The environmental prevalence of antibiotic residues poses a potential threat to gut health and may thereby disrupt brain function through the microbiota-gut-brain axis. However, little is currently known about the impacts of antibiotics on gut health and neurotransmitters along the microbiota-gut-brain axis in fish species. Taking enrofloxacin (ENR) as a representative, the impacts of antibiotic exposure on the gut structural integrity, intestinal microenvironment, and neurotransmitters along the microbiota-gut-brain axis were evaluated in zebrafish in this study. Data obtained demonstrated that exposure of zebrafish to 28-day environmentally realistic levels of ENR (6 and 60 μg/L) generally resulted in marked elevation of two intestinal integrity biomarkers (diamine oxidase (DAO) and malondialdehyde (MDA), upregulation of genes that encode inter-epithelial tight junction proteins, and histological alterations in gut as well as increase of lipopolysaccharide (LPS) in plasma, indicating an evident impairment of the structural integrity of gut. Moreover, in addition to significantly altered neurotransmitters, markedly higher levels of LPS while less amount of two short-chain fatty acids (SCFAs), namely acetic acid and valeric acid, were detected in the gut of ENR-exposed zebrafish, suggesting a disruption of gut microenvironment upon ENR exposure. Along with corresponding changes detected in gut, significant disruption of neurotransmitters in brain indicated by marked alterations in the contents of neurotransmitters, the activity of acetylcholin esterase (AChE), and the expression of neurotransmitter-related genes were also observed. These findings suggest exposure to environmental antibiotic residues may impair gut health and disrupt neurotransmitters along the microbiota-gut-brain axis in zebrafish. Considering the prevalence of antibiotic residues in environments and the high homology of zebrafish to other vertebrates including human, the risk of antibiotic exposure to the health of wild animals as well as human deserves more attention. © 2024 Elsevier Ltd

The ubiquitous presence of plastic particles in water bodies poses a potential threat to aquatic species. Although numerous adverse effects of microplastics (MPs) and nanoplastics (NPs) have been documented, their effects on fish feeding, one of the most important behaviors of animals, are far from being fully understood. In this study, the effects of MPs and NPs (at environmentally realistic levels) on fish food consumption and feeding behavior were assessed using goldfish (Carassius auratus) and polystyrene (PS) particles as representatives. In addition, to reveal the potential mechanisms, the effects of MPs and NPs on peripheral and central regulation of appetite were evaluated by examining appetite-regulation related intestinal, serous, and hypothalamic parameters. The results obtained indicated that the 28-day MP- and NP-exposure significantly impaired goldfish feeding by disrupting peripheral and central appetite regulation. Based on differences observed in their effects on the abovementioned behavioral, histological, and physiological parameters, MPs and NPs may interfere with appetite regulation in a size-dependent manner. Blocking the gastrointestinal tract and causing histopathological and functional damage to inner organs may be the main routes through which MPs and NPs disrupt appetite regulation. Our findings suggested that plastic particles exposure may have far-reaching effects on fish species through impaired feeding, which warrants further attention. © 2024 Elsevier B.V.

In recent years, aquatic ecosystems have been exposed to various stressors such as hypoxia and acidification, which has become an issue of significant concern. Many studies in fish have investigated the regulatory mechanisms of the response to hypoxia and acidification stress at the molecular level. However, molecular studies on hypoxia acidification dual stress conditions in rhubarb fish are less. For this study, the juvenile large yellow croaker was used as the study object. Four experimental groups were established, including the control group (normal group, N107; DO= 7.0 mg/L, pH= 8.1), hypoxia group (H107; DO= 3.5 mg/L, pH= 8.1), acidification group (A107; DO = 7.0 mg/L, pH = 7.3), and hypoxia-acidification group (dual stress group, D107; DO = 3.5 mg/L, pH = 7.3). Study of its response mechanism under hypoxia acidification conditions by transcriptome and proteome analysis. The present study revealed that the number of quantifiable proteins was 6303. Five pairwise comparisons between experimental groups demonstrated that a total of 265 DEGs/DEPs showed associations between the transcriptome and proteome at the level of quantitative and differential expression. Comparative proteomic and transcriptomic analyses were performed to identify differentially expressed genes/proteins in juvenile Larimichthys crocea under hypoxia and acidification stress. The GO term enrichment analysis showed that hypoxia had a greater effect on the organism. The KEGG pathway enrichment analysis showed that pathways associated with the extracellular matrix ECM-receptor interaction and protein digestion and absorption pathways were notably affected by hypoxia and acidification stress. Among these, the protein digestion and absorption pathway was significantly affected in all five pairwise comparisons between experimental groups. The ECM-receptor interaction pathway was significantly enriched under dual stress, indicating that dual stress had a greater detrimental effect on fish growth than single stressors. The study provides valuable insights into the potential combined effects of decreased pH and DO in Sciaenidae and elucidates the mechanism underlying the response of L. crocea to simultaneous hypoxia-acidification stress during early development.

Theubiquitous environmental presence of tris(2-chloroethyl) phosphate(TCEP) poses a potential threat to animals; however, little is knownabout its hepatotoxicity. In this study, the effects of TCEP exposure(0.5 and 5.0 mu g/L for 28 days) on liver health and the potentialunderlying toxification mechanisms were investigated in zebrafish.Our results demonstrated that TCEP exposure led to hepatic tissuelesions and resulted in significant alterations in liver-injury-specificmarkers. Moreover, TCEP-exposed fish had significantly lower levelsof thyrotropin-releasing hormone and thyroid-stimulating hormone inthe brain, evidently less triiodothyronine whereas more thyroxinein plasma, and markedly altered expressions of genes from the hypothalamic-pituitary-thyroid(HPT) axis in the brain or liver. In addition, a significantly higherproportion of Bacteroidetes in the gut microbiota, an elevated bacterialsource endotoxin lipopolysaccharide (LPS) in the plasma, upregulatedexpression of LPS-binding protein and Toll-like receptor 4 in theliver, and higher levels of proinflammatory cytokines in the liverwere detected in TCEP-exposed zebrafish. Furthermore, TCEP-exposedfish also suffered severe oxidative damage, possibly due to disruptionof the antioxidant system. These findings suggest that TCEP may exerthepatotoxic effects on zebrafish by disrupting the HPT and gut-liveraxes and thereafter inducing hepatic inflammation and oxidative stress.Tris(2-chloroethyl) phosphate, one ofthe most frequentlydetected organophosphate esters in environments, exerted hepatