Exposure Effects of Environmentally Relevant Concentrations of the Tricyclic Antidepressant Amitriptyline in Early Life Stage Zebrafish.

Antidepressants are one of the most globally prescribed classes of pharmaceuticals, and drug target conservation across phyla means that nontarget organisms may be at risk from the effects of exposure. Here, we address the knowledge gap for the effects of chronic exposure (28 days) to the tricyclic antidepressant amitriptyline (AMI) on fish, including for concentrations with environmental relevance, using zebrafish (Danio rerio) as our experimental model. AMI was found to bioconcentrate in zebrafish, was readily transformed to its major active metabolite nortriptyline, and induced a pharmacological effect (downregulation of the gene encoding the serotonin transporter; slc6a4a) at environmentally relevant concentrations (0.03 µg/L and above). Exposures to AMI at higher concentrations accelerated the hatch rate and reduced locomotor activity, the latter of which was abolished after a 14 day period of depuration. The lack of any response on the features of physiology and behavior we measured at concentrations found in the environment would indicate that AMI poses a relatively low level of risk to fish populations. The pseudopersistence and likely presence of multiple drugs acting via the same mechanism of action, however, together with a global trend for increased prescription rates, mean that this risk may be underestimated using current ecotoxicological assessment paradigms.

Pleiotropic contribution of rbfox1 to psychiatric and neurodevelopmental phenotypes in two zebrafish models.

RBFOX1 is a highly pleiotropic gene that contributes to several psychiatric and neurodevelopmental disorders. Both rare and common variants in RBFOX1 have been associated with several psychiatric conditions, but the mechanisms underlying the pleiotropic effects of RBFOX1 are not yet understood. Here we found that, in zebrafish, rbfox1 is expressed in spinal cord, mid- and hindbrain during developmental stages. In adults, expression is restricted to specific areas of the brain, including telencephalic and diencephalic regions with an important role in receiving and processing sensory information and in directing behaviour. To investigate the contribution of rbfox1 to behaviour, we used rbfox1sa15940, a zebrafish mutant line with TL background. We found that rbfox1sa15940 mutants present hyperactivity, thigmotaxis, decreased freezing behaviour and altered social behaviour. We repeated these behavioural tests in a second rbfox1 mutant line with a different genetic background (TU), rbfox1del19, and found that rbfox1 deficiency affects behaviour similarly in this line, although there were some differences. rbfox1del19 mutants present similar thigmotaxis, but stronger alterations in social behaviour and lower levels of hyperactivity than rbfox1sa15940 fish. Taken together, these results suggest that mutations in rbfox1 lead to multiple behavioural changes in zebrafish that might be modulated by environmental, epigenetic and genetic background effects, and that resemble phenotypic alterations present in Rbfox1-deficient mice and in patients with different psychiatric conditions. Our study, thus, highlights the evolutionary conservation of rbfox1 function in behaviour and paves the way to further investigate the mechanisms underlying rbfox1 pleiotropy on the onset of neurodevelopmental and psychiatric disorders.

Continuous co-treatment of mine drainage with municipal wastewater.

Acid mine drainage (AMD) and municipal wastewater (MWW) are commonly co-occurring waste streams in mining regions. Co-treating AMD at existing wastewater facilities represents an innovative solution for simultaneous AMD reclamation and improved MWW treatment. However, unknowns related to biological processes and continuous treatment performance block full-scale use. The overarching goal of this work was to address questions related to efficacy and performance of continuous processing of AMD in a biological MWW treatment system. Synthetic AMD was co-treated with synthetic MWW in a continuously-operating bench-scale sequencing batch reactor (SBR). SBRs treated MWW with two strengths of AMD (91 and 720 mg/L as CaCO3 Acidity) to capture the variations of coal AMD chemistry and strength observed in the field. Each co-treatment phases lasted 40+ days, during which clarified effluent and settled sludge quality was routinely monitored to determine impacts of co-treatment relative to conventional MWW treatment performance. Co-treatment produced effluent that met key standards for secondary treatment including biochemical oxygen demand (BOD) < 5 mg/L, total suspended solids (TSS) < 20 mg/L, and pH ∼7.0. Addition of AMD also improved treatment performance, increasing Phosphate (PO4) removal by >60% and pathogen removal by an order of magnitude. Furthermore, AMD co-treatment did not exhibit any major impacts on the overall diversity of the wastewater microbial community. Co-treatment sludge had slightly higher settleability and a lower bound water content, but notable changes in sludge morphology was observed. This study demonstrates co-treatment allows for continuous mitigation of AMD without adversely impacting MWW treatment performance in conventional biological MWW processes.

