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The longevity of sperm in teleost such as zebrafish and medaka is short when isolated even in saline-balanced solution at a physiological temperature. In contrast, some internal fertilizers exhibit the long-term storage of sperm, >10 months, in the female reproductive tract. This evidence implies that sperm in teleost possesses the ability to survive for a long time under suitable conditions; however, these conditions are not well understood. In this study, we show that the sperm of zebrafish can survive and maintain fertility in L-15-based storage medium supplemented with bovine serum albumin, fetal bovine serum, glucose, and lactic acid for 28 days at room temperature. The fertilized embryos developed to normal fertile adults. This storage medium was effective in medaka sperm stored for 7 days at room temperature. These results suggest that sperm from external fertilizer zebrafish and medaka has the ability to survive for at least 4 and 1 week, respectively, in the body fluid-like medium at a physiological temperature. This sperm storage method allows researchers to ship sperm by low-cost methods and to investigate key factors for motility and fertile ability in those sperm.

Inducible gene expression systems are valuable tools for studying biological processes. We previously developed an optogenetic gene expression system called TAEL that is optimized for use in zebrafish. When illuminated with blue light, TAEL transcription factors dimerize and activate gene expression downstream of the TAEL-responsive C120 promoter. By using light as the inducing agent, the TAEL/C120 system overcomes limitations of traditional inducible expression systems by enabling fine spatial and temporal regulation of gene expression. In this study, we describe ongoing efforts to improve the TAEL/C120 system. We made modifications to both the TAEL transcriptional activator and the C120 regulatory element, collectively referred to as TAEL 2.0. We demonstrate that TAEL 2.0 consistently induces higher levels of reporter gene expression and at a faster rate, but with comparable background and toxicity as the original TAEL system. With these improvements, we were able to create functional stable transgenic lines to express the TAEL 2.0 transcription factor either ubiquitously or with a tissue-specific promoter. We demonstrate that the ubiquitous line in particular can be used to induce expression at late embryonic and larval stages, addressing a major deficiency of the original TAEL system. This improved optogenetic expression system will be a broadly useful resource for the zebrafish community.

Animal models are an important tool for studying noncommunicable diseases (NCDs) as they provide a unique opportunity to investigate real-time changes that occur in the onset of, and during, the diseased state. This is of particular importance given that the global prevalence of NCDs, such as type 2 diabetes mellitus (T2DM), is rising at an alarming rate. In South Africa, which has one of the highest levels of HIV in the world, the incidence of T2DM is thought to be associated, in part, with exposure to combination antiretrovirals. We report on the establishment of both nonobese and obese zebrafish models of T2DM, as well as associated changes in mRNA expression of preproinsulin and phosphoenolpyruvate carboxykinase (pck ) 1 and 2. The diabetic state was achieved by either immersing adult zebrafish in a 2% glucose solution for 40 days or by overfeeding adult zebrafish for 10 weeks. Glucose immersion resulted in significantly elevated fasting blood glucose levels twice as high as control, whereas bodyweight did not change significantly (nonobese model). Overfeeding led to both significantly elevated fasting blood glucose and bodyweight compared with control (obese model). Both models were characterized by significantly increased preproinsulin mRNA expression indicating insulin resistance; mRNA expression of metabolic enzymes PCK 1 and 2 was also significantly upregulated, as seen in diabetic patients. These candidate gene expression changes, similar in both zebrafish models, establish a baseline that can be utilized to investigate the underlying mechanisms driving the increased T2DM incidence, using an excellent alternative to traditional rodent models.

Hydrocephalus is a cerebrospinal fluid-related disease that usually manifests as abnormal dilation of the ventricles, with a triad of clinical findings including walking difficulty, reduced attention span, and urinary frequency or incontinence. The onset of congenital hydrocephalus is closely related to mutations in genes that regulate brain development. Currently, our understanding of the mechanisms of congenital hydrocephalus remains limited, and the prognosis of existing treatments is unsatisfactory. Additionally, there are no suitable or dedicated model organisms for congenital hydrocephalus. Therefore, it is significant to determine the mechanism and develop special animal models of congenital hydrocephalus. Recently, zebrafish have emerged as a popular model organism in many fields, including developmental biology, genetics, and toxicology. Its genome shares high similarity with that of humans, and it has fast and low-cost reproduction. These advantages make it suitable for studying the pathogenesis and therapeutic approaches for various diseases, specifically congenital diseases. This study explored the possibility of using zebrafish as a model organism for congenital hydrocephalus. This review describes the characteristics of zebrafish and discusses specific congenital hydrocephalus models. The advantages and limitations of using zebrafish for hydrocephalus research are highlighted, and insights for further model development are provided.

