Use of Zebrafish in Drug Discovery Toxicology.

Unpredicted human safety events in clinical trials for new drugs are costly in terms of human health and money. The drug discovery industry attempts to minimize those events with diligent preclinical safety testing. Current standard practices are good at preventing toxic compounds from being tested in the clinic; however, false negative preclinical toxicity results are still a reality. Continual improvement must be pursued in the preclinical realm. Higher-quality therapies can be brought forward with more information about potential toxicities and associated mechanisms. The zebrafish model is a bridge between in vitro assays and mammalian in vivo studies. This model is powerful in its breadth of application and tractability for research. In the past two decades, our understanding of disease biology and drug toxicity has grown significantly owing to thousands of studies on this tiny vertebrate. This Review summarizes challenges and strengths of the model, discusses the 3Rs value that it can deliver, highlights translatable and untranslatable biology, and brings together reports from recent studies with zebrafish focusing on new drug discovery toxicology.

The effects of intensive feeding on reproductive performance in laboratory zebrafish (Danio rerio).

The zebrafish (Danio rerio) is among the most widely used model animals in scientific research. Historically, these fish have been reared in the laboratory using simple methods developed by home aquarists. For laboratories with high demand for breeding and generation turn-over, however, there has been a shift away from this approach towards one that leverages techniques, tools, and feeds from commercial aquaculture to help accelerate growth rates and decrease generation times. While these advances have improved efficiency, the effects of feeding zebrafish diets that are designed to grow disparately related cold-water fish species to market size quickly are not well-understood. To explore the impacts that intensive feeding protocols may have on this species, groups of zebrafish larvae from two different wild-type lines were stocked into treatment tanks at a standard density of 10 fish per liter and were administered either a "high" or "low" food diet for a maximum of 63 days. During their growth phase, the "high" food diet group received at least 8x more rotifers and at least 2x more Artemia than the "low" food diet group. Growth, survival, and reproductive performance (fecundity and viability) were measured in these fish and in their offspring. We found that fish that were fed more grew more rapidly and were able to reproduce earlier than fish that were fed less, but they were also more likely to produce higher proportions of non-viable embryos.

An evaluation of programs designed to increase representation of diverse faculty at academic medical centers.

PURPOSE: Minority physicians have been persistently underrepresented in medicine (URiM) when compared with their representation in the general U.S. POPULATION: There is evidence that diversifying the physician workforce would have a positive impact on healthcare delivery. While programs have been implemented to diversify the physician workforce, there has been less emphasis and progress in diversifying academic medical centers (AMCs) at the faculty level. This review sought to provide an update in the literature on the published outcomes and components of programs designed to increase the racial/ethnic diversity of faculty at AMCs. METHOD: A scoping review study design was used. Search terms-academic medical faculty, diversity, and recruitment or retention-were used to search literature published from August 2012 through February 4, 2021. Eligible studies (1) evaluate structured organization/institutional programs to increase the representation of faculty who identify as URiM; (2) include faculty who identify as URiM as defined by the Association of American Medical Colleges (individuals who identify as African Americans and/or Black, Hispanic/Latino, Native American (American Indians, Alaska Natives, and Native Hawaiians), Pacific Islander, and mainland Puerto Rican); (3) describe interventions for the academic medicine setting; and (4) report either quantitative or qualitative data. RESULTS: Ten papers describing eight programs were included in the review. Program components were heterogeneous. Most studies described funded research-focused programs and fewer studies were institution-wide efforts or included institutional culture (i.e., rules, values, beliefs, behaviors, and customs that shape how people behave within an organization) or climate (i.e., perception regarding the culture) efforts. Four studies reported outcomes that included changes in faculty representation and six included faculty retention efforts such as promotion, leadership positions, grants, and scholarly productivity. CONCLUSIONS: This review summarizes and updates the literature on programs to improve the diversity of faculty at AMCs. It includes specific recommendations for components that can provide a foundation for programs to improve faculty diversity. Future research should use high quality methods to compare different interventions to improve the diversity of faculty in AMCs.

