylvaganam, Ravi; Yoder, Jeffrey A; Blackburn, Jessica S; Sadreyev, Ruslan I; Ceol, Craig J; North, Trista E; Langenau, David M

2016-05-30

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4(+) cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2(E450fs) mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4(+) cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2(E450fs) mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4(+)/CD8(+) cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. © 2016 Moore et al.

Beta-glucan enhances the response to SVCV infection in zebrafish.

PubMed

M Medina-Gali, Regla; Ortega-Villaizan, María Del Mar; Mercado, Luis; Novoa, Beatriz; Coll, Julio; Perez, Luis

2018-07-01

The antiviral effects of beta-glucan, an immunostimulatory agent were studied in zebrafish both in vitro and in vivo. Here we show that zebrafish ZF4 cells as well as whole fish primed with yeast β-glucan zymosan exhibited increased cytokine expression and elevated response to spring viremia of carp virus (SVCV) infection. In vitro, previous treatment of β-glucan enhanced ZF4 cell viability against SVCV infection which is associated to the activation of interferon signaling pathway and inflammatory cytokines gene expression. In vivo, the SVCV-infected fish primed with β-glucan had a higher survival rate (≈73%) than the control SVCV-infected group (≈33%). Additionally, up-regulation of the expression of a set of genes involved in innate immune response was detected in zebrafish intraperitoneally injected of β-glucan: il1b, il6, il8, il10 and tnfa transcripts showed increased expression that appear to be rapid (2 days) but not long-lived (less than 2 weeks). The present study is, to our knowledge, the first to combine cell culture and in vivo approaches to describe host response to β-glucan stimulation and viral infection in zebrafish. Copyright © 2018 Elsevier Ltd. All rights reserved.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish

PubMed Central

Garcia, Elaine G.; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C.; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C.; Garcia, Amaris J.; Mylvaganam, Ravi; Yoder, Jeffrey A.; Blackburn, Jessica S.; Sadreyev, Ruslan I.; Ceol, Craig J.; North, Trista E.

2016-01-01

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4+ cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2E450fs mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4+ cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2E450fs mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4+/CD8+ cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb. In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. PMID:27139488

Shoaling develops with age in Zebrafish (Danio rerio)

PubMed Central

Buske, Christine; Gerlai, Robert

2010-01-01

The biological mechanisms of human social behavior are complex. Animal models may facilitate the understanding of these mechanisms and may help one to develop treatment strategies for abnormal human social behavior, a core symptom in numerous clinical conditions. The zebrafish is perhaps the most social vertebrate among commonly used laboratory species. Given its practical features and the numerous genetic tools developed for it, it should be a promising tool. Zebrafish shoal, i.e. form tight multimember groups, but the ontogenesis of this behavior has not been described. Analyzing the development of shoaling is a step towards discovering the mechanisms of this behavior. Here we study age-dependent changes of shoaling in zebrafish from day 7 post fertilization to over 5 months of age by measuring the distance between all pairs of fish in freely swimming groups of ten subjects. Our longitudinal (repeated measure within subject) and cross sectional (non-repeated measure between subject) analyses both demonstrated a significant increase of shoaling with age (decreased distance between shoal members). Given the sophisticated genetic and developmental biology methods already available for zebrafish, we argue that our behavioral results open a new avenue towards the understanding of the development of vertebrate social behavior and of its mechanisms and abnormalities. PMID:20837077

Study of effects of radio-wave frequency radiation emitted from cellular telephones on embryonic development of danio rerio

NASA Astrophysics Data System (ADS)

Vagula, Mary; Harkless, Ryan

2013-05-01

Radio wave frequency (RF) radiation emitted from cellular telephones has become increasingly ubiquitous as a result of the popularity of these phones. With the increasing and unavoidable exposure to RF radiation a reality, it is imperative that the effects of such radiation on living tissue be well understood. In particular, it is critical to understand any effects that RF radiation may have as a carcinogen and on embryonic development, as pregnant women are not exempt from such exposure. As a model organism, zebrafish (Danio rerio) have been studied extensively, and their value in studies of gene expression cannot be overstated. This study observed the effects of RF radiation on the embryonic development of zebrafish. The expression of two genes, shha and hoxb9a, that are key to the early development of the fish was examined. Both genes have homologs in humans as well as in other model organisms. Preliminary results suggest that exposure to cell phone radiation might have an effect on the expression of shha in zebrafish embryos, causing under expression. More trials are necessary to validate these results.

Oral vaccination of fish: Lessons from humans and veterinary species.

PubMed

Embregts, Carmen W E; Forlenza, Maria

2016-11-01

The limited number of oral vaccines currently approved for use in humans and veterinary species clearly illustrates that development of efficacious and safe oral vaccines has been a challenge not only for fish immunologists. The insufficient efficacy of oral vaccines is partly due to antigen breakdown in the harsh gastric environment, but also to the high tolerogenic gut environment and to inadequate vaccine design. In this review we discuss current approaches used to develop oral vaccines for mass vaccination of farmed fish species. Furthermore, using various examples from the human and veterinary vaccine development, we propose additional approaches to fish vaccine design also considering recent advances in fish mucosal immunology and novel molecular tools. Finally, we discuss the pros and cons of using the zebrafish as a pre-screening animal model to potentially speed up vaccine design and testing for aquaculture fish species. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Reproductive and Developmental Toxicity of Dioxin in Fish1

PubMed Central

King-Heiden, Tisha C.; Mehta, Vatsal; Xiong, Kong M.; Lanham, Kevin A.; Antkiewicz, Dagmara S.; Ganser, Alissa; Heideman, Warren

2011-01-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is a global environmental contaminant and the prototypical ligand for investigating aryl hydrocarbon receptor (AHR)-mediated toxicity. Environmental exposure to TCDD results in developmental and reproductive toxicity in fish, birds and mammals. To resolve the ecotoxicological relevance and human health risks posed by exposure to dioxin-like AHR agonists, a vertebrate model is needed that allows for toxicity studies at various levels of biological organization, assesses adverse reproductive and developmental effects and establishes appropriate integrative correlations between different levels of effects. Here we describe the reproductive and developmental toxicity of TCDD in feral fish species and summarize how using the zebrafish model to investigate TCDD toxicity has enabled us to characterize the AHR signaling in fish and to better understand how dioxin-like chemicals induce toxicity. We propose that such studies can be used to predict the risks that AHR ligands pose to feral fish populations and provide a platform for integrating risk assessments for both ecologically relevant organisms and humans. PMID:21958697

Blocking VEGF signaling delays development of replacement teeth in zebrafish.

PubMed

Crucke, J; Huysseune, A

2015-01-01

The dentition in zebrafish is extremely and richly vascularized, but the function of the vasculature, in view of the continuous replacement of the teeth, remains elusive. Through application of SU5416, a vascular endothelial growth factor receptor inhibitor, we studied the role of the blood vessels in the dentition of the zebrafish. We were unable to show an effect on the development of first-generation teeth as well as first tooth replacement. However, in juvenile fish, a delay was observed in the developmental state of the replacement tooth compared with what was expected based on the maturation state of the functional tooth. Furthermore, we observed a difference between treated and nontreated fish in the distance between blood vessels and developing replacement teeth. In conclusion, our results provide support for a nutritive, rather than an inductive, function of the vasculature in the process of tooth development and replacement. © International & American Associations for Dental Research 2014.

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

PubMed

Christianson-Heiska, I; Wahteristo, P; Kastilan, E-L; Bergelin, E; Bylund, G; Isomaa, B

2004-05-01

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.

Neurobehavioral impairments produced by developmental lead exposure persisted for generations in zebrafish (Danio rerio).

PubMed

Xu, Xiaojuan; Weber, Daniel; Burge, Rebekah; VanAmberg, Kelsey

2016-01-01

The zebrafish has become a useful animal model for studying the effects of environmental contaminants on neurobehavioral development due to its ease of breeding, high number of eggs per female, short generation times, and a well-established avoidance conditioning paradigm. Using avoidance conditioning as the behavioral paradigm, the present study investigated the effects of embryonic exposure to lead (Pb) on learning in adult zebrafish and the third (F3) generation of those fish. In Experiment 1, adult zebrafish that were developmentally exposed to 0.0, 0.1, 1.0 or 10.0μM Pb (2-24h post fertilization) as embryos were trained and tested for avoidance responses. The results showed that adult zebrafish hatched from embryos exposed to 0.0 or 0.1μM Pb learned avoidance responses during training and displayed significantly increased avoidance responses during testing, while those hatched from embryos exposed to 1.0 or 10.0μM Pb displayed no significant increases in avoidance responses from training to testing. In Experiment 2, the F3 generation of zebrafish that were developmentally exposed to an identical exposure regimen as in Experiment 1 were trained and tested for avoidance responses. The results showed that the F3 generation of zebrafish developmentally exposed as embryos to 0.0 or 0.1μM Pb learned avoidance responses during training and displayed significantly increased avoidance responses during testing, while the F3 generation of zebrafish developmentally exposed as embryos to 1.0 or 10.0μM Pb displayed no significant changes in avoidance responses from training to testing. Thus, developmental Pb exposure produced learning impairments that persisted for at least three generations, demonstrating trans-generational effects of embryonic exposure to Pb. Copyright © 2015. Published by Elsevier B.V.

Identification of Zebrafish Fxyd11a Protein that is Highly Expressed in Ion-Transporting Epithelium of the Gill and Skin and its Possible Role in Ion Homeostasis

PubMed Central

Saito, Kaori; Nakamura, Nobuhiro; Ito, Yusuke; Hoshijima, Kazuyuki; Esaki, Masahiro; Zhao, Boqiang; Hirose, Shigehisa

2010-01-01

FXYD proteins, small single-transmembrane proteins, have been proposed to be auxiliary regulatory subunits of Na+–K+-ATPase and have recently been implied in ion osmoregulation of teleost fish. In freshwater (FW) fish, numerous ions are actively taken up through mitochondrion-rich cells (MRCs) of the gill and skin epithelia, using the Na+ electrochemical gradient generated by Na+–K+-ATPase. In the present study, to understand the molecular mechanism for the regulation of Na+–K+-ATPase in MRCs of FW fish, we sought to identify FXYD proteins expressed in MRCs of zebrafish. Reverse-transcriptase PCR studies of adult zebrafish tissues revealed that, out of eight fxyd genes found in zebrafish database, only zebrafish fxyd11 (zfxyd11) mRNA exhibited a gill-specific expression. Double immunofluorescence staining showed that zFxyd11 is abundantly expressed in MRCs rich in Na+–K+-ATPase (NaK-MRCs) but not in those rich in vacuolar-type H+-transporting ATPase. An in situ proximity ligation assay demonstrated its close association with Na+–K+-ATPase in NaK-MRCs. The zfxyd11 mRNA expression was detectable at 1 day postfertilization, and its expression levels in the whole larvae and adult gills were regulated in response to changes in environmental ionic concentrations. Furthermore, knockdown of zFxyd11 resulted in a significant increase in the number of Na+–K+-ATPase–positive cells in the larval skin. These results suggest that zFxyd11 may regulate the transport ability of NaK-MRCs by modulating Na+–K+-ATPase activity, and may be involved in the regulation of body fluid and electrolyte homeostasis. PMID:21423371

The sensitivity and reproducibility of the zebrafish (Danio rerio) embryo test for the screening of waste water quality and for testing the toxicity of chemicals.

PubMed

Lahnsteiner, Franz

2008-07-01

The sensitivity of the zebrafish embryo test, a test proposed for routine waste water control, was compared with the acute fish toxicity test, in the determination of six types of waste water and ten different chemicals. The waste water was sampled from the following industrial processes: paper and cardboard production, hide tanning, metal galvanisation, carcass treatment and utilisation, and sewage treatment. The chemicals tested were: dimethylacetamide, dimethylsulphoxide, cadmium chloride, cyclohexane, hydroquinone, mercuric chloride, nickel chloride, nonylphenol, resmethrin and sodium nitrite. For many of the test substances, the zebrafish embryo test and the acute fish toxicity test results showed high correlations. However, there were certain environmentally-relevant substances for which the results of the zebrafish embryo test and the acute fish toxicity test differed significantly, up to 10,000-fold (Hg(2+) > 150-fold difference; NO(2)(-) > 300-fold; Cd(2+) > 200-fold; resmethrin > 10,000-fold). For the investigated waste water samples and chemicals, the survival rate of the zebrafish embryos showed high variations between different egg samples, within the range of the EC50 concentration. Subsequently, 5-6 parallel assays were deemed to be the appropriate number necessary for the precise evaluation of the toxicity of the test substances. Also, it was found that the sensitivities of different ontogenetic stages to chemical exposure differed greatly. During the first 12 hours after fertilisation (4-cell stage to the 5-somite stage), the embryos reacted most sensitively to test substance exposure, whereas the later ontogenetic stages showed only slight or no response, indicating that the test is most sensitive during the first 24 hours post-fertilisation.

Transcriptomic characterization of cold acclimation in larval zebrafish

PubMed Central

2013-01-01

Background Temperature is one of key environmental parameters that affect the whole life of fishes and an increasing number of studies have been directed towards understanding the mechanisms of cold acclimation in fish. However, the adaptation of larvae to cold stress and the cold-specific transcriptional alterations in fish larvae remain largely unknown. In this study, we characterized the development of cold-tolerance in zebrafish larvae and investigated the transcriptional profiles under cold stress using RNA-seq. Results Pre-exposure of 96 hpf zebrafish larvae to cold stress (16°C) for 24 h significantly increased their survival rates under severe cold stress (12°C). RNA-seq generated 272 million raw reads from six sequencing libraries and about 92% of the processed reads were mapped to the reference genome of zebrafish. Differential expression analysis identified 1,431 up- and 399 down-regulated genes. Gene ontology enrichment analysis of cold-induced genes revealed that RNA splicing, ribosome biogenesis and protein catabolic process were the most highly overrepresented biological processes. Spliceosome, proteasome, eukaryotic ribosome biogenesis and RNA transport were the most highly enriched pathways for genes up-regulated by cold stress. Moreover, alternative splicing of 197 genes and promoter switching of 64 genes were found to be regulated by cold stress. A shorter isoform of stk16 that lacks 67 amino acids at the N-terminus was specifically generated by skipping the second exon in cold-treated larvae. Alternative promoter usage was detected for per3 gene under cold stress, which leading to a highly up-regulated transcript encoding a truncated protein lacking the C-terminal domains. Conclusions These findings indicate that zebrafish larvae possess the ability to build cold-tolerance under mild low temperature and transcriptional and post-transcriptional regulations are extensively involved in this acclimation process. PMID:24024969

SideRack: A Cost-Effective Addition to Commercial Zebrafish Housing Systems

PubMed Central

Burg, Leonard; Gill, Ryan; Balciuniene, Jorune

2014-01-01

Abstract Commercially available aquatic housing systems provide excellent and relatively trouble-free hardware for rearing and housing juvenile as well as adult zebrafish. However, the cost of such systems is quite high and potentially prohibitive for smaller educational and research institutions. The need for tank space prompted us to experiment with various additions to our existing Aquaneering system. We also noted that high water exchange rates typical in commercial systems are suboptimal for quick growth of juvenile fish. We devised a housing system we call “SideRack,” which contains 20 large tanks with air supply and slow water circulation. It enables cost-effective expansion of existing fish facility, with a key additional benefit of increased growth and maturation rates of juvenile fish. PMID:24611601

Genotoxic and teratogenic effect of freshwater sediment samples from the Rhine and Elbe River (Germany) in zebrafish embryo using a multi-endpoint testing strategy.

PubMed

Garcia-Käufer, M; Gartiser, S; Hafner, C; Schiwy, S; Keiter, S; Gründemann, C; Hollert, H

2015-11-01

The embryotoxic potential of three model sediment samples with a distinct and well-characterized pollutant burden from the main German river basins Rhine and Elbe was investigated. The Fish Embryo Contact Test (FECT) in zebrafish (Danio rerio) was applied and submitted to further development to allow for a comprehensive risk assessment of such complex environmental samples. As particulate pollutants are constructive constituents of sediments, they underlay episodic source-sink dynamics, becoming available to benthic organisms. As bioavailability of xenobiotics is a crucial factor for ecotoxicological hazard, we focused on the direct particle-exposure pathway, evaluating throughput-capable endpoints and considering toxicokinetics. Fish embryo and larvae were exposed toward reconstituted (freeze-dried) sediment samples on a microcosm-scale experimental approach. A range of different developmental embryonic stages were considered to gain knowledge of potential correlations with metabolic competence during the early embryogenesis. Morphological, physiological, and molecular endpoints were investigated to elucidate induced adverse effects, placing particular emphasis on genomic instability, assessed by the in vivo comet assay. Flow cytometry was used to investigate the extent of induced cell death, since cytotoxicity can lead to confounding effects. The implementation of relative toxicity indices further provides inter-comparability between samples and related studies. All of the investigated sediments represent a significant ecotoxicological hazard by disrupting embryogenesis in zebrafish. Beside the induction of acute toxicity, morphological and physiological embryotoxic effects could be identified in a concentration-response manner. Increased DNA strand break frequency was detected after sediment contact in characteristic non-monotonic dose-response behavior due to overlapping cytotoxic effects. The embryonic zebrafish toxicity model along with the in vivo comet assay and molecular biomarker analysis should prospectively be considered to assess the ecotoxicological potential of sediments allowing for a comprehensive hazard ranking. In order to elucidate mode of action, novel techniques such as flow cytometry have been adopted and proved to be valuable tools for advanced risk assessment and management.

Zebrafish vitamin K epoxide reductases: expression in vivo, along extracellular matrix mineralization and under phylloquinone and warfarin in vitro exposure.

PubMed

Fernández, Ignacio; Vijayakumar, Parameswaran; Marques, Carlos; Cancela, M Leonor; Gavaia, Paulo J; Laizé, Vincent

2015-06-01

Vitamin K (VK) acts as a cofactor driving the biological activation of VK-dependent proteins and conferring calcium-binding properties to them. As a result, VK is converted into VK epoxide, which must be recycled by VK epoxide reductases (Vkors) before it can be reused. Although VK has been shown to play a central role in fish development, particularly during skeletogenesis, pathways underlying VK actions are poorly understood, while good and reliable molecular markers for VK cycle/homeostasis are still lacking in fish. In the present work, expression of 2 zebrafish vkor genes was characterized along larval development and in adult tissues through qPCR analysis. Zebrafish cell line ZFB1 was used to evaluate in vitro regulation of vkors and other VK cycle-related genes during mineralization and upon 24 h exposure to 0.16 and 0.8 µM phylloquinone (VK1), 0.032 µM warfarin, or a combination of both molecules. Results showed that zebrafish vkors are differentially expressed during larval development, in adult tissues, and during cell differentiation/mineralization processes. Further, several VK cycle intermediates were differentially expressed in ZFB1 cells exposed to VK1 and/or warfarin. Present work provides data identifying different developmental stages and adult tissues where VK recycling is probably highly required, and shows how genes involved in VK cycle respond to VK nutritional status in skeletal cells. Expression of vkor genes can represent a reliable indicator to infer VK nutritional status in fish, while ZFB1 cells could represent a suitable in vitro tool to get insights into the mechanisms underlying VK action on fish bone.

Developmental exposure of zebrafish (Danio rerio) to 17α-ethinylestradiol affects non-reproductive behavior and fertility as adults, and increases anxiety in unexposed progeny.

PubMed

Volkova, Kristina; Reyhanian Caspillo, Nasim; Porseryd, Tove; Hallgren, Stefan; Dinnétz, Patrik; Porsch-Hällström, Inger

2015-07-01

Exposure to estrogenic endocrine disruptors (EDCs) during development affects fertility, reproductive and non-reproductive behavior in mammals and fish. These effects can also be transferred to coming generations. In fish, the effects of developmental EDC exposure on non-reproductive behavior are less well studied. Here, we analyze the effects of 17α-ethinylestradiol (EE2) on anxiety, shoaling behavior and fertility in zebrafish after developmental treatment and remediation in clean water until adulthood. Zebrafish embryos were exposed from day 1 to day 80 post fertilization to actual concentrations of 1.2 and 1.6ng/L EE2. After remediation for 82days non-reproductive behavior and fertilization success were analyzed in both sexes. Males and females from the 1.2ng/L group, as well as control males and females, were bred, and behavior of the untreated F1 offspring was tested as adults. Developmental treatment with 1.2 and 1.6ng/L EE2 significantly increased anxiety in the novel tank test and increased shoaling intensity in both sexes. Fertilization success was significantly reduced by EE2 in both sexes when mated with untreated fish of opposite sex. Progeny of fish treated with 1.2ng/L EE2 showed increased anxiety in the novel tank test and increased light avoidance in the scototaxis test compared to control offspring. In conclusion, developmental exposure of zebrafish to low doses of EE2 resulted in persistent changes in behavior and fertility. The behavior of unexposed progeny was affected by their parents' exposure, which might suggest transgenerational effects. Copyright © 2015. Published by Elsevier Inc.

The effects of rearing light level and duration differences on the optic nerve, brain, and associated structures in developing zebrafish larvae: a light and transmission electron microscope study.

PubMed

Chapman, George B; Tarboush, Rania; Connaughton, Victoria P

2012-03-01

The ultrastructure of the optic nerve, brain, and some associated structures of larval zebrafish, grown under three different light regimens were studied. Fish grown under cyclic light (control), constant dark (CD), and constant light (CL) were studied for 4 and 8 days postfertilization (dpf). We also studied the control and CD fish at 15 dpf. The brains of the control and CL fish were larger at 4 dpf than at 8 dpf. In all 4 dpf fish, the brain occupied the entire expanse between the two retinas and the optic nerve extended the shortest distance between the retina and the brain. The 15 dpf zebrafish had the smallest brain size. Groups of skeletal muscle cells associated with the optic nerves became visible in all older larvae. In the 15 dpf larvae, bulges and dilations in the optic nerve occurred as it reached the brain and optic chiasms occurred proximal to the brain. Electron microscopy yielded information about myelinated and unmyelinated axons in the optic nerve, the dimensions of neurotubules, neurofilaments, and myofilaments, including a unique variation in actin myofilaments, and a confirmation of reported myosin myofilament changes (but with dimensions). We also describe the ultrastructure of a sheath-like structure that is confluent over the optic nerve and the brain, which has not been described before in zebrafish. Also presented are images of associated fibroblasts, epithelial cells lining the mouth, cartilage plates, blood vessels, nerve bundles, and skeletal muscle cells, most of which have not been previously described in the literature. Copyright © 2012 Wiley Periodicals, Inc.

Interaction of environmental contaminants with zebrafish organic anion transporting polypeptide, Oatp1d1 (Slco1d1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popovic, Marta; Zaja, Roko; Fent, Karl

Polyspecific transporters from the organic anion transporting polypeptide (OATP/Oatp) superfamily mediate the uptake of a wide range of compounds. In zebrafish, Oatp1d1 transports conjugated steroid hormones and cortisol. It is predominantly expressed in the liver, brain and testes. In this study we have characterized the transport of xenobiotics by the zebrafish Oatp1d1 transporter. We developed a novel assay for assessing Oatp1d1 interactors using the fluorescent probe Lucifer yellow and transient transfection in HEK293 cells. Our data showed that numerous environmental contaminants interact with zebrafish Oatp1d1. Oatp1d1 mediated the transport of diclofenac with very high affinity, followed by high affinity towardsmore » perfluorooctanesulfonic acid (PFOS), nonylphenol, gemfibrozil and 17α-ethinylestradiol; moderate affinity towards carbaryl, diazinon and caffeine; and low affinity towards metolachlor. Importantly, many environmental chemicals acted as strong inhibitors of Oatp1d1. A strong inhibition of Oatp1d1 transport activity was found by perfluorooctanoic acid (PFOA), chlorpyrifos-methyl, estrone (E1) and 17β-estradiol (E2), followed by moderate to low inhibition by diethyl phthalate, bisphenol A, 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4 tetrahydronapthalene and clofibrate. In this study we identified Oatp1d1 as a first Solute Carrier (SLC) transporter involved in the transport of a wide range of xenobiotics in fish. Considering that Oatps in zebrafish have not been characterized before, our work on zebrafish Oatp1d1 offers important new insights on the understanding of uptake processes of environmental contaminants, and contributes to the better characterization of zebrafish as a model species. - Highlights: • We optimized a novel assay for determination of Oatp1d1 interactors • Oatp1d1 is the first SLC characterized fish xenobiotic transporter • PFOS, nonylphenol, diclofenac, EE2, caffeine are high affinity Oatp1d1substrates • PFOA, chlorpyrifos-methyl, E1, E2 are strong inhibitors of Oatp1d1 • PFOA and diclofenac can block Oatp1d1 binding of DHEAS, E3S and E17ß-glucuronide.« less

Autonomic innervation of the fish gut.

PubMed

Olsson, Catharina

2009-01-01

The enteric nervous system follows a similar overall arrangement in all vertebrate groups. In fish, the majority of nerve cell bodies are found in the myenteric plexus, innervating muscles, blood vessels and glands. In this review, I describe similarities and differences in size, shape and transmitter content in enteric neurons in different fish species and also in comparison with other vertebrates, foremost mammals. The use of different histological and immunochemical methods is reviewed in a historical perspective including advantages and disadvantages of different methods. Lately, zebrafish have become an important model species for developmental studies of the nervous system, including the enteric nervous system, and this is briefly discussed. Finally, examples of how the enteric nervous system controls gut activity in fish is presented, focussing on the effect on gastrointestinal motility.

Dechorionation of Zebrafish Embryos on Day 1 Post Fertilization Alters Response to an Acute Chemical Challenge at 6 Days Post Fertilization

EPA Science Inventory

Dechorionation is a method used to enable image acquisition in embryonic and larval zebrafish studies. As it is assumed that dechorionation has no long-term effects on fish embryo development, it is important to determine if that assumption is correct. The present study explored ...

Altered Gene Expression in the Brain and Ovaries of Zebrafish Exposed to the Aromatase Inhibitor Fadrosole: Microarray Analysis for Hypothesis Generation

EPA Science Inventory

A part of an overall program of research aimed at examining system-wide responses of the hypothalamic-pituitary-gonadal axis in fish to endocrine active chemicals acting through a variety of modes of action, we exposed zebrafish (Danio rerio) to the aromatase inhibitor fadrozole ...

Stability of citrate-capped silver nanoparticles in exposure media and their effects on the development of embryonic zebrafish (Danio rerio)

PubMed Central

Park, Kwangsik; Tuttle, George; Sinche, Federico; Harper, Stace L.