Global warming and extreme weather events pose a significant threat to global biodiversity, with rising water temperatures exerting a profound influence on fish conservation and fishery development. In this study, we used zebrafish as a model organism to explore the impact of a heat acclimation period on their survival rates. The results demonstrated that a 2-month heat acclimation period almost completely mitigated heat stress-induced mortality in zebrafish. Subsequent analysis of the surviving zebrafish revealed a predominance of hepatic mitochondria in a fission state. Remarkably, a short-term fasting regimen, which induced hepatic mitochondrial fission, mirrored the outcomes of the protective effect of heat acclimation and augmented animal survival under heat stress. Conversely, treatment with a mitochondrial fission inhibitor within the fasting group attenuated the elevated survival rate. Furthermore, zebrafish embryos subjected to brief heat acclimation also exhibited increased heat resistance, a trait diminished by a chemical intervention inhibiting mitochondrial fission. This suggests a shared mechanism for heat resistance between embryos and adult zebrafish. These findings underscore the potential use of inducing mitochondrial fission to enhance heat resistance in zebrafish, offering promise for fish biodiversity conservation in the face of global warming.

Various methods have been used in rodents to evaluate learning and memory. Although much less frequently used, the zebrafish emerges as an alternative model organism in this context. For example, it allows assessing potential behavioral deficits because of neurodevelopmental disorders or environmental neurotoxins. A variety of learning tasks have been employed in previous studies that required extensive habituation and training sessions. Here, we introduce a simpler and faster method to evaluate learning and memory of zebrafish with minimum habituation. A new apparatus, a transparent L-shaped tube, was developed in which we trained each zebrafish to swim through a long arm and measured the time to swim through this arm. We demonstrate that in this task, zebrafish could acquire both short-term (1 h) and long-term memory (4 days). We also studied learning and memory of a gene knockout (KO) zebrafish that showed social impairments related to autism. We found KO mutant zebrafish to show a quantitative impairment in habituation, learning, and memory performance compared with wild-type control fish. In conclusion, we established a novel learning apparatus and sensitive paradigm that allowed us to evaluate learning and memory of adult zebrafish that required only a brief habituation period and minimal training.

Soybean meal (SBM) has become a common dietary replacement for fish meal (FM) in aquafeed. However, at high inclusions, SBM has been shown to have negative impacts presenting as reduced feed intake and intestinal inflammation. Medicinal plant extracts, namely essential oils, have been used to promote growth performance and immune response. The objective of this study was to investigate the potential therapeutic effects of oregano ( Origanum vulgare ) essential oil (OEO) inclusion on utilization of a high-inclusion SBM diet using zebrafish as a model. Five diets were used in this study: reference-FM-based diet, control-55.7% inclusion SBM diet, and three experimental SBM-based diets OEO1, OEO2, and OEO3 that were supplemented with 1%, 2%, or 3% of oregano oil, respectively. The FM group had overall better growth performance when compared with the other treatment groups; however, the OEO3 mean weight and feed conversion ratio were not significantly different from the FM group ( p > 0.05) and were significantly improved compared with the SBM group ( p < 0.05). Similarly, OEO2 total length was not significantly different from FM ( p > 0.05) but significantly higher than the SBM group ( p < 0.05). Expression of inflammation-related genes did not significantly differ between the OEO groups and the SBM-only group. However, the OEO2 and OEO3 groups displayed improved growth performance compared with the SBM group, suggesting that inclusion of OEO at or above 2% inclusion may help to alleviate common symptoms induced by a high-inclusion SBM diet.

Over the years, scientific research with fish models has grown at a rapid pace, and issues such as animal welfare are becoming increasingly important in various areas of animal husbandry and experimentation. Here, we evaluated whether Danio rerio behavior is affected by long-term maintenance (75 days) in an enriched environment or a chronic stress (CS) situation. In addition, we evaluated some biochemical parameters related to redox status. We concluded that long-term maintenance of zebrafish in enriched environment might induce an anxiety-like behavior pattern when these fish are faced with an acute subsequent stressor. These anxiety results, the increased school cohesion, and the absence of oxidative damage allow us to hypothesize that the fish maintained in environmental enrichment (EE) situation is more reactive, showing a strong protective reaction to the stress. From an applicable perspective, we show that both too much stress and too little stress are not ideal for zebrafish stocks. In CS situations, fish can habituate and might not respond optimally to test conditions. In opposite, the low stress promoted by environmental enrichment also renders the fish incapable of dealing with occasional stressors optimally, because now even normal conditions appear stressful to them and may elicit fear behaviors they normally would not exhibit.