Space microgravity improves proliferation of human iPSC-derived cardiomyocytes.

In microgravity, cells undergo profound changes in their properties. However, how human cardiac progenitors respond to space microgravity is unknown. In this study, we evaluated the effect of space microgravity on differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiac progenitors compared with 1G cultures on the International Space Station (ISS). Cryopreserved 3D cardiac progenitors were cultured for 3 weeks on the ISS. Compared with 1G cultures, the microgravity cultures had 3-fold larger sphere sizes, 20-fold higher counts of nuclei, and increased expression of proliferation markers. Highly enriched cardiomyocytes generated in space microgravity showed improved Ca2+ handling and increased expression of contraction-associated genes. Short-term exposure (3 days) of cardiac progenitors to space microgravity upregulated genes involved in cell proliferation, survival, cardiac differentiation, and contraction, consistent with improved microgravity cultures at the late stage. These results indicate that space microgravity increased proliferation of hiPSC-cardiomyocytes, which had appropriate structure and function.

A fish is not a mouse: understanding differences in background genetics is critical for reproducibility.

Poorly controlled background genetics in animal models contributes to the lack of reproducibility that is increasingly recognized in biomedical research. The laboratory zebrafish, Danio rerio, has been an important model organism for decades in many research areas, yet inbred strains and traditionally managed outbred stocks are not available for this species. Sometimes incorrectly referred to as 'inbred strains' or 'strains', zebrafish wild-type lines possess background genetics that are often not well characterized, and breeding practices for these lines have not been consistent over time or among institutions. In this Perspective, we trace key milestones in the history of one of the most widely used genetic backgrounds, the AB line, to illustrate the dynamic complexity within an example background that is largely invisible when reading the scientific literature. Failure to adequately control for genetic background compromises the validity of experimental outcomes. We therefore propose that authors provide as much specific detail about the origin and genetic makeup of zebrafish lines as is reasonable and possible, and that the terms used to describe background genetics be applied in a way that is consistent with other fish and mammalian model organisms. We strongly encourage the adoption of genetic monitoring for the characterization of existing zebrafish lines, to help detect genetic contamination in breeding colonies and to verify the level of genetic heterogeneity in breeding colonies over time. Careful attention to background genetics will improve transparency and reproducibility, therefore improving the utility of the zebrafish as a model organism.

Pleistophora hyphessobryconis (Microsporidia) infecting zebrafish Danio rerio in research facilities.

Zebrafish Danio rerio are important models for biomedical research, and thus, there is an increased concern about diseases afflicting them. Here we describe infections by Pleistophora hyphessobryconis (Microsporidia) in zebrafish from 3 laboratories. As reported in other aquarium fishes, affected zebrafish exhibited massive infections in the skeletal muscle, with no involvement of smooth or cardiac muscle. In addition, numerous spores within macrophages were observed in the visceral organs, including the ovaries. Transmission studies and ribosomal RNA (rRNA) gene sequence comparisons confirmed that the parasite from zebrafish was P. hyphessobryconis as described from neon tetra Paracheirodon innesi. Ten 15 d old zebrafish were exposed to P. hyphessobryconis collected from 1 infected neon tetra, and 7 of 10 fish became infected. Comparison of P. hyphessobryconis small subunit rRNA gene sequence from neon tetra with that obtained from zebrafish was nearly identical, with < 1% difference. Given the severity of infections, P. hyphessobryconis should be added to the list of pathogens that should be avoided in zebrafish research facilities, and it would be prudent to avoid mixing zebrafish used in research with other aquarium fishes.

Exploring the advantages and limitations of sampling methods commonly used in research facilities for zebrafish health inspections.