2014-01-01

The stability of citrate-capped silver nanoparticles (AgNPs) and the embryonic developmental toxicity were evaluated in the fish test water. Serious aggregation of AgNPs was observed in undiluted fish water (DM-100) in which high concentration of ionic salts exist. However, AgNPs were found to be stable for 7 days in DM-10, prepared by diluting the original fish water (DM-100) with deionized water to 10%. The normal physiology of zebrafish embryos were evaluated in DM-10 to see if DM-10 can be used as a control vehicle for the embryonic fish toxicity test. As results, DM-10 without AgNPs did not induce any significant adverse effects on embryonic development of zebrafish determined by mortality, hatching, malformations and heart rate. When embryonic toxicity of AgNPs was tested in both DM-10 and in DM-100, AgNPs showed higher toxicity in DM-10 than in DM-100. This means that the big-sized aggregates of AgNPs were low toxic compared to the nano-sized AgNPs. AgNPs induced delayed hatching, decreased heart rate, pericardial edema, and embryo death. Accumulation of AgNPs in the embryo bodies was also observed. Based on this study, citrate-capped AgNPs are not aggregated in DM-10 and it can be used as a control vehicle in the toxicity test of fish embryonic development. PMID:23325492

The Mammalian “Obesogen” Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish

PubMed Central

Lyssimachou, Angeliki; Santos, Joana G.; André, Ana; Soares, Joana; Lima, Daniela; Guimarães, Laura; Almeida, C. Marisa R.; Teixeira, Catarina; Castro, L. Filipe C.; Santos, Miguel M.

2015-01-01

Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an “obesogenic” phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound. PMID:26633012

Effects of non-steroidal anti-inflammatory drugs on hormones and genes of the hypothalamic-pituitary-gonad axis, and reproduction of zebrafish.

PubMed

Ji, Kyunghee; Liu, Xiaoshan; Lee, Saeram; Kang, Sungeun; Kho, Younglim; Giesy, John P; Choi, Kyungho

2013-06-15

This study was conducted in two experiments, to identify non-steroidal anti-inflammatory drugs (NSAIDs) with high endocrine disruption potentials, and to understand consequences of exposure to such NSAIDs in fish. In the first experiment, the effects of five NSAIDs on hormones and gene transcriptions of the hypothalamic-pituitary-gonad (HPG) axis were evaluated after 14 d exposure of adult zebrafish. Ibuprofen and mefenamic acids were identified to increase the concentrations of 17β-estradiol and testosterone in females significantly, while decreased those of testosterone among male fish. Significant up-regulation of fshβ, lhβ, fshr and lhr were observed in females, whereas down-regulation was observed in males exposed to each NSAID. In the second experiment, ibuprofen was chosen as a model chemical. Adult zebrafish pairs were exposed to ibuprofen for 21 d, and the effects on reproduction and development of offspring were examined. The egg production was significantly decreased at ≥1 μg/L ibuprofen, and parental exposure resulted in delayed hatching even when they were transferred to clean water for hatching. The results demonstrated that ibuprofen could modulate hormone production and related gene transcription of the HPG axis in a sex-dependent way, which could cause adverse effects on reproduction and the development of offspring. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of dietary exposure to brominated flame retardant BDE-47 on thyroid condition, gonadal development and growth of zebrafish

USGS Publications Warehouse

Torres, Leticia; Orazio, Carl E.; Peterman, Paul H.; Patino, Reynaldo

2013-01-01

Little is known about the effects of brominated flame retardants in teleosts and some of the information currently available is inconsistent. This study examined effects of dietary exposure to 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) on thyroid condition, body mass and size, and gonadal development of zebrafish. Pubertal, 49-day-old (posthatch) fish were fed diets without BDE-47 (control) or with 1, 5 or 25 μg/g BDE-47/diet. Treatments were conducted in triplicate 30-L tanks each containing 50 zebrafish, and 15 fish per treatment (5 per tank) were sampled at days 40, 80 and 120 of exposure. Measurements were taken of body mass, standard length, head depth and head length. Sex (at 40–120 days of exposure), germ cell stage (at 40 days) and thyroid condition (at 120 days; follicular cell height, colloid depletion, angiogenesis) were histologically determined. Whole-body BDE-47 levels at study completion were within the high end of levels reported in environmentally exposed (wild) fishes. Analysis of variance was used to determine differences among treatments at each sampling time. No effects were observed on thyroid condition or germ cell stage in either sex. Reduced head length was observed in females exposed to BDE-47 at 80 days but not at 40 or 120 days. In males, no apparent effects of BDE-47 were observed at 40 and 80 days, but fish exposed to 25 μg/g had lower body mass at 120 days compared to control fish. These observations suggest that BDE-47 at environmentally relevant whole-body concentrations does not affect thyroid condition or pubertal development of zebrafish but does affect growth during the juvenile-to-adult transition, especially in males.

Pigment pattern in jaguar/obelix zebrafish is caused by a Kir7.1 mutation: implications for the regulation of melanosome movement.

PubMed

Iwashita, Motoko; Watanabe, Masakatsu; Ishii, Masaru; Chen, Tim; Johnson, Stephen L; Kurachi, Yoshihisa; Okada, Norihiro; Kondo, Shigeru

2006-11-24

Many animals have a variety of pigment patterns, even within a species, and these patterns may be one of the driving forces of speciation. Recent molecular genetic studies on zebrafish have revealed that interaction among pigment cells plays a key role in pattern formation, but the mechanism of pattern formation is unclear. The zebrafish jaguar/obelix mutant has broader stripes than wild-type fish. In this mutant, the development of pigment cells is normal but their distribution is altered, making these fish ideal for studying the process of pigment pattern formation. Here, we utilized a positional cloning method to determine that the inwardly rectifying potassium channel 7.1 (Kir7.1) gene is responsible for pigment cell distribution among jaguar/obelix mutant fish. Furthermore, in jaguar/obelix mutant alleles, we identified amino acid changes in the conserved region of Kir7.1, each of which affected K(+) channel activity as demonstrated by patch-clamp experiments. Injection of a bacterial artificial chromosome containing the wild-type Kir7.1 genomic sequence rescued the jaguar/obelix phenotype. From these results, we conclude that mutations in Kir7.1 are responsible for jaguar/obelix. We also determined that the ion channel function defect of melanophores expressing mutant Kir7.1 altered the cellular response to external signals. We discovered that mutant melanophores cannot respond correctly to the melanosome dispersion signal derived from the sympathetic neuron and that melanosome aggregation is constitutively activated. In zebrafish and medaka, it is well known that melanosome aggregation and subsequent melanophore death increase when fish are kept under constant light conditions. These observations indicate that melanophores of jaguar/obelix mutant fish have a defect in the signaling pathway downstream of the alpha2-adrenoceptor. Taken together, our results suggest that the cellular defect of the Kir7.1 mutation is directly responsible for the pattern change in the jaguar/obelix mutant.

Pigment Pattern in jaguar/obelix Zebrafish Is Caused by a Kir7.1 Mutation: Implications for the Regulation of Melanosome Movement

PubMed Central

Iwashita, Motoko; Watanabe, Masakatsu; Ishii, Masaru; Chen, Tim; Johnson, Stephen L; Kurachi, Yoshihisa; Okada, Norihiro; Kondo, Shigeru

2006-01-01

Many animals have a variety of pigment patterns, even within a species, and these patterns may be one of the driving forces of speciation. Recent molecular genetic studies on zebrafish have revealed that interaction among pigment cells plays a key role in pattern formation, but the mechanism of pattern formation is unclear. The zebrafish jaguar/obelix mutant has broader stripes than wild-type fish. In this mutant, the development of pigment cells is normal but their distribution is altered, making these fish ideal for studying the process of pigment pattern formation. Here, we utilized a positional cloning method to determine that the inwardly rectifying potassium channel 7.1 (Kir7.1) gene is responsible for pigment cell distribution among jaguar/obelix mutant fish. Furthermore, in jaguar/obelix mutant alleles, we identified amino acid changes in the conserved region of Kir7.1, each of which affected K+ channel activity as demonstrated by patch-clamp experiments. Injection of a bacterial artificial chromosome containing the wild-type Kir7.1 genomic sequence rescued the jaguar/obelix phenotype. From these results, we conclude that mutations in Kir7.1 are responsible for jaguar/obelix. We also determined that the ion channel function defect of melanophores expressing mutant Kir7.1 altered the cellular response to external signals. We discovered that mutant melanophores cannot respond correctly to the melanosome dispersion signal derived from the sympathetic neuron and that melanosome aggregation is constitutively activated. In zebrafish and medaka, it is well known that melanosome aggregation and subsequent melanophore death increase when fish are kept under constant light conditions. These observations indicate that melanophores of jaguar/obelix mutant fish have a defect in the signaling pathway downstream of the α2-adrenoceptor. Taken together, our results suggest that the cellular defect of the Kir7.1 mutation is directly responsible for the pattern change in the jaguar/obelix mutant. PMID:17121467

Toward developmental models of psychiatric disorders in zebrafish

PubMed Central

Norton, William H. J.

2013-01-01

Psychiatric disorders are a diverse set of diseases that affect all aspects of mental function including social interaction, thinking, feeling, and mood. Although psychiatric disorders place a large economic burden on society, the drugs available to treat them are often palliative with variable efficacy and intolerable side-effects. The development of novel drugs has been hindered by a lack of knowledge about the etiology of these diseases. It is thus necessary to further investigate psychiatric disorders using a combination of human molecular genetics, gene-by-environment studies, in vitro pharmacological and biochemistry experiments, animal models, and investigation of the non-biological basis of these diseases, such as environmental effects. Many psychiatric disorders, including autism spectrum disorder, attention-deficit/hyperactivity disorder, mental retardation, and schizophrenia can be triggered by alterations to neural development. The zebrafish is a popular model for developmental biology that is increasingly used to study human disease. Recent work has extended this approach to examine psychiatric disorders as well. However, since psychiatric disorders affect complex mental functions that might be human specific, it is not possible to fully model them in fish. In this review, I will propose that the suitability of zebrafish for developmental studies, and the genetic tools available to manipulate them, provide a powerful model to study the roles of genes that are linked to psychiatric disorders during neural development. The relative speed and ease of conducting experiments in zebrafish can be used to address two areas of future research: the contribution of environmental factors to disease onset, and screening for novel therapeutic compounds. PMID:23637652

Chronic effects of clofibric acid in zebrafish (Danio rerio): a multigenerational study.

PubMed

Coimbra, Ana M; Peixoto, Maria João; Coelho, Inês; Lacerda, Ricardo; Carvalho, António Paulo; Gesto, Manuel; Lyssimachou, Angeliki; Lima, Daniela; Soares, Joana; André, Ana; Capitão, Ana; Castro, Luís Filipe C; Santos, Miguel M

2015-03-01

Clofibric acid (CA) is an active metabolite of the blood lipid lowering agent clofibrate, a pharmaceutical designed to work as agonist of peroxisome proliferator-activated receptor alpha (PPARa). It is the most commonly reported fibrate in aquatic environments with low degradation rate and potential environmental persistence. Previous fish exposures showed that CA may impact spermatogenesis, growth and the expression of fat binding protein genes. However, there are limited data on the effects of chronic multigenerational CA exposures. Here, we assessed chronic multigenerational effects of CA exposure using zebrafish (Danio rerio) as a teleost model. Zebrafish were exposed through the diet to CA (1 and 10mg/g) during their whole lifetime. Growth, reproduction-related parameters and embryonic development were assessed in the exposed fish (F1 generation) and their offspring (F2 generation), together with muscle triglyceride content and gonad histology. In order to study the potential underlying mechanisms, the transcription levels of genes coding for enzymes involved in lipid metabolism pathways were determined. The results show that chronic life-cycle exposure to CA induced a significant reduction in growth of F1 generation and lowered triglyceride muscle content (10mg/g group). Also, an impact in male gonad development was observed together with a decrease in the fecundity (10mg/g group) and higher frequency of embryo abnormalities in the offspring of fish exposed to the lowest CA dose. The profile of the target genes was sex- and tissue-dependent. In F1 an up-regulation of male hepatic pparaa, pparb and acox transcript levels was observed, suggesting an activation of the fatty acid metabolism (provided that transcript level change indicates also a protein level change). Interestingly, the F2 generation, raised with control diet, displayed a response pattern different from that observed in F1, showing an increase in weight in the descendants of CA exposed fish, in comparison with control animals, which points to a multigenerational effect. Copyright © 2015 Elsevier B.V. All rights reserved.

Bisphenol A causes reproductive toxicity, decreases dnmt1 transcription, and reduces global DNA methylation in breeding zebrafish (Danio rerio)

PubMed Central

Laing, L. V.; Viana, J.; Dempster, E. L.; Trznadel, M.; Trunkfield, L. A.; Uren Webster, T. M.; van Aerle, R.; Paull, G. C.; Wilson, R. J.; Mill, J.; Santos, E. M.

2016-01-01

ABSTRACT Bisphenol A (BPA) is a commercially important high production chemical widely used in epoxy resins and polycarbonate plastics, and is ubiquitous in the environment. Previous studies demonstrated that BPA activates estrogenic signaling pathways associated with adverse effects on reproduction in vertebrates and that exposure can induce epigenetic changes. We aimed to investigate the reproductive effects of BPA in a fish model and to document its mechanisms of toxicity. We exposed breeding groups of zebrafish (Danio rerio) to 0.01, 0.1, and 1 mg/L BPA for 15 d. We observed a significant increase in egg production, together with a reduced rate of fertilization in fish exposed to 1 mg/L BPA, associated with significant alterations in the transcription of genes involved in reproductive function and epigenetic processes in both liver and gonad tissue at concentrations representing hotspots of environmental contamination (0.1 mg/L) and above. Of note, we observed reduced expression of DNA methyltransferase 1 (dnmt1) at environmentally relevant concentrations of BPA, along with a significant reduction in global DNA methylation, in testes and ovaries following exposure to 1 mg/L BPA. Our findings demonstrate that BPA disrupts reproductive processes in zebrafish, likely via estrogenic mechanisms, and that environmentally relevant concentrations of BPA are associated with altered transcription of key enzymes involved in DNA methylation maintenance. These findings provide evidence of the mechanisms of action of BPA in a model vertebrate and advocate for its reduction in the environment. PMID:27120497

Use of a highly transparent zebrafish mutant for investigations in the development of the vertebrate auditory system (Conference Presentation)

NASA Astrophysics Data System (ADS)

Wisniowiecki, Anna M.; Mattison, Scott P.; Kim, Sangmin; Riley, Bruce; Applegate, Brian E.

2016-03-01

Zebrafish, an auditory specialist among fish, offer analogous auditory structures to vertebrates and is a model for hearing and deafness in vertebrates, including humans. Nevertheless, many questions remain on the basic mechanics of the auditory pathway. Phase-sensitive Optical Coherence Tomography has been proven as valuable technique for functional vibrometric measurements in the murine ear. Such measurements are key to building a complete understanding of auditory mechanics. The application of such techniques in the zebrafish is impeded by the high level of pigmentation, which develops superior to the transverse plane and envelops the auditory system superficially. A zebrafish double mutant for nacre and roy (mitfa-/- ;roya-/- [casper]), which exhibits defects for neural-crest derived melanocytes and iridophores, at all stages of development, is pursued to improve image quality and sensitivity for functional imaging. So far our investigations with the casper mutants have enabled the identification of the specialized hearing organs, fluid-filled canal connecting the ears, and sub-structures of the semicircular canals. In our previous work with wild-type zebrafish, we were only able to identify and observe stimulated vibration of the largest structures, specifically the anterior swim bladder and tripus ossicle, even among small, larval specimen, with fully developed inner ears. In conclusion, this genetic mutant will enable the study of the dynamics of the zebrafish ear from the early larval stages all the way into adulthood.

Matrix metalloproteinase-9 plays a role in protecting zebrafish from lethal infection with Listeria monocytogenes by enhancing macrophage migration.

PubMed

Shan, Ying; Zhang, Yikai; Zhuo, Xunhui; Li, Xiaoliang; Peng, Jinrong; Fang, Weihuan

2016-07-01

Zebrafish could serve as an alternative animal model for pathogenic bacteria in multiple infectious routes. Our previous study showed that immersion infection in zebrafish with Listeria monocytogenes did not cause lethality but induced transient expression of several immune response genes. We used an Affymetrix gene chip to examine the expression profiles of genes of zebrafish immersion-infected with L. monocytogenes. A total of 239 genes were up-regulated and 56 genes down-regulated compared with uninfected fish. Highest expression (>20-fold) was seen with the mmp-9 gene encoding the matrix metalloproteinase-9 (Mmp-9) known to degrade the extracellular matrix proteins. By morpholino knockdown of mmp-9, we found that the morphants showed rapid death with much higher bacterial load after intravenous or intraventricular (brain ventricle) infection with L. monocytogenes. Macrophages in mmp-9-knockdown morphants had significant defect in migrating to the brain cavity upon intraventricular infection. Decreased migration of murine macrophages with knockdown of mmp-9 and cd44 was also seen in transwell inserts with 8-μm pore polycarbonate membrane, as compared with the scrambled RNA. These findings suggest that Mmp-9 is a protective molecule against infection by L. monocytogenes by engaging in migration of zebrafish macrophages to the site of infection via a non-proteolytic role. Further work is required on the molecular mechanisms governing Mmp-9-driven macrophage migration in zebrafish. Copyright © 2016 Elsevier Ltd. All rights reserved.

BDNF Expression in Larval and Adult Zebrafish Brain: Distribution and Cell Identification

PubMed Central

Cacialli, Pietro; Gueguen, Marie-Madeleine; Coumailleau, Pascal; D’Angelo, Livia; Kah, Olivier; Lucini, Carla; Pellegrini, Elisabeth

2016-01-01

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has emerged as an active mediator in many essential functions in the central nervous system of mammals. BDNF plays significant roles in neurogenesis, neuronal maturation and/or synaptic plasticity and is involved in cognitive functions such as learning and memory. Despite the vast literature present in mammals, studies devoted to BDNF in the brain of other animal models are scarse. Zebrafish is a teleost fish widely known for developmental genetic studies and is emerging as model for translational neuroscience research. In addition, its brain shows many sites of adult neurogenesis allowing higher regenerative properties after traumatic injuries. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the distribution of bdnf mRNAs in the larval and adult zebrafish brain and to characterize the phenotype of cells expressing bdnf mRNAs by means of double staining studies. Our results showed that bdnf mRNAs were widely expressed in the brain of 7 days old larvae and throughout the whole brain of mature female and male zebrafish. In adults, bdnf mRNAs were mainly observed in the dorsal telencephalon, preoptic area, dorsal thalamus, posterior tuberculum, hypothalamus, synencephalon, optic tectum and medulla oblongata. By combining immunohistochemistry with in situ hybridization, we showed that bdnf mRNAs were never expressed by radial glial cells or proliferating cells. By contrast, bdnf transcripts were expressed in cells with neuronal phenotype in all brain regions investigated. Our results provide the first demonstration that the brain of zebrafish expresses bdnf mRNAs in neurons and open new fields of research on the role of the BDNF factor in brain mechanisms in normal and brain repairs situations. PMID:27336917

A rapid throughput approach identifies cognitive deficits in adult zebrafish from developmental exposure to polybrominated flame retardants.

PubMed

Truong, Lisa; Mandrell, David; Mandrell, Rick; Simonich, Michael; Tanguay, Robert L

2014-07-01

A substantial body of evidence has correlated the human body burdens of some polybrominated diphenyl ether (PBDE) flame retardants with cognitive and other behavioral deficits. Adult zebrafish exhibit testable learning and memory, making them an increasingly attractive model for neurotoxicology. Our goal was to develop a rapid throughput means of identifying the cognitive impact of developmental exposure to flame retardants in the zebrafish model. We exposed embryos from 6h post fertilization to 5 days post fertilization to either PBDE 47 (0.1μM), PBDE 99 (0.1μM) or PBDE 153 (0.1μM), vehicle (0.1% DMSO), or embryo medium (EM). The larvae were grown to adulthood and evaluated for the rate at which they learned an active-avoidance response in an automated shuttle box array. Zebrafish developmentally exposed to PBDE 47 learned the active avoidance paradigm significantly faster than the 0.1% DMSO control fish (P<0.0001), but exhibited significantly poorer performance when retested suggestive of impaired memory retention or altered neuromotor activity. Learning in the PBDE 153 group was not significantly different from the DMSO group. Developmental exposure to 0.1% DMSO impaired adult active avoidance learning relative to the sham group (n=39; P<0.0001). PBDE 99 prevented the DMSO effect, yielding a learning rate not significantly different from the sham group (n=36; P>0.9). Our results underscore the importance of vehicle choice in accurately assessing chemical effects on behavior. Active avoidance response in zebrafish is an effective model of learning that, combined with automated shuttle box testing, will provide a highly efficient platform for evaluating persistent neurotoxic hazard from many chemicals. Copyright © 2014 Elsevier Inc. All rights reserved.

Zebrafish cdc6 hypomorphic mutation causes Meier-Gorlin syndrome-like phenotype.

PubMed

Yao, Likun; Chen, Jing; Wu, Xiaotong; Jia, Shunji; Meng, Anming

2017-11-01

Cell Division Cycle 6 (Cdc6) is a component of pre-replicative complex (preRC) forming on DNA replication origins in eukaryotes. Recessive mutations in ORC1, ORC4, ORC6, CDT1 or CDC6 of the preRC in human cause Meier-Gorlin syndrome (MGS) that is characterized by impaired post-natal growth, short stature and microcephaly. However, vertebrate models of MGS have not been reported. Through N-ethyl-N-nitrosourea mutagenesis and Cas9 knockout, we generate several cdc6 mutant lines in zebrafish. Loss-of-function mutations of cdc6, as manifested by cdc6tsu4305 and cdc6tsu7cd mutants, lead to embryonic lethality due to cell cycle arrest at the S phase and extensive apoptosis. Embryos homozygous for a cdc6 hypomorphic mutation, cdc6tsu21cd, develop normally during embryogenesis. Later on, compared with their wild-type (WT) siblings, cdc6tsu21cd mutant fish show growth retardation, and their body weight and length in adulthood are greatly reduced, which resemble human MGS. Surprisingly, cdc6tsu21cd mutant fish become males with a short life and fail to mate with WT females, suggesting defective reproduction. Overexpression of Cdc6 mutant forms, which mimic human CDC6(T323R) mutation found in a MGS patient, in zebrafish cdc6tsu4305 mutant embryos partially represses cell death phenotype, suggesting that the human CDC6(T323R) mutation is a hypomorph. cdc6tsu21cd mutant fish will be useful to detect more tissue defects and develop medical treatment strategies for MGS patients. © The Author 2017. Published by Oxford University Press.

Zebrafish cdc6 hypomorphic mutation causes Meier-Gorlin syndrome-like phenotype

PubMed Central

Yao, Likun; Chen, Jing; Wu, Xiaotong; Jia, Shunji; Meng, Anming

2017-01-01

Abstract Cell Division Cycle 6 (Cdc6) is a component of pre-replicative complex (preRC) forming on DNA replication origins in eukaryotes. Recessive mutations in ORC1, ORC4, ORC6, CDT1 or CDC6 of the preRC in human cause Meier-Gorlin syndrome (MGS) that is characterized by impaired post-natal growth, short stature and microcephaly. However, vertebrate models of MGS have not been reported. Through N-ethyl-N-nitrosourea mutagenesis and Cas9 knockout, we generate several cdc6 mutant lines in zebrafish. Loss-of-function mutations of cdc6, as manifested by cdc6tsu4305 and cdc6tsu7cd mutants, lead to embryonic lethality due to cell cycle arrest at the S phase and extensive apoptosis. Embryos homozygous for a cdc6 hypomorphic mutation, cdc6tsu21cd, develop normally during embryogenesis. Later on, compared with their wild-type (WT) siblings, cdc6tsu21cd mutant fish show growth retardation, and their body weight and length in adulthood are greatly reduced, which resemble human MGS. Surprisingly, cdc6tsu21cd mutant fish become males with a short life and fail to mate with WT females, suggesting defective reproduction. Overexpression of Cdc6 mutant forms, which mimic human CDC6(T323R) mutation found in a MGS patient, in zebrafish cdc6tsu4305 mutant embryos partially represses cell death phenotype, suggesting that the human CDC6(T323R) mutation is a hypomorph. cdc6tsu21cd mutant fish will be useful to detect more tissue defects and develop medical treatment strategies for MGS patients. PMID:28985365

In vivo alternative assessment of the chemicals that interfere with anterior pituitary POMC expression and interrenal steroidogenesis in POMC: EGFP transgenic zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Lingli; Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072; Xu Wei

2010-11-01

Adrenocorticotropin (ACTH) has been considered a classic adrenocorticotropic hormone and the key pituitary-derived peptide controlling steroidogenesis in the adult adrenal. ACTH is encoded by the propiomelanocortin (POMC) gene, and its active form is mainly synthesized and processed from the POMC-encoded multihormone precursor in the anterior pituitary. The ACTH level has always been precisely controlled in the signaling cascade of the hypothalamo-pituitary-adrenal (HPA) axis due to its central role. The purpose of this study was to investigate whether the transgenic zebrafish line with EGFP driven by the POMC promoter can be used as a surrogate marker to detect the interference effectsmore » on anterior pituitary POMC expression caused by chemicals in teleost. The Tg (POMC:EGFP) fish treated for 4 days with the known adrenergic agents, dexamethasone (Dex) or aminoglutethimide (AG), exhibited altered levels of EGFP and POMC expression in the anterior domain of pituitary corticotrophs. Whole-mount in situ hybridization revealed impaired patterns of expression of the zebrafish ftz-fl gene (ff1b), a key molecular marker for early interrenal development. Next, several chemicals and six commonly used organophosphorus compounds (OPs) were tested for their effects on anterior pituitary POMC expression and early interrenal development. Our preliminary screening analyses indicated that simazine and 3,3',4,4'5-pentachlorobiphenyl (PCB126) could interfere with anterior pituitary POMC expression and interrenal development in fish. In summary, our results demonstrated that the Tg (POMC:EGFP) zebrafish line might be employed as a specific and reproductive in vivo assessment model for the effects of endocrine disruption on HPA signaling.« less

Developmental Deltamethrin Exposure Causes Persistent Changes in Dopaminergic Gene Expression, Neurochemistry, and Locomotor Activity in Zebrafish.

PubMed

Kung, Tiffany S; Richardson, Jason R; Cooper, Keith R; White, Lori A

2015-08-01

Pyrethroids are commonly used insecticides that are considered to pose little risk to human health. However, there is an increasing concern that children are more susceptible to the adverse effects of pesticides. We used the zebrafish model to test the hypothesis that developmental exposure to low doses of the pyrethroid deltamethrin results in persistent alterations in dopaminergic gene expression, neurochemistry, and locomotor activity. Zebrafish embryos were treated with deltamethrin (0.25-0.50 μg/l), at concentrations below the LOAEL, during the embryonic period [3-72 h postfertilization (hpf)], after which transferred to fresh water until the larval stage (2-weeks postfertilization). Deltamethrin exposure resulted in decreased transcript levels of the D1 dopamine (DA) receptor (drd1) and increased levels of tyrosine hydroxylase at 72 hpf. The reduction in drd1 transcripts persisted to the larval stage and was associated with decreased D2 dopamine receptor transcripts. Larval fish, exposed developmentally to deltamethrin, had increased levels of homovanillic acid, a DA metabolite. Since the DA system is involved in locomotor activity, we measured the swim activity of larval fish following a transition to darkness. Developmental exposure to deltamethrin significantly increased larval swim activity which was attenuated by concomitant knockdown of the DA transporter. Acute exposure to methylphenidate, a DA transporter inhibitor, increased swim activity in control larva, while reducing swim activity in larva developmentally exposed to deltamethrin. Developmental exposure to deltamethrin causes locomotor deficits in larval zebrafish, which is likely mediated by dopaminergic dysfunction. This highlights the need to understand the persistent effects of low-dose neurotoxicant exposure during development. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Developmental Deltamethrin Exposure Causes Persistent Changes in Dopaminergic Gene Expression, Neurochemistry, and Locomotor Activity in Zebrafish

PubMed Central

Kung, Tiffany S.; Richardson, Jason R.; Cooper, Keith R.; White, Lori A.

