Exposure of Danio rerio to environmental sulfamethoxazole may contribute to neurobehavioral abnormalities via gut microbiome disturbance.

The neurotoxic effects and mechanisms of low-dose and long-term sulfamethoxazole (SMZ) exposure remain unknown. This study exposed zebrafish to environmental SMZ concentrations and observed behavioral outcomes. SMZ exposure increased hyperactivity and altered the transcript levels of 17 genes associated with neurological function. It impaired intestinal function by reducing the number of intestinal goblet cells and lipid content. Metabolomic results indicated that the contents of several lipids and amino acids in the gut were altered, which might affect the expression levels of neurological function-related genes. Metagenomic results demonstrated that SMZ exposure substantially altered the composition of the gut microbiome. Zebrafish receiving a transplanted fecal microbiome from the SMZ group were also found to exhibit abnormal behavior, suggesting that the gut microbiome is an important target for SMZ exposure-induced neurobehavioral abnormalities. Multi-omics correlation analysis revealed that gut micrometabolic function was related to differential gut metabolite levels, which may affect neurological function through the gut-brain-axis. Reduced abundance of Lefsonia and Microbacterium was strongly correlated with intestinal metabolic function and may be the key bacterial genera in neurobehavioral changes. This study confirms for the first time that SMZ-induced neurotoxicity in zebrafish is closely mediated by alterations in the gut microbiome.

Early environmental exposure to oxytetracycline in Danio rerio may contribute to neurobehavioral abnormalities in adult zebrafish.

The common antibiotic oxytetracycline (OTC) is nowadays commonly found in natural aquatic environments. However, the underlying mechanisms of low-dose OTC exposure and its neurotoxic effects on aquatic animals remain unknown. In this study, we exposed zebrafish larvae to environmental concentrations of OTC in early life and performed neurobehavioral, 16S rRNA gene sequencing, and transcriptomic analyses. OTC exposure resulted in hyperactivity of larvae and a significant reduction in the number of neurons in the midbrain. The expression levels of 15 genes related to neural function changed. Additionally, the composition of 65 genera of the gut microbiota of larvae was altered, which may be one of the reasons for the abnormal neural development. We further studied the long-term outcomes among adult fish long after cessation of OTC exposure. OTC treatment caused adult fish to be depressive and impulsive, symbolizing bipolar disorder. Adult fish exposed to OTC had significantly fewer neurons and their gut bacteria composition did not recover 104 days after terminating OTC exposure. Finally, we analyzed the correlation between the gut microbiota of larvae, genes related to neural function, and metabolites of adult fish brain tissue. The results showed that the abundance of several members of the biome in larvae was related to the transcription levels of genes related to neural function, which were related to the metabolic levels in the adult brain. In conclusion, our study showed that early-life exposure to environmental concentrations of OTC can lead to persistent neurobehavioral abnormalities until adulthood through dysbiosis in the gut microbiota.

Association of long-term effects of low-level sulfamethoxazole with ovarian lipid and amino acid metabolism, sex hormone levels, and oocyte maturity in zebrafish.

Sulfamethoxazole (SMZ) is an important antibiotic used to prevent and treat infections in both clinical settings and animal husbandry. High levels of SMZ may exhibit endocrine toxicity. Environmental SMZ enters the human body via food and water; however, the toxicity of environmental doses of SMZ and its effects on reproductive health are unknown. In the present study, zebrafish were exposed to low concentrations of SMZ (1000 and 5000 ng/L) from 2 h post-fertilization to 120 d post-fertilization. Consequently, the proportion of mature oocytes in adult female zebrafish ovarian tissue increased by 98.2 %, indicating that SMZ promotes ovarian maturation. Metabolomics analysis revealed significant changes in ovarian lipid and amino acid levels after SMZ treatment. An enzyme-linked immunoassay used to detect sex hormones in the ovaries showed that SMZ exposure significantly increased the levels of estradiol, a follicle-stimulating hormone, and of luteinizing hormone. Furthermore, an association analysis showed that most of the differentially expressed metabolites in the ovary were strongly correlated with the levels of sex hormones secreted by the pituitary gland. Therefore, significantly increased transcript levels of gonadotropin-releasing hormone (GnRH) and follicle-stimulating hormone detected in brain tissue suggested that SMZ may exhibit ovarian toxicity via the hypothalamus. In vitro experiments were performed to demonstrate that SMZ targets neurons in the hypothalamus. Exposure to SMZ significantly increased the GnRH content in GnRH neurons. Finally, molecular docking simulations indicated the potential interaction of SMZ with G protein-coupled receptor 54; this molecular binding can activate, synthesize, and release GnRH in neurons. In conclusion, long-term environmental exposure to SMZ may induce ovarian toxicity by affecting the hypothalamus-pituitary-gonad axis.