Xenotransplantation of neuroblastoma cells into larval zebrafish allows the characterization of their in vivo tumorigenic abilities and high-throughput treatment screening. This established preclinical model traditionally relies on microinjection into the yolk or perivitelline space, leaving the engraftment ability of cells at the hindbrain ventricle (HBV) and pericardial space (PCS), sites valuable for evaluating metastasis, angiogenesis, and the brain microenvironment, unknown. To address this gap in knowledge, Casper zebrafish at 48 h postfertilization were microinjected with approximately 200 Kelly, Be(2)-C, SK-N-AS, or SY5Y cells into either the HBV or PCS. Fish were imaged at 1, 3, and 6 days postinjection and tumor growth was monitored at each timepoint. We hypothesized that engraftment ability and location preference would be cell line dependent. Kelly and SK-N-AS cells were able to engraft at both the HBV and PCS, with a near doubling in size of tumor volume during the 6 days observation period, with cells appearing to grow better in the HBV. Be(2)-C tumors remained static while SY5Y tumors decreased in size, with almost complete loss of volume at both sites. Therefore, the capability of neuroblastoma cell engraftment in zebrafish larvae is cell line dependent with a location preference.

Retinal markers with high quality and specificity are important for the observation of pathologic changes of retinal cells during retinal development, degeneration, and regeneration. The zpr-3 antibody is widely used to label rods in zebrafish, but the exact antigen is still unknown. In this study, we provided evidence to demonstrate that the antigen gene of zpr-3 is rho, which encodes the rod opsin, and the exact epitope of zpr-3 is the 320-354 region of Rho protein. More importantly, our immunofluorescence assays indicated that zpr-3 labels both the outer segments of rods and green cones on zebrafish retinal sections, probably due to the cross-reaction with the green-cone opsin. Our work is valuable for the scientific community to interpret the experimental data involving the zpr-3 antibody.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been applied to edit genomes in a wide variety of model systems. Although this process can be quite efficient, editing at precise locations in the genome remains difficult without a suitable single guide RNA (sgRNA). We have developed a method for screening sgRNA function in vitro, using reagents that most zebrafish laboratories are already using. The results from our in vitro assay correlate with function in vivo in every sgRNA that we have examined so far. When combined with endonucleases with alternative protospacer adjacent motif site specificities and alternative sgRNAs, this method will streamline genome editing at almost any locus.

Parkinson's disease (PD) is a neurodegenerative disorder that is clinically assessed by motor symptoms associated with the loss of midbrain dopaminergic neurons affecting the quality of life for over 8.5 million people worldwide. The neurotoxin 6-hydroxydopamine (6-OHDA) has been used to chemically induce a PD-like state in zebrafish larvae by several laboratories; however, highly variable concentration, methodology, and reagents have resulted in conflicting results suggesting a need to investigate these issues of reproducibility. We propose a protocol that addresses the differences in methodology and induces changes in 6 days postfertilization (dpf) larvae utilizing a 24-h exposure at 3 dpf with 30 μM 6-OHDA. Despite ∼50% lethality, no morphological or development differences in surviving fish are observed. Definition of our model is defined by downregulation of the expression of th1 by reverse transcriptase-quantitative polymerase chain reaction, a marker for dopaminergic neurons and a reduction in movement. Additionally, we observed a downregulation of pink1 and an upregulation of sod1 and sod2 , indicators of mitochondrial dysfunction and response to reactive oxygen species, respectively.

Invasive species control is important for ecological and agricultural management. Genetic methods can provide species specificity for population control. We developed heritable maternal effect embryo lethality (HMEL), a novel strategy allowing negative population pressure from HMEL individuals to be transmitted within a population across generations. We demonstrate the HMEL technique in zebrafish through genome-integrated CRISPR/Cas targeted mutagenic disruption of nucleoplasmin 2b (npm2b) , a female-specific essential maternal effect gene, causing heritable sex-limited disruption of reproduction. HMEL -induced high-efficiency mutation of npm2b in females suppresses population, while males transmit the HMEL allele across generations. HMEL could be easily modified to target other genes causing sex-specific sterility, or generalized to control invasive fish or other vertebrate species for environmental conservation or agricultural protection.

An effective method for tissue-specific ablation in zebrafish is the nitroreductase (NTR)/metronidazole (MTZ) system. Expressing bacterial NTR in the presence of nitroimidazole compounds causes apoptotic cell death, which can be useful for understanding many biological processes. However, this requires tissue-specific expression of the NTR enzyme, and many tissues have yet to be targeted with transgenic lines that express NTR. We generated a transgenic zebrafish line expressing NTR in differentiated skeletal muscle. Treatment of embryos with MTZ caused muscle specific cell ablation. We demonstrate this line can be used to monitor muscle regeneration in whole embryos and in transplanted transgenic cells.