2015-01-01

Pyrethroids are commonly used insecticides that are considered to pose little risk to human health. However, there is an increasing concern that children are more susceptible to the adverse effects of pesticides. We used the zebrafish model to test the hypothesis that developmental exposure to low doses of the pyrethroid deltamethrin results in persistent alterations in dopaminergic gene expression, neurochemistry, and locomotor activity. Zebrafish embryos were treated with deltamethrin (0.25–0.50 μg/l), at concentrations below the LOAEL, during the embryonic period [3–72 h postfertilization (hpf)], after which transferred to fresh water until the larval stage (2-weeks postfertilization). Deltamethrin exposure resulted in decreased transcript levels of the D1 dopamine (DA) receptor (drd1) and increased levels of tyrosine hydroxylase at 72 hpf. The reduction in drd1 transcripts persisted to the larval stage and was associated with decreased D2 dopamine receptor transcripts. Larval fish, exposed developmentally to deltamethrin, had increased levels of homovanillic acid, a DA metabolite. Since the DA system is involved in locomotor activity, we measured the swim activity of larval fish following a transition to darkness. Developmental exposure to deltamethrin significantly increased larval swim activity which was attenuated by concomitant knockdown of the DA transporter. Acute exposure to methylphenidate, a DA transporter inhibitor, increased swim activity in control larva, while reducing swim activity in larva developmentally exposed to deltamethrin. Developmental exposure to deltamethrin causes locomotor deficits in larval zebrafish, which is likely mediated by dopaminergic dysfunction. This highlights the need to understand the persistent effects of low-dose neurotoxicant exposure during development. PMID:25912032

Embryonic exposure to benzo(a)pyrene inhibits reproductive capability in adult female zebrafish and correlation with DNA methylation.

PubMed

Gao, Dongxu; Lin, Jing; Ou, Kunlin; Chen, Ying; Li, Hongbin; Dai, Qinhua; Yu, Zhenni; Zuo, Zhenghong; Wang, Chonggang

2018-05-09

This study was conducted to investigate the effects of embryonic short-term exposure to benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, on ovarian development and reproductive capability in adult female zebrafish. In 1-year-old fish after embryonic exposure to BaP for 96 h, the gonadosomatic indices and the percentage of mature oocytes were significantly decreased in the 0.5, 5 and 50 nmol/L treatments. The spawned egg number, the fertilization rate and the hatching success were significantly reduced, while the malformation rate of the F1 unexposed larvae were increased. The mRNA levels of follicle-stimulating hormone, luteinizing hormone, ovarian cytochrome P450 aromatase cyp19a1a and cyp19b, estrogen receptor esr1 and esr2, and hepatic vitellogenin vtg1 and vtg2 genes, were down-regulated in adult female zebrafish that were exposed to BaP during embryonic stage. Both 17β-estradiol and testosterone levels were reduced in the ovary of adult females. The methylation levels of the gonadotropin releasing hormone (GnRH) gene gnrh3 were significantly increased in the adult zebrafish brain, and those of the GnRH receptor gene gnrhr3 were elevated both in the larvae exposed to BaP and in the adult brain, which might cause the down-regulation of the mRNA levels of gnrh3 and gnrhr3. This epigenetic change caused by embryonic exposure to BaP might be a reason for physiological changes along the brain-pituitary-gonad axis. These results suggest that short-term exposure in early life should be included and evaluated in any risk assessment of pollutant exposure to the reproductive health of fish. Copyright © 2018 Elsevier Ltd. All rights reserved.

Nicotine increases fear responses and brain acetylcholinesterase activity in a context-dependent manner in zebrafish.

PubMed

Ziani, Paola R; Müller, Talise E; Stefanello, Flavia V; Fontana, Barbara D; Duarte, Tâmie; Canzian, Julia; Rosemberg, Denis B

2018-07-01

Nicotine is an alkaloid with positive effects on learning and memory processes. Exposure to conspecific alarm substance (CAS) elicits fear responses in zebrafish, but the effects of nicotine on aversive behaviors and associative learning in this species remain unclear. Here, we evaluated whether nicotine enhances contextual fear responses in zebrafish and investigated a putative involvement of brain acetylcholinesterase (AChE) in associative learning. Fish were exposed to 1 mg/L nicotine for 3 min and then kept in non-chlorinated water for 20 min. Later, animals were transferred to experimental tanks in the absence or presence of 3.5 mL/L CAS for 5 min (training session). After 24 h, fish were tested in tanks with similar or altered context in the absence of CAS (post-training session) and brain AChE activity was further assessed. At training, CAS increased freezing, erratic movements, and decreased the time spent in top area, while nicotine abolished the effects of CAS on erratic movements. Nicotine/CAS group tested in a similar context showed exacerbated freezing and reduced transitions to top area. Moreover, a decrease in distance traveled was observed in control, nicotine, and nicotine/CAS groups at post-training. Nicotine also stimulated brain AChE activity in CAS-exposed animals reintroduced in tanks with similar context. Although freezing bouts and time spent in top could serve as behavioral endpoints that reflect CAS-induced sensitization, the effects of nicotine occurred in a context-dependent manner. Collectively, our data suggest an involvement of cholinergic signaling in aversive learning, reinforcing the growing utility of zebrafish models to explore the neurobehavioral effects of nicotine in vertebrates. Copyright © 2018 Elsevier Inc. All rights reserved.

Light directs zebrafish period2 expression via conserved D and E boxes.

PubMed

Vatine, Gad; Vallone, Daniela; Appelbaum, Lior; Mracek, Philipp; Ben-Moshe, Zohar; Lahiri, Kajori; Gothilf, Yoav; Foulkes, Nicholas S

2009-10-01

For most species, light represents the principal environmental signal for entraining the endogenous circadian clock. The zebrafish is a fascinating vertebrate model for studying this process since unlike mammals, direct exposure of most of its tissues to light leads to local clock entrainment. Importantly, light induces the expression of a set of genes including certain clock genes in most zebrafish cell types in vivo and in vitro. However, the mechanism linking light to gene expression remains poorly understood. To elucidate this key mechanism, here we focus on how light regulates transcription of the zebrafish period2 (per2) gene. Using transgenic fish and stably transfected cell line-based assays, we define a Light Responsive Module (LRM) within the per2 promoter. The LRM lies proximal to the transcription start site and is both necessary and sufficient for light-driven gene expression and also for a light-dependent circadian clock regulation. Curiously, the LRM sequence is strongly conserved in other vertebrate per2 genes, even in species lacking directly light-sensitive peripheral clocks. Furthermore, we reveal that the human LRM can substitute for the zebrafish LRM to confer light-regulated transcription in zebrafish cells. The LRM contains E- and D-box elements that are critical for its function. While the E-box directs circadian clock regulation by mediating BMAL/CLOCK activity, the D-box confers light-driven expression. The zebrafish homolog of the thyrotroph embryonic factor binds efficiently to the LRM D-box and transactivates expression. We demonstrate that tef mRNA levels are light inducible and that knock-down of tef expression attenuates light-driven transcription from the per2 promoter in vivo. Together, our results support a model where a light-dependent crosstalk between E- and D-box binding factors is a central determinant of per2 expression. These findings extend the general understanding of the mechanism whereby the clock is entrained by light and how the regulation of clock gene expression by light has evolved in vertebrates.

Drug Discovery in Fish, Flies, and Worms

PubMed Central

Strange, Kevin

2016-01-01

Abstract Nonmammalian model organisms such as the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the zebrafish Danio rerio provide numerous experimental advantages for drug discovery including genetic and molecular tractability, amenability to high-throughput screening methods and reduced experimental costs and increased experimental throughput compared to traditional mammalian models. An interdisciplinary approach that strategically combines the study of nonmammalian and mammalian animal models with diverse experimental tools has and will continue to provide deep molecular and genetic understanding of human disease and will significantly enhance the discovery and application of new therapies to treat those diseases. This review will provide an overview of C. elegans, Drosophila, and zebrafish biology and husbandry and will discuss how these models are being used for phenotype-based drug screening and for identification of drug targets and mechanisms of action. The review will also describe how these and other nonmammalian model organisms are uniquely suited for the discovery of drug-based regenerative medicine therapies. PMID:28053067

Effects of simulated weightlessness on fish otolith growth: Clinostat versus Rotating-Wall Vessel

NASA Astrophysics Data System (ADS)

Brungs, Sonja; Hauslage, Jens; Hilbig, Reinhard; Hemmersbach, Ruth; Anken, Ralf

2011-09-01

Stimulus dependence is a general feature of developing sensory systems. It has been shown earlier that the growth of inner ear heavy stones (otoliths) of late-stage Cichlid fish ( Oreochromis mossambicus) and Zebrafish ( Danio rerio) is slowed down by hypergravity, whereas microgravity during space flight yields an opposite effect, i.e. larger than 1 g otoliths, in Swordtail ( Xiphophorus helleri) and in Cichlid fish late-stage embryos. These and related studies proposed that otolith growth is actively adjusted via a feedback mechanism to produce a test mass of the appropriate physical capacity. Using ground-based techniques to apply simulated weightlessness, long-term clinorotation (CR; exposure on a fast-rotating Clinostat with one axis of rotation) led to larger than 1 g otoliths in late-stage Cichlid fish. Larger than normal otoliths were also found in early-staged Zebrafish embryos after short-term Wall Vessel Rotation (WVR; also regarded as a method to simulate weightlessness). These results are basically in line with the results obtained on Swordtails from space flight. Thus, the growth of fish inner ear otoliths seems to be an appropriate parameter to assess the quality of "simulated weightlessness" provided by a particular simulation device. Since CR and WVR are in worldwide use to simulate weightlessness conditions on ground using small-sized specimens, we were prompted to directly compare the effects of CR and WVR on otolith growth using developing Cichlids as model organism. Animals were simultaneously subjected to CR and WVR from a point of time when otolith primordia had begun to calcify both within the utricle (gravity perception) and the saccule (hearing); the respective otoliths are the lapilli and the sagittae. Three such runs were subsequently carried out, using three different batches of fish. The runs were discontinued when the animals began to hatch. In the course of all three runs performed, CR led to larger than normal lapilli, whereas WVR had no effect on the growth of these otoliths. Regarding sagittae, CR resulted in larger than normal stones in one of the three runs. The other CR runs and all WVR runs had no effect on sagittal growth. These results clearly indicate that CR rather than WVR can be regarded as a device to simulate weightlessness using the Cichlid as model organism. Since WVR has earlier been shown to affect otolith growth in Zebrafish, the lifestyle of an animal (mouth-breeding versus egg-laying) seems to be of considerable importance. Further studies using a variety of simulation techniques (including, e.g. magnetic levitation and random positioning) and various species are needed in order to identify the most appropriate technique to simulate weightlessness regarding a particular model organism.

The oxidized state of the nanocomposite Carbo-Iron® causes no adverse effects on growth, survival and differential gene expression in zebrafish.

PubMed

Weil, Mirco; Meißner, Tobias; Busch, Wibke; Springer, Armin; Kühnel, Dana; Schulz, Ralf; Duis, Karen

2015-10-15

For degradation of halogenated chemicals in groundwater Carbo-Iron®, a composite of activated carbon and nano-sized Fe(0), was developed (Mackenzie et al., 2012). Potential effects of this nanocomposite on fish were assessed. Beyond the contaminated zone Fe(0) can be expected to have oxidized and Carbo-Iron was used in its oxidized form in ecotoxicological tests. Potential effects of Carbo Iron in zebrafish (Danio rerio) were investigated using a 48 h embryo toxicity test under static conditions, a 96 h acute test with adult fish under semi-static conditions and a 34 d fish early life stage test (FELST) in a flow-through system. Particle diameters in test suspensions were determined via dynamic light scattering (DLS) and ranged from 266 to 497 nm. Particle concentrations were measured weekly in samples from the FELST using a method based on the count rate in DLS. Additionally, uptake of particles into test organisms was investigated using microscopic methods. Furthermore, effects of Carbo-Iron on gene expression were investigated by microarray analysis in zebrafish embryos. In all tests performed, no significant lethal effects were observed. Furthermore, Carbo-Iron had no significant influence on weight and length of fish as determined in the FELST. In the embryo test and the early life stage test, growth of fungi on the chorion was observed at Carbo-Iron concentrations between 6.3 and 25mg/L. Fungal growth did not affect survival, hatching success and growth. In the embryo test, no passage of Carbo-Iron particles into the perivitelline space or the embryo was observed. In juvenile and adult fish, Carbo-Iron was detected in the gut at the end of exposure. In juvenile fish exposed to Carbo-Iron for 29 d and subsequently kept for 5d in control water, Carbo-Iron was no longer detectable in the gut. Global gene expression in zebrafish embryos was not significantly influenced by Carbo-Iron. Copyright © 2015 Elsevier B.V. All rights reserved.

In Vivo Time-Lapse Imaging in the Zebrafish Lateral Line: A Flexible, Open-Ended Research Project for an Undergraduate Neurobiology Laboratory Course.

PubMed

Marra, Molly H; Tobias, Zachary J C; Cohen, Hannah R; Glover, Greta; Weissman, Tamily A

2015-01-01

The lateral line sensory system in fish detects movements in the water and allows fish to respond to predators, prey, and other stimuli. As the lateral line forms in the first two days of zebrafish development, axons extend caudally along the lateral surface of the fish, eventually forming synapses with hair cells of neuromasts. Growing lateral line axons are located superficially under the skin and can be labeled in living zebrafish using fluorescent protein expression. This system provides a relatively straightforward approach for in vivo time-lapse imaging of neuronal development in an undergraduate setting. Here we describe an upper-level neurobiology laboratory module in which students investigate aspects of axonal development in the zebrafish lateral line system. Students learn to handle and image living fish, collect time-lapse videos of moving mitochondria, and quantitatively measure mitochondrial dynamics by generating and analyzing kymographs of their movements. Energy demands may differ between axons with extending growth cones versus axons that have already reached their targets and are forming synapses. Since relatively little is known about this process in developing lateral line axons, students generate and test their own hypotheses regarding how mitochondrial dynamics may differ at two different time points in axonal development. Students also learn to incorporate into their analysis a powerful yet accessible quantitative tool, the kymograph, which is used to graph movement over time. After students measure and quantify dynamics in living fish at 1 and 2 days post fertilization, this module extends into independent projects, in which students can expand their studies in a number of different, inquiry-driven directions. The project can also be pared down for courses that wish to focus solely on the quantitative analysis (without fish handling), or vice versa. This research module provides a useful approach for the design of open-ended laboratory research projects that integrate the scientific process into undergraduate Biology courses, as encouraged by the AAAS and NSF Vision and Change Initiative.

Chemical Ototoxicity of the Fish Inner Ear and Lateral Line.

PubMed

Coffin, Allison B; Ramcharitar, John

2016-01-01

Hair cell-driven mechanosensory systems are crucial for successful execution of a number of behaviors in fishes, and have emerged as good models for exploring questions relevant to human hearing. This review focuses on ototoxic effects in the inner ear and lateral line system of fishes. We specifically examine studies where chemical ototoxins such as aminoglycoside antibiotics have been employed as tools to disable the lateral line. Lateral line ablation results in alterations to feeding behavior and orientation to water current in a variety of species. However, neither behavior is abolished in the presence of additional sensory cues, supporting the hypothesis that many fish behaviors are driven by multisensory integration. Within biomedical research, the larval zebrafish lateral line has become an important model system for understanding signaling mechanisms that contribute to hair cell death and for developing novel pharmacological therapies that protect hair cells from ototoxic damage. Furthermore, given that fishes robustly regenerate damaged hair cells, ototoxin studies in fishes have broadened our understanding of the molecular and genetic events in an innately regenerative system, offering potential targets for mammalian hair cell regeneration. Collectively, studies of fish mechanosensory systems have yielded insight into fish behavior and in mechanisms of hair cell death, protection, and regeneration.

Zebrafish take their cue from temperature but not photoperiod for the seasonal plasticity of thermal performance.

PubMed

Condon, Catriona H; Chenoweth, Stephen F; Wilson, Robbie S

2010-11-01

Organisms adjust to seasonal variability in the environment by responding to cues that indicate environmental change. As most studies of seasonal phenotypic plasticity test only the effect of a single environmental cue, how animals may integrate information from multiple cues to fine-tune plastic responses remains largely unknown. We examined the interaction between correlated (seasonally matching) and conflicting (seasonally opposite) temperature and photoperiod cues on the acclimation of performance traits in male zebrafish, Danio rerio. We acclimated fish for 8 weeks and then tested the change in thermal dependence of maximum burst swimming and feeding rate between 8 and 38°C. We predicted that correlated environmental cues should induce a greater acclimation response than uncorrelated cues. However, we found that only temperature was important for the seasonal acclimation of performance traits in zebrafish. Thermal acclimation shifted the thermal performance curve of both traits. For maximum burst swimming, performance increased for each group near the acclimation temperature and reduced in environments that were far from their acclimation temperature. The feeding rate of cold-acclimated zebrafish was reduced across the test temperature range compared with that of warm-acclimated fish. Our study is the first that has found no effect of the covariation between temperature and photoperiod acclimation cues on locomotor performance in fishes. Our results support the intuitive idea that photoperiod may be a less important seasonal cue for animals living at lower latitudes.

Preliminary Evaluation on the Effects of Feeds on the Growth and Early Reproductive Performance of Zebrafish (Danio rerio)

PubMed Central

2012-01-01

This study evaluated the effects of several commercially available feeds and different feeding regimes on the growth and early reproductive performance of zebrafish (Danio rerio). Juvenile zebrafish (n= 20; 5.06 ± 0.69 mg) were stocked into each of 24 tanks (volume, 2 L); 3 tanks were assigned to each of 8 feeding combinations for a period of 60 d. At the end of 60 d, 2 male and 2 female fish from each tank were pooled by dietary treatment (n = 6) and used to evaluate the effects of feeding combinations on early reproductive performance. Zebrafish fed dietary treatments 3 and 7 had significantly greater weight gain than zebrafish fed diet 5. Mean spawning success was significantly greater in zebrafish fed the control diet (Artemiaonly) than in those fed diet 1. Mean hatch rates were greater in zebrafish fed the control feed and diets 1, 2, 3, 5, and 6 than zebrafish fed diet 4. Additional results suggest that female zebrafish are sexually mature after 90 d post fertilization and that fertilization rates are the limiting factor in early reproduction. PMID:23043806

A novel keratin18 promoter that drives reporter gene expression in the intrahepatic and extrahepatic biliary system allows isolation of cell-type specific transcripts from zebrafish liver

PubMed Central

Wilkins, Benjamin J.; Gong, Weilong; Pack, Michael

2015-01-01

Heritable and acquired biliary disorders are an important cause of acute and chronic human liver disease. Biliary development and physiology have been studied extensively in rodent models and more recently, zebrafish have been used to uncover pathogenic mechanisms and potential therapies for these conditions. Here we report development of novel transgenic lines labeling the intrahepatic and extrahepatic biliary system of zebrafish larvae that can be used for lineage tracing and isolation of biliary-specific RNAs from mixed populations of liver cells. We show that GFP expression driven by a 4.4 kilobase promoter fragment from the zebrafish keratin18 (krt18) gene allows visualization of all components of the developing biliary system as early as 3 days post-fertilization. In addition, expression of a ribosomal fusion protein (EGFP-Rpl10a) in krt18:TRAP transgenic fish allows for enrichment of translated biliary cell mRNAs via translating ribosome affinity purification (TRAP). Future studies utilizing these reagents will enhance our understanding of the morphologic and molecular processes involved in biliary development and disease. PMID:24394404

There and Back Again: Development and Regeneration of the Zebrafish Lateral Line System

PubMed Central

Thomas, Eric D.; Cruz, Ivan A.; Hailey, Dale W.; Raible, David W.

2014-01-01

The zebrafish lateral line is a sensory system used to detect changes in water flow. It is comprised of clusters of mechanosensory hair cells called neuromasts. The lateral line is initially established by a migratory group of cells, called a primordium, that deposits neuromasts at stereotyped locations along the surface of the fish. Wnt, FGF, and Notch signaling are all important regulators of various aspects of lateral line development, from primordium migration to hair cell specification. As zebrafish age, the organization of the lateral line becomes more complex in order to accommodate the fish’s increased size. This expansion is regulated by many of the same factors involved in the initial development. Furthermore, unlike mammalian hair cells, lateral line hair cells have the capacity to regenerate after damage. New hair cells arise from the proliferation and differentiation of surrounding support cells, and the molecular and cellular pathways regulating this are beginning to be elucidated. All in all, the zebrafish lateral line has proven to be an excellent model in which to study a diverse array of processes, including collective cell migration, cell polarity, cell fate, and regeneration. PMID:25330982

Establishment of Three Francisella Infections in Zebrafish Embryos at Different Temperatures

PubMed Central

Brudal, Espen; Ulanova, Lilia S.; O. Lampe, Elisabeth; Rishovd, Anne-Lise; Winther-Larsen, Hanne C.

2014-01-01

Francisella spp. are facultative intracellular pathogens identified in increasingly diverse hosts, including mammals. F. noatunensis subsp. orientalis and F. noatunensis subsp. noatunensis infect fish inhabiting warm and cold waters, respectively, while F. tularensis subsp. novicida is highly infectious for mice and has been widely used as a model for the human pathogen F. tularensis. Here, we established zebrafish embryo infection models of fluorescently labeled F. noatunensis subsp. noatunensis, F. noatunensis subsp. orientalis, and F. tularensis subsp. novicida at 22, 28, and 32°C, respectively. All infections led to significant bacterial growth, as shown by reverse transcription-quantitative PCR (RT-qPCR), and to a robust proinflammatory immune response, dominated by increased transcription of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). F. noatunensis subsp. orientalis was the most virulent, F. noatunensis subsp. noatunensis caused chronic infection, and F. tularensis subsp. novicida showed moderate virulence and led to formation of relatively small granuloma-like structures. The use of transgenic zebrafish strains with enhanced green fluorescent protein (EGFP)-labeled immune cells revealed their detailed interactions with Francisella species. All three strains entered preferentially into macrophages, which eventually assembled into granuloma-like structures. Entry into neutrophils was also observed, though the efficiency of this event depended on the route of infection. The results demonstrate the usefulness of the zebrafish embryo model for studying infections caused by different Francisella species at a wide range of temperatures and highlight their interactions with immune cells. PMID:24614659

ZeBase: an open-source relational database for zebrafish laboratories.

PubMed

Hensley, Monica R; Hassenplug, Eric; McPhail, Rodney; Leung, Yuk Fai

2012-03-01

Abstract ZeBase is an open-source relational database for zebrafish inventory. It is designed for the recording of genetic, breeding, and survival information of fish lines maintained in a single- or multi-laboratory environment. Users can easily access ZeBase through standard web-browsers anywhere on a network. Convenient search and reporting functions are available to facilitate routine inventory work; such functions can also be automated by simple scripting. Optional barcode generation and scanning are also built-in for easy access to the information related to any fish. Further information of the database and an example implementation can be found at http://zebase.bio.purdue.edu.

Developmental Localization of Nephrin in Zebrafish and Medaka Pronephric Glomerulus

PubMed Central

Ichimura, Koichiro; Fukuyo, Yayoi; Nakamura, Tomomi; Powell, Rebecca; Sakai, Tatsuo; Janknecht, Ralf

2013-01-01

Slit diaphragm (SD) is a highly specialized intercellular junction between podocyte foot processes and plays a crucial role in the formation of the filtration barrier. In this study, we examined the developmental localization of Nephrin, an essential component of SD, in the pronephric glomerulus of zebrafish and medaka. In the mature glomerulus of both fish, Nephrin is localized along the glomerular basement membrane as seen in mammals, indicating that Nephrin is localized at the SD. Interestingly, Nephrin was detected already in immature podocytes before the SD and foot processes started to form in both fish. Nephrin was localized along the cell surface of immature podocytes but as different localization patterns. In zebrafish, Nephrin signal bordered the lateral membrane of podocytes, which were columnar in shape, as in rat immature podocytes. However, in medaka immature podocytes, Nephrin was localized in a punctate pattern among podocyte cell bodies. These findings suggest that Nephrin needs to be integrated to the membrane before the formation of the SD and then moves to the proper site to form the SD. Furthermore, a podocyte-specific marker, such as Nephrin, should be a useful tool for the future analysis of pronephric glomerular development in fish mutants and morphants. PMID:23324868

Successful Replacement of Artemia salina nauplii with Marine Rotifers (Brachionus plicatilis) in the Diet of Preadult Zebrafish (Danio rerio).

PubMed

Lawrence, Christian; James, Althea; Mobley, Scottie

2015-10-01

Previously established rearing protocols for zebrafish begin feeding with marine rotifers (Brachionus plicatilis), followed by Artemia nauplii until the fish reach subadult stage, the developmental time point at which they can be most easily transitioned onto a processed diet. However, the inclusion of Artemia is less than ideal, given its fluctuating availability and high costs. We tested whether or not we could replace Artemia with rotifers during our normal rearing sequence and still meet published performance standards for (i) weaning fish onto a processed diet by 25 days postfertilization (dpf) and (ii) successful breeding by 60 dpf. Here, we present the results of trials where wild-type and casper zebrafish were fed exclusively with rotifers (R) or rotifers followed by Artemia (RA) for the first 25 dpf after which point all fish were transitioned to a processed diet (Gemma Micro 300). We measured growth and survival at days 25 and 60, and tested for reproductive capability at 60 dpf. While growth performance was significantly better in the RA groups, we were still able to meet goals for both weaning and generation time in the R groups without compromising survival or sex ratios.

Exposure to gemfibrozil and atorvastatin affects cholesterol metabolism and steroid production in zebrafish (Danio rerio).

PubMed

Al-Habsi, Aziz A; Massarsky, Andrey; Moon, Thomas W

2016-09-01

The commonly used lipid-lowering pharmaceuticals gemfibrozil (GEM) and atorvastatin (ATV) are detected in the aquatic environment; however, their potential effects on non-target fish species are yet to be fully understood. This study examined the effects of GEM and/or ATV on female and male adult zebrafish after a 30d dietary exposure. The exposure led to changes in several biochemical parameters, including reduction in cholesterol, triglycerides, cortisol, testosterone, and estradiol. Changes in cholesterol and triglycerides were also associated with changes in transcript levels of key genes involved with cholesterol and lipid regulation, including SREBP2, HMGCR1, PPARα, and SREBP1. We also noted higher CYP3A65 and atrogin1 mRNA levels in drug-treated male fish. Sex differences were apparent in some of the examined parameters at both biochemical and molecular levels. This study supports these drugs affecting cholesterol metabolism and steroid production in adult zebrafish. We conclude that the reduction in cortisol may impair the ability of these fish to mount a suitable stress response, whereas the reduction of sex steroids may negatively affect reproduction. Copyright © 2015 Elsevier Inc. All rights reserved.