Examining zebrafish populations for the presence of disease is an integral component of managing fish health in research facilities. Currently, many different strategies are used for zebrafish fish health inspections, which is a scenario that may result in subjective and biased diagnostic evaluations. The goal of this study was to compare the success of pathogen detection between a sample size of randomly selected fish (n = 60) that provides 95% confidence in pathogen detection based on a presumed pathogen prevalence level ≥5%, and other subpopulations and sample numbers commonly submitted for diagnostic testing within a 1000 tank, 30,000 fish, recirculating research system. This included fish collected from a sump tank (n = 53), sentinel fish (n = 11), and fish that were found moribund or freshly dead (n = 18). Additionally, five fish from each subpopulation were collected for histopathologic examination. A second study used retrospective data to examine pathogen distribution between systems (n = 2-5) in multi-system facilities (n = 5) using a sample size of 60 fish per system. For the pathogens detected, results supported the use of representative sample numbers rather than smaller numbers of populations considered more at risk. The exception to this is for the moribund/mortality group, which may be a resource for targeted surveillance of select pathogens. Each system within multi-system facilities should be considered separate units in terms of fish health inspections and biosecurity. Development of these evidence-based standards for fish health inspections in zebrafish systems enhances fish welfare, provides identification of potentially zoonotic pathogens, and ensures scientific integrity and reproducibility of research results.

The Influence of Behavioral, Social, and Environmental Factors on Reproducibility and Replicability in Aquatic Animal Models.

The publication of reproducible, replicable, and translatable data in studies utilizing animal models is a scientific, practical, and ethical necessity. This requires careful planning and execution of experiments and accurate reporting of results. Recognition that numerous developmental, environmental, and test-related factors can affect experimental outcomes is essential for a quality study design. Factors commonly considered when designing studies utilizing aquatic animal species include strain, sex, or age of the animal; water quality; temperature; and acoustic and light conditions. However, in the aquatic environment, it is equally important to consider normal species behavior, group dynamics, stocking density, and environmental complexity, including tank design and structural enrichment. Here, we will outline normal species and social behavior of 2 commonly used aquatic species: zebrafish (Danio rerio) and Xenopus (X. laevis and X. tropicalis). We also provide examples as to how these behaviors and the complexity of the tank environment can influence research results and provide general recommendations to assist with improvement of reproducibility and replicability, particularly as it pertains to behavior and environmental complexity, when utilizing these popular aquatic models.

Proximal Correlates of Suicidal Ideation and Behaviors: A Test of the Interpersonal-Psychological Theory of Suicide.

OBJECTIVE: The interpersonal-psychological theory of suicide hypothesizes proximal causes of suicidal ideation and behaviors; however, past studies have generally tested distal relations. The present study tested the proximal nature of the theory's hypotheses. METHODS: A repeated-measures design collected daily survey data on the theory constructs over 90 days from 206 (150 women) college students with previous histories of suicidal ideation. Participants completed 7,342 (39.6%) of the 18,540 surveys sent. RESULTS: Thwarted belongingness and perceived burdensomeness both positively associated with passive suicidal ideation at the daily level. A statistical trend revealed that perceived burdensomeness was associated with daily active suicidal ideation only at high levels of thwarted belongingness and hopelessness. Active suicidal ideation, but not capability for suicide, was positively associated with suicidal behavior at the daily level. CONCLUSIONS: These results support the theory's predictions regarding passive and active suicidal ideation, with limited support regarding suicidal behaviors. The proximal associations of the IPTS constructs with daily suicidal ideation suggest areas for potential intervention with suicidal clients.

A Preliminary Validation of the Suicidal Behavior Exposure Scale.