Longitudinal analysis of exposure to a low concentration of oxytetracycline on the zebrafish gut microbiome.

Oxytetracycline, a widely produced and administered antibiotic, is uncontrollably released in low concentrations in various types of environments. However, the impact of exposure to such low concentrations of antibiotics on the host remains poorly understood. In this study, we exposed zebrafish to a low concentration (5,000 ng/L) of oxytetracycline for 1 month, collected samples longitudinally (Baseline, and Days 3, 6, 9, 12, 24, and 30), and elucidated the impact of exposure on microbial composition, antibiotic resistance genes, mobile genetic elements, and phospholipid metabolism pathway through comparison of the sequenced data with respective sequence databases. We identified Pseudomonas aeruginosa, a well-known pathogen, to be significantly positively associated with the duration of oxytetracycline exposure (Adjusted P = 5.829e-03). Several tetracycline resistance genes (e.g., tetE) not only showed significantly higher abundance in the exposed samples but were also positively associated with the duration of exposure (Adjusted P = 1.114e-02). Furthermore, in the exposed group, the relative abundance of genes involved in phospholipid metabolism had also decreased. Lastly, we characterized the impact of exposure on zebrafish intestinal structure and found that the goblet cell counts were decreased (~82%) after exposure. Overall, our results show that a low concentration of oxytetracycline can increase the abundance of pathogenic bacteria and lower the abundance of key metabolic pathways in the zebrafish gut microbiome that can render them prone to bacterial infections and health-associated complications.

Genome-Resolved Characterization of Structure and Potential Functions of the Zebrafish Stool Microbiome.

Zebrafish have been used as a model organism for more than 50 years and are considered an excellent model for studying host-microbiome interactions. However, this largely depends on our understanding of the zebrafish gut microbiome itself. Despite advances in sequencing and data analysis methods, the zebrafish gut microbiome remains highly understudied. This study performed the de novo metagenome assembly and recovery of the metagenome-assembled genomes (MAGs) through genome binning (and refinement) of the contigs assembled from the zebrafish stool. The results indicate that majority of the MAGs had excellent quality i.e. high completeness (≥90%) and low contamination levels (≤5%). MAGs mainly belong to the taxa that are known to be members of the core zebrafish stool microbiome, including the phylum Proteobacteria, Fusobacteriota, and Actinobacteriota. However, most of the MAGs remained unclassified at the species level and reflected previously unexplored microbial taxa and their potential novelty. These MAGs also contained genes with predicted functions associated with diverse metabolic pathways that included carbohydrate, amino acid, and lipid metabolism pathways. Lastly, we performed a comparative analysis of Paucibacter MAGs and reference genomes that highlighted the presence of novel Paucibacter species and enriched metabolic potential in the recovered MAGs.

Long-term low-dose oxytetracycline potentially leads to neurobehavioural changes.

Trace levels of oxytetracycline (OTC)-a veterinary antibiotic and feed additive-are widespread in the environment. Studies revealed that OTC potentially impairs thyroid function, which may affect neurobehaviour; however, the impact of exposure to environmental concentrations of OTC on adult neurobehaviour is unknown. In this study, the effects of OTC on zebrafish after 30-day exposure were investigated. The total swimming distance was significantly increased under vibration and light/dark stimulation, while time spent in the white area was prolonged during the black/white preference test, indicating that the zebrafish became bolder and more impulsive under low OTC exposure. Additionally, monoamine neurotransmitter (5-hydroxytryptamine, dopamine, norepinephrine) levels were decreased and gene expression of monoamine oxidase (mao) involved in neurotransmitter metabolism was upregulated at the transcription level after OTC exposure. Because triiodothyronine (T3) levels were enhanced following exposure to OTC, we speculated that T3 may mediate OTC damage to the nervous system. Our simulated molecular docking analysis showed that OTC combined with the sodium iodide cotransporter protein may result in excessive T3 synthesis. We further exposed zebrafish to T3, and they exhibited similar behaviour to the OTC exposure group. In conclusion, environmental OTC may activate monoamine oxidase and enhance the metabolism of monoaminergic neurotransmitters via T3, thereby inducing abnormal neurobehaviour.

Environmental concentrations of antibiotics alter the zebrafish gut microbiome structure and potential functions.