Influence of polyethylene microplastic beads on the uptake and localization of silver in zebrafish (Danio rerio).

PubMed

Khan, Farhan R; Syberg, Kristian; Shashoua, Yvonne; Bury, Nicolas R

2015-11-01

This study aimed to determine whether the uptake and localization of Ag in zebrafish was affected by the presence of polyethylene microplastic beads (PE MPBs). Zebrafish were exposed to 1 μg Ag L(-1) (radiolabelled with (110m)Ag) for 4 and 24 h in the presence or absence of PE MPBs (10, 100 or 1000 MPBs mL(-1)), and one treatment in which MPBs (1000 MPBs mL(-1)) were incubated with Ag to promote adsorption. The presence of MPBs, at any of the tested doses, had no effect on the uptake or localization of Ag. However, exposure to the Ag-incubated MPBs (∽75% of the Ag bound to MPBs) significantly reduced Ag uptake at both time points and also significantly increased the proportion of intestinal Ag. This study demonstrates that microplastics can alter the bioavailability and uptake route of a metal contaminant in a model fish species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals.

PubMed

Hanisch, Karen; Küster, Eberhard; Altenburger, Rolf; Gündel, Ulrike

2010-01-01

Studies using embryos of the zebrafish Danio rerio (DarT) instead of adult fish for characterising the (eco-) toxic potential of chemicals have been proposed as animal replacing methods. Effect analysis at the molecular level might enhance sensitivity, specificity, and predictive value of the embryonal studies. The present paper aimed to test the potential of toxicoproteomics with zebrafish eleutheroembryos for sensitive and specific toxicity assessment. 2-DE-based toxicoproteomics was performed applying low-dose (EC(10)) exposure for 48 h with three-model substances Rotenone, 4,6-dinitro-o-cresol (DNOC) and Diclofenac. By multivariate "pattern-only" PCA and univariate statistical analyses, alterations in the embryonal proteome were detectable in nonetheless visibly intact organisms and treatment with the three substances was distinguishable at the molecular level. Toxicoproteomics enabled the enhancement of sensitivity and specificity of the embryonal toxicity assay and bear the potency to identify protein markers serving as general stress markers and early diagnosis of toxic stress.

Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals

PubMed Central

Hanisch, Karen; Küster, Eberhard; Altenburger, Rolf; Gündel, Ulrike

2010-01-01

Studies using embryos of the zebrafish Danio rerio (DarT) instead of adult fish for characterising the (eco-) toxic potential of chemicals have been proposed as animal replacing methods. Effect analysis at the molecular level might enhance sensitivity, specificity, and predictive value of the embryonal studies. The present paper aimed to test the potential of toxicoproteomics with zebrafish eleutheroembryos for sensitive and specific toxicity assessment. 2-DE-based toxicoproteomics was performed applying low-dose (EC10) exposure for 48 h with three-model substances Rotenone, 4,6-dinitro-o-cresol (DNOC) and Diclofenac. By multivariate “pattern-only” PCA and univariate statistical analyses, alterations in the embryonal proteome were detectable in nonetheless visibly intact organisms and treatment with the three substances was distinguishable at the molecular level. Toxicoproteomics enabled the enhancement of sensitivity and specificity of the embryonal toxicity assay and bear the potency to identify protein markers serving as general stress markers and early diagnosis of toxic stress. PMID:22084678

Toxicity test of xanthone from mangosteen on zebrafish embryos

NASA Astrophysics Data System (ADS)

Noordin, Muhammad Akram Mohd; Noor, Mahanem Mat; Kamaruddin, Wan Mohd Aizat Wan; Lazim, Azwan Mat; Fazry, Shazrul

2016-11-01

Xanthone is a chemical compound identified in mangosteen pericarp. A previous study showed that xanthone has anti-proliferating effect on cancer cells. In this study we investigate the toxicity level of xanthone in zebrafish embryo to for future reference on other animal model. We employed Fish Embryo Toxicity (FET) assay to determine the toxicity level of different concentrations of xanthone. Embryos were observed at 24, 48 and 72 hours post fertilization (hpf) under microscope at 4× magnification. The extract showed toxicity effect on embryo at concentrations of 250, 125 and 62.5 µg/mL. Concentrations at 15.63, 7.81 and 3.91 µg / mL of xanthone did not harm the embryos and showed 100% of survival.

Assessment of the lethal and sublethal effects of 20 environmental chemicals in zebrafish embryos and larvae by using OECD TG 212.

PubMed

Horie, Yoshifumi; Yamagishi, Takahiro; Takahashi, Hiroko; Shintaku, Youko; Iguchi, Taisen; Tatarazako, Norihisa

2017-10-01

Fish embryo toxicity tests are used to assess the lethal and sublethal effects of environmental chemicals in aquatic organisms. Previously, we used a short-term toxicity test published by the Organization for Economic Co-operation and Development (test no. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages [OECD TG 212]) to assess the lethal and sublethal effects of aniline and several chlorinated anilines in zebrafish embryos and larvae. To expand upon this previous study, we used OECD TG 212 in zebrafish embryos and larvae to assess the lethal and sublethal effects of 20 additional environmental chemicals that included active pharmaceutical ingredients, pesticides, metals, aromatic compounds or chlorinated anilines. Zebrafish embryos (Danio rerio) were exposed to the test chemicals until 8 days post-fertilization. A delayed lethal effect was induced by 16 of the 20 test chemicals, and a positive correlation was found between heart rate turbulence and mortality. We also found that exposure to the test chemicals at concentrations lower than the lethal concentration induced the sublethal effects of edema, body curvature and absence of swim-bladder inflation. In conclusion, the environmental chemicals assessed in the present study induced both lethal and sublethal effects in zebrafish embryos and larvae, as assessed by using OECD TG 212. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Axonal regeneration in zebrafish spinal cord

PubMed Central

Hui, Subhra Prakash

2018-01-01

Abstract In the present review we discuss two interrelated events—axonal damage and repair—known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals. PMID:29721326

Effects of BPF on steroid hormone homeostasis and gene expression in the hypothalamic-pituitary-gonadal axis of zebrafish.

PubMed

Yang, Qian; Yang, Xianhai; Liu, Jining; Ren, Wenjuan; Chen, Yingwen; Shen, Shubao

2017-09-01

Bisphenol F (BPF) has been frequently detected in various environmental compartments, and previous studies found that BPF exhibits similar estrogenic and anti-androgenic effects on the mammalian endocrine system to those of bisphenol A (BPA). However, the potential disrupting effects of BPF on aquatic organisms and the underling disrupting mechanisms have not been investigated. In this study, the potential disrupting mechanisms of BPF on the hypothalamic-pituitary-gonadal (HPG) axis and liver were probed by employing the OECD 21-day short-term fecundity assay in zebrafish. The results show that BPF exposure (1 mg/L) impaired the reproductive function of zebrafish, as exemplified by alterations to testicular and ovarian histology of the treated zebrafish. Homogenate testosterone (T) levels in male zebrafish decreased in a concentration-dependent manner, and 17β-estradiol (E2) levels increased significantly when fish were exposed to 0.1 and 1 mg/L BPF. The real-time polymerase chain reaction was performed to examine gene expression in the HPG axis and liver. Hepatic vitellogenin expression was significantly upregulated in males, suggesting that BPF possesses estrogenic activity. The disturbed hormone balance was enhanced by the significant changes in gene expression along the HPG axis. These alterations suggest that BPF leads to adverse effects on the endocrine system of teleost fish, and that these effects were more prominent in males than in females.

Multiple zebrafish atoh1 genes specify a diversity of neuronal types in the zebrafish cerebellum.

PubMed

Kidwell, Chelsea U; Su, Chen-Ying; Hibi, Masahiko; Moens, Cecilia B

2018-06-01

A single Atoh1 basic-helix-loop-helix transcription factor specifies multiple neuron types in the mammalian cerebellum and anterior hindbrain. The zebrafish genome encodes three paralagous atoh1 genes whose functions in cerebellum and anterior hindbrain development we explore here. With use of a transgenic reporter, we report that zebrafish atoh1c-expressing cells are organized in two distinct domains that are separated both by space and developmental time. An early isthmic expression domain gives rise to an extracerebellar population in rhombomere 1 and an upper rhombic lip domain gives rise to granule cell progenitors that migrate to populate all four granule cell territories of the fish cerebellum. Using genetic mutants we find that of the three zebrafish atoh1 paralogs, atoh1c and atoh1a are required for the full complement of granule neurons. Surprisingly, the two genes are expressed in non-overlapping granule cell progenitor populations, indicating that fish use duplicate atoh1 genes to generate granule cell diversity that is not detected in mammals. Finally, live imaging of granule cell migration in wildtype and atoh1c mutant embryos reveals that while atoh1c is not required for granule cell specification per se, it is required for granule cells to delaminate and migrate away from the rhombic lip. Copyright © 2018 Elsevier Inc. All rights reserved.

Axonal regeneration in zebrafish spinal cord.

PubMed

Ghosh, Sukla; Hui, Subhra Prakash

2018-03-01

In the present review we discuss two interrelated events-axonal damage and repair-known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals.

Maintenance of Zebrafish Lines at the European Zebrafish Resource Center.

PubMed

Geisler, Robert; Borel, Nadine; Ferg, Marco; Maier, Jana Viktoria; Strähle, Uwe

2016-07-01

We have established a European Zebrafish Resource Center (EZRC) at the KIT. This center not only maintains and distributes a large number of existing mutant and transgenic zebrafish lines but also gives zebrafish researchers access to screening services and technologies such as imaging and high-throughput sequencing, provided by the Institute of Toxicology and Genetics (ITG). The EZRC maintains and distributes the stock collection of the Nüsslein-Volhard laboratory, comprising over 2000 publicly released mutations, as frozen sperm samples. Within the framework of the ZF-HEALTH EU project, the EZRC distributes over 10,000 knockout mutations from the Sanger Institute (United Kingdom), as well as over 100 mutant and transgenic lines from other sources. In this article, we detail the measures we have taken to ensure the health of our fish, including hygiene, quarantine, and veterinary inspections.

Altered Gene Expression in the Brain and Ovaries of Zebrafish Exposed to the Aromatase Inhibitor Fadrozole: Microarray analysis and Hypothesis Generation

EPA Science Inventory

As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine active chemicals (EACs) with different modes of action, we exposed zebrafish (Danio rerio) to 25 or 100 ìg/L of the aromatase inhibitor fadrozole for...

Sox-2 in taste bud and lateral line system of zebrafish during development.

PubMed

Germanà, A; Montalbano, G; Guerrera, M C; Laura, R; Levanti, M; Abbate, F; de Carlos, F; Vega, J A; Ciriaco, E

2009-12-18

The Sox-2 is a transcription factor involved in adult neurogenesis in different vertebrate species, including fishes. Sox-2 also participates in growth and renewal on sensory cells in neuromasts of the fish lateral line system, and it is essential for development of taste buds in mammals. Using immunohistochemistry and Western blot we have investigated the occurrence and localization of Sox-2 taste buds and neuromast of zebrafish from 10 days post-fertilization to adult stage (1 year). The antibody used identifies two protein bands with estimated molecular weights of 34 and 37kDa which are consistent with those predicted for Sox-2. Sensory cells in taste buds displayed Sox-2 immunoreactivity at all the ages sampled, whereas in the neuromasts Sox-2 expression was restricted to the basal non-sensory cells. Interestingly Sox-2 immunoreactivity was observed in epithelial cells associated with both taste buds and neuromasts. Present results demonstrate that Sox-2 expressed in taste buds and neuromasts of zebrafish during the whole lifespan. Nevertheless, whereas the role of Sox-2 in taste buds of zebrafish remains to be established, the results in neuromast suggest that Sox-2 could participate in cell renewal of the mechanosensory cells.

Elucidating the mechanism of action of tributyltin (TBT) in zebrafish.

PubMed

McGinnis, Courtney L; Crivello, Joseph F

2011-05-01

Tributyltin (TBT), an antifouling agent, has been implicated in the masculinization of fish species worldwide, but the masculinizing mechanism is not fully understood. We have examined the actions of TBT as an endocrine disruptor in zebrafish (Danio rerio). In HeLa cells transiently co-transfected with plasmid constructs containing the zebrafish estrogen receptors (zfERα, zfERβ(1) and zfERβ(2)) and the zebrafish estrogen response element (zfERE-tk-luc), ethinyl estradiol (EE2) induced luciferase activity 4 to 6-fold and was inhibited by TBT. In HeLa cells transiently co-transfected with the zebrafish androgen receptor (zfAR) and the murine androgen receptor response element (ARE-slp-luc), testosterone induced luciferase activity was not inhibited by TBT. In HeLa cells co-transfected with zfERα, zfERβ(1) and zfERβ(2) and a plasmid containing zebrafish aromatase (zfCyp19b-luc), TBT inhibited luciferase activity. In zebrafish exposed to 1mg/kg and 5mg/kg TBT in vivo, there was a increase in liver sulfotransferase and a decrease acyl-CoA testosterone acyltransferase activity. Real-time PCR analysis of sexual differentiation markers in fish exposed to TBT in vivo revealed a tissue-specific response. In brain there was increased production of Sox9, Dax1, and SF1 mRNA, an androgenizing effect, while in the liver there was increased production of Dax1, Cyp19a and zfERβ(1) mRNA but decreased production of Sox9 mRNA, a feminizing effect. In the gonads there was increased production of zfERα and zfCyp19a mRNA, again a feminizing effect. TBT has an overall masculinizing effect but the masculinizing effect is tempered by a feminizing effect on gene transcription in certain tissues. These results are discussed in the context of TBT as an endocrine disruptor in zebrafish. Copyright © 2011 Elsevier B.V. All rights reserved.

(Eco)toxicological effects of 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) in zebrafish (Danio rerio) and permanent fish cell cultures.

PubMed

Vincze, Krisztina; Gehring, Martin; Braunbeck, Thomas

2014-01-01

2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) is a high-production volume chemical used in paper, ink, pesticide, and adhesive industries as a wetting and anti-foaming agent. The physicochemical properties and slow biodegradation rate of TMDD indicate a low bioaccumulation potential but a high prevalence in the environment. As a consequence, TMDD has been detected in several European rivers in the nanogram per liter and lower microgram per liter range; however, its environmental risk to aquatic organisms is considered low. Recent studies almost exclusively focused on acute effects by TMDD, little is known about cytotoxic and genotoxic effects, reproduction and developmental toxicity, endocrine disruption, and any kind of long-term toxicity and carcinogenicity so far. The present study aims to provide more specific baseline information on the ecotoxicological effects of TMDD in fish. For this end, cyto- and genotoxicity assays were carried out in vitro with the permanent fish cell line RTL-W1; in addition, in vivo studies were conducted with the early life stages of zebrafish (Danio rerio) in order to fill the data gaps in developmental toxicity and endocrine disruption. TMDD showed a cytotoxic and slight genotoxic potential in fish cell lines; moreover, various sublethal and lethal effects could be detected in developing zebrafish embryos. There was no evidence of endocrine-disrupting effects by TMDD; however, mortality following prolonged exposure to TMDD during fish sexual development test was clearly higher than mortality in the fish embryo test after 96-h exposure. Our results thus confirmed previous findings of laboratory screening tests, suggesting short-term toxic effects of TMDD in the intermediate, and long-term effects in the lower milligram per liter range.

Automated processing of zebrafish imaging data: a survey.

PubMed

Mikut, Ralf; Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A; Kausler, Bernhard X; Ledesma-Carbayo, María J; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

2013-09-01

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines.

Automated Processing of Zebrafish Imaging Data: A Survey

PubMed Central

Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A.; Kausler, Bernhard X.; Ledesma-Carbayo, María J.; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

2013-01-01

Abstract Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines. PMID:23758125

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hinfray, N., E-mail: nathalie.hinfray@ineris.fr

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase genemore » under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures. - Highlights: • Combined effects of EE2 and LNG were assessed on ER-dependent cyp19a1b expression. • EE2 and LNG alone induced brain aromatase in zebrafish specific bioassays. • Experimental ray design allowed complete concentration-response surfaces modeling. • EE2 and LNG exerted additive effects on brain aromatase in radial glial cells.« less

Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos

PubMed Central

2012-01-01

Background Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein). Results The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2), the synthetic oestrogen 17α- ethinyloestradiol (EE2), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures). For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish). We also demonstrate that our construct works in medaka, another model fish test species, suggesting the transient assay is applicable for testing oestrogenic chemicals in fish generally. Conclusion Our results indicate that the transient expression assay system can be used as a rapid integrated testing system for environmental oestrogens and to detect the oestrogenic target sites in developing fish embryos. PMID:22726887

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays.

PubMed

Hinfray, N; Tebby, C; Garoche, C; Piccini, B; Bourgine, G; Aït-Aïssa, S; Kah, O; Pakdel, F; Brion, F

2016-09-15

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase gene under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures. Copyright © 2016 Elsevier Inc. All rights reserved.

Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies.

PubMed

Villarreal, Michael A; Biediger, Nicole M; Bonner, Natalie A; Miller, Jennifer N; Zepeda, Samantha K; Ricard, Benjamin J; García, Dana M; Lewis, Karen A

2017-08-01

Antibodies raised against mammalian proteins may exhibit cross-reactivity with zebrafish proteins, making these antibodies useful for fish studies. However, zebrafish may express multiple paralogues of similar sequence and size, making them difficult to distinguish by traditional Western blot analysis. To identify the zebrafish proteins that are recognized by an antimammalian antibody, we developed a system to screen putative epitopes by cloning the sequences between the yeast SUMO protein and a C-terminal 6xHis tag. The recombinant fusion protein was expressed in Escherichia coli and analyzed by Western blot to conclusively identify epitopes that exhibit cross-reactivity with the antibodies of interest. This approach can be used to determine the species cross-reactivity and epitope specificity of a wide variety of peptide antigen-derived antibodies.

Several synthetic progestins disrupt the glial cell specific-brain aromatase expression in developing zebra fish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cano-Nicolau, Joel

The effects of some progestins on fish reproduction have been recently reported revealing the hazard of this class of steroidal pharmaceuticals. However, their effects at the central nervous system level have been poorly studied until now. Notwithstanding, progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. Herein, we investigated the effects of a large set of synthetic ligands of the nuclear progesterone receptor on the glial-specific expression of the zebrafish brain aromatase (cyp19a1b) using zebrafish mechanism-based assays. Progesterone and 24 progestins were first screened on transgenic cyp19a1b-GFP zebrafish embryos. Wemore » showed that progesterone, dydrogesterone, drospirenone and all the progesterone-derived progestins had no effect on GFP expression. Conversely, all progestins derived from 19-nortesterone induced GFP in a concentration-dependent manner with EC{sub 50} ranging from the low nM range to hundreds nM. The 19-nortestosterone derived progestins levonorgestrel (LNG) and norethindrone (NET) were further tested in a radial glial cell context using U251-MG cells co-transfected with zebrafish ER subtypes (zfERα, zfERβ1 or zfERβ2) and cyp19a1b promoter linked to luciferase. Progesterone had no effect on luciferase activity while NET and LNG induced luciferase activity that was blocked by ICI 182,780. Zebrafish-ERs competition assays showed that NET and LNG were unable to bind to ERs, suggesting that the effects of these compounds on cyp19a1b require metabolic activation prior to elicit estrogenic activity. Overall, we demonstrate that 19-nortestosterone derived progestins elicit estrogenic activity by inducing cyp19a1b expression in radial glial cells. Given the crucial role of radial glial cells and neuro-estrogens in early development of brain, the consequences of exposure of fish to these compounds require further investigation. - Highlights: • P4 + 24 progestins were tested on embryonic brain aromatase expression in zebrafish. • 19 nor-testosterone derivatives induced cyp19a1b expression. • cyp19a1b up-regulation involved functional zfERs. • 19 nor-testosterone derivatives are pro-estrogenic compounds. • Effect of progestins should be further investigated at the brain level.« less

Differences in toxicity of anionic and cationic PAMAM and PPI dendrimers in zebrafish embryos and cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bodewein, Lambert

Dendrimers are an emerging class of polymeric nanoparticles with beneficial biomedical applications like early diagnostics, in vitro gene transfection or controlled drug delivery. However, the potential toxic impact of exposure on human health or the environment is often inadequately defined. Thus, polyamidoamine (PAMAM) dendrimers of generations G3.0, 3.5, 4.0, 4.5 and 5.0 and polypropylenimine (PPI) dendrimers G3.0, 4.0 and 5.0 were tested in zebrafish embryos for 96 h and human cancer cell lines for 24 h, to assess and compare developmental in vivo toxicity with cytotoxicity. The zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time, withmore » EC50 values ranging from 0.16 to just below 1.7 μM at 24 and 48 hpf. The predominant effects were mortality, plus reduced heartbeat and blood circulation for PPI dendrimers. Apoptosis in the embryos increased in line with the general toxicity concentration-dependently. Hatch and dechorionation of the embryos increased the toxicity, suggesting a protective role of the chorion. Lower generation dendrimers were more toxic in the embryos whereas the toxicity in the HepG2 and DU145 cell lines increased with increasing generation of cationic PAMAMs and PPI dendrimers. HepG2 were less sensitive than DU145 cells, with IC50 values ≥ 402 μM (PAMAMs) and ≤ 240 μM (PPIs) for HepG2 and ≤ 13.24 μM (PAMAMs) and ≤ 12.84 μM (PPIs) for DU145. Neither in fish embryos nor cells toxicity thresholds were determinable for anionic PAMAM G3.5 and G4.5. The study demonstrated that the cytotoxicity underestimated the in-vivo toxicity of the dendrimers in the fish embryos. - Highlights: • Zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time. • Zebrafish embryo toxicity of cationic dendrimers did not increase with generation. • Cationic dendrimers induced apoptosis in zebrafish embryos. • Toxicity of cationic dendrimers was lower in HepG2 and DU145 than zebrafish embryos. • Anionic PAMAM dendrimers showed little to no toxicity in fish embryos and cells.« less

Chevron formation of the zebrafish muscle segments

PubMed Central

Rost, Fabian; Eugster, Christina; Schröter, Christian; Oates, Andrew C.; Brusch, Lutz

2014-01-01

The muscle segments of fish have a folded shape, termed a chevron, which is thought to be optimal for the undulating body movements of swimming. However, the mechanism shaping the chevron during embryogenesis is not understood. Here, we used time-lapse microscopy of developing zebrafish embryos spanning the entire somitogenesis period to quantify the dynamics of chevron shape development. By comparing such time courses with the start of movements in wildtype zebrafish and analysing immobile mutants, we show that the previously implicated body movements do not play a role in chevron formation. Further, the monotonic increase of chevron angle along the anteroposterior axis revealed by our data constrains or rules out possible contributions by previously proposed mechanisms. In particular, we found that muscle pioneers are not required for chevron formation. We put forward a tension-and-resistance mechanism involving interactions between intra-segmental tension and segment boundaries. To evaluate this mechanism, we derived and analysed a mechanical model of a chain of contractile and resisting elements. The predictions of this model were verified by comparison with experimental data. Altogether, our results support the notion that a simple physical mechanism suffices to self-organize the observed spatiotemporal pattern in chevron formation. PMID:25267843

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

PubMed

O'Connor, Marie N; Salles, Isabelle I; Cvejic, Ana; Watkins, Nicholas A; Walker, Adam; Garner, Stephen F; Jones, Chris I; Macaulay, Iain C; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H; Deckmyn, Hans; Stemple, Derek L; Ouwehand, Willem H

2009-05-07

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

PubMed Central

O'Connor, Marie N.; Salles, Isabelle I.; Cvejic, Ana; Watkins, Nicholas A.; Walker, Adam; Garner, Stephen F.; Jones, Chris I.; Macaulay, Iain C.; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L.; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H.; Stemple, Derek L.; Ouwehand, Willem H.

2009-01-01

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis. PMID:19109564

Changes in Brain Monoamines Underlie Behavioural Disruptions after Zebrafish Diet Exposure to Polycyclic Aromatic Hydrocarbons Environmental Mixtures

PubMed Central

Vignet, Caroline; Trenkel, Verena M.; Vouillarmet, Annick; Bricca, Giampiero; Bégout, Marie-Laure; Cousin, Xavier

2017-01-01

Zebrafish were exposed through diet to two environmentally relevant polycyclic aromatic hydrocarbons (PAHs) mixtures of contrasted compositions, one of pyrolytic (PY) origin and one from light crude oil (LO). Monoamine concentrations were quantified in the brains of the fish after six month of exposure. A significant decrease in noradrenaline (NA) was observed in fish exposed to both mixtures, while a decrease in serotonin (5HT) and dopamine (DA) was observed only in LO-exposed fish. A decrease in metabolites of 5HT and DA was observed in fish exposed to both mixtures. Several behavioural disruptions were observed that depended on mixtures, and parallels were made with changes in monoamine concentrations. Indeed, we observed an increase in anxiety in fish exposed to both mixtures, which could be related to the decrease in 5HT and/or NA, while disruptions of daily activity rhythms were observed in LO fish, which could be related to the decrease in DA. Taken together, these results showed that (i) chronic exposures to PAHs mixtures disrupted brain monoamine contents, which could underlie behavioural disruptions, and that (ii) the biological responses depended on mixture compositions. PMID:28273853

A zebrafish model of diabetes mellitus and metabolic memory.

PubMed

Intine, Robert V; Olsen, Ansgar S; Sarras, Michael P

2013-02-28

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined. We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta.

PubMed

Gioia, Roberta; Tonelli, Francesca; Ceppi, Ilaria; Biggiogera, Marco; Leikin, Sergey; Fisher, Shannon; Tenedini, Elena; Yorgan, Timur A; Schinke, Thorsten; Tian, Kun; Schwartz, Jean-Marc; Forte, Fabiana; Wagener, Raimund; Villani, Simona; Rossi, Antonio; Forlino, Antonella

2017-08-01

Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment. © The Author 2017. Published by Oxford University Press.

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta

PubMed Central

Gioia, Roberta; Tonelli, Francesca; Ceppi, Ilaria; Biggiogera, Marco; Leikin, Sergey; Fisher, Shannon; Tenedini, Elena; Yorgan, Timur A.; Schinke, Thorsten; Tian, Kun; Schwartz, Jean-Marc; Forte, Fabiana; Wagener, Raimund; Villani, Simona; Rossi, Antonio; Forlino, Antonella

2017-01-01

Abstract Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment. PMID:28475764

Maternal nodal and zebrafish embryogenesis.

PubMed

Bennett, James T; Stickney, Heather L; Choi, Wen-Yee; Ciruna, Brian; Talbot, William S; Schier, Alexander F

2007-11-08

In fish and amphibians, the dorsal axis is specified by the asymmetric localization of maternally provided components of the Wnt signalling pathway. Gore et al. suggest that the Nodal signal Squint (Sqt) is required as a maternally provided dorsal determinant in zebrafish. Here we test their proposal and show that the maternal activities of sqt and the related Nodal gene cyclops (cyc) are not required for dorsoventral patterning.