Phosphorus uptake and release patterns in overwintering constructed floating wetlands.

Understanding nutrient cycling patterns in plants deployed within constructed floating wetlands (CFWs) is critical for improving CFWs' design and management practices. This study evaluated phosphorus (P) uptake and release patterns during fall/winter plant senescence and spring regrowth. Two mesocosm-scale CFW experiments were conducted characterizing plant growth, plant tissue P levels, and water quality (nutrients and phytoplankton). Experiment 1 quantified P uptake during spring regrowth after overwintering, and experiment 2 quantified P release during fall senescence. Plant treatments (CFWs with Pontederia cordata or Juncus effusus) were compared to an open-water control. In spring, J. effusus removed 0.056 g P m-2 d-1 (19.4% of the load), P. cordata removed 0.034 g P m-2 d-1 (10%), and the open-water control removed 0.03 g P m-2 d-1 (10%). In fall, J. effusus fixed 0.008 g P m-2 d-1 (2.1% of the load), P. cordata released 0.014 g P m-2 d-1 (-2.1%), and controls fixed 0.023 g P m-2 d-1 (6.3%). P was consistently released during the fall experiment and occasionally released in the spring experiment, likely from senescing plant tissues (fall) and from roots sloughing after new root growth (spring). Results demonstrate the potential for multi-season deployment of CFWs using J. effusus for reducing P loads year-round.

A novel portable flip-phone based visual behaviour assay for zebrafish.

The optokinetic reflex (OKR) serves as a vital index for visual system development in early life, commonly observed within the first six months post-birth in humans. Zebrafish larvae offer a robust and convenient model for OKR studies due to their rapid development and manageable size. Existing OKR assays often involve cumbersome setups and offer limited portability. In this study, we present an innovative OKR assay that leverages the flexible screen of the Samsung Galaxy Z Flip to optimize setup and portability. We conducted paired slow-phase velocity measurements in 5-day post-fertilization (dpf) zebrafish larvae (n = 15), using both the novel flip-phone-based assay and a traditional liquid-crystal display (LCD) arena. Utilizing Bland-Altman plots, we assessed the agreement between the two methods. Both assays were efficacious in eliciting OKR, with eye movement analysis indicating high tracking precision in the flip-phone-based assay. No statistically significant difference was observed in slow-phase velocities between the two assays (p = 0.40). Our findings underscore the feasibility and non-inferiority of the flip-phone-based approach, offering streamlined assembly, enhanced portability, and the potential for cost-effective alternatives. This study contributes to the evolution of OKR assay methodologies, aligning them with emerging research paradigms.

adgrl3.1-deficient zebrafish show noradrenaline-mediated externalizing behaviors, and altered expression of externalizing disorder-candidate genes, suggesting functional targets for treatment.

Externalizing disorders (ED) are a cause of concern for public health, and their high heritability makes genetic risk factors a priority for research. Adhesion G-Protein-Coupled Receptor L3 (ADGRL3) is strongly linked to several EDs, and loss-of-function models have shown the impacts of this gene on several core ED-related behaviors. For example, adgrl3.1-/- zebrafish show high levels of hyperactivity. However, our understanding of the mechanisms by which this gene influences behavior is incomplete. Here we characterized, for the first time, externalizing behavioral phenotypes of adgrl3.1-/- zebrafish and found them to be highly impulsive, show risk-taking in a novel environment, have attentional deficits, and show high levels of hyperactivity. All of these phenotypes were rescued by atomoxetine, demonstrating noradrenergic mediation of the externalizing effects of adgrl3.1. Transcriptomic analyses of the brains of adgrl3.1-/- vs. wild-type fish revealed several differentially expressed genes and enriched gene clusters that were independent of noradrenergic manipulation. This suggests new putative functional pathways underlying ED-related behaviors, and potential targets for the treatment of ED.

angptl4 gene expression as a marker of adaptive homeostatic response to social isolation across the lifespan in zebrafish.