This study addresses the challenge of collecting blood samples from zebrafish for biochemical analysis. Traditional methods are cumbersome due to low blood flow and rapid coagulation. Based on a previously published technique, we simplified the process by applying an anticoagulant solution directly to the incision site. The modified protocol involves immersing the fish in an ice bath, making a cross-sectional incision, and immediately applying anticoagulant solution. Centrifugation of the specimens provides a streamlined and efficient approach to zebrafish fluid sample collection, compatible with classic biochemical marker analyses.

Myocardial infarction results in scar tissue and irreversible loss of ventricular function. Unlike humans, zebrafish has the capacity to remove scar tissue after injury. To assess ventricular function during repair, we synchronized microelectrocardiogram (μECG) signals with a high-frequency ultrasound pulsed-wave (PW) Doppler to interrogate cardiac hemodynamics. μECG signals allowed for identification of PW Doppler signals for passive (early [E]-wave velocity) and active ventricular filling (atrial [A]-wave velocity) during diastole. The A wave (9.0±1.2 cm·s(-1)) is greater than the E wave (1.1±0.4 cm·s(-1)), resulting in an E/A ratio

The transgenic (TG) zebrafish allows researchers to bio-image specific biological phenomena in cells and tissues in vivo . We established TG lines to monitor changes in the ovaries of live fish. The original TG line with ovarian fluorescence was occasionally established. Although the cDNA integrated into the line was constructed for the expression of enhanced green fluorescent protein (EGFP) driven by the medaka β- actin promoter, the expression of EGFP is restricted to the oocytes and gills in adult fish. Furthermore, we found that germinal vesicles (GVs) in oocytes of the established line can be observed by relatively strong fluorescence around the GV. In this study, we tried to capture the dynamic processes of germinal vesicle breakdown (GVBD) during meiotic cell division using the GV fluorescent oocytes. As a result, GV migration and GVBD could be monitored in real time. We also succeeded in observing actin filaments involved in the migration of GV to the animal pole. This strain can be used for education in the process of oocyte meiotic cell division.

Rising in popularity as a model organism in the classroom, zebrafish have numerous characteristics that make them ideal for teaching. In this study, we describe an experiment that helps students better understand the concept of tissue regeneration and the genes that control it. This experiment utilizes a dominant negative transgene for fgfr1 and allows students to observe the consequences of its activation. The first part of the laboratory is hands-on, and includes details of the amputation of caudal fins, heat shocking, general fish care, and visual observations. Over the course of a week, students observed the differences between the activated and unactivated transgene in the zebrafish. The second part was literature based, in which students tried to determine which gene is responsible for inhibiting regeneration. This encouraged students to sharpen their skills of deductive reasoning and critical thinking as they conduct research based on the information they receive about dominant negative receptors and transgenes. Having both a hands-on and critical thinking component in the laboratory helped synthesize the learning goals and allowed students to actively participate.

Fipronil is a broad-spectrum insecticide that has off-target effects in developing vertebrate embryos. In this study, we investigate treatment of zebrafish embryos with fipronil over the course of 5 days and examine the effects on body length, the cardiovascular system, and craniofacial morphology. We found the insecticide caused shorter body length and a decrease in eye size. By examining specific heart chamber morphology, as well as jaw angle and length, we quantified defects including enlargement of the heart and increases in jaw length and width. Further studies are needed to assess the mechanisms of fipronil's effect on vertebrate development for both environmental and human health concerns.

Research-based education at the undergraduate level is ideal for fostering the training of future scientists. In an undergraduate Developmental Biology course, this learning strategy requires the availability of model species and enough research reagents, not only for technique training but also for the development of student original projects. This might be challenging in most countries, where resources are limited. Hence, there is a need to develop low-cost solutions for use in the classroom. In this study, we describe the optimization and use of two low-cost protocols in zebrafish embryos for hands-on practical sessions and project-based learning in a Developmental Biology undergraduate course in Ecuador. These protocols were designed for the practical and experimental learning of vertebrate meroblastic cleavage, gastrulation, and neural crest differentiation. The proposed protocols have been previously described in the literature and use silver nitrate and alcian blue, two relatively inexpensive reagents, to label cell membranes and cartilage. The silver nitrate protocol allows the study of cell contact formation during cleavage and the identification of cellular changes during gastrulation, including yolk internalization and epiboly. The alcian blue staining allows the analysis of cranial mesenchymal differentiation into cartilage. These protocols are ideal for practical sessions due to their ease of application, quick results, adaptability to the class schedule, and robustness in the hands of beginning researchers. Finally, these protocols are adaptable for research-based class projects.