OBJECTIVE: Research on suicide contagion has suggested that exposure to suicide-related thoughts and behaviors of others promotes one's personal risk for suicide. However, our understanding of the role suicidal exposure plays in the development of suicide risk is underdeveloped. This limited understanding may be due, in part, to the lack of a validated measure of exposure to suicidal behavior. The present study aimed to develop and validate a comprehensive self-report instrument of exposure to suicidal behaviors. METHOD: Using two independent convenience samples of undergraduate students, exploratory structural equation modeling (ESEM) was performed on a newly developed measure of suicidal exposure. RESULTS: Data supported a three-factor model of suicidal exposure: exposure to suicidal communication, direct exposure to suicide deaths and attempts, and indirect exposure to suicide deaths and attempts. Each factor demonstrated positive bivariate associations with depressive symptoms, posttraumatic stress disorder symptoms, suicide ideation, and suicide attempt history, suggesting convergent validity. Analyses demonstrated generally acceptable internal consistency. CONCLUSION: This instrument will facilitate the study of suicidal exposure. Further validation of this scale, with attention to its reliability and invariance across gender and other demographic variables in clinically relevant samples, will be important for advancing the assessment of suicidal exposure and understanding its relation to suicide risk.

The risk-benefit ratio of studying psychiatric symptoms via daily diary methods.

Ethics committee members and researchers have deliberated about the risk-benefit ratio of researching sensitive issues such as psychiatric symptoms. Although research has suggested that inquiring about psychiatric symptoms in research generally does not cause harm, these findings have primarily arisen from cross-sectional studies. We examined whether this generalized to repeated, daily assessments of psychiatric symptoms. We collected daily survey data on psychiatric symptoms over 90 days from a sample of 206 college students. A subset of the sample (n = 80) provided reactions to study participation administered on the 90th day. Individuals who did not complete the 90th day survey reported higher levels of suicidal ideation and hopelessness than those who did. For individuals who completed the 90th, final assessment, reactions primarily fell within the neutral to positive range, with variation depending on their baseline levels of psychiatric symptoms and identification as religious. This study adds to past work by demonstrating that individuals who remained in the study had neutral to positive experiences. However, participants with greater suicidal ideation and hopelessness were likely to attrit, warranting caution in assuming a low risk-benefit ratio of these studies. Management of risks involved in repeated assessment studies may be informed by this work.

Modeling human muscle disease in zebrafish.

Zebrafish reproduce in large quantities, grow rapidly, and are transparent early in development. For these reasons, zebrafish have been used extensively to model vertebrate development and disease. Like mammals, zebrafish express dystrophin and many of its associated proteins early in development and these proteins have been shown to be vital for zebrafish muscle stability. In dystrophin-null zebrafish, muscle degeneration becomes apparent as early as 3 days post-fertilization (dpf) making the zebrafish an excellent organism for large-scale screens to identify other genes involved in the disease process or drugs capable of correcting the disease phenotype. Being transparent, developing zebrafish are also an ideal experimental model for monitoring the fate of labeled transplanted cells. Although zebrafish dystrophy models are not meant to replace existing mammalian models of disease, experiments requiring large numbers of animals may be best performed in zebrafish. Results garnered from using this model could lead to a better understanding of the pathogenesis of the muscular dystrophies and the development of future therapies.

Effects of Diet and Social Housing on Reproductive Success in Adult Zebrafish, Danio rerio.

Zebrafish (Danio rerio) have proven their efficiency as an animal model for genetics and development, but their nutrition and housing requirements continue to elude researchers. Diet and housing density were predicted to affect weight change and reproductive success in 120 days postfertilization (dpf) zebrafish, and growth performance of their progeny. Fish were fed one of four diets, each utilizing a different primary protein source (fish meal [Zeigler™], algae, or insect), while being housed 3.3 or 6.6 fish/L for 3 weeks. Clutch size, viability, and larval development of their progeny were monitored out to 10 dpf. All diets were sent out for proximate nutrient analysis and fatty acid profiles to understand how diet compositions affect reproduction. We found that diet and housing proximity affected adult fish weight and larvae growth; diets composed of higher levels of protein and polyunsaturated fatty acids (specifically arachidonic acid [AA] and eicosapentanoic acid) allowed fish to gain weight and produce healthy larvae. Fish housed at higher densities produced smaller embryos, but larger larvae than those housed at lower densities. These findings imply that significant effects of a modified stimulus are exhibited after relatively short periods.

Workshop Report: Zebrafish and Other Fish Models-Description of Extrinsic Environmental Factors for Rigorous Experiments and Reproducible Results.