Social isolation has detrimental health effects, but the underlying mechanisms are unclear. Here, we investigated the impact of 2 weeks of isolation on behavior and gene expression in the central nervous system at different life stages of zebrafish. Results showed that socially deprived young adult zebrafish experienced increased anxiety, accompanied by changes in gene expression. Most gene expression patterns returned to normal within 24 hours of reintroduction to a social environment, except angptl4, which was upregulated after reintroduction, suggesting an adaptive mechanism. Similarly, aging zebrafish displayed heightened anxiety and increased central nervous system expression of angptl4 during isolation, but effects were reversed upon reintroduction to a social group. The findings imply that angptl4 plays a homeostatic role in response to social isolation, which varies across the lifespan. The study emphasizes the importance of social interactions for psychological well-being and highlights the negative consequences of isolation, especially in older individuals. Further research may unravel how social isolation affects angptl4 expression and its developmental and aging effects.

Eye movement defects in KO zebrafish reveals SRPK3 as a causative gene for an X-linked intellectual disability.

Intellectual disability (ID) is a common neurodevelopmental disorder characterized by significantly impaired intellectual and adaptive functioning. X-linked ID (XLID) disorders, caused by defects in genes on the X chromosome, affect 1.7 out of 1,000 males. Employing exome sequencing, we identified three missense mutations (c.475C>G; p.H159D, c.1373C>A; p.T458N, and c.1585G>A; p.E529K) in the SRPK3 gene in seven XLID patients from three independent families. Clinical features common to the patients are intellectual disability, agenesis of the corpus callosum, abnormal smooth pursuit eye movement, and ataxia. SRPK proteins are known to be involved in mRNA processing and, recently, synaptic vesicle and neurotransmitter release. In order to validate SRPK3 as a novel XLID gene, we established a knockout (KO) model of the SRPK3 orthologue in zebrafish. In day 5 of larval stage, KO zebrafish showed significant defects in spontaneous eye movement and swim bladder inflation. In adult KO zebrafish, we found agenesis of cerebellar structures and impairments in social interaction. These results suggest an important role of SRPK3 in eye movements, which might reflect learning problems, intellectual disability, and other psychiatric disorders.

A simple multilevel sampler for synchronous collection of stratified waters.

Stratified water collection plays a crucial role in water quality monitoring, as most water bodies are not perfectly mixed systems. In order to precisely collect stratified waters, we developed an inexpensive, simple, and high-resolution sampler to simultaneously collect and measure physical and chemical parameters along vertical water profiles. The water sampler predominantly consists of two parts: (1) an apparatus for measuring sampling depth below the water and (2) water sampling units secured below the water. Proof of concept water sampling was performed in Caohai wetland (Southwest China) at 40 cm intervals, as sampling depth and interval are adjustable. Stratified waters in four sampling sites were characterized by markedly different levels of major and trace elements as well as physicochemical parameters. Results indicate this simple multilevel sampler to be a cheap, precise, and portable option for simultaneously collecting water samples at different depths in a wide array of water body types.

Novel non-stimulants rescue hyperactive phenotype in an adgrl3.1 mutant zebrafish model of ADHD.