Currently, in Brazil, all researchers involved in animal experimentation must undergo training in laboratory animal science to stay updated on biology, methodology, ethics, and legal considerations related to the use of animals. The training program presented in this study not only aims to fulfill a legal obligation but also intends to train students and professionals to effectively care for their biomodels. It seeks to help them understand the importance of this care, both for the welfare of the animals and for the results of their projects. In total, 58 participants were present at the event (pre-event and full-time course). These participants consisted students and professionals from 11 institutions and 5 different countries. These numbers demonstrate the successful attainment of the desired capillarity in the scientific community and the posterior dissemination of knowledge. Through this course, it was possible to train the participants and raise their awareness about the importance of applying scientific knowledge in their daily practices to maintain the animals, ensuring the welfare of the models and refining the research. Finally, the program presented in this study, as well as the strategies adopted, can serve as a model for other institutions aiming to achieve similar results.

This study outlines a 2-week laboratory module for an authentic cell biology undergraduate research experience that uses zebrafish ( Danio rerio ), a popular model organism for research. Previous research has indicated that course-based undergraduate research experiences such as this one increase student confidence, active learning, and retention. During this research experience, students investigate variations in pigmentation in the caudal fins of wild type (WT) and transgenic fish [ Tg(mitfa:GNAQ Q209L )]. The transgenic fish express a hyperactive Gα protein, GNAQ Q209L , under the melanocyte-specific mitfa promoter, offering insights into uveal melanoma, a common eye cancer. Students specifically analyze the black pigmented cells, melanophores, within the caudal fin. We determined that the transgenic zebrafish have increased pigmentation in their caudal fins, but smaller melanophores. These results suggest there are more melanophores in the Tg(mitfa:GNAQ Q209L ) fish compared to the WT. Future undergraduate research could investigate these cellular differences. This research experience imparts microscopy and image analysis skills and instills the ability to grapple with large datasets, statistical tests, and data interpretation in alignment with biology education principles. Post-laboratory surveys reveal students attain confidence in the above skills and in handling animals, along with a deeper appreciation for model organism research and its relevance to cancer cell biology.

Tjp1a and other tight junction and adherens proteins play important roles in cell-cell adhesion, scaffolding, and forming seals between cells in epithelial and endothelial tissues. In this study, we labeled Tjp1a of zebrafish with the monomeric red fluorescent protein (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled polymerase chain reaction (PCR) generated templates. Labeling Tjp1a with RFP allowed us to follow membrane and junctional dynamics of epithelial and endothelial cells throughout zebrafish embryo development. For targeted integration, we used short 35 bp homology arms on each side of the Cas9 genomic target site at the C-terminal of the coding sequence in tjp1a . Through PCR using 5' biotinylated primers containing the homology arms, we generated a double-stranded template for homology directed repair containing a flexible linker followed by RFP. Cas9 protein was complexed with the tjp1a gRNA before mixing with the repair template and microinjected into one-cell zebrafish embryos. We confirmed and recovered a precise integration allele at the desired site at the tjp1a C-terminus. Examination of fluorescence reveals RFP cell-cell junctional labeling using confocal imaging. We are currently using this stable tjp1a-mRFP is86 line to examine the behavior and interactions between cells during vascular formation in zebrafish.

Lectins are carbohydrate-binding proteins with specific affinity to glycoconjugates expressed in various tissues. Lectins are of substantial utility as research, histochemical, and diagnostic tools in mammalian systems. Reactivity of 12 commonly used plant-based lectins was studied in zebrafish liver. Four lectins, tomato lectin (TL), wheat germ agglutinin, concanavalin A, and Jacalin showed strong reactivity to hepatic parenchymal structures. Importantly, TL reacted to glycoconjugates within segments of the larval and adult intrahepatic biliary network, from canaliculi to bile ducts. We provide evidence that lectins can serve as important histochemical tools to investigate the structural and functional characteristics of the zebrafish liver.

Zebrafish have been used as an education tool for students of all ages and can be used in many learning environments to teach different fields of science. In this study, we focus on the biology of zebrafish. We describe an educational program within a weeklong science camp for students between 12 and 14 years old. The methodology described is based on running annual science camps over an 11-year period. In these camps, students learnt about the developmental stages of zebrafish, as well as general zebrafish biology, husbandry, ecology, behavior, and reproduction. This article describes how to provide students and educators with an educational program to explore, discover, and contribute to the ever-evolving landscape of biological understanding through active and visual learning. We describe the methodology, the evaluation, revisions to our program over time, and future directions for expansion.