Current standards for husbandry and maintenance of zebrafish and other aquatic species in the laboratory are diverse, and are subject to laboratory performance, engineering, and practice standards (the Guide), institutional interpretation, national animal welfare laws, and cultural differences. Consequently, it is difficult, and probably not advantageous, to establish a single standard in view of the hardy nature of zebrafish and the diversity of research requirements it is used to address. Based on their natural habitat, zebrafish can thrive in a variety of environmental conditions, which is a specific advantage for working with this laboratory organism. However, it also makes reporting and reproducibility difficult, because variations in the husbandry and environmental conditions, including the environmental conditions before and during experiments, are often underreported in the scientific literature. This lack of consistency presents a potential problem for research reproducibility. To begin addressing this emerging scientific gap, the National Institutes of Health's (NIH) Office of Research Infrastructure Programs (ORIP), Division of Construction and Instruments (DCI), hosted a workshop in late 2017, entitled "Zebrafish and Other Aquatic Models: Reporting of Environmental Husbandry Conditions for Rigorous Experiments and Reproducible Results," that was attended by ∼60 participants. The objectives of the workshop were to bring together a diverse group of stakeholders-researchers, facility managers, veterinarians, journal editors, commercial vendors, and others to (1) review current husbandry and environmental management practices for the care of zebrafish and other aquatic organisms in the laboratory and to (2) propose a process for the development of a minimal set of environmental parameters that should be reported in publications to ensure rigor and robustness of experiments and reproducible outcomes. The participants also discussed how these recommendations, as an initial step, might be collected, disseminated, implemented, and improved upon after future iteration.

Fluorescent light exposure incites acute and prolonged immune responses in zebrafish (Danio rerio) skin.

Artificial light produces an emission spectrum that is considerably different than the solar spectrum. Artificial light has been shown to affect various behavior and physiological processes in vertebrates. However, there exists a paucity of data regarding the molecular genetic effects of artificial light exposure. Previous studies showed that one of the commonly used fluorescent light source (FL; 4100K or "cool white") can affect signaling pathways related to maintenance of circadian rhythm, cell cycle progression, chromosome segregation, and DNA repair/recombination in the skin of male Xiphophorus maculatus. These observations raise questions concerning the kinetics of the FL induced gene expression response, and which biological functions become modulated at various times after light exposure. To address these questions, we exposed zebrafish to 4100K FL and utilized RNA-Seq to assess gene expression changes in skin at various times (1 to 12h) after FL exposure. We found 4100K FL incites a robust early (1-2h) transcriptional response, followed by a more protracted late response (i.e., 4-12h). The early transcriptional response involves genes associated with cell migration/infiltration and cell proliferation as part of an overall increase in immune function and inflammation. The protracted late transcriptional response occurs within gene sets predicted to maintain and perpetuate the inflammatory response, as well as suppression of lipid, xenobiotic, and melatonin metabolism.

An open source microcontroller based flume for evaluating swimming performance of larval, juvenile, and adult zebrafish.

Zebrafish are a preferred vertebrate model for delineating genotype-phenotype relationships. One of the most studied features of zebrafish is their exceptional swimming ability. By 7 days postfertilization (dpf), zebrafish spend over two-thirds of their time engaged in spontaneous swimming activity and several months later they are capable of attaining some of the fastest swimming velocities relative to body length ever recorded in the laboratory. However, laboratory-assembled flumes capable of achieving the slow flow velocities characteristics of larvae as well as the relatively fast maximal velocities of adults have not been described in sufficient detail to allow easy replication. Here we describe an easily assembled, open-source zebrafish-scaled flume for assessing swimming performance. The flume uses two independent spherical-impeller pumps modulated by a microcontroller to achieve flow velocities ranging from 1 to 70 cm s-1. The microcontroller also monitors water temperature and flow velocity and sends these data to a personal computer for real-time display and storage. Incremental protocols for assessing maximal swimming speed (Umax) were developed, stored in custom software, and then uploaded to the microcontroller in order to assess performance of larval (14, 21, 28 dpf), juvenile (35, 42 dpf), and adult (8, 22 month) zebrafish. The flume had sufficient range and sensitivity to detect developmental changes in Umax of larvae and juveniles, an 18-24% faster Umax of adult males vs. females, and a 14-20% age-related reduction in Umax for the oldest zebrafish. Detailed information is provided to assemble and operate this low-cost, versatile, and reliable tool for assessing zebrafish swimming performance.