ADHD is a highly prevalent neurodevelopmental disorder. The first-line therapeutic for ADHD, methylphenidate, can cause serious side effects including weight loss, insomnia, and hypertension. Therefore, the development of non-stimulant-based therapeutics has been prioritized. However, many of these also cause other effects, most notably somnolence. Here, we have used a uniquely powerful genetic model and unbiased drug screen to identify novel ADHD non-stimulant therapeutics. We first found that adgrl3.1 null (adgrl3.1-/-) zebrafish larvae showed a robust hyperactive phenotype. Although the hyperactivity was rescued by three ADHD non-stimulant therapeutics, all interfered significantly with sleep. Second, we used wild-type zebrafish larvae to characterize a simple behavioral phenotype generated by atomoxetine and screened the 1200 compound Prestwick Chemical Library® for a matching behavioral profile resulting in 67 hits. These hits were re-assayed in the adgrl3.1-/-. Using the previously identified non-stimulants as a positive control, we identified four compounds that matched the effect of atomoxetine: aceclofenac, amlodipine, doxazosin, and moxonidine. We additionally demonstrated cognitive effects of moxonidine in mice using a T-maze spontaneous alternation task. Moxonidine, has high affinity for imidazoline 1 receptors. We, therefore, assayed a pure imidazoline 1 agonist, LNP599, which generated an effect closely matching other non-stimulant ADHD therapeutics suggesting a role for this receptor system in ADHD. In summary, we introduce a genetic model of ADHD in zebrafish and identify five putative therapeutics. The findings offer a novel tool for understanding the neural circuits of ADHD, suggest a novel mechanism for its etiology, and identify novel therapeutics.

Zebrafish Optokinetic Reflex: Minimal Reporting Guidelines and Recommendations.

Optokinetic reflex (OKR) assays in zebrafish models are a valuable tool for studying a diverse range of ophthalmological and neurological conditions. Despite its increasing popularity in recent years, there are no clear reporting guidelines for the assay. Following reporting guidelines in research enhances reproducibility, reduces bias, and mitigates underreporting and poor methodologies in published works. To better understand optimal reporting standards for an OKR assay in zebrafish, we performed a systematic literature review exploring the animal, environmental, and technical factors that should be considered. Using search criteria from three online databases, a total of 109 research papers were selected for review. Multiple crucial factors were identified, including larval characteristics, sample size, fixing method, OKR set-up, distance of stimulus, detailed stimulus parameters, eye recording, and eye movement analysis. The outcome of the literature analysis highlighted the insufficient information provided in past research papers and the lack of a systematic way to present the parameters related to each of the experimental factors. To circumvent any future errors and champion robust transparent research, we have created the zebrafish optokinetic (ZOK) reflex minimal reporting guideline.

Modelling ADHD-Like Phenotypes in Zebrafish.

The use of multiple species to model complex human psychiatric disorders, such as ADHD, can give important insights into conserved evolutionary patterns underlying multidomain behaviors (e.g., locomotion, attention, and impulsivity). Here we discuss the advantages and challenges in modelling ADHD-like phenotypes in zebrafish (Danio rerio), a vertebrate species that has been widely used in neuroscience and behavior research. Moreover, multiple behavioral tasks can be used to model the core symptoms of ADHD and its comorbidities. We present a critical review of current ADHD studies in zebrafish, and how this species might be used to accelerate the discovery of new drug treatments for this disorder.

Transcriptomic underpinnings of high and low mirror aggression zebrafish behaviours.

BACKGROUND: Aggression is an adaptive behaviour that animals use to protect offspring, defend themselves and obtain resources. Zebrafish, like many other animals, are not able to recognize themselves in the mirror and typically respond to their own reflection with aggression. However, mirror aggression is not an all-or-nothing phenomenon, with some individuals displaying high levels of aggression against their mirror image, while others show none at all. In the current work, we have investigated the genetic basis of mirror aggression by using a classic forward genetics approach - selective breeding for high and low mirror aggression zebrafish (HAZ and LAZ). RESULTS: We characterized AB wild-type zebrafish for their response to the mirror image. Both aggressive and non-aggressive fish were inbred over several generations. We found that HAZ were on average more aggressive than the corresponding LAZ across generations and that the most aggressive adult HAZ were less anxious than the least aggressive adult LAZ after prolonged selective breeding. RNAseq analysis of these fish revealed that hundreds of protein-encoding genes with important diverse biological functions such as arsenic metabolism (as3mt), cell migration (arl4ab), immune system activity (ptgr1), actin cytoskeletal remodelling (wdr1), corticogenesis (dgcr2), protein dephosphorylation (ublcp1), sialic acid metabolism (st6galnac3) and ketone body metabolism (aacs) were differentially expressed between HAZ and LAZ, suggesting a strong genetic contribution to this phenotype. DAVID pathway analysis showed that a number of diverse pathways are enriched in HAZ over LAZ including pathways related to immune function, oxidation-reduction processes and cell signalling. In addition, weighted gene co-expression network analysis (WGCNA) identified 12 modules of highly correlated genes that were significantly associated with aggression duration and/or experimental group. CONCLUSIONS: The current study shows that selective breeding based of the mirror aggression phenotype induces strong, heritable changes in behaviour and gene expression within the brain of zebrafish suggesting a strong genetic basis for this behaviour. Our transcriptomic analysis of fish selectively bred for high and low levels of mirror aggression revealed specific transcriptomic signatures induced by selective breeding and mirror aggression and thus provides a large and novel resource of candidate genes for future study.