The goal of the University of Wisconsin-Milwaukee WInSTEP SEPA program is to provide valuable and relevant research experiences to students and instructors in diverse secondary educational settings. Introducing an online experience allows the expansion of a proven instructional research program to a national scale and removes many common barriers. These can include lack of access to zebrafish embryos, laboratory equipment, and modern classroom facilities, which often deny disadvantaged and underrepresented students from urban and rural school districts valuable inquiry-based learning opportunities. An online repository of zebrafish embryo imagery was developed in the Carvan laboratory to assess the effects of environmental chemicals. The WInSTEP SEPA program expanded its use as an accessible online tool, complementing the existing classroom experience of our zebrafish module. This virtual laboratory environment contains images of zebrafish embryos grown in the presence of environmental toxicants (ethanol, caffeine, and nicotine), allowing students to collect data on 19 anatomical endpoints and generate significant amounts of data related to developmental toxicology and environmental health. This virtual laboratory offers students and instructors the choice of data sets that differ in the independent variables of chemical concentration and duration of postfertilization exposure. This enables students considerable flexibility in establishing their own experimental design to match the curriculum needs of each instructor.

The Ala Wai Canal is an artificial waterway in the tourist district of Waikiki in Honolulu, HI. Originally built to collect runoff from industrial, residential, and green spaces dedicated to recreation, the Ala Wai Canal has since experienced potent levels of toxicity due to this runoff entering the watershed and making it hazardous for both marine life and humans at current concentration, including Danio rerio (zebrafish). A community of learners at educations levels from high school to postbaccalaureate from Oahu, HI was connected through the Consortium for Increasing Research and Collaborative Learning Experiences (CIRCLE) distance research program. This team conducted research with an Investigator and team from Mayo Clinic in Rochester, MN, with the Ala Wai Canal as its primary subject. Through CIRCLE, research trainees sent two 32 oz bottles of Ala Wai- acquired water to a partnered laboratory at the Mayo Clinic in which zebrafish embryos were observed at differing concentrations of the sampled water against a variety of developmental and behavioral assays. Research trainees also created atlases of developmental outcomes in zebrafish following exposure to environmental toxins and tables of potential pesticide contaminants to enable the identification of the substances linked to structural defects and enhanced stress during Ala Wai water exposure experiments.

The superfamily of Cys-loop ionotropic neurotransmitter receptors includes those that detect GABA, glutamate, glycine, and acetylcholine. There is ample evidence that many Cys-loop receptor subunit genes include alternatively spliced exons. In this study, we report a novel example of alternative splicing (AS): we show that the 68-bp exon 3 in the zebrafish gabrr2b gene-which codes for the ρ2b GABA A R subunit-is an alternative cassette exon. Skipping of gabrr2b exon 3 results in a downstream frame shift and a premature termination codon (PTC). We provide evidence in larval zebrafish that transcripts containing the PTC are subject to degradation through nonsense-mediated decay. We also compile reports of AS of homologous exons in other Cys-loop receptor genes in multiple species. Our data add to a large body of research demonstrating that exon 3 in Cys-loop receptor genes is a conserved site for AS, the effects of which can vary from novel splice-isoform generation to downregulation of gene expression through transcript degradation.

The zebrafish is a powerful model organism for studying development and regeneration. However, there is a lack of a standardized reference diet for developmental and regeneration experiments. Most studies evaluate the rate of growth, survival, and fecundity. In this study, we compare three diets and their effects on growth and regeneration after a spinal cord injury (SCI). Fish were fed daily for 1 week with daily measurements of overall length and width of spinal injury. Fish fed a live rotifer diet grew 32%, whereas a commercially available diet only led to a 4% increase in body length. Similarly, differences in rate of regeneration were observed with over 80% of rotifer-fed larvae forming a glial bridge after injury compared to <10% of zebrafish fed with the commercial diet. Our data highlight the need for establishing a standardized diet for regeneration studies to improve research reproducibility.

We have developed a one-credit semester-long research experience for undergraduate students that involves the use of CRISPR/Cas9 to edit genes in zebrafish. The course is available to students at all stages of their undergraduate training and can be taken up to four times. Students select a gene of interest to edit as the basis of their semester-long project. To select a gene, exploration of developmental processes and human disease is encouraged. As part of the course, students use basic bioinformatic tools, design guide RNAs, inject zebrafish embryos, and analyze both the molecular consequences of gene editing and phenotypic outcomes. Over the 10 years we have offered the course, enrollment has grown from less than 10 students to more than 60 students per semester. Each year, we choose a different gene editing strategy to explore based on recent publications of gene editing methodologies. These have included making CRISPants, targeted integrations, and large gene deletions. In this study, we present how we structure the course and our assessment of the course over the past 3 years.