Molecular and functional characterization of the scavenger receptor CD36 in zebrafish and common carp.

PubMed

Fink, Inge R; Benard, Erica L; Hermsen, Trudi; Meijer, Annemarie H; Forlenza, Maria; Wiegertjes, Geert F

2015-02-01

CD36 is a scavenger receptor which has been studied closely in mammals where it is expressed by many different cell types and plays a role in highly diverse processes, both homeostatic and pathologic. It is among other things important in the innate immune system, in angiogenesis, and in clearance of apoptotic cells, and it is also involved in lipid metabolism and atherosclerosis. Recently, in the cephalochordate amphioxus a primitive CD36 family member was described, which was present before the divergence of CD36 from other scavenger receptor B family members, SCARB1 and SCARB2. Not much is known on the Cd36 molecule in teleost fish. We therefore studied Cd36 in both zebrafish and common carp, two closely related cyprinid fish species. Whereas a single cd36 gene is present in zebrafish, carp has two cd36 genes, and all show conserved synteny compared to mammalian CD36. The gene expression of carp cd36 is high in brain, ovary and testis but absent in immune organs. Although in mammals CD36 expression in erythrocytes, monocytes and macrophages is high, gene expression studies in leukocyte subtypes of adult carp and zebrafish larvae, including thrombocytes and macrophages provided no indication for any substantial expression of cd36 in immune cell types. Surprisingly, analysis of the cd36 promoter region does show the presence of several binding sites for transcription factors known to regulate immune responses. Overexpression of carp cd36 locates the receptor on the cell surface of mammalian cell lines consistent with the predicted topology of cyprinid Cd36 with a large extracellular domain, two transmembrane domains, and short cytoplasmic tails at both ends. Gene expression of cd36 is down-regulated during infection of zebrafish with Mycobacterium marinum, whereas knockdown of cd36 in zebrafish larvae led to higher bacterial burden upon such infection. We discuss the putative role for Cd36 in immune responses of fish in the context of other members of the scavenger receptor class B family. Copyright © 2014 Elsevier Ltd. All rights reserved.

Single Low-Dose Ionizing Radiation Induces Genotoxicity in Adult Zebrafish and its Non-Irradiated Progeny.

PubMed

Lemos, J; Neuparth, T; Trigo, M; Costa, P; Vieira, D; Cunha, L; Ponte, F; Costa, P S; Metello, L F; Carvalho, A P

2017-02-01

This study investigated to what extent a single exposure to low doses of ionizing radiation can induce genotoxic damage in irradiated adult zebrafish (Danio rerio) and its non-irradiated F1 progeny. Four groups of adult zebrafish were irradiated with a single dose of X-rays at 0 (control), 100, 500 and 1000 mGy, respectively, and couples of each group were allowed to reproduce following irradiation. Blood of parental fish and whole-body offspring were analysed by the comet assay for detection of DNA damage. The level of DNA damage in irradiated parental fish increased in a radiation dose-dependent manner at day 1 post-irradiation, but returned to the control level thereafter. The level of DNA damage in the progeny was directly correlated with the parental irradiation dose. Results highlight the genotoxic risk of a single exposure to low-dose ionizing radiation in irradiated individuals and also in its non-irradiated progeny.

Food and conspecific chemical cues modify visual behavior of zebrafish, Danio rerio.

PubMed

Stephenson, Jessica F; Partridge, Julian C; Whitlock, Kathleen E

2012-06-01

Animals use the different qualities of olfactory and visual sensory information to make decisions. Ethological and electrophysiological evidence suggests that there is cross-modal priming between these sensory systems in fish. We present the first experimental study showing that ecologically relevant chemical mixtures alter visual behavior, using adult male and female zebrafish, Danio rerio. Neutral-density filters were used to attenuate the light reaching the tank to an initial light intensity of 2.3×10(16) photons/s/m2. Fish were exposed to food cue and to alarm cue. The light intensity was then increased by the removal of one layer of filter (nominal absorbance 0.3) every minute until, after 10 minutes, the light level was 15.5×10(16) photons/s/m2. Adult male and female zebrafish responded to a moving visual stimulus at lower light levels if they had been first exposed to food cue, or to conspecific alarm cue. These results suggest the need for more integrative studies of sensory biology.

Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone.

PubMed

Orn, Stefan; Holbech, Henrik; Madsen, Trine H; Norrgren, Leif; Petersen, Gitte I

2003-12-10

In a partial life-cycle test, the impact of 17alpha-ethinylestradiol (EE2) and 17alpha-methyltestosterone (MT) on juvenile zebrafish was evaluated by use of vitellogenin measurements and gonadal development. Exposure to EE2 (1-25 ng/l) resulted in a dose-dependent increase in vitellogenin production starting at 2 ng/l. Significant changes in sex ratios in female direction were detected at 1 ng/l, with complete sex reversal taking place after exposure to 2 ng/l. No intersex fish were observed after exposure to EE2. Exposure to MT resulted in decreased vitellogenin concentrations. Complete sex reversal was detected in all MT concentrations used (26-1000 ng/l). A large proportion of intersex fish was observed after exposure to 1000 ng MT/l. The period of gonadal sex reversal in non-exposed zebrafish was also studied. The main morphological features of the transformation of ovaries into testis were observed 4-5 weeks after hatching.

Environmental estrogen(s) induced swimming behavioural alterations in adult zebrafish (Danio rerio).

PubMed

Goundadkar, Basavaraj B; Katti, Pancharatna

2017-09-01

The present study is an attempt to investigate the effects of long-term (75days) exposure to environmental estrogens (EE) on the swimming behaviour of zebrafish (Danio rerio). Adult zebrafish were exposed semi-statically to media containing commonly detected estrogenic water contaminants (EE2, DES and BPA) at a concentration (5ng/L) much lower than environmentally recorded levels. Time spent in swimming, surface preference, patterns and path of swimming were recorded (6mins) for each fish using two video cameras on day 15, 30 60 and 75. Video clips were analysed using a software program. Results indicate that chronic exposure to EE leads to increased body weight and size of females, reduced (P<0.05) swimming time, delay in latency, increased (P<0.05) immobility, erratic movements and freezing episodes. We conclude that estrogenic contamination of natural aquatic systems induces alterations in locomotor behaviour and associated physiological disturbances in inhabitant fish fauna. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification and expression analysis of zebrafish glypicans during embryonic development.

PubMed

Gupta, Mansi; Brand, Michael

2013-01-01

Heparan sulfate Proteoglycans (HSPG) are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG's, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.

The antiviral innate immune response in fish: evolution and conservation of the IFN system.

PubMed

Langevin, Christelle; Aleksejeva, Elina; Passoni, Gabriella; Palha, Nuno; Levraud, Jean-Pierre; Boudinot, Pierre

2013-12-13

Innate immunity constitutes the first line of the host defense after pathogen invasion. Viruses trigger the expression of interferons (IFNs). These master antiviral cytokines induce in turn a large number of interferon-stimulated genes, which possess diverse effector and regulatory functions. The IFN system is conserved in all tetrapods as well as in fishes, but not in tunicates or in the lancelet, suggesting that it originated in early vertebrates. Viral diseases are an important concern of fish aquaculture, which is why fish viruses and antiviral responses have been studied mostly in species of commercial value, such as salmonids. More recently, there has been an interest in the use of more tractable model fish species, notably the zebrafish. Progress in genomics now makes it possible to get a relatively complete image of the genes involved in innate antiviral responses in fish. In this review, by comparing the IFN system between teleosts and mammals, we will focus on its evolution in vertebrates. © 2013 Elsevier Ltd. All rights reserved.

Characterization of zebrafish dysferlin by morpholino knockdown

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawahara, Genri; Serafini, Peter R.; Myers, Jennifer A.

2011-09-23

Highlights: {yields} cDNAs of zebrafish dysferlin were cloned (6.3 kb). {yields} The dysferlin expression was detected in skeletal muscle, heart and eye. {yields} Injection of antisense morpholinos to dysferlin caused marked muscle disorganization. {yields} Zebrafish dysferlin expression may be involved in stabilizing muscle structures. -- Abstract: Mutations in the gene encoding dysferlin cause two distinct muscular dystrophy phenotypes: limb-girdle muscular dystrophy type 2B (LGMD-2B) and Miyoshi myopathy (MM). Dysferlin is a large transmembrane protein involved in myoblast fusion and membrane resealing. Zebrafish represent an ideal animal model to use for studying muscle disease including abnormalities of dysferlin. cDNAs of zebrafishmore » dysferlin were cloned (6.3 kb) and the predicted amino acid sequences, showed 68% similarity to predicted amino acid sequences of mammalian dysferlin. The expression of dysferlin was mainly in skeletal muscle, heart and eye, and the expression could be detected as early as 11 h post fertilization (hpf). Three different antisense oligonucleotide morpholinos were targeted to inhibit translation of this dysferlin mRNA and the morpholino-injected fish showed marked muscle disorganization which could be detected by birefringence assay. Western blot analysis using dysferlin antibodies showed that the expression of dysferlin was reduced in each of the three morphants. Dysferlin expression was shown to be reduced at the myosepta of zebrafish muscle using immunohistochemistry, although the expression of other muscle membrane components, dystrophin, laminin, {beta}-dystroglycan were detected normally. Our data suggest that zebrafish dysferlin expression is involved in stabilizing muscle structures and its downregulation causes muscle disorganization.« less

Microfluidic EmbryoSort technology: towards in flow analysis, sorting and dispensing of individual vertebrate embryos

NASA Astrophysics Data System (ADS)

Fuad, Nurul M.; Wlodkowic, Donald

2013-12-01

The demand to reduce the numbers of laboratory animals has facilitated the emergence of surrogate models such as tests performed on zebrafish (Danio rerio) or African clawed frog's (Xenopus levis) eggs, embryos and larvae. Those two model organisms are becoming increasingly popular replacements to current adult animal testing in toxicology, ecotoxicology and also in drug discovery. Zebrafish eggs and embryos are particularly attractive for toxicological analysis due their size (diameter 1.6 mm), optical transparency, large numbers generated per fish and very straightforward husbandry. The current bottleneck in using zebrafish embryos for screening purposes is, however, a tedious manual evaluation to confirm the fertilization status and subsequent dispensing of single developing embryos to multitier plates to perform toxicity analysis. Manual procedures associated with sorting hundreds of embryos are very monotonous and as such prone to significant analytical errors due to operator's fatigue. In this work, we present a proofof- concept design of a continuous flow embryo sorter capable of analyzing, sorting and dispensing objects ranging in size from 1.5 - 2.5 mm. The prototypes were fabricated in polymethyl methacrylate (PMMA) transparent thermoplastic using infrared laser micromachining. The application of additive manufacturing processes to prototype Lab-on-a-Chip sorters using both fused deposition manufacturing (FDM) and stereolithography (SLA) were also explored. The operation of the device was based on a revolving receptacle capable of receiving, holding and positioning single fish embryos for both interrogation and subsequent sorting. The actuation of the revolving receptacle was performed using a DC motor and/or microservo motor. The system was designed to separate between fertilized (LIVE) and non-fertilized (DEAD) eggs, based on optical transparency using infrared (IR) emitters and receivers.

Adaptive differences in the structure and macromolecular compositions of the air and water corneas of the “four-eyed” fish (Anableps anableps)

PubMed Central

SWAMYNATHAN, SHIVALINGAPPA K.; CRAWFORD, MARY A.; ROBISON, W. GERALD; KANUNGO, JYOTSHNABALA; PIATIGORSKY, JORAM

2018-01-01

The water meniscus bisects the eyes of the “four-eyed” fish Anableps anableps, resulting in simultaneous vision in air and water. We compare the structure and macromolecular compositions of the Anableps dorsal (air) and ventral (water) corneas with the fully aquatic zebrafish cornea. The Anableps dorsal corneal epithelium is thicker (>20 cell layers), flatter (~1.94 mm radius of curvature), and contains ~15-fold more glycogen (0.16 μg/μg water-soluble protein) than the ventral corneal epithelium (5–7 cell layers; ~1.63 mm radius of curvature; 0.01 μg glycogen/μg water-soluble protein), which resembles the zebrafish corneal epithelium. Gelsolin is the major water-soluble protein in the zebrafish (~50%) and Anableps dorsal (~38%) and ventral (~21%) corneal epithelia, suggesting that gelsolin was recruited for high corneal expression before these two species diverged at least 100 million years ago and that abundant corneal gelsolin is not limited to aquatic vision. Anableps gelsolin, deduced from its cDNA, is 57% identical to zebrafish gelsolin. Paucity of Anableps corneal F-actin (consistent with high gelsolin) was confirmed by the absence of rhodaminephalloidin staining. We suggest amphibious refraction and protection from UV irradiation and desiccation in air as selective constraints for the specializations of the Anableps dorsal cornea.—Swamynathan, S. K., Craw-ford, M. A., Robison, W. G., Jr., Kanungo, J., Piatigorsky, J. Adaptive differences in the structure and macromolecular compositions of the air and water corneas of the “four-eyed” fish (Anableps anableps). PMID:14597669

Characterization of a hypoxia-response element in the Epo locus of the pufferfish, Takifugu rubripes.

PubMed

Kulkarni, Rashmi P; Tohari, Sumanty; Ho, Adrian; Brenner, Sydney; Venkatesh, Byrappa

2010-06-01

Animals respond to hypoxia by increasing synthesis of the glycoprotein hormone erythropoietin (Epo) which in turn stimulates the production of red blood cells. The gene encoding Epo has been recently cloned in teleost fishes such as the pufferfish Takifugu rubripes (fugu) and zebrafish (Danio rerio). It has been shown that the transcription levels of Epo in teleost fishes increase in response to anemia or hypoxia in a manner similar to its human ortholog. However, the cis-regulatory element(s) mediating the hypoxia response of Epo gene in fishes has not been identified. In the present study, using the human hepatoma cell line (Hep3B), we have identified and characterized a hypoxia response element (HRE) in the fugu Epo locus. The sequence of the fugu HRE (ACGTGCTG) is identical to that of the HRE in the human EPO locus. However, unlike the HRE in the mammalian Epo locus, which is located in the 3' region of the gene, the fugu HRE is located in the 5' flanking region and on the opposite strand of DNA. This HRE is conserved in other teleosts such as Tetraodon and zebrafish in a similar location. A 365-bp fragment containing the fugu HRE was able to drive GFP expression in the liver of transgenic zebrafish. However, we could not ascertain if the expression of transgene is induced by hypoxia in vivo due to the low and variable levels of GFP expression in transgenic zebrafish. Our investigations also revealed that the Epo locus has experienced extensive rearrangements during vertebrate evolution. Copyright © 2010 Elsevier B.V. All rights reserved.

Effects of ammonium perchlorate on the reproductive performance and thyroid follicle histology of zebrafish

USGS Publications Warehouse

Patino, R.; Wainscott, M.R.; Cruz-Li, E. I.; Balakrishnan, S.; McMurry, C.; Blazer, V.S.; Anderson, T.A.

2003-01-01

Adult zebrafish were reared up to eight weeks in control water or in water containing ammonium perchlorate (AP) at measured perchlorate concentrations of 18 (environmentally relevant, high) and 677 ppm. Groups of eight females were paired with four males on a weekly basis to assess AP effects on spawned egg volume, an index of reproductive performance. All treatments were applied to four to five spawning replicates. At 677 ppm, spawn volume was reduced within one week and became negligible after four weeks. At 18 ppm, spawn volume was unaffected even after eight weeks. Also, perchlorate at 18 ppm did not affect percentage egg fertilization. Fish were collected at the end of the exposures (677 ppm, four weeks; control and 18 ppm, eight weeks) for whole-body perchlorate content and thyroid histopathological analysis. Fish perchlorate levels were about one-hundredth of those of treatment water levels, indicating that waterborne perchlorate does not accumulate in whole fish. At 677 ppm for four weeks, perchlorate caused thyroid follicle cell (nuclear) hypertrophy and angiogenesis, whereas at 18 ppm for eight weeks, its effects were more pronounced and included hypertrophy, angiogenesis, hyperplasia, and colloid depletion. In conclusion, an eight-week exposure of adult zebrafish to 18 ppm perchlorate (high environmentally relevant concentrations) affected the histological condition of their thyroid follicles but not their reproductive performance. The effect of 677 ppm perchlorate on reproduction may be due to extrathyroidal toxicity. Further research is needed to determine if AP at lower environmentally relevant concentrations also affects the thyroid follicles of zebrafish.

Endocrine disruption and reproduction impairment in zebrafish after long-term exposure to DE-71.

PubMed

Yu, Liqin; Liu, Chunsheng; Chen, Qi; Zhou, Bingsheng

2014-06-01

The objective of the present study was to investigate the impact of polybrominated diphenyl ethers (PBDEs) on fish reproduction over 2 generations. Zebrafish (Danio rerio) embryos (F0) were exposed to low concentrations (3 µg/L, 10 µg/L, and 30 µg/L) of the PBDE mixture DE-71 until they were sexually mature, and steroid hormone production, expression of genes involved in steroidogenesis, gonadal development, and gamete characteristics were examined. Exposure of female zebrafish to DE-71 resulted in lower estradiol production and downregulation of cytochrome P450 aromatase mRNA. In males, exposure to DE-71 resulted in greater testosterone production and greater cytochrome P450 c17 α-hydroxylase,17,20-lase mRNA expression. Moreover, hepatic vitellogenin mRNA and estrogenic receptor β gene transcription were downregulated in females and males. Expression of the follicle-stimulating hormone β gene in the pituitary was upregulated, and the expression of luteinizing hormone β was downregulated in both sexes. Histological examination showed inhibition of oocyte maturation in females and retarded spermiation in males. The average number of eggs (F1) produced was also reduced. Additionally, exposure of F0 embryos to DE-71 did not result in developmental toxicity, whereas delayed hatching, reduced survival, and decreased growth were observed in the F1 embryos derived from parent fish exposed to DE-71. Therefore, long-term exposure to low concentrations of PBDEs in zebrafish could cause reproductive impairment, suggesting that PBDEs might have significant adverse effects on fish population in the highly PBDEs-contaminated aquatic environment. © 2014 SETAC.

Establishment and Characterization of a New Muscle Cell Line of Zebrafish (Danio rerio) as an In Vitro Model for Gene Expression Studies.

PubMed

Kumar, Amit; Singh, Neha; Goswami, Mukunda; Srivastava, J K; Mishra, Akhilesh K; Lakra, W S

2016-01-01

A new continuous fibroblast cell line was established from the muscle tissue of healthy juvenile Danio rerio (Zebrafish) through explant method. Fish cell lines serve as useful tool for investigating basic fish biology, as a model for bioassay of environmental toxicant, toxicity ranking, and for developing molecular biomarkers. The cell line was continuously subcultured for a period of 12 months (61 passages) and maintained at 28 °C in L-15 medium supplemented with 10% FBS and 10 ng/mL of basic fibroblastic growth factor (bFGF) without use of antibiotics. Its growth rate was proportional to the FBS concentration, with optimum growth at 15% FBS. DNA barcoding (16SrRNA and COX1) was used to authenticate the cell line. Cells were incubated with propidium iodide and sorted via flow cytometry to calculate the DNA content to confirm the genetic stability. Significant green fluorescent protein (GFP) signals confirmed the utility of cell line in transgenic and genetic manipulation studies. In vitro assay was performed with MTT to examine the growth potential of the cell line. The muscle cell line would provide a novel invaluable in vitro model to identify important genes to understand regulatory mechanisms that govern the molecular regulation of myogenesis and should be useful in biomedical research.

European network using fish as osteoporosis research models (ENFORM)

NASA Astrophysics Data System (ADS)

Goerlich, R.; Renn, J.; Alestrom, P.; Nouizadeh-Lillabadi, R.; Schartl, M.; Winkler, C.; Muller, M.; Midtyng, P. J.; Eberius, M.; Slenzka, K.

2005-08-01

Osteoporosis, characterised by loss of bone density, is one of the most important bone diseases of humans worldwide. It causes problems in post-menopausal women, in astronauts during long-term spaceflights and in industrial animal production. Bone alterations leading to osteoporosis are well-documented at the cellular level, but the underlying molecular events are still poorly understood and most of our knowledge is derived from in vitro studies using cell culture systems. Recent findings indicate a remarkable conservation of the key regulators of bone development and homeostasis between mammals and fish. Medaka (Oryzias latipes) and zebrafish (Danio rerio) offer experimental advantages that can be exploited for bone research.

Species-specific functional evolution of neuroglobin.

PubMed

Wakasugi, Keisuke; Takahashi, Nozomu; Uchida, Hiroyuki; Watanabe, Seiji

2011-09-01

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Human Ngb is involved in neuroprotection under oxidative stress conditions such as ischemia and reperfusion. We previously demonstrated that, on the one hand, human ferric Ngb binds to the α-subunit of heterotrimeric G proteins (Gα(i)) and acts as a guanine nucleotide dissociation inhibitor (GDI) for Gα(i). On the other hand, zebrafish Ngb does not exhibit GDI activity. By using wild-type and Ngb mutants, we demonstrated that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. The crucial residues for both GDI and neuroprotective activity, corresponding to Glu53, Arg97, Glu118, and Glu151 of human Ngb, are conserved among boreotheria of mammalia. Recently, we found that zebrafish, but not human, Ngb can translocate into cells and clarified that module M1 of zebrafish Ngb is important for protein transduction. By performing site-directed mutagenesis, we showed that Lys7, Lys9, Lys21, and Lys23 of zebrafish Ngb are crucial for protein transduction activity. Because these residues are conserved among fishes, but not among mammals, birds, reptilians, or amphibians, the ability to penetrate cell membranes may be a unique characteristic of fish Ngb proteins. Moreover, we clarified that zebrafish Ngb interacts with negatively charged cell-surface glycosaminoglycan. Taken together, these results suggest that the function of Ngb proteins has been changing dynamically throughout the evolution of life. Copyright © 2011 Elsevier B.V. All rights reserved.

A stochastic vision-based model inspired by zebrafish collective behaviour in heterogeneous environments

PubMed Central

Collignon, Bertrand; Séguret, Axel; Halloy, José

2016-01-01

Collective motion is one of the most ubiquitous behaviours displayed by social organisms and has led to the development of numerous models. Recent advances in the understanding of sensory system and information processing by animals impels one to revise classical assumptions made in decisional algorithms. In this context, we present a model describing the three-dimensional visual sensory system of fish that adjust their trajectory according to their perception field. Furthermore, we introduce a stochastic process based on a probability distribution function to move in targeted directions rather than on a summation of influential vectors as is classically assumed by most models. In parallel, we present experimental results of zebrafish (alone or in group of 10) swimming in both homogeneous and heterogeneous environments. We use these experimental data to set the parameter values of our model and show that this perception-based approach can simulate the collective motion of species showing cohesive behaviour in heterogeneous environments. Finally, we discuss the advances of this multilayer model and its possible outcomes in biological, physical and robotic sciences. PMID:26909173

The utility of zebrafish to study the mechanisms by which ethanol affects social behavior and anxiety during early brain development.

PubMed

Parker, Matthew O; Annan, Leonette V; Kanellopoulos, Alexandros H; Brock, Alistair J; Combe, Fraser J; Baiamonte, Matteo; Teh, Muy-Teck; Brennan, Caroline H

2014-12-03

Exposure to moderate levels of ethanol during brain development has a number of effects on social behavior but the molecular mechanisms that mediate this are not well understood. Gaining a better understanding of these factors may help to develop therapeutic interventions in the future. Zebrafish offer a potentially useful model in this regard. Here, we introduce a zebrafish model of moderate prenatal ethanol exposure. Embryos were exposed to 20mM ethanol for seven days (48hpf-9dpf) and tested as adults for individual social behavior and shoaling. We also tested their basal anxiety with the novel tank diving test. We found that the ethanol-exposed fish displayed reductions in social approach and shoaling, and an increase in anxiety in the novel tank test. These behavioral differences corresponded to differences in hrt1aa, slc6a4 and oxtr expression. Namely, acute ethanol caused a spike in oxtr and ht1aa mRNA expression, which was followed by down-regulation at 7dpf, and an up-regulation in slc6a4 at 72hpf. This study confirms the utility of zebrafish as a model system for studying the molecular basis of developmental ethanol exposure. Furthermore, it proposes a putative developmental mechanism characterized by ethanol-induced OT inhibition leading to suppression of 5-HT and up-regulation of 5-HT1A, which leads, in turn, to possible homeostatic up-regulation of 5-HTT at 72hpf and subsequent imbalance of the 5-HT system. Copyright © 2014 Elsevier Inc. All rights reserved.

Development and implementation of a three-choice serial reaction time task for zebrafish (Danio rerio).

PubMed

Parker, Matthew O; Millington, Mollie E; Combe, Fraser J; Brennan, Caroline H

2012-02-01

Zebrafish are an established and widely utilized developmental genetic model system, but limitations in developed behavioral assays have meant that their potential as a model in behavioral neuroscience has yet to be fully realized. Here, we describe the development of a novel operant behavioral assay to examine a variety of aspects of stimulus control in zebrafish using a 3 choice serial reaction time task (3 CSRTT). Fish were briefly exposed to three spatially distinct, but perceptually identical stimuli, presented in a random order after a fixed-time inter-trial interval (ITI). Entries to the correct response aperture either during the stimulus presentation, or within a brief limited hold period following presentation, were reinforced with illumination of the magazine light and delivery of a small food reward. Following training, premature responding was probed with a long-ITI session three times; once at baseline, once following a saline injection and once following an injection of a low dose of amphetamine (AMPH; 0.025 mg/kg). We predicted that if premature responding was related to impulsivity (as in rodents) it would be reduced following the AMPH injection. Results confirmed that zebrafish could learn to perform a complex operant task similar to tasks developed for rodents which are used to probe sustained attention and impulsivity, but the results from the AMPH trials were inconclusive. This study provides the foundations for development and further validation of this species as a model for some aspects of human attentional and impulse control disorders, such as substance abuse disorder. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of microalgae on intestinal inflammation triggered by soybean meal and bacterial infection in zebrafish

PubMed Central

Solís, Camila J.; Ulloa, Pilar E.; Hedrera, Manuel; Pizarro-Guajardo, Marjorie; Paredes-Sabja, Daniel

2017-01-01

Soybean meal has been used in many commercial diets for farm fish; despite this component inducing intestinal inflammation. On the other hand, microalgae have increasingly been used as dietary supplements in fish feed. Nevertheless, the vast quantity of microalgae species means that many remain under- or unstudied, thus limiting wide scale commercial application. In this work, we evaluated the effects to zebrafish (Danio rerio) of including Tetraselmis sp (Ts); Phaeodactylum tricornutum (Pt); Chlorella sp (Ch); Nannochloropsis oculata (No); or Nannochloropsis gaditana (Ng) as additives in a soybean meal-based diet on intestinal inflammation and survival after Edwardsiella tarda infection. In larvae fed a soybean meal diet supplemented with Ts, Pt, Ch, or Ng, the quantity of neutrophils present in the intestine drastically decreased as compared to larvae fed only the soybean meal diet. Likewise, Ts or Ch supplements in soybean meal or fishmeal increased zebrafish survival by more than 20% after being challenged. In the case of Ts, the observed effect correlated with an increased number of neutrophils present at the infection site. These results suggest that the inclusion of Ts or Ch in fish diets could allow the use of SBM and at the same time improve performance against pathogen. PMID:29117213

Social Eavesdropping in Zebrafish: Tuning of Attention to Social Interactions

PubMed Central

Abril-de-Abreu, Rodrigo; Cruz, José; Oliveira, Rui F.