Deficiency of the ywhaz gene, involved in neurodevelopmental disorders, alters brain activity and behaviour in zebrafish.

Genetic variants in YWHAZ contribute to psychiatric disorders such as autism spectrum disorder and schizophrenia, and have been related to an impaired neurodevelopment in humans and mice. Here, we have used zebrafish to investigate the mechanisms by which YWHAZ contributes to neurodevelopmental disorders. We observed that ywhaz expression was pan-neuronal during developmental stages and restricted to Purkinje cells in the adult cerebellum, cells that are described to be reduced in number and size in autistic patients. We then performed whole-brain imaging in wild-type and ywhaz CRISPR/Cas9 knockout (KO) larvae and found altered neuronal activity and connectivity in the hindbrain. Adult ywhaz KO fish display decreased levels of monoamines in the hindbrain and freeze when exposed to novel stimuli, a phenotype that can be reversed with drugs that target monoamine neurotransmission. These findings suggest an important role for ywhaz in establishing neuronal connectivity during development and modulating both neurotransmission and behaviour in adults.

Pollutant co-attenuation via in-stream interactions between mine drainage and municipal wastewater.

Municipal wastewater (MWW) and mine drainage (MD) are common co-occurring sources of freshwater pollution in mining regions. The physicochemical interactions that occur after mixing MWW and MD in a waterway may improve downstream water quality of an impaired reach by reducing downstream concentrations of nutrients and metals (i.e., "co-attenuation"). A first-order stream (Bradley Run in central Pennsylvania), with coal MD and secondarily treated MWW entering the stream in the same location, was systematically monitored to determine in-stream water-quality dynamics. Monitored constituents included pH, nutrients (i.e., phosphorus and nitrogen), and metals (e.g., iron, aluminum, manganese). Mixing of the MWW, MD, and upstream water decreased concentrations of phosphate, aluminum, and iron by 94%, 91%, and 98%, respectively, relative to conservative mixtures at the 1400-m-downstream site. The pollutant co-attenuation resulted in water quality equivalent to that upstream of the pollutant sources and improved the phosphorus-based trophic status of the stream. Geochemical models indicate the primary mechanisms for P attenuation in the studied stream were precipitation as variscite (AlPO4:2H2O) or amorphous AlPO4 plus adsorption to hydrous ferric oxide, despite a much greater abundance of hydrous aluminum oxide. The results presented in this study suggest that in-stream mixing of MD with untreated or secondarily treated MWW may be an important, overlooked factor affecting downstream transport of common pollutants in mining regions. Decreased metals loading and increased pH resulting from natural attenuation and remediation of MD could affect the potential for retention of phosphate by stream sediment and could lead to the release of nutrients from legacy accumulations, highlighting the potential need to address high-nutrient discharges (e.g., improved MWW treatment) in concert with MD remediation.

Changes in supramolecular organization of cyanobacterial thylakoid membrane complexes in response to far-red light photoacclimation.