Coordinated signaling pathway activity directs early patterning to set up the vertebrate body plan. Perturbations in the timing or location of signal molecule expression impacts embryo morphology and organ formation. In this study, we present a laboratory course to use zebrafish for studying the role of Wnt signaling in specifying the early embryonic axes. Students are exposed to basic techniques in molecular and developmental biology, including embryo manipulation, fluorescence microscopy, image processing, and data analysis. Furthermore, this course incorporates student-designed experiments to stimulate independent inquiry and improve scientific learning, providing an experience resembling graduate-level laboratory research. Students appreciated following vertebrate development in real-time, and principles of embryogenesis were reinforced by observing the morphological changes that arise due to signaling alterations. Scientific and research skills were enhanced through practice in experimental design, interpretation, and presentation.

Project BioEYES celebrated 20 years in K12 schools during the 2022-2023 school year. Using live zebrafish ( Danio rerio ) during week-long science experiments, sparks the interest of students and teachers from school districts, locally and globally. Over the past two decades, BioEYES has been replicated in different ways based on the interest and capacity of our partners. This article discusses several of the successful models, the common challenges, and how each BioEYES site has adopted guiding principles to help foster their success. The core principles of (a) reinforcing content that students are expected to learn in schools, while focusing on the students BECOMING scientists through hands-on experimentation and (b) establishing trust and buy-in from collaborating teachers and partners are what has led to BioEYES being sustained and replicated over the past two decades.

Obesity is a public health concern resulting in a variety of health complications, including heart disease and insulin resistance. Estrogens have been associated with a reduced risk of obesity, but this relationship remains incompletely understood. We assessed the role of 17β-estradiol (E2) in mitigating complications associated with obesity by supplementing E2 in the diets of overfed zebrafish. We report that dietary E2 supplementation protects against weight gain and modulates de novo cholesterol synthesis in a sex-specific manner. Our studies lead us to propose a model in which E2 regulates hmgcr expression independently of unsaturated fat consumption. These data can be used to develop sex-specific treatments for obesity-related health conditions.

Early research experiences positively affect students' interest in STEM careers, and develop practical science and critical thinking skills. However, outreach opportunities are not equally accessible for all students. In states like West Virginia, where many students live in rural Appalachian communities, opportunities for engaging in STEM experiences are limited. In addition, rural teachers may not be equipped to provide authentic research experiences for students due to lack of resources or support. For many students in West Virginia, the Health Sciences and Technology Academy (HSTA) is a major opportunity for STEM engagement. Since its inception in 1998, HSTA has spread to 26 of 55 counties in West Virginia. The program recruits first-generation, low-socioeconomic status, rurally living, and African American high school students who are under-represented in STEM fields. Our research laboratory partnered with HSTA to implement an innovative, hands-on research camp using zebrafish for students participating in their annual junior-level biomedical sciences summer camp. Our camp was held in-person and adapted to an online format during the Covid-19 pandemic. We used pre-post surveys in both camps to assess impacts on science confidence and to collect information about general perceptions of zebrafish, research, and STEM fields. We found that students participating in the in-person and online camps experienced similar overall gains in science confidence. We also identified strong interest in zebrafish, research, and STEM degrees among online students. Online students did not prefer virtual learning experiences; however, they still enjoyed our camp. We also surveyed high school teachers volunteering for HSTA to identify factors that would encourage use of zebrafish in classrooms. The most prominent needs include classroom supplies, experience, and funding. Our successful science-education partnership demonstrates that zebrafish research experiences foster positive outcomes for under-represented students, and can inform future outreach efforts and collaborations with teachers.

Microscopes are essential for research and education in science. Unlike computers and online learning tools, however, microscopes are not currently a fixed element in K-12 classrooms, due to steep cost, needless complexity, and often requiring a prohibitive level of staff training to effectively deploy. In a collaboration with Area 10 Labs, Integrated Science Education Outreach (InSciEd Out) developed a state-of-the-art alternative microscope, the InSciEdRS View, to reduce the financial barrier, prohibitive per-student cost, unnecessary complexity, and extensive staff training. Utilizing a 1080p camera and a lunchbox-style case, this Wi-Fi- and USB-connectable microscope comes with all necessary components for visualization of microscopic specimens (10 × -50 × magnification). While built to handle the rigors of classroom use, its imaging capability and battery-operation can make it flexible for a laboratory or fieldwork as well. We further highlight here K-12 curricula that we have developed using larval zebrafish to enable teachers, science outreach leaders, and parents to support active hands-on science observations. The InSciEdRS View microscope and the InSciEd Out curricula are readily scalable, translatable, and accessible for traditional and neurodiverse students and integrating these in various settings can be an efficient way to achieve better outcomes in science education.