Effects of the prebiotic mannan-oligosaccharide on the stress response of feed deprived zebrafish (Danio rerio).

Feed deprivation has deleterious effects on fish behavior and stress physiology which may susceptible them to disease outbreak. Functional ingredients in diets may substantially impact the physiology and stress responses of host organisms. Here, we hypothesized that the administration of a dietary prebiotic might attenuate the negative influences of feed deprivation on the behavioral profile of anxiety and physiological responses to stress in zebrafish (Danio rerio). Fish were fed with either basal or mannan-oligosaccharide supplemented (0.4% MOS/kg diet) diets, once per day (normal-control: CN, and normal-prebiotic: PN) or once every other day (starved-control: CS, and starved-prebiotic: PS) for 8weeks. Afterwards, fish were subjected to a novel tank test to measure anxiety. Fish from the CS treatment exhibited more pronounced bottom-dwelling behavior than the other treatments. The number of transitions from the bottom to the top third of the novel tank was significantly higher in PN fish than the CS specimens. No significant differences were found between the CN and PS treatments in all of the anxiety behaviors. CS fish showed higher baseline cortisol levels than the other treatments, which was in line with higher expression of CRH gene in fish subjected to this treatment. Cortisol levels and CRH gene expression of the subjects were also measured after induction of two routine aquaculture stressors. CN and PS fish exhibited similar patterns of cortisol responses at most of the sampling times after stress, and PN specimens showed a significantly lower concentration of cortisol than the other treatments in most cases. Expression of the CRH gene was higher in feed deprived fish immediately after stress induction. Overall, the results show that feed deprivation in some cases influenced anxiety-like behaviors and elevated stress response in zebrafish juveniles; however, the addition of MOS to the diet helped deprived fish exhibit behaviors more typical of normally fed animals.

Comparison of Antemortem and Environmental Samples for Zebrafish Health Monitoring and Quarantine.

Molecular diagnostic assays offer both exquisite sensitivity and the ability to test a wide variety of sample types. Various types of environmental sample, such as detritus and concentrated water, might provide a useful adjunct to sentinels in routine zebrafish health monitoring. Similarly, antemortem sampling would be advantageous for expediting zebrafish quarantine, without euthanasia of valuable fish. We evaluated the detection of Mycobacterium chelonae, M. fortuitum, M. peregrinum, Pseudocapillaria tomentosa, and Pseudoloma neurophilia in zebrafish, detritus, pooled feces, and filter membranes after filtration of 1000-, 500-, and 150-mL water samples by real-time PCR analysis. Sensitivity varied according to sample type and pathogen, and environmental sampling was significantly more sensitive than zebrafish sampling for detecting Mycobacterium spp. but not for Pseudocapillaria neurophilia or Pseudoloma tomentosa. The results of these experiments provide strong evidence of the utility of multiple sample types for detecting pathogens according to each pathogen's life cycle and ecological niche within zebrafish systems. In a separate experiment, zebrafish subclinically infected with M. chelonae, M. marinum, Pleistophora hyphessobryconis, Pseudocapillaria tomentosa, or Pseudoloma neurophilia were pair-spawned and individually tested with subsets of embryos from each clutch that received no rinse, a fluidizing rinse, or were surface-disinfected with sodium hypochlorite. Frequently, one or both parents were subclinically infected with pathogen(s) that were not detected in any embryo subset. Therefore, negative results from embryo samples may not reflect the health status of the parent zebrafish.

The Vital Relationship Between Nutrition and Health in Zebrafish.

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.