A paradoxical impact of high rates of production and consumption of antibiotics is their widespread release in the environment. Consequently, low concentrations of antibiotics and their byproducts have been routinely identified from various environmental settings especially from aquatic environments. However, the impact of such low concentrations of antibiotics on the exposed host especially in early life remains poorly understood. We exposed zebrafish to two different environmental concentrations of oxytetracycline and sulfamethoxazole, from larval stage to adulthood (∼120 days) and characterized their impact on the taxonomic diversity, antibiotic resistance genes, and metabolic pathways of the gut microbiome using metagenomic shotgun sequencing and analysis. Long term exposure of environmental concentrations of oxytetracycline and sulfamethoxazole significantly impacted the taxonomic composition and metabolic pathways of zebrafish gut microbiome. The antibiotic exposed samples exhibited significant enrichment of multiple flavobacterial species, including Flavobacterium sp. F52, Flavobacterium johnsoniae and Flavobacterium sp. Fl, which are well known pathogenic bacteria. The relative abundance of antibiotic resistance genes, especially several tetratcycline and sulfonamide resistance genes were significantly higher in the exposed samples and showed a linear correlation with the antibiotic concentrations. Furthermore, several metabolic pathways, including folate biosynthesis, oxidative phosphorylation, and biotin metabolism pathways, showed significant enrichment in the antibiotic exposed samples. Collectively, our results suggest that early life exposure of the environmental concentrations of antibiotics can increase the abundance of unfavorable bacteria, antibiotic resistance genes and associated pathways in the gut microbiome of zebrafish.

Low-dose effects on thyroid disruption in zebrafish by long-term exposure to oxytetracycline.

As a feed additive in agriculture, the antibiotic oxytetracycline (OTC) has become widely distributed in the natural environment, leading to the exposure of many organisms to low doses of OTC. Although OTC is clinically contraindicated in children because of its multiple side effects, the effect of exposure to low doses of environmental OTC on children is unknown, particularly during development. In this study, we investigated the effects of OTC on the thyroid endocrine system in zebrafish, through determinations of the whole-body contents of triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH) by enzyme-linked immunosorbent assay, and analysis of the mRNA expression of regulatory genes involved in the hypothalamus-pituitary-thyroid (HPT) axis using quantitative real-time polymerase chain reaction. Zebrafish embryos were exposed to OTC at environmentally relevant concentrations from 2 h to 120 days post-fertilisation. After exposure to OTC at 1,000 and 5,000 ng/L, T3 contents were significantly enhanced (37.8% and 45.1%, respectively) and TSH contents were reduced (16% and 16.3%, respectively) compared with those in the controls. The OTC-driven increase in the transcription of genes involved in thyroid synthesis (tpo and nis) may be responsible for the altered T3 levels. These data indicate that OTC may cause thyroid dysfunction and lead to reduced TSH secretion owing to enhanced negative feedback control of the HPT axis. Meanwhile, a decrease in body length, weight, and BMI and an increase in heart rate were observed with increasing OTC exposure. In conclusion, our results indicate that long-term exposure to low concentrations of OTC may alter the transcription of key genes involved in the HPT axis, as well as T3 and TSH contents, thereby disrupting the thyroid system and affecting the growth and development of zebrafish.

Efficient genome engineering of Toxoplasma gondii using the TALEN technique.

BACKGROUND: Aromatic amino acid hydroxylase 2 (AAH2) is a bradyzoite-specific upregulated protein that may alter host behaviour by altering the host dopaminergic pathway. To better understand the role of the parasite's AAH2 in host-parasite interactions, we generated an AAH2 fluorescent marker strain of T. gondii using the TALEN technique. METHODS: We generated an AAH2 fluorescent marker strain of T. gondii, which was designated PRU/AAH2-eGFP, using the TALEN technique. This strain stably expressed pyrimethamine resistance for screening and expressed enhanced green fluorescent protein (eGFP)-tagged AAH2 in the bradyzoite stage. The bradyzoite conversion of PRU/AAH2-eGFP was observed both in vitro and in vivo. The fluorescence localization of AAH2 in mouse models of chronic infection was observed by a Bruker in vivo imaging system. RESULTS: Transgenic T. gondii was successfully generated by the TALEN system. The eGFP-tagged AAH2 could be detected by in vivo imaging. CONCLUSIONS: This study verified the feasibility of using TALEN technology for T. gondii research and provided an in vivo imaging method for in vivo research of bradyzoite-stage proteins.