Zebrafish ( Danio rerio ) is a widely used vertebrate animal for modeling genetic diseases by targeted editing strategies followed by gross phenotypic and biomarker characterization. While larval transparency permits microscopic detection of anatomical defects, histological adult screening for organ-level defects remains invasive, tedious, inefficient, and subject to technical artifact. Here, we describe a noninvasive magnetic resonance imaging (MRI) approach to systematically screen adult zebrafish for anatomical growth defects. An anatomical atlas of wild-type (WT) zebrafish at 5-31 months post-fertilization was created by ex vivo MRI with a 9.4 T magnet. Volumetric growth over time was measured of animals and major organs, including the brain, spinal cord, heart, eyes, optic nerve, ear, liver, kidneys, and swim bladder. Subsequently, surf1 -/- , fbxl4 -/- , and opa1 +/- mitochondrial disease mutant adult zebrafish were quantitatively studied to compare organ volumes with age-matched WT zebrafish. Results demonstrated that MRI enabled noninvasive, high-resolution, rapid screening of mutant adult zebrafish for overall and organ-specific growth abnormalities. Detailed volumetric analyses of three mitochondrial disease mutants delineated specific organ differences, including significantly increased brain growth in surf1 -/- and opa1 +/- , and marginally significant decreased heart and spinal cord volumes in surf1 -/- mutants. This is interesting as we know neurological involvement can be seen in SURF1 - /- patients with ataxia, dystonia, and lesions in basal ganglia, as well as in OPA1 +/- patients with spasticity, ataxia, and hyperreflexia indicative of neuropathology. Similarly, cardiomyopathy is a known sequelae of cardiac pathology in patients with SURF1 - /- -related disease. Future studies will define MRI signaling patterns of organ dysfunction to further delineate specific pathology.

Eigenmannia is a highly diverse genus within the Sternopygidae family, comprising 30 species. Due to its complex taxonomy, molecular analyses have been crucial for species delimitation within this group. Therefore, the present study presents a genetic analysis using sequences of the mitochondrial gene cytochrome c oxidase subunit 1 ( COI ) in specimens previously identified through alpha taxonomy as E. correntes (with unpublished data), E. virescens , and E. trilineata , originating from various locations within the Upper Paraná and Paraguay River basins in Brazil. The molecular data confirm the taxonomic complexity of the genus, as individuals morphologically identified as E. virescens and E. trilineata shared the same haplotype (H52). Furthermore, the results of the species delimitation tests suggest that specimens morphologically identified as E. virescens belong to the species E. trilineata . In addition, samples morphologically identified as E. correntes may correspond to more than one Operational Taxonomic Units ( OTUs ). Furthermore, the intraspecific Kimura-2-parameter ( K2P ) distances within the different studied populations are significant. This study has contributed valuable information about genetic diversity in Eigenmannia , emphasizing the importance of using integrative analyses to resolve taxonomic conflicts within the group. It also supports biogeographical studies and assists in biodiversity conservation efforts.

The marine medaka is emerging as a potential behavioral model organism for ocean studies, namely on marine ecotoxicology. However, not much is known on the behavior of the species and behavioral assays lack standardization. This study assesses the marine medaka as a potential model for chemical communication. We investigated how short exposure to visual and chemical cues mediated the stress response to social isolation with the light/dark preference test (LDPT) and the open field test (OFT). After a 5-day isolation period, and 1 h before testing, isolated fish were randomly assigned to one of four groups: (1) placed in visual contact with conspecifics; (2) exposed to a flow of holding water from a group of conspecifics; (3) exposed to both visual and chemical cues from conspecifics; or (4) not exposed to any stimuli (controls). During the LDPT, the distance traveled and transitions between zones were more pronounced in animals exposed to the conspecific's chemical stimuli. The time spent in each area did not differ between the groups, but a clear preference for the bright area in all animals indicates robust phototaxis. During the OFT, animals exposed only to chemical cues initially traveled more than those exposed to visual or both stimuli, and displayed lower thigmotaxis. Taken together, results show that chemical cues play a significant role in exploratory behavior in this species and confirm the LDPT and OFT as suitable tests for investigating chemical communication in this species.