2015-01-01

Group living animals may eavesdrop on signalling interactions between conspecifics in order to collect adaptively relevant information obtained from others, without incurring in the costs of first-hand information acquisition. This ability (aka social eavesdropping) is expected to impact Darwinian fitness, and hence predicts the evolution of cognitive processes that enable social animals to use public information available in the environment. These adaptive specializations in cognition may have evolved both at the level of learning and memory mechanisms, and at the level of input mechanisms, such as attention, which select the information that is available for learning. Here we used zebrafish to test if attention in a social species is tuned to the exchange of information between conspecifics. Our results show that zebrafish are more attentive towards interacting (i.e. fighting) than towards non-interacting pairs of conspecifics, with the exposure to fighting not increasing activity or stress levels. Moreover, using video playbacks to manipulate form features of the fighting fish, we show that during the assessment phase of the fight, bystanders’ attention is more driven by form features of the interacting opponents; whereas during the post-resolution phase, it is driven by biological movement features of the dominant fish chasing the subordinate fish. PMID:26242246

Brief embryonic cadmium exposure induces a stress response and cell death in the developing olfactory system followed by long-term olfactory deficits in juvenile zebrafish.

PubMed

Blechinger, Scott R; Kusch, Robin C; Haugo, Kristine; Matz, Carlyn; Chivers, Douglas P; Krone, Patrick H

2007-10-01

The toxic effects of cadmium and other metals have been well established. A primary target of these metals is known to be the olfactory system, and fish exposed to a number of different waterborne metals display deficiencies in olfaction. Importantly, exposure over embryonic/larval development periods can cause deficits in chemosensory function in juvenile fish, but the specific cell types affected are unknown. We have previously characterized a transgenic zebrafish strain expressing the green fluorescent protein (eGFP) gene linked to the hsp70 gene promoter, and shown it to be a useful tool for examining cell-specific toxicity in living embryos and larvae. Here we show that the hsp70/eGFP transgene is strongly and specifically upregulated within the olfactory sensory neurons (OSNs) of transgenic zebrafish larvae following a brief 3-h exposure to water-borne cadmium. This molecular response was closely correlated to an endpoint for tissue damage within the olfactory placode, namely cell death. Furthermore, cadmium-induced olfactory cytotoxicity in zebrafish larvae gives rise to more permanent effects. Juvenile zebrafish briefly exposed to cadmium during early larval development display deficits in olfactory-dependent predator avoidance behaviors 4-6 weeks after a return to clean water. Lateral line neuromasts of exposed zebrafish larvae also activate both the endogenous hsp70 gene and the hsp70/eGFP transgene. The data reveal that even a very brief exposure period that gives rise to cell death within the developing olfactory placode results in long-term deficits in olfaction, and that hsp70/eGFP may serve as an effective indicator of sublethal cadmium exposure in sensory cells.

Brief embryonic cadmium exposure induces a stress response and cell death in the developing olfactory system followed by long-term olfactory deficits in juvenile zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blechinger, Scott R.; Toxicology Group, University of Saskatchewan, Saskatoon, Saskatchewan; Kusch, Robin C.

2007-10-01

The toxic effects of cadmium and other metals have been well established. A primary target of these metals is known to be the olfactory system, and fish exposed to a number of different waterborne metals display deficiencies in olfaction. Importantly, exposure over embryonic/larval development periods can cause deficits in chemosensory function in juvenile fish, but the specific cell types affected are unknown. We have previously characterized a transgenic zebrafish strain expressing the green fluorescent protein (eGFP) gene linked to the hsp70 gene promoter, and shown it to be a useful tool for examining cell-specific toxicity in living embryos and larvae.more » Here we show that the hsp70/eGFP transgene is strongly and specifically upregulated within the olfactory sensory neurons (OSNs) of transgenic zebrafish larvae following a brief 3-h exposure to water-borne cadmium. This molecular response was closely correlated to an endpoint for tissue damage within the olfactory placode, namely cell death. Furthermore, cadmium-induced olfactory cytotoxicity in zebrafish larvae gives rise to more permanent effects. Juvenile zebrafish briefly exposed to cadmium during early larval development display deficits in olfactory-dependent predator avoidance behaviors 4-6 weeks after a return to clean water. Lateral line neuromasts of exposed zebrafish larvae also activate both the endogenous hsp70 gene and the hsp70/eGFP transgene. The data reveal that even a very brief exposure period that gives rise to cell death within the developing olfactory placode results in long-term deficits in olfaction, and that hsp70/eGFP may serve as an effective indicator of sublethal cadmium exposure in sensory cells.« less

Effects of the UV filter benzophenone-3 (oxybenzone) at low concentrations in zebrafish (Danio rerio)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blüthgen, Nancy; University of Basel, Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, Klingelbergstrasse 50, CH-4056 Basel; Zucchi, Sara

Organic UV filters including benzophenone-3 (BP-3) are widely used to protect humans and materials from damage by UV irradiation. Despite the environmental occurrence of BP-3 in the aquatic environment, little is known about its effects and modes of action. In the present study we assess molecular and physiological effects of BP-3 in adult male zebrafish (Danio rerio) and in eleuthero-embryos by a targeted gene expression approach focusing on the sex hormone system. Fish and embryos are exposed for 14 days and 120 hours post fertilization, respectively, to 2.4–312 μg/L and 8.2–438 μg/L BP-3. Chemical analysis of water and fish demonstratesmore » that BP-3 is partly transformed to benzophenone-1 (BP-1) and both compounds are accumulated in adult fish. Biotransformation to BP-1 is absent in eleuthero-embryos. BP-3 exposure leads to similar alterations of gene expression in both adult fish and eleuthero-embryos. In the brain of adult males esr1, ar and cyp19b are down-regulated at 84 μg/L BP-3. There is no induction of vitellogenin expression by BP-3, both at the transcriptional and protein level. An overall down-regulation of the hsd3b, hsd17b3, hsd11b2 and cyp11b2 transcripts is observed in the testes, suggesting an antiandrogenic activity. No histological changes were observed in the testes after BP-3 treatment. The study leads to the conclusion that low concentrations of BP-3 exhibit similar multiple hormonal activities at the transcription level in two different life stages of zebrafish. Forthcoming studies should show whether this translates to additional physiological effects. Highlights: ► Activity of UV filter benzophenone-3 (BP-3) is assessed in zebrafish. ► BP-3 is partly metabolized to benzophenone-1 by adult fish but not embryos. ► Alterations of gene expression are similar in adult males and embryos. ► Gene expression alterations point to multiple hormonal activity of BP-3.« less

Toxicogenomics to Evaluate Endocrine Disrupting Effects of Environmental Chemicals Using the Zebrafish Model

PubMed Central

Caballero-Gallardo, Karina; Olivero-Verbel, Jesus; Freeman, Jennifer L.

2016-01-01

The extent of our knowledge on the number of chemical compounds related to anthropogenic activities that can cause damage to the environment and to organisms is increasing. Endocrine disrupting chemicals (EDCs) are one group of potentially hazardous substances that include natural and synthetic chemicals and have the ability to mimic endogenous hormones, interfering with their biosynthesis, metabolism, and normal functions. Adverse effects associated with EDC exposure have been documented in aquatic biota and there is widespread interest in the characterization and understanding of their modes of action. Fish are considered one of the primary risk organisms for EDCs. Zebrafish (Danio rerio) are increasingly used as an animal model to study the effects of endocrine disruptors, due to their advantages compared to other model organisms. One approach to assess the toxicity of a compound is to identify those patterns of gene expression found in a tissue or organ exposed to particular classes of chemicals, through new technologies in genomics (toxicogenomics), such as microarrays or whole-genome sequencing. Application of these technologies permit the quantitative analysis of thousands of gene expression changes simultaneously in a single experiment and offer the opportunity to use transcript profiling as a tool to predict toxic outcomes of exposure to particular compounds. The application of toxicogenomic tools for identification of chemicals with endocrine disrupting capacity using the zebrafish model system is reviewed. PMID:28217008

Exploring Uncoupling Proteins and Antioxidant Mechanisms under Acute Cold Exposure in Brains of Fish

PubMed Central

Lucassen, Magnus; Schmidt, Maike M.; Dringen, Ralf; Abele, Doris; Hwang, Pung-Pung

2011-01-01

Exposure to fluctuating temperatures accelerates the mitochondrial respiration and increases the formation of mitochondrial reactive oxygen species (ROS) in ectothermic vertebrates including fish. To date, little is known on potential oxidative damage and on protective antioxidative defense mechanisms in the brain of fish under cold shock. In this study, the concentration of cellular protein carbonyls in brain was significantly increased by 38% within 1 h after cold exposure (from 28°C to 18°C) of zebrafish (Danio rerio). In addition, the specific activity of superoxide dismutase (SOD) and the mRNA level of catalase (CAT) were increased after cold exposure by about 60% (6 h) and by 60%–90% (1 and 24 h), respectively, while the specific glutathione content as well as the ratio of glutathione disulfide to glutathione remained constant and at a very low level. In addition, cold exposure increased the protein level of hypoxia-inducible factor (HIF) by about 50% and the mRNA level of the glucose transporter zglut3 in brain by 50%–100%. To test for an involvement of uncoupling proteins (UCPs) in the cold adaptation of zebrafish, five UCP members were annotated and identified (zucp1-5). With the exception of zucp1, the mRNA levels of the other four zucps were significantly increased after cold exposure. In addition, the mRNA levels of four of the fish homologs (zppar) of the peroxisome proliferator-activated receptor (PPAR) were increased after cold exposure. These data suggest that PPARs and UCPs are involved in the alterations observed in zebrafish brain after exposure to 18°C. The observed stimulation of the PPAR-UCP axis may help to prevent oxidative damage and to maintain metabolic balance and cellular homeostasis in the brains of ectothermic zebrafish upon cold exposure. PMID:21464954

Reproduction impairment and endocrine disruption in female zebrafish after long-term exposure to MC-LR: A life cycle assessment.

PubMed

Hou, Jie; Li, Li; Wu, Ning; Su, Yujing; Lin, Wang; Li, Guangyu; Gu, Zemao

2016-01-01

Microcystin-LR (MC-LR) has been found to cause reproductive and developmental impairments as well as to disrupt sex hormone homeostasis of fish during acute and sub-chronic toxic experiments. However, fish in natural environments are continuously exposed to MC-LR throughout their entire life cycle as opposed to short-term exposure. Here, we tested the hypothesis that the mechanism by which MC-LR harms female fish reproduction and development within natural water bodies is through interference of the reproductive endocrine system. In the present study, zebrafish hatchlings (5 d post-fertilization) were exposed to 0, 0.3, 3 and 30 μg/L MC-LR for 90 d until reaching sexual maturity. Female zebrafish were selected, and the changes in growth and developmental indicators, ovarian ultrastructure as well as the levels of gonadal steroid hormones and vitellogenin (VTG) were examined along with the transcription of related genes in the hypothalamic-pituitary-gonadal-liver axis (HPGL-axis). The results showed for the first time, a life cycle exposure to MC-LR caused growth inhibition, decreased ovary weight and ovarian ultra-pathological lesions. Decreased ovarian testosterone levels indicated that MC-LR disrupted sex steroid hormone balance. Significantly up-regulated transcription of brain FSHβ and LHβ along with ovarian ERα, FSHR and LHR suggested positive feedback regulation in the HPGL-axis was induced as a compensatory mechanism for MC-LR damage. It was also noted that ovarian VTG content and hepatic ERα and VTG1 expression were all down-regulated, which might be responsible for reduced vitellus storage noted in our histological observations. Our findings indicate that a life cycle exposure to MC-LR impairs the development and reproduction of female zebrafish by disrupting the transcription of related HPGL-axis genes, suggesting that MC-LR has potential adverse effects on fish reproduction and thus population dynamics in MCs-contaminated aquatic environment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Gene expression profiling of the androgen receptor antagonists flutamide and vinclozolin in zebrafish (Danio rerio) gonads.

PubMed

Martinović-Weigelt, Dalma; Wang, Rong-Lin; Villeneuve, Daniel L; Bencic, David C; Lazorchak, Jim; Ankley, Gerald T

2011-01-25

The studies presented in this manuscript focus on characterization of transcriptomic responses to anti-androgens in zebrafish (Danio rerio). Research on the effects of anti-androgens in fish has been characterized by a heavy reliance on apical endpoints, and molecular mechanisms of action (MOA) of anti-androgens remain poorly elucidated. In the present study, we examined effects of a short term exposure (24-96h) to the androgen receptor antagonists flutamide (FLU) and vinclozolin (VZ) on gene expression in gonads of sexually mature zebrafish, using commercially available zebrafish oligonucleotide microarrays (4×44K platform). We found that VZ and FLU potentially impact reproductive processes via multiple pathways related to steroidogenesis, spermatogenesis, and fertilization. Observed changes in gene expression often were shared by VZ and FLU, as demonstrated by overlap in differentially-expressed genes and enrichment of several common key pathways including: (1) integrin and actin signaling, (2) nuclear receptor 5A1 signaling, (3) fibroblast growth factor receptor signaling, (4) polyamine synthesis, and (5) androgen synthesis. This information should prove useful to elucidating specific mechanisms of reproductive effects of anti-androgens in fish, as well as developing biomarkers for this important class of endocrine-active chemicals. 2010 Elsevier B.V. All rights reserved.

Enantio-alteration of gene transcription associated with bioconcentration in adult zebrafish (Danio rerio) exposed to chiral PCB149

NASA Astrophysics Data System (ADS)

Chai, Tingting; Cui, Feng; Mu, Pengqian; Yang, Yang; Xu, Nana; Yin, Zhiqiang; Jia, Qi; Yang, Shuming; Qiu, Jing; Wang, Chengju

2016-01-01

Enantioselective enrichment of chiral PCB149 (2,2’,3,4’,5’,6-hexachlorobiphenyl) was analysed in adult zebrafish (Danio rerio) exposed to the racemate, (-)-PCB149, and (+)-PCB149. Greater enrichment of (-)-PCB149 compared to (+) PCB149 was observed following 0.5 ng/L exposure; however, as the exposure time and concentration increased, racemic enrichment was observed in adult fish exposed to the racemate. No biotransformation between the two isomers was observed in fish exposed to single enantiomers. When zebrafish were exposed to different forms of chiral PCB149, enantioselective expression of genes associated with polychlorinated biphenyls (PCBs) was observed in brain and liver tissues and enantioselective correlations between bioconcentration and target gene expression levels were observed in brain and liver tissues. The strong positive correlations between expression levels of target genes (alox5a and alox12) and PCB149 bioconcentration suggest that prolonged exposure to the racemate of chiral PCB149 may result in inflammation-associated diseases. Prolonged exposure to (-)-PCB149 may also affect metabolic pathways such as dehydrogenation and methylation in the brain tissues of adult zebrafish. Hepatic expression levels of genes related to the antioxidant system were significantly negatively correlated with bioconcentration following exposure to (+)-PCB149.

Pharmacological analysis of zebrafish lphn3.1 morphant larvae suggests that saturated dopaminergic signaling could underlie the ADHD-like locomotor hyperactivity.

PubMed

Lange, Merlin; Froc, Cynthia; Grunwald, Hannah; Norton, William H J; Bally-Cuif, Laure

2018-06-08

Polymorphisms in the gene coding for the adhesion G-protein coupled receptor LPHN3 are a risk factor for attention-deficit/hyperactivity disorder (ADHD). Transient down-regulation of latrophilin3.1 (lphn3.1), the zebrafish LPHN3 homologue, causes hyperactivity. Zebrafish injected with a lphn3.1-specific morpholino are hyperactive and display an impairment in dopaminergic neuron development. In the present study we used lphn3.1 morphants to further characterize the changes to dopaminergic signaling that trigger hyperactivity. We applied dopamine agonists (Apomorphine, Quinpirole, SKF-38393) and antagonists (Haloperidol, Eticlopride, SCH-23390) to Lphn3.1 morpholino-injected or control-injected animals. The percentage of change in locomotor activity was then determined at three different time periods (10-20 min, 30-40 min and 60-70 min). Our results show that drugs targeting dopamine receptors appear to elicit similar effects on locomotion in zebrafish larvae and mammals. In addition, we observed that lphn3.1 morphants have an overall hyposensitivity to dopamine agonists and antagonists compared to control fish. These results are compatible with a model whereby dopaminergic neurotransmission is saturated in lphn3.1 morphants. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The Proprotein Convertase Subtilisin/Kexin FurinA Regulates Zebrafish Host Response against Mycobacterium marinum

PubMed Central

Ojanen, Markus J. T.; Turpeinen, Hannu; Cordova, Zuzet M.; Hammarén, Milka M.; Harjula, Sanna-Kaisa E.; Parikka, Mataleena; Rämet, Mika

2015-01-01

Tuberculosis is a chronic bacterial disease with a complex pathogenesis. An effective immunity against Mycobacterium tuberculosis requires both the innate and adaptive immune responses, including proper T helper (Th) type 1 cell function. FURIN is a proprotein convertase subtilisin/kexin (PCSK) enzyme, which is highly expressed in Th1 type cells. FURIN expression in T cells is essential for maintaining peripheral immune tolerance, but its role in the innate immunity and infections has remained elusive. Here, we utilized Mycobacterium marinum infection models in zebrafish (Danio rerio) to investigate how furin regulates host responses against mycobacteria. In steady-state furinAtd204e/+ fish reduced furinA mRNA levels associated with low granulocyte counts and elevated Th cell transcription factor expressions. Silencing furin genes reduced the survival of M. marinum-infected zebrafish embryos. A mycobacterial infection upregulated furinA in adult zebrafish, and infected furinAtd204e/+ mutants exhibited a proinflammatory phenotype characterized by elevated tumor necrosis factor a (tnfa), lymphotoxin alpha (lta) and interleukin 17a/f3 (il17a/f3) expression levels. The enhanced innate immune response in the furinAtd204e/+ mutants correlated with a significantly decreased bacterial burden in a chronic M. marinum infection model. Our data show that upregulated furinA expression can serve as a marker for mycobacterial disease, since it inhibits early host responses and consequently promotes bacterial growth in a chronic infection. PMID:25624351

Tributyltin impaired reproductive success in female zebrafish through disrupting oogenesis, reproductive behaviors and serotonin synthesis.

PubMed

Xiao, Wei-Yang; Li, Ying-Wen; Chen, Qi-Liang; Liu, Zhi-Hao

2018-07-01

Tributyltin (TBT), an organotin acting as aromatase (Cyp19a1) inhibitor, has been found to disrupt gametogenesis and reproductive behaviors in several fish species. However, few studies addressing the mechanisms underlying the impaired gametogenesis and reproduction have been reported. In this study, female adults of zebrafish (Danio rerio) were continuously exposed to two nominal concentrations of TBT (100 and 500 ng/L, actual concentrations: 90.8 ± 1.3 ng/L and 470.3 ± 2.7 ng/L, respectively) for 28 days. After exposures, TBT decreased the total egg number, reduced the hatchability and elevated the mortality of the larvae. Decreased gonadosomatic index (GSI) and altered percentages of follicles in different developmental stages (increased early-stage follicles and reduced mid/late-stage follicles) were also observed in the ovary of TBT-treated fish. TBT also lowered the plasma level of 17β-estradiol and suppressed the expressions of cyp19a1a in the ovary. In treated fish, up-regulated expressions of aldhla2, sycp3 and dmc1 were present in the ovary, indicating an enhanced level of meiosis. The mRNA level of vtg1 was dramatically suppressed in the liver of TBT-treated fish, suggesting an insufficient synthesis of Vtg protein, consistent with the decreased percentage of mid/late-stage follicles in the ovaries. Moreover, TBT significantly suppressed the reproductive behaviors of the female fish (duration of both sexes simultaneously in spawning area, the frequency of meeting and the visit in spawning area) and down-regulated the mRNA levels of genes involved in the regulation of reproductive behaviors (cyp19a1b, gnrh-3 and kiss 2) in the brain. In addition, TBT significantly suppressed the expressions of serotonin-related genes, such as tph2 (encoding serotonin synthase), pet1 (marker of serotonin neuron) and kiss 1 (the modulator of serotonin synthesis), suggesting that TBT might disrupt the non-reproductive behaviors of zebrafish. The present study demonstrated that TBT may impair the reproductive success of zebrafish females probably through disrupting oogenesis, disturbing reproductive behaviors and altering serotonin synthesis. The present study greatly extends our understanding on the reproductive toxicity of TBT on fish. Copyright © 2018 Elsevier B.V. All rights reserved.

DMSO modifies the permeability of the zebrafish (Danio rerio) chorion-implications for the fish embryo test (FET).

PubMed

Kais, B; Schneider, K E; Keiter, S; Henn, K; Ackermann, C; Braunbeck, T

2013-09-15

Since 2007, when REACH came into force, the fish embryo test has received increasing attention as a potential alternative for the acute fish test. Due to its low toxicity and the ability to permeate biological membranes without significant damage to their structural integrity, dimethyl sulfoxide (DMSO) is a commonly used solvent in the fish embryo test. Little is known, however, about the membrane penetration properties of DMSO, the impact of different concentrations of DMSO on the potential barrier function of the zebrafish chorion and on changes in the uptake of chemicals into the embryo. Therefore, in the present study, the fluorescent dyes fluorescein (mol wt 332; Pow 3.4) and 2,7-dichlorofluorescein (mol wt 401; Pow 4.7), both substances with limited water solubility, were used to visualize the uptake into the egg as well as the accumulation in the embryo of the zebrafish depending on different concentrations of DMSO. The distribution of fluorescein within the egg compartments varied with DMSO concentration: When dissolved in 0.01% DMSO, fluorescein did not pass the chorion. In contrast, concentrations ≥ 0.1% DMSO increasingly facilitated the uptake into the perivitelline space. In contrast, the uptake of 2,7-dichlorofluorescein was not substantially increased with rising DMSO concentrations, indicating the importance of factors other than the solvent (e.g. mol wt). With respect to the fish embryo test, results indicate that DMSO may be used without complications as a solvent, however, only at a maximum concentration of 0.01% (0.1 mL/L) as already indicated in the OECD difficult substances paper (OECD, 2000). Copyright © 2013 Elsevier B.V. All rights reserved.

Architecture of GnRH-Gonadotrope-Vasculature Reveals a Dual Mode of Gonadotropin Regulation in Fish.

PubMed

Golan, Matan; Zelinger, Einat; Zohar, Yonathan; Levavi-Sivan, Berta

2015-11-01

The function and components of the hypothalamic-pituitary axis are conserved among vertebrates; however, in fish, a neuroglandular mode of delivery (direct contact between axons and endocrine cells) was considered dominant, whereas in tetrapods hypothalamic signals are relayed to their targets via the hypophysial portal blood system (neurovascular delivery mode). By using a transgenic zebrafish model we studied the functional and anatomical aspects of gonadotrope regulation thus revisiting the existing model. FSH cells were found to be situated close to the vasculature whereas the compact organization of LH cells prevented direct contact of all cells with the circulation. GnRH3 fibers formed multiple boutons upon reaching the pituitary, but most of these structures were located in the neurohypophysis rather than adjacent to gonadotropes. A close association was observed between FSH cells and GnRH3 boutons, but only a fifth of the LH cells were in direct contact with GnRH3 axons, suggesting that FSH cells are more directly regulated than LH cells. GnRH3 fibers closely followed the vasculature in the neurohypophysis and formed numerous boutons along these tracts. These vessels were found to be permeable to relatively large molecules, suggesting the uptake of GnRH3 peptides. Our findings have important implications regarding the differential regulation of LH and FSH and contradict the accepted notion that fish pituitary cells are mostly regulated directly by hypothalamic fibers. Instead, we provide evidence that zebrafish apply a dual mode of gonadotrope regulation by GnRH3 that combines both neuroglandular and neurovascular components.

A novel zebrafish mutant with wavy-notochord: an effective biological index for monitoring the copper pollution of water from natural resources.

PubMed

Chen, Yau-Hung; Lin, Ji-Sheng

2011-02-01

We identified a novel zebrafish mutant that has wavy-notochord phenotypes, such as severely twisted notochord and posterior malformations, but has normal melanocytes. Histological evidences showed that proliferating vacuolar cells extended their growth to the muscle region, and consequently caused the wavy-notochord phenotypes. Interestingly, those malformations can be greatly reversed by exposure with copper, suggesting that copper plays an important role on wavy-notochord phenotypes. In addition, after long-term copper exposure, the surviving larvae derived from wavy-notochord mutants displayed bone malformations, such as twisted axial skeleton and osteophyte. These phenotypic changes and molecular evidences of wavy-notochord mutants are highly similar to those embryos whose lysyl oxidases activities have been inactivated. Taken together, we propose that (i) the putative mutated genes of this wavy-notochord mutant might be highly associated with the lysyl oxidase genes in zebrafish; and (ii) this fish model is an effective tool for monitoring copper pollution of water from natural resources. Copyright © 2009 Wiley Periodicals, Inc.

Transformation of tributyltin in zebrafish eleutheroembryos (Danio rerio).

PubMed

Borges, Aline Rocha; López-Serrano Oliver, Ana; Gallego-Gallegos, Mercedes; Muñoz-Olivas, Riansares; Rodrigues Vale, Maria Goreti; Cámara, Carmen

2014-12-01

Organotin compounds are highly versatile group of organometallic chemicals used in industrial and agricultural applications. Their endocrine-disrupting effects are well known and their extensive uses as biocide materials, e.g., in antifouling paints, for many years have led to serious environmental problems. So far, attention has mainly been given to tributyltin pollution in water, sediments, and marine organisms because of its highly toxic effects and high accumulation levels at very low concentrations. In this study, we will focus on the conversion of tributyltin after it is absorbed by zebrafish eleutheroembryos, presented here as an alternative model to adult fish for describing bioconcentration. A simplified analytical extraction procedure based on the use of an assisted ultrasonic probe and derivatization by ethylation, followed by gas chromatography with a flame photometric detector (GC-FPD) is proposed. This classical methodology for organotin determination has been validated by inductively coupled plasma mass spectrometry (ICP-MS) and Zeeman graphite furnace atomic absorption spectrometry (ZGF-AAS) in terms of total tin content. The speciation analysis results show that zebrafish eleutheroembryos absorb high amounts of tributyltin and convert it into monobutyltin and likely in inorganic tin.

Optimal Anesthetic Regime for Motionless Three-Dimensional Image Acquisition During Longitudinal Studies of Adult Nonpigmented Zebrafish.