Cyanobacteria are ubiquitous in nature and have developed numerous strategies that allow them to live in a diverse range of environments. Certain cyanobacteria synthesize chlorophylls d and f to acclimate to niches enriched in far-red light (FRL) and incorporate paralogous photosynthetic proteins into their photosynthetic apparatus in a process called FRL-induced photoacclimation (FaRLiP). We characterized the macromolecular changes involved in FRL-driven photosynthesis and used atomic force microscopy to examine the supramolecular organization of photosystem I associated with FaRLiP in three cyanobacterial species. Mass spectrometry showed the changes in the proteome of Chroococcidiopsis thermalis PCC 7203 that accompany FaRLiP. Fluorescence lifetime imaging microscopy and electron microscopy reveal an altered cellular distribution of photosystem complexes and illustrate the cell-to-cell variability of the FaRLiP response.

Mine drainage precipitates attenuate and conceal wastewater-derived phosphate pollution in stream water.

Hydrous ferric-oxide (HFO) coatings on streambed sediments may attenuate dissolved phosphate (PO4) concentrations at acidic to neutral pH conditions, limiting phosphorus (P) transport and availability in aquatic ecosystems. Mesh-covered tiles on which "natural" HFO from abandoned mine drainage (AMD) had precipitated were exposed to treated municipal wastewater (MWW) effluent or a mixture of stream water and effluent. Between 42 and 99% of the dissolved P in effluent was removed from the water to a thin coating (~2 µm) of HFO on the mesh. Geochemical equilibrium model results predicted the removal of 76 to 99% of PO4 from the water by adsorption to the HFO, depending on the HFO quantity, initial PO4 concentration, and pH. The measurements and model results indicated the capacity for P removal decreased as the concentration of P associated with the HFO increased. Continuing accumulation of HFO from upstream AMD sources replenish the in-stream capacity for P attenuation below the MWW discharge. This indicates AMD pollution may conceal P inputs and limit the amount of dissolved P transported to downstream ecosystems. However, HFO-rich sediments also represent a potential source of "legacy" P that could confound management practices intended to decrease nutrient and metal loadings.

Using zebrafish (Danio rerio) models to understand the critical role of social interactions in mental health and wellbeing.

Social behavior represents a beneficial interaction between conspecifics that is critical for maintaining health and wellbeing. Dysfunctional or poor social interaction are associated with increased risk of physical (e.g., vascular) and psychiatric disorders (e.g., anxiety, depression, and substance abuse). Although the impact of negative and positive social interactions is well-studied, their underlying mechanisms remain poorly understood. Zebrafish have well-characterized social behavior phenotypes, high genetic homology with humans, relative experimental simplicity and the potential for high-throughput screens. Here, we discuss the use of zebrafish as a candidate model organism for studying the fundamental mechanisms underlying social interactions, as well as potential impacts of social isolation on human health and wellbeing. Overall, the growing utility of zebrafish models may improve our understanding of how the presence and absence of social interactions can differentially modulate various molecular and physiological biomarkers, as well as a wide range of other behaviors.

The potential for adverse effects in fish exposed to antidepressants in the aquatic environment.

Antidepressants are one of the most commonly prescribed pharmaceutical classes for the treatment of psychiatric conditions. They act via modulation of brain monoaminergic signaling systems (predominantly serotonergic, adrenergic, dopaminergic) that show a high degree of structural conservation across diverse animal phyla. A reasonable assumption, therefore, is that exposed fish and other aquatic wildlife may be affected by antidepressants released into the natural environment. Indeed, there are substantial data reported for exposure effects in fish, albeit most are reported for exposure concentrations exceeding those occurring in natural environments. From a critical analysis of the available evidence for effects in fish, risk quotients (RQs) were derived from laboratory-based studies for a selection of antidepressants most commonly detected in the aquatic environment. We conclude that the likelihood for effects in fish on standard measured end points used in risk assessment (i.e., excluding effects on behavior) is low for levels of exposure occurring in the natural environment. Nevertheless, some effects on behavior have been reported for environmentally relevant exposures, and antidepressants can bioaccumulate in fish tissues. Limitations in the datasets used to calculate RQs revealed important gaps in which future research should be directed to more accurately assess the risks posed by antidepressants to fish. Developing greater certainty surrounding risk of antidepressants to fish requires more attention directed toward effects on behaviors relating to individual fitness, the employment of environmentally realistic exposure levels, on chronic exposure scenarios, and on mixtures analyses, especially given the wide range of similarly acting compounds released into the environment.