PubMed

Lockwood, Nicola; Parker, Jennifer; Wilson, Carole; Frankel, Paul

2017-04-01

With many live imaging techniques, it is crucial that a deep level of anesthesia is reached and maintained throughout image acquisition without reducing zebrafish viability. This is particularly true for three-dimensional tomographic imaging modalities. Currently, the most commonly used anesthetic in the zebrafish community, MS-222 (tricaine methanesulfonate), does not allow this. We show, using a combination of both MS-222 and isoflurane, that we can significantly improve the anesthetic regime required for motionless image acquisition of live adult zebrafish. We have benchmarked this against the requirements of our novel quantitative imaging platform, compressive sensing optical projection tomography. Using nonpigmented transgenic zebrafish, we show that a combination of 175 ppm of both anesthetics improves the maintenance of deep anesthesia for prolonged periods of time and it can be used repeatedly to enable longitudinal imaging. Importantly, it does not affect the health or viability of the adult zebrafish. We also show that nonpigmented fish, with a mutated form of the gene transparent, took significantly longer to reach deep anesthesia. The anesthetic regime presented in this study should lead to significant improvements in accuracy and information achievable from imaging live adult zebrafish and in its application to longitudinal studies.

Identification and characterization of a novel zebrafish (Danio rerio) pentraxin-carbonic anhydrase.

PubMed

Patrikainen, Maarit S; Tolvanen, Martti E E; Aspatwar, Ashok; Barker, Harlan R; Ortutay, Csaba; Jänis, Janne; Laitaoja, Mikko; Hytönen, Vesa P; Azizi, Latifeh; Manandhar, Prajwol; Jáger, Edit; Vullo, Daniela; Kukkurainen, Sampo; Hilvo, Mika; Supuran, Claudiu T; Parkkila, Seppo

2017-01-01

Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H + ions. Vertebrate genomes generally contain gene loci for 15-21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs. We isolated and sequenced zebrafish ( Danio rerio ) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI-PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI-PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae. These findings provide novel insights into the evolution, structure, and function of this unique CA form.

Strain differences in the collective behaviour of zebrafish (Danio rerio) in heterogeneous environment

PubMed Central

Collignon, Bertrand

2016-01-01

Recent studies show differences in individual motion and shoaling tendency between strains of the same species. Here, we analyse collective motion and response to visual stimuli in two morphologically different strains (TL and AB) of zebrafish. For both strains, we observed 10 groups of 5 and 10 zebrafish swimming freely in a large experimental tank with two identical landmarks (cylinders or discs) for 1 h. We tracked the positions of the fish by an automated tracking method and compute several metrics at the group level. First, the probability of the presence shows that both strains avoid free space and are more likely to swim in the vicinity of the walls of the tank and the landmarks. Second, the analysis of landmarks occupancy shows that AB zebrafish are more present in their vicinity than TL ones and that both strains regularly transit from one to the other one with no preference on the long duration. Finally, TL zebrafish show a higher cohesion than AB zebrafish. Thus, environmental heterogeneity and duration of the trials allow to reveal individual and collective behavioural variabilities among different strains of zebrafish. These results provide a new insight into the need to take into account individual variability of zebrafish strains for studying collective behaviour. PMID:27853558

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish.

PubMed

Callard, G V; Tchoudakova, A V; Kishida, M; Wood, E

2001-12-01

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromB>A) and ovary (P450aromA>B) and have a different developmental program (B>A) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24-48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (alpha, beta, and gamma). The 5'-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (a>b) are opposite to fish pituitary (b>a). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30-48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.

Development of a flow-through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio).

PubMed

Lammer, E; Kamp, H G; Hisgen, V; Koch, M; Reinhard, D; Salinas, E R; Wendler, K; Zok, S; Braunbeck, Th

2009-10-01

The acute fish test is still a mandatory component in chemical hazard and risk assessment. However, one of the objectives of the new European chemicals policy (REACH - Registration, Evaluation, Authorization and Restriction of Chemicals) is to promote non-animal testing. For whole effluent testing in Germany, the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) has been an accepted and mandatory replacement of the fish test since January 2005. For chemical testing, however, further optimization of the FET is required to improve the correlation between the acute fish test and the alternative FET. Since adsorption of the test chemical to surfaces may reduce available exposure concentrations, a flow-through system for the FET using modified commercially available polystyrene 24-well microtiter plates was developed, thus combining the advantages of the standard FET with those of continuous delivery of test substances. The advantages of the design presented include: small test footprint, availability of adequate volumes of test solution for subsequent chemical analysis, and sufficient flow to compensate for effects of non-specific adsorption within 24h. The flow-through test system can also be utilized to conduct longer-term embryo larval fish tests, thus offering the possibility for teratogenicity testing.

Species-specific considerations in using the fish embryo test as an alternative to identify endocrine disruption.

PubMed

Schiller, Viktoria; Zhang, Xiaowei; Hecker, Markus; Schäfers, Christoph; Fischer, Rainer; Fenske, Martina

2014-10-01

A number of regulations have been implemented that aim to control the release of potentially adverse endocrine disrupters into the aquatic environment based on evidence from laboratory studies. Currently, such studies rely on testing approaches with adult fish because reliable alternatives have not been validated so far. Fish embryo tests have been proposed as such an alternative, and here we compared two species (medaka and zebrafish) to determine their suitability for the assessment of substances with estrogenic and anti-androgenic activity. Changes in gene expression (in here the phrase gene expression is used synonymously to gene transcription, although it is acknowledged that gene expression is additionally regulated, e.g., by translation and protein stability) patterns between the two species were compared in short term embryo exposure tests (medaka: 7-day post fertilization [dpf]; zebrafish: 48 and 96h post fertilization [hpf]) by using relative quantitative real-time RT-PCR. The tested genes were related to the hypothalamic-gonadal-axis and early steroidogenesis. Test chemicals included 17α-ethinylestradiol and flutamide as estrogenic and anti-androgenic reference compounds, respectively, as well as five additional substances with endocrine activities, namely bisphenol A, genistein, prochloraz, linuron and propanil. Estrogenic responses were comparable in 7-dpf medaka and 48/96-hpf zebrafish embryos and included transcriptional upregulation of aromatase b, vitellogenin 1 as well as steroidogenic genes, suggesting that both species reliably detected exposure to estrogenic compounds. However, anti-androgenic responses differed between the two species, with each species providing specific information concerning the mechanism of anti-androgenic disruption in fish embryos. Although small but significant changes in the expression of selected genes was observed in 48-hpf zebrafish embryos, exposure prolonged to 96hpf was necessary to obtain a response indicative of anti-androgenic activity. In contrast, for medaka clear anti-androgenic response, e.g. transcriptional downregulation of 11β-hydroxylase, 3β-hydroxysteroid-dehydrogenase, gonadotropin-releasing hormone receptor 2, was already observed at the pre-hatch stage. Together, this data suggests that medaka and zebrafish embryos would provide a beneficial alternative testing platform for endocrine disruption that involves additive information on interspecies and exposure time variability when using both species. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection and signal amplification in zebrafish RNA FISH.

PubMed

Hauptmann, Giselbert; Lauter, Gilbert; Söll, Iris

2016-04-01

In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts. Copyright © 2016 Elsevier Inc. All rights reserved.

Metagenomics analysis of gut microbiota and immune modulation in zebrafish (Danio rerio) fed chitosan silver nanocomposites.

PubMed

Udayangani, R M C; Dananjaya, S H S; Nikapitiya, Chamilani; Heo, Gang-Joon; Lee, Jehee; De Zoysa, Mahanama

2017-07-01

In this study, we evaluated the effects of chitosan silver nanocomposites (CAgNCs) supplemented diet on gut microbial community, goblet cell density, gut morphometry and mRNA expression of immune related and mucin encoding genes in zebrafish. Zebrafish gut microbiota analysis results clearly showed the reduction of phylum Proteobacteria. However, they remained as the major bacterial group in gut with CAgNCs supplemented diet, while the abundance of phylum Fusobacteria and phylum Bacteroidetes were increased notably compared to the control diet fed fish. Total goblet cell density was significantly increased at 30 and 60 days in CAgNCs supplemented group (1.6-fold and 2.0-fold, respectively) compared to the control group indicating enhanced immune function in the gut. CAgNCs supplementation has also increased villi height significantly in the zebrafish mid gut at the end of 30 (95.5 ± 3.7 μm) and 60 days (144.40 ± 4.8 μm) compared to control diet fed fish at 30 (86.90 ± 3.7 μm) and 60 days (96.2 ± 4.8 μm). Furthermore, mRNA expression of immune related genes such as TNF-α (6.2-fold), IL-10 (5.0-fold), IL-12 (9.2-fold), IRF-1 (5.2-fold), Defbl1 (3-fold), Lyz (5.1-fold) and mucin encoding genes were significantly upregulated (above 2-fold) compared to that of control group. The current study revealed that CAgNCs supplemented diet engenders promising effects on fish gut immunity by enhancing beneficial microbial populations, goblet cell density, villi length, and transcriptional regulation of immune related and mucin encoding genes. Copyright © 2017 Elsevier Ltd. All rights reserved.

A genomic view of the NOD-like receptor family in teleost fish: Identification of a novel NLR subfamily in zebrafish

USGS Publications Warehouse

Laing, K.J.; Purcell, M.K.; Winton, J.R.; Hansen, J.D.

2008-01-01

Background. A large multigene family of NOD-like receptor (NLR) molecules have been described in mammals and implicated in immunity and apoptosis. Little information, however, exists concerning this gene family in non-mammalian taxa. This current study, therefore, provides an in-depth investigation of this gene family in lower vertebrates including extensive phylogenetic comparison of zebrafish NLRs with orthologs in tetrapods, and analysis of their tissue-specific expression. Results. Three distinct NLR subfamilies were identified by mining genome databases of various non-mammalian vertebrates; the first subfamily (NLR-A) resembles mammalian NODs, the second (NLR-B) resembles mammalian NALPs, while the third (NLR-C) appears to be unique to teleost fish. In zebrafish, NLR-A and NLR-B subfamilies contain five and six genes respectively. The third subfamily is large, containing several hundred NLR-C genes, many of which are predicted to encode a C-terminal B30.2 domain. This subfamily most likely evolved from a NOD3-like molecule. Gene predictions for zebrafish NLRs were verified using sequence derived from ESTs or direct sequencing of cDNA. Reverse-transcriptase (RT)-PCR analysis confirmed expression of representative genes from each subfamily in selected tissues. Conclusion. Our findings confirm the presence of multiple NLR gene orthologs, which form a large multigene family in teleostei. Although the functional significance of the three major NLR subfamilies is unclear, we speculate that conservation and abundance of NLR molecules in all teleostei genomes, reflects an essential role in cellular control, apoptosis or immunity throughout bony fish. ?? 2008 Laing et al; licensee BioMed Central Ltd.

Exposure to perchlorate induces the formation of macrophage aggregates in the trunk kidney of zebrafish and mosquitofish

USGS Publications Warehouse

Capps, T.; Mukhi, S.; Rinchard, J.J.; Theodorakis, C.W.; Blazer, V.S.; Patino, R.

2004-01-01

Environmental contamination of ground and surface waters by perchlorate, derived from ammonium perchlorate (AP) and other perchlorate salts, is of increasing concern. Exposure to perchlorate can impair the thyroid endocrine system, which is thought to modulate renal and immune function in vertebrates. This study with zebrafish Danio rerio and eastern mosquitofish Gambusia holbrooki examined the histological effects of perchlorate on the trunk kidney, which in teleosts serves excretory and hemopoietic functions and therefore may be a target of perchlorate effects. Adult zebrafish of both sexes were exposed in the laboratory to waterborne, AP-derived perchlorate at measured concentrations of 18 mg/L for 8 weeks. Adult male mosquitofish were exposed to waterborne sodium perchlorate at measured perchlorate concentrations of 1-92 mg/L for 8 weeks. Control fish were kept in untreated water. The region of the body cavity containing the trunk kidney was processed from each fish for histological analysis. Macrophage aggregates (MAs), possible markers of contaminant exposure or immunotoxic effect, were present in the hemopoietic region of the kidney in both species exposed to perchlorate. The estimated percent area of kidney sections occupied by MAs was greater in zebrafish exposed to perchlorate at 18 mg/L (P < 0.05) than in controls. In male mosquitofish, the incidence of renal MAs increased proportionally with sodium perchlorate concentration and was significantly different from that of controls at 92 mg/L (P < 0.05). These observations confirm that in fish the kidney is affected by exposure to perchlorate. The concentrations of perchlorate at which the effects were noted are relatively high but within the range reported in some contaminated habitats.

Ecological Support of Larval Fish During Multigenerational Studies on Space Station

NASA Technical Reports Server (NTRS)

Taub, Frieda B.

1998-01-01

Live, microscopic food is required by larval Zebrafish, Danio rerio, which are candidates for the Aquatic Habitat of the Space Station Biological Research Project (SSBRP). Zebrafish have proven to be convenient research animals, and their embryology and genetics are extensively documented. Their ability to mature at 3 months of age, and the transparent eggs which hatches in 2 days, are attractive attributes for space research. Among the goals of the SSBRP Aquatic Habitat is the ability to study three generations, with the objective of maintaining adults, their offspring, and the maintaining of these offspring through maturity and spawning. For Zebrafish, it is anticipated that sexually mature fish (PI) would be delivered to Space Station and spawned in space. The challenge would be it to provide appropriate microscopic foods for the offspring (FI), and 3 months later for the next generation (F2); if these were raised to maturity and bred, live foods would be required at approximately 6 months. In laboratories where Zebrafish are traditionally reared, the larval foods are the protozoan Parameciwn micromultinucleatwn and later brine shrimp Artemia nauplii. Under normal laboratory conditions, the rearing of these foods are relatively easy, although time consuming because of the food organisms must be separated from their rearing medium which is discarded. A freshwater food chain that would ensur-e healthy on- orbit research animals is needed. ne food chain should (a) be reared in conditions that are compatible with the larval fish (water chemistry, pH, temperature and light), (b) assist in maintaining water quality (by removing ammonia, nitrate, phosphate, carbon dioxide, and bacteria) and (c) be convenient for the space crew (minimize handling and waste production).

Toxicity of chlorine to zebrafish embryos

PubMed Central

Kent, Michael L.; Buchner, Cari; Barton, Carrie; Tanguay, Robert L.

2014-01-01

Surface disinfection of fertilized fish eggs is widely used in aquaculture to reduce extraovum pathogens that may be released from brood fish during spawning, and this is routinely used in zebrafish (Danio rerio) research laboratories. Most laboratories use approximately 25 – 50 ppm unbuffered chlorine solution for 5 – 10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages of protozoa, it has reduced efficacy against cyst or spore stages of protozoa and certain Mycobacterium spp. Therefore, we evaluated the toxicity of unbufferred and buffered chlorine solution to embryos exposed at 6 or 24 hours post-fertilization (hpf) to determine if higher concentrations can be used for treating zebrafish embryos. Most of our experiments entailed using an outbred line (5D), with both mortality and malformations as endpoints. We found that 6 hpf embryos consistently were more resistant than 24 hpf embryos to the toxic effects of chlorine. Chlorine is more toxic and germicidal at lower pHs, and chlorine causes elevated pH. Consistent with this, we found that unbufferred chlorine solutions (pH ca 8–9) were less toxic at corresponding concentrations than solutions buffered to pH 7. Based on our findings here, we recommend treating 6 hpf embryos for 10 min and 24 hpf for 5 min with unbuffered chlorine solution at 100 ppm. One trial indicated that AB fish, a popular outbred line, are more susceptible to toxicity than 5Ds. This suggests that variability between zebrafish lines occurs, and researchers should evaluate each line or strain under their particular laboratory conditions for selection of the optimum chlorine treatment procedure. PMID:24429474

How does predation affect the bioaccumulation of hydrophobic organic compounds in aquatic organisms?

PubMed

Xia, Xinghui; Li, Husheng; Yang, Zhifeng; Zhang, Xiaotian; Wang, Haotian

2015-04-21

It is well-known that the body burden of hydrophobic organic compounds (HOCs) increases with the trophic level of aquatic organisms. However, the mechanism of HOC biomagnification is not fully understood. To fill this gap, this study investigated the effect of predation on the bioaccumulation of polycyclic aromatic hydrocarbons (PAHs), one type of HOC, in low-to-high aquatic trophic levels under constant freely dissolved PAH concentrations (1, 5, or 10 μg L(-1)) maintained by passive dosing systems. The tested PAHs included phenanthrene, anthracene, fluoranthene, and pyrene. The test organisms included zebrafish, which prey on Daphnia magna, and cichlids, which prey on zebrafish. The results revealed that for both zebrafish and cichlids, predation elevated the uptake and elimination rates of PAHs. The increase of uptake rate constant ranged from 20.8% to 39.4% in zebrafish with the amount of predation of 5 daphnids per fish per day, and the PAH uptake rate constant increased with the amount of predation. However, predation did not change the final bioaccumulation equilibrium; the equilibrium concentrations of PAHs in fish only depended on the freely dissolved concentration in water. Furthermore, the lipid-normalized water-based bioaccumulation factor of each PAH was constant for fish at different trophic levels. These findings infer that the final bioaccumulation equilibrium of PAHs is related to a partition between water and lipids in aquatic organisms, and predation between trophic levels does not change bioaccumulation equilibrium but bioaccumulation kinetics at stable freely dissolved PAH concentrations. This study suggests that if HOCs have not reached bioaccumulation equilibrium, biomagnification occurs due to enhanced uptake rates caused by predation in addition to higher lipid contents in higher trophic organisms. Otherwise, it is only due to the higher lipid contents in higher trophic organisms.

Dynamic and differential expression of the gonadal aromatase during the process of sexual differentiation in a novel transgenic cyp19a1a-eGFP zebrafish line.

PubMed

Hinfray, Nathalie; Sohm, Frédéric; Caulier, Morgane; Chadili, Edith; Piccini, Benjamin; Torchy, Camille; Porcher, Jean-Marc; Guiguen, Yann; Brion, François

2018-05-15

In zebrafish, there exists a clear need for new tools to study sex differentiation dynamic and its perturbation by endocrine disrupting chemicals. In this context, we developed and characterized a novel transgenic zebrafish line expressing green fluorescent protein (GFP) under the control of the zebrafish cyp19a1a (gonadal aromatase) promoter. In most gonochoristic fish species including zebrafish, cyp19a1a, the enzyme responsible for the synthesis of estrogens, has been shown to play a critical role in the processes of reproduction and sexual differentiation. This novel cyp19a1a-eGFP transgenic line allowed a deeper characterization of expression and localization of cyp19a1a gene in zebrafish gonads both at the adult stage and during development. At the adult stage, GFP expression was higher in ovaries than in testis. We showed a perfect co-expression of GFP and endogenous Cyp19a1a protein in gonads that was mainly localized in the cytoplasm of peri-follicular cells in the ovary and of Leydig and germ cells in the testis. During development, GFP was expressed in all immature gonads of 20 dpf-old zebrafish. Then, GFP expression increased in early differentiated female at 30 and 35dpf to reach a high GFP intensity in well-differentiated ovaries at 40dpf. On the contrary, males consistently displayed low GFP expression as compared to female whatever their stage of development, resulting in a clear dimorphic expression between both sexes. Interestingly, fish that undergoes ovary-to-testis transition (35 and 40dpf) presented GFP levels similar to males or intermediate between females and males. In this transgenic line our results confirm that cyp19a1a is expressed early during development, before the histological differentiation of the gonads, and that the down-regulation of cyp19a1a expression is likely responsible for the testicular differentiation. Moreover, we show that although cyp19a1a expression exhibits a clear dimorphic expression pattern in gonads during sexual differentiation, its expression persists whatever the sex suggesting that estradiol synthesis is important for gonadal development of both sexes. Monitoring the expression of GFP in control and exposed-fish will help determine the sensitivity of this transgenic line to EDCs and to refine mechanistic based-assays for the study of EDCs. In fine, this transgenic zebrafish line will be a useful tool to study physiological processes such as reproduction and sexual differentiation, and their perturbations by EDCs. Copyright © 2017 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dynan, William S.

The goal of this study was to examine long-term effects of low-dose radiation exposure. One of the hypotheses was that radiation exposure would accelerate the normal aging process. The study was jointly funded by NASA and examined both low-LET radiation (γ-rays) and high-LET radiation (1000 MeV/nucleon 56Fe ions) at doses of 0.1 Gy and up. The work used the Japanese medaka fish (Oryzias latipes), as a vertebrate model organism that can be maintained in large numbers at low cost for lifetime studies. Like other small laboratory fish, Japanese medaka share many anatomical and histological characteristics with other vertebrates, and amore » variety of genetic and genomic resources are available. Some work also used the zebrafish (Danio rerio), another widely used laboratory model organism.« less

'Fish matters': the relevance of fish skin biology to investigative dermatology.

PubMed

Rakers, Sebastian; Gebert, Marina; Uppalapati, Sai; Meyer, Wilfried; Maderson, Paul; Sell, Anne F; Kruse, Charli; Paus, Ralf

2010-04-01

Fish skin is a multi-purpose tissue that serves numerous vital functions including chemical and physical protection, sensory activity, behavioural purposes or hormone metabolism. Further, it is an important first-line defense system against pathogens, as fish are continuously exposed to multiple microbial challenges in their aquatic habitat. Fish skin excels in highly developed antimicrobial features, many of which have been preserved throughout evolution, and infection defense principles employed by piscine skin are still operative in human skin. This review argues that it is both rewarding and important for investigative dermatologists to revive their interest in fish skin biology, as it provides insights into numerous fundamental issues that are of major relevance to mammalian skin. The basic molecular insights provided by zebrafish in vivo-genomics for genetic, regeneration and melanoma research, the complex antimicrobial defense systems of fish skin and the molecular controls of melanocyte stem cells are just some of the fascinating examples that illustrate the multiple potential uses of fish skin models in investigative dermatology. We synthesize the essentials of fish skin biology and highlight selected aspects that are of particular comparative interest to basic and clinically applied human skin research.

Zebrafish as a model to study the role of DNA methylation in environmental toxicology.

PubMed

Kamstra, Jorke H; Aleström, Peter; Kooter, Jan M; Legler, Juliette

2015-11-01

Environmental epigenetics is a rapidly growing field which studies the effects of environmental factors such as nutrition, stress, and exposure to compounds on epigenetic gene regulation. Recent studies have shown that exposure to toxicants in vertebrates is associated with changes in DNA methylation, a major epigenetic mechanism affecting gene transcription. Zebra fish, a well-known model in toxicology and developmental biology, are emerging as a model species in environmental epigenetics despite their evolutionary distance to rodents and humans. In this review, recent insights in DNA methylation during zebra fish development are discussed and compared to mammalian models in order to evaluate zebra fish as a model to study the role of DNA methylation in environmental toxicology. Differences exist in DNA methylation reprogramming during early development, whereas in later developmental stages, tissue distribution of both 5-methylcytosine and 5-hydroxymethylcytosine seems more conserved between species, as well as basic DNA (de)methylation mechanisms. All DNA methyl transferases identified so far in mammals are present in zebra fish, as well as a number of major demethylation pathways. However, zebra fish appear to lack some methylation pathways present in mammals, such as parental imprinting. Several studies report effects on DNA methylation in zebra fish following exposure to environmental contaminants, such as arsenic, benzo[a]pyrene, and tris(1,3-dichloro-2-propyl)phosphate. Though more research is needed to examine heritable effects of contaminant exposure on DNA methylation, recent data suggests the usefulness of the zebra fish as a model in environmental epigenetics.

Zebrafish and relational memory: Could a simple fish be useful for the analysis of biological mechanisms of complex vertebrate learning?

PubMed

Gerlai, Robert

2017-08-01

Analysis of the zebrafish allows one to combine two distinct scientific approaches, comparative ethology and neurobehavioral genetics. Furthermore, this species arguably represents an optimal compromise between system complexity and practical simplicity. This mini-review focuses on a complex form of learning, relational learning and memory, in zebrafish. It argues that zebrafish are capable of this type of learning, and it attempts to show how this species may be useful in the analysis of the mechanisms and the evolution of this complex brain function. The review is not intended to be comprehensive. It is a short opinion piece that reflects the author's own biases, and it draws some of its examples from the work coming from his own laboratory. Nevertheless, it is written in the hope that it will persuade those who have not utilized zebrafish and who may be interested in opening their research horizon to this relatively novel but powerful vertebrate research tool. Copyright © 2017 Elsevier B.V. All rights reserved.

Relationships among msx gene structure and function in zebrafish and other vertebrates.

PubMed

Ekker, M; Akimenko, M A; Allende, M L; Smith, R; Drouin, G; Langille, R M; Weinberg, E S; Westerfield, M

1997-10-01

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.

Molecular aspect of silver nanoparticles regulated embryonic development in Zebrafish (Danio rerio) by Oct-4 expression.

PubMed

Sarkar, Biplab; Verma, Suresh K; Akhtar, Javed; Netam, Surya Prakash; Gupta, Sanjay Kr; Panda, Pritam Kumar; Mukherjee, Koel

2018-09-01

With the enhancement of commercial manifestation of silver nanoparticles, concerned has risen on their accumulation in aquatic system and consequent effects on fish development and metabolism. In this study, experiments were conducted to assess the impacts of silver nanoparticles on early life cycles of fish considering Zebrafish (Danio rerio) as experimental model. Silver nanoparticles were synthesized through chemical reduction method and characterized through UV-visible spectroscopy, dynamic light scattering (DLS), and HR-TEM. Different sub lethal doses of nanosilver were applied (13.6, 21.6, 42.4, 64, and 128 μgL -1 ) to post-fertilization phases of Zebrafish embryos and their interaction effects were monitored up to six days period. No significant morphological variations were observed at 13.6, 21.6, 42.4 μgL -1 dose of silver nanoparticles, whereas 64 and 128 μgL -1 exposure dose exhibited bending in myotome, deformity in tail region, somites, notochord and swelling in anterior and posterior region of embryos and larva. Hatching performances analysis elicited highest hatching success in 13.6 and 21.6 μgL -1 doses of silver nanoparticles followed by positive and negative control, whereas exposure dose of 64 and 128 μgL -1 exhibited comparatively lower success. Western blot analysis were conducted on developing hatchlings with Oct4 antibody and at 13.6 and 21.6 μgL -1 dose,it showed over expression elucidating stimulatory role of nanosilver in these mentioned doses. In silico analysis depicted a firm interaction of nanosilver with Oct4 revealing their key role in growth stimulation of developing embryos. The study demonstrates the function of nanosilver as a growth promoter rather only as a toxicant in fish metabolic system. Copyright © 2018 Elsevier Ltd. All rights reserved.

High-throughput behavioral screening method for detecting auditory response defects in zebrafish.

PubMed

Bang, Pascal I; Yelick, Pamela C; Malicki, Jarema J; Sewell, William F

2002-08-30

We have developed an automated, high-throughput behavioral screening method for detecting hearing defects in zebrafish. Our assay monitors a rapid escape reflex in response to a loud sound. With this approach, 36 adult zebrafish, restrained in visually isolated compartments, can be simultaneously assessed for responsiveness to near-field 400 Hz sinusoidal tone bursts. Automated, objective determinations of responses are achieved with a computer program that obtains images at precise times relative to the acoustic stimulus. Images taken with a CCD video camera before and after stimulus presentation are subtracted to reveal a response to the sound. Up to 108 fish can be screened per hour. Over 6500 fish were tested to validate the reliability of the assay. We found that 1% of these animals displayed hearing deficits. The phenotypes of non-responders were further assessed with radiological analysis for defects in the gross morphology of the auditory system. Nearly all of those showed abnormalities in conductive elements of the auditory system: the swim bladder or Weberian ossicles. Copyright 2002 Elsevier Science B.V.

Effects of oxytetracycline and amoxicillin on development and biomarkers activities of zebrafish (Danio rerio).

PubMed

Oliveira, Rhaul; McDonough, Sakchai; Ladewig, Jessica C L; Soares, Amadeu M V M; Nogueira, António J A; Domingues, Inês

2013-11-01

Antibiotics have been widely used in human and veterinary medicine to treat or prevent diseases. Residues of antibiotics have been found in aquatic environments, but their effects on fish have been not properly investigated. This work aimed to assess the sub-lethal effects of oxytetracycline and amoxicillin on zebrafish development and biomarkers. Embryos and adults were exposed during 96 h to amoxicillin and oxytetracycline following OECD guidelines. Tissues of adults and pools of embryos were used for catalase, glutathione-S-transferases and lactate dehydrogenase determinations. Amoxicillin caused premature hatching (48 h-EC50=132.4 mg/l) whereas oxytetracycline cause delayed hatching of embryos (72 h-EC50=127.6 mg/l). Moreover, both antibiotics inhibited catalase and induced glutathione-S-transferases in zebrafish adults. However, only oxytetracycline induced lactate dehydrogenase. Short-term effects of antibiotics were observed at high doses (mg/l) indicating that physiological impairment in fish populations is unlike to occur. However, effects of chronic exposures to low doses of ABs must be investigated. Copyright © 2013 Elsevier B.V. All rights reserved.

Chevron formation of the zebrafish muscle segments.

PubMed

Rost, Fabian; Eugster, Christina; Schröter, Christian; Oates, Andrew C; Brusch, Lutz

2014-11-01

The muscle segments of fish have a folded shape, termed a chevron, which is thought to be optimal for the undulating body movements of swimming. However, the mechanism shaping the chevron during embryogenesis is not understood. Here, we used time-lapse microscopy of developing zebrafish embryos spanning the entire somitogenesis period to quantify the dynamics of chevron shape development. By comparing such time courses with the start of movements in wildtype zebrafish and analysing immobile mutants, we show that the previously implicated body movements do not play a role in chevron formation. Further, the monotonic increase of chevron angle along the anteroposterior axis revealed by our data constrains or rules out possible contributions by previously proposed mechanisms. In particular, we found that muscle pioneers are not required for chevron formation. We put forward a tension-and-resistance mechanism involving interactions between intra-segmental tension and segment boundaries. To evaluate this mechanism, we derived and analysed a mechanical model of a chain of contractile and resisting elements. The predictions of this model were verified by comparison with experimental data. Altogether, our results support the notion that a simple physical mechanism suffices to self-organize the observed spatiotemporal pattern in chevron formation. © 2014. Published by The Company of Biologists Ltd.

Dasatinib as a treatment for Duchenne muscular dystrophy.

PubMed

Lipscomb, Leanne; Piggott, Robert W; Emmerson, Tracy; Winder, Steve J

2016-01-15

Identification of a systemically acting and universal small molecule therapy for Duchenne muscular dystrophy would be an enormous advance for this condition. Based on evidence gained from studies on mouse genetic models, we have identified tyrosine phosphorylation and degradation of β-dystroglycan as a key event in the aetiology of Duchenne muscular dystrophy. Thus, preventing tyrosine phosphorylation and degradation of β-dystroglycan presents itself as a potential therapeutic strategy. Using the dystrophic sapje zebrafish, we have investigated the use of tyrosine kinase and other inhibitors to treat the dystrophic symptoms in this model of Duchenne muscular dystrophy. Dasatinib, a potent and specific Src tyrosine kinase inhibitor, was found to decrease the levels of β-dystroglycan phosphorylation on tyrosine and to increase the relative levels of non-phosphorylated β-dystroglycan in sapje zebrafish. Furthermore, dasatinib treatment resulted in the improved physical appearance of the sapje zebrafish musculature and increased swimming ability as measured by both duration and distance of swimming of dasatinib-treated fish compared with control animals. These data suggest great promise for pharmacological agents that prevent the phosphorylation of β-dystroglycan on tyrosine and subsequent steps in the degradation pathway as therapeutic targets for the treatment of Duchenne muscular dystrophy. © The Author 2015. Published by Oxford University Press.

Effects of ozonated sewage effluent on reproduction and behavioral endpoints in zebrafish (Danio rerio).

PubMed

Pohl, Johannes; Björlenius, Berndt; Brodin, Tomas; Carlsson, Gunnar; Fick, Jerker; Larsson, D G Joakim; Norrgren, Leif; Örn, Stefan

2018-04-25

Pharmaceutical residues and other micro-contaminants may enter aquatic environments through effluent from sewage treatment plants (STPs) and could cause adverse effects in wild fish. One strategy to alleviate this situation is to improve wastewater treatment by ozonation. To test the effectiveness of full-scale wastewater effluent ozonation at a Swedish municipal STP, the added removal efficiency was measured for 105 pharmaceuticals. In addition, gene expression, reproductive and behavioral endpoints were analyzed in zebrafish (Danio rerio) exposed on-site over 21 days to ozonated or non-ozonated effluents as well as to tap water. Ozone treatment (7 g O 3 /m 3 ) removed pharmaceuticals by an average efficiency of 77% in addition to the conventional treatment, leaving 11 screened pharmaceuticals above detection limits. Differences in biological responses of the exposure treatments were recorded in gene expression, reproduction and behavior. Hepatic vitellogenin gene expression was higher in male zebrafish exposed to the ozonated effluent compared to the non-ozonated effluent and tap water treatments. The reproductive success was higher in fish exposed to ozonated effluent compared to non-ozonated effluent and to tap water. The behavioral measurements showed that fish exposed to the ozonated STP effluent were less active in swimming the first minute after placed in a novel vessel. Ozonation is a capable method for removing pharmaceuticals in effluents. However, its implementation should be thoroughly evaluated for any potential biological impact. Future research is needed for uncovering the factors which produced the in vivo responses in fish. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of a new screening assay to identify proteratogenic substances using zebrafish danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT).

PubMed

Busquet, François; Nagel, Roland; von Landenberg, Friedrich; Mueller, Stefan O; Huebler, Nicole; Broschard, Thomas H

2008-07-01

The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays, for example, in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were cocultured at 2 hpf (hours postfertilization) with the test material at varying concentrations, induced male rat liver microsomes and nicotinamide adenine dinucleotide phosphate (reduced) for 60 min at 32 degrees C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterward fish embryos were transferred individually into 24-well plates filled with fish medium for 48 h at 26 degrees C with a 12-h light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, that is, without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when coincubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the D. rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.

Developmental lead exposure causes startle response deficits in zebrafish.

PubMed

Rice, Clinton; Ghorai, Jugal K; Zalewski, Kathryn; Weber, Daniel N

2011-10-01

Lead (Pb(2+)) exposure continues to be an important concern for fish populations. Research is required to assess the long-term behavioral effects of low-level concentrations of Pb(2+) and the physiological mechanisms that control those behaviors. Newly fertilized zebrafish embryos (<2h post fertilization; hpf) were exposed to one of three concentrations of lead (as PbCl(2)): 0, 10, or 30 nM until 24 hpf. (1) Response to a mechanosensory stimulus: Individual larvae (168 hpf) were tested for response to a directional, mechanical stimulus. The tap frequency was adjusted to either 1 or 4 taps/s. Startle response was recorded at 1000 fps. Larvae responded in a concentration-dependent pattern for latency to reaction, maximum turn velocity, time to reach V(max) and escape time. With increasing exposure concentrations, a larger number of larvae failed to respond to even the initial tap and, for those that did respond, ceased responding earlier than control larvae. These differences were more pronounced at a frequency of 4 taps/s. (2) Response to a visual stimulus: Fish, exposed as embryos (2-24 hpf) to Pb(2+) (0-10 μM) were tested as adults under low light conditions (≈ 60 μW/m(2)) for visual responses to a rotating black bar. Visual responses were significantly degraded at Pb(2+) concentrations of 30 nM. These data suggest that zebrafish are viable models for short- and long-term sensorimotor deficits induced by acute, low-level developmental Pb(2+) exposures. Copyright © 2011 Elsevier B.V. All rights reserved.

Asian medaka fishes offer new models for studying mechanisms of seawater adaptation.

PubMed

Inoue, Koji; Takei, Yoshio

2003-12-01

Japanese medaka (Oryzias latipes) is a freshwater (FW) teleost that is popular throughout the world for laboratory use. In this paper, we discuss the utility of Japanese medaka and related species for studying mechanisms of seawater (SW) adaptation. In addition to general advantages as an experimental animal such as their daily spawning activity, transparency of embryos, short generation time and established transgenic techniques, Japanese medaka have some adaptability to SW unlike the strictly stenohaline zebrafish (Danio rerio). Since other species in the genus Oryzias exhibit different degrees of adaptability to SW, comparative studies between Japanese medaka, where molecular-biological and genetic information is abundant, and other Oryzias species are expected to present varying approaches to solving the problems of SW adaptation. We introduce some examples of interspecies comparison for SW adaptabilities both in adult fish and in embryos. Oryzias species are good models for evolutionary, ecological and zoogeographical studies and a relationship between SW adaptability and geographic distribution has been suggested. Medaka fishes may thus deliver new insights into our understanding of how fish have expanded their distribution to a wide variety of osmotic environments.

Biotransformation in the zebrafish embryo -temporal gene transcription changes of cytochrome P450 enzymes and internal exposure dynamics of the AhR binding xenobiotic benz[a]anthracene.

PubMed

Kühnert, Agnes; Vogs, Carolina; Seiwert, Bettina; Aulhorn, Silke; Altenburger, Rolf; Hollert, Henner; Küster, Eberhard; Busch, Wibke

2017-11-01

Not much is known about the biotransformation capability of zebrafish (Danio rerio) embryos. For understanding possible toxicity differences to adult fish, it might be crucial to understand the biotransformation of chemicals in zebrafish embryos i.e. as part of toxicokinetics. The biotransformation capabilities were analysed for two different stages of zebrafish embryos in conjunction with the internal concentrations of a xenobiotic. Zebrafish embryos of the late cleavage/early blastula period (2-26 hpf) and the early pharyngula period (26-50 hpf) were exposed for 24 h to the AhR binding compound benz[a]anthracene (BaA). Time dependent changes in cyp transcription (cyp1a, cyp1b1, cyp1c1 and cyp1c2) as well as concentration & time-dependent courses of BaA in the fish embryo and the exposure medium were analysed. Additionally, the CYP mediated formation of biotransformation products was investigated. We found correlations between transcriptional responses and the internal concentration for both exposure types. These correlations were depending on the start of the exposure i.e. the age of the exposed embryo. While no significant induction of the examined gene transcripts was observed in the first 12 h of exposure beginning in the blastula period a correlation was apparent when exposure started later i.e. in the pharyngula period. A significant induction of cyp1a was detected already after 1.5 h of BaA exposure. Gene transcripts for cyp1b1, cyp1c1 and cyp1c2 showed expressions distinctly different from cyp1a and were, in general, less inducible by BaA in both exposure windows. The toxicokinetic analysis showed that the biotransformation capability was fivefold higher in the older fish embryos. Biotransformation products of phase I reactions were found between 32 hpf and 50 hpf and were tentatively identified as benz[a]anthracene-phenol and benz[a]anthracene-dihydrodiol-epoxide. In conclusion, not only duration but also onset of exposure in relation to the developmental stage of zebrafish embryos is important in the analysis and interpretation of effects due to different biotransformation capabilities. Copyright © 2017 Elsevier Ltd. All rights reserved.

Prolactin-dependent modulation of organogenesis in the vertebrate: Recent discoveries in zebrafish.

PubMed

Nguyen, Nhu; Stellwag, Edmund J; Zhu, Yong

2008-11-01

The scientific literature is replete with evidence of the multifarious functions of the prolactin (PRL)/growth hormone (GH) superfamily in adult vertebrates. However, little information is available on the roles of PRL and related hormones prior to the adult stage of development. A limited number of studies suggest that GH functions to stimulate glucose transport and protein synthesis in mouse blastocytes and may be involved during mammalian embryogenesis. In contrast, the evidence for a role of PRL during vertebrate embryogenesis is limited and controversial. Genes encoding GH/PRL hormones and their respective receptors are actively transcribed and translated in various animal models at different time points, particularly during tissue remodeling. We have addressed the potential function of GH/PRL hormones during embryonic development in zebrafish by the temporary inhibition of in vivo PRL translation. This treatment caused multiple morphological defects consistent with a role of PRL in embryonic-stage organogenesis. The affected organs and tissues are known targets of PRL activity in fish and homologous structures in mammalian species. Traditionally, the GH/PRL hormones are viewed as classical endocrine hormones, mediating functions through the circulatory system. More recent evidence points to cytokine-like actions of these hormones through either an autocrine or a paracrine mechanism. In some situations they could mimic actions of developmentally regulated genes as suggested by experiments in multiple organisms. In this review, we present similarities and disparities between zebrafish and mammalian models in relation to PRL and PRLR activity. We conclude that the zebrafish could serve as a suitable alternative to the rodent model to study PRL functions in development, especially in relation to organogenesis.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

PubMed

Busquet, François; Strecker, Ruben; Rawlings, Jane M; Belanger, Scott E; Braunbeck, Thomas; Carr, Gregory J; Cenijn, Peter; Fochtman, Przemyslaw; Gourmelon, Anne; Hübler, Nicole; Kleensang, André; Knöbel, Melanie; Kussatz, Carola; Legler, Juliette; Lillicrap, Adam; Martínez-Jerónimo, Fernando; Polleichtner, Christian; Rzodeczko, Helena; Salinas, Edward; Schneider, Katharina E; Scholz, Stefan; van den Brandhof, Evert-Jan; van der Ven, Leo T M; Walter-Rohde, Susanne; Weigt, Stefan; Witters, Hilda; Halder, Marlies

2014-08-01

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Navigational strategies underlying phototaxis in larval zebrafish.

PubMed

Chen, Xiuye; Engert, Florian

2014-01-01

Understanding how the brain transforms sensory input into complex behavior is a fundamental question in systems neuroscience. Using larval zebrafish, we study the temporal component of phototaxis, which is defined as orientation decisions based on comparisons of light intensity at successive moments in time. We developed a novel "Virtual Circle" assay where whole-field illumination is abruptly turned off when the fish swims out of a virtually defined circular border, and turned on again when it returns into the circle. The animal receives no direct spatial cues and experiences only whole-field temporal light changes. Remarkably, the fish spends most of its time within the invisible virtual border. Behavioral analyses of swim bouts in relation to light transitions were used to develop four discrete temporal algorithms that transform the binary visual input (uniform light/uniform darkness) into the observed spatial behavior. In these algorithms, the turning angle is dependent on the behavioral history immediately preceding individual turning events. Computer simulations show that the algorithms recapture most of the swim statistics of real fish. We discovered that turning properties in larval zebrafish are distinctly modulated by temporal step functions in light intensity in combination with the specific motor history preceding these turns. Several aspects of the behavior suggest memory usage of up to 10 swim bouts (~10 sec). Thus, we show that a complex behavior like spatial navigation can emerge from a small number of relatively simple behavioral algorithms.

Recombinant expression and purification of the RNA-binding LARP6 proteins from fish genetic model organisms.

PubMed

Castro, José M; Horn, Daniel A; Pu, Xinzhu; Lewis, Karen A

2017-06-01

The RNA-binding proteins that comprise the La-related protein (LARP) superfamily have been implicated in a wide range of cellular functions, from tRNA maturation to regulation of protein synthesis. To more expansively characterize the biological function of the LARP6 subfamily, we have recombinantly expressed the full-length LARP6 proteins from two teleost fish, platyfish (Xiphophorus maculatus) and zebrafish (Danio rerio). The yields of the recombinant proteins were enhanced to >2 mg/L using a tandem approach of an N-terminal His 6 -SUMO tag and an iterative solubility screening assay to identify structurally stabilizing buffer components. The domain topologies of the purified fish proteins were probed with limited proteolysis. The fish proteins contain an internal, protease-resistant 40 kDa domain, which is considerably more stable than the comparable domain from the human LARP6 protein. The fish proteins are therefore a lucrative model system in which to study both the evolutionary divergence of this family of La-related proteins and the structure and conformational dynamics of the domains that comprise the LARP6 protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Valerenic acid and Valeriana officinalis extracts delay onset of Pentylenetetrazole (PTZ)-Induced seizures in adult Danio rerio (Zebrafish).

PubMed

Torres-Hernández, Bianca A; Del Valle-Mojica, Lisa M; Ortíz, José G

2015-07-14

Anticonvulsant properties have been attributed to extracts of the herbal medicine Valeriana officinalis. Our aims were to examine the anticonvulsant properties of valerenic acid and valerian extracts and to determine whether valerian preparations interact with the activity of other anti-epileptic drugs (phenytoin or clonazepam). To achieve these goals, we validated the adult zebrafish, Danio rerio, as an animal model for studying anticonvulsant drugs. All drug treatments were administered by immersion in water containing the drug. For assays of anticonvulsant activity, zebrafish were pretreated with: anti-epileptic drugs, valerenic acid, aqueous or ethanolic valerian extracts, or mixtures (phenytoin or clonazepam with valerenic acid or valerian extracts). Seizures were then induced with pentylenetetrazole (PTZ). A behavioral scale was developed for scoring PTZ-induced seizures in adult zebrafish. The seizure latency was evaluated for all pretreatments and control, untreated fish. Valerenic acid and both aqueous and ethanolic extracts of valerian root were also evaluated for their ability to improve survival after pentylenetetrazole-challenge. The assay was validated by comparison with well-studied anticonvulsant drugs (phenytoin, clonazepam, gabapentin and valproate). One-way ANOVA followed by Tukey post-hoc test was performed, using a p < 0.05 level of significance. All treatments were compared with the untreated animals and with the other pretreatments. After exposure to pentylenetetrazole, zebrafish exhibited a series of stereotypical behaviors prior to the appearance of clonic-like movements--convulsions. Both valerenic acid and valerian extracts (aqueous and ethanolic) significantly extended the latency period to the onset of seizure (convulsion) in adult zebrafish. The ethanolic valerian extract was a more potent anticonvulsant than the aqueous extract. Valerenic acid and both valerian extracts interacted synergistically with clonazepam to extended the latency period to the onset of seizure. Phenytoin showed interaction only with the ethanolic valerian extracts. Valerenic acid and valerian extracts have anticonvulsant properties in adult zebrafish. Valerian extracts markedly enhanced the anticonvulsant effect of both clonazepam and phenytoin, and could contribute to therapy of epileptic patients.

Effect of tributyltin on antioxidant ability and immune responses of zebrafish (Danio rerio).

PubMed

Zhang, Chun-Nuan; Zhang, Ji-Liang; Ren, Hong-Tao; Zhou, Bian-Hua; Wu, Qiu-Jue; Sun, Ping

2017-04-01

Tributyltin (TBT) is a toxic compound released into aquatic ecosystems through antifouling paints. This study was designed to examine the effects of TBT on antioxidant ability and immune responses of zebrafish (Danio rerio). Three hundred sixty healthy zebrafish were randomly grouped into four groups and exposed to different doses of TBT (0, 1, 10 and 100ngL -1 ). At the end of 8 weeks, the fish were sampled, and antioxidant capability, immune parameters and immune-related genes were assessed. The results showed that with an increase in TBT dose, the concentration of malonaldehyde in the liver was significantly increased (p<0.05), whereas the activities of total superoxide dismutase, catalase and glutathione peroxidase were significantly decreased (p<0.05) compared to the control. The activity and expression of lysozyme and the content of immunoglobulin M were significantly decreased compared to those of the fish exposed to 0ngL -1 TBT (p<0.05). However, the expression of the HSP70, HSP90, tumor necrosis factor-α(TNF-α), interleukins (IL-1β, IL-6), and nuclear factor-kappa B p65 (NF-κ B p65) genes were all enhanced with an increase in TBT dose. The results indicated that TBT induced oxidative stress and had immunotoxic effects on zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential maturation of rhythmic clock gene expression during early development in medaka (Oryzias latipes).

PubMed

Cuesta, Ines H; Lahiri, Kajori; Lopez-Olmeda, Jose Fernando; Loosli, Felix; Foulkes, Nicholas S; Vallone, Daniela

2014-05-01

One key challenge for the field of chronobiology is to identify how circadian clock function emerges during early embryonic development. Teleosts such as the zebrafish are ideal models for studying circadian clock ontogeny since the entire process of development occurs ex utero in an optically transparent chorion. Medaka (Oryzias latipes) represents another powerful fish model for exploring early clock function with, like the zebrafish, many tools available for detailed genetic analysis. However, to date there have been no reports documenting circadian clock gene expression during medaka development. Here we have characterized the expression of key clock genes in various developmental stages and in adult tissues of medaka. As previously reported for other fish, light dark cycles are required for the emergence of clock gene expression rhythms in this species. While rhythmic expression of per and cry genes is detected very early during development and seems to be light driven, rhythmic clock and bmal expression appears much later around hatching time. Furthermore, the maturation of clock function seems to correlate with the appearance of rhythmic expression of these positive elements of the clock feedback loop. By accelerating development through elevated temperatures or by artificially removing the chorion, we show an earlier onset of rhythmicity in clock and bmal expression. Thus, differential maturation of key elements of the medaka clock mechanism depends on the developmental stage and the presence of the chorion.

Indomethacin treatment prior to pentylenetetrazole-induced seizures downregulates the expression of il1b and cox2 and decreases seizure-like behavior in zebrafish larvae.

PubMed

Barbalho, Patrícia Gonçalves; Lopes-Cendes, Iscia; Maurer-Morelli, Claudia Vianna

2016-03-09

It has been demonstrated that the zebrafish model of pentylenetetrazole (PTZ)-evoked seizures and the well-established rodent models of epilepsy are similar pertaining to behavior, electrographic features, and c-fos expression. Although this zebrafish model is suitable for studying seizures, to date, inflammatory response after seizures has not been investigated using this model. Because a relationship between epilepsy and inflammation has been established, in the present study we investigated the transcript levels of the proinflammatory cytokines interleukin-1 beta (il1b) and cyclooxygenase-2 (cox2a and cox2b) after PTZ-induced seizures in the brain of zebrafish 7 days post fertilization. Furthermore, we exposed the fish to the nonsteroidal anti-inflammatory drug indomethacin prior to PTZ, and we measured its effect on seizure latency, number of seizure behaviors, and mRNA expression of il1b, cox2b, and c-fos. We used quantitative real-time PCR to assess the mRNA expression of il1b, cox2a, cox2b, and c-fos, and visual inspection was used to monitor seizure latency and the number of seizure-like behaviors. We found a short-term upregulation of il1b, and we revealed that cox2b, but not cox2a, was induced after seizures. Indomethacin treatment prior to PTZ-induced seizures downregulated the mRNA expression of il1b, cox2b, and c-fos. Moreover, we observed that in larvae exposed to indomethacin, seizure latency increased and the number of seizure-like behaviors decreased. This is the first study showing that il1b and cox-2 transcripts are upregulated following PTZ-induced seizures in zebrafish. In addition, we demonstrated the anticonvulsant effect of indomethacin based on (1) the inhibition of PTZ-induced c-fos transcription, (2) increase in seizure latency, and (3) decrease in the number of seizure-like behaviors. Furthermore, anti-inflammatory effect of indomethacin is clearly demonstrated by the downregulation of the mRNA expression of il1b and cox2b. Our results are supported by previous evidences suggesting that zebrafish is a suitable alternative for studying inflammation, seizures, and the effect of anti-inflammatory compounds on seizure suppression.

Protective effect of Uncaria tomentosa extract against oxidative stress and genotoxicity induced by glyphosate-Roundup® using zebrafish (Danio rerio) as a model.

PubMed

Santo, Glaucia Dal; Grotto, Alan; Boligon, Aline A; Da Costa, Bárbara; Rambo, Cassiano L; Fantini, Emily A; Sauer, Elisa; Lazzarotto, Luan M V; Bertoncello, Kanandra T; Júnior, Osmar Tomazelli; Garcia, Solange C; Siebel, Anna M; Rosemberg, Denis B; Magro, Jacir Dal; Conterato, Greicy M M; Zanatta, Leila

2018-04-01

Oxidative stress and DNA damage are involved in the glyphosate-based herbicide toxicity. Uncaria tomentosa (UT; Rubiaceae) is a plant species from South America containing bioactive compounds with known beneficial properties. The objective of this work was to evaluate the antioxidant and antigenotoxic potential of UT extract in a model of acute exposure to glyphosate-Roundup® (GR) in zebrafish (Danio rerio). We showed that UT (1.0 mg/mL) prevented the decrease of brain total thiols, the increase of lipid peroxidation in both brain and liver, and the decrease of liver GPx activity caused after 96 h of GR (5.0 mg/L) exposure. In addition, UT partially protected against the increase of micronucleus frequency induced by GR exposure in fish brain. Overall, our results indicate that UT protects against damage induced by a glyphosate-based herbicide by providing antioxidant and antigenotoxic effects, which may be related to the phenolic compounds identified in the extract.

Fish in the matrix: motor learning in a virtual world.

PubMed

Engert, Florian

2012-01-01

One of the large remaining challenges in the field of zebrafish neuroscience is the establishment of techniques and preparations that permit the recording and perturbation of neural activity in animals that can interact meaningfully with the environment. Since it is very difficult to do this in freely behaving zebrafish, I describe here two alternative approaches that meet this goal via tethered preparations. The first uses head-fixation in agarose in combination with online imaging and analysis of tail motion. In the second method, paralyzed fish are suspended with suction pipettes in mid-water and nerve root recordings serve as indicators for intended locomotion. In both cases, fish can be immersed into a virtual environment and allowed to interact with this virtual world via real or fictive tail motions. The specific examples given in this review focus primarily on the role of visual feedback~- but the general principles certainly extend to other modalities, including proprioception, hearing, balance, and somatosensation.

Fish in the matrix: motor learning in a virtual world

PubMed Central

Engert, Florian

2013-01-01

One of the large remaining challenges in the field of zebrafish neuroscience is the establishment of techniques and preparations that permit the recording and perturbation of neural activity in animals that can interact meaningfully with the environment. Since it is very difficult to do this in freely behaving zebrafish, I describe here two alternative approaches that meet this goal via tethered preparations. The first uses head-fixation in agarose in combination with online imaging and analysis of tail motion. In the second method, paralyzed fish are suspended with suction pipettes in mid-water and nerve root recordings serve as indicators for intended locomotion. In both cases, fish can be immersed into a virtual environment and allowed to interact with this virtual world via real or fictive tail motions. The specific examples given in this review focus primarily on the role of visual feedback~– but the general principles certainly extend to other modalities, including proprioception, hearing, balance, and somatosensation. PMID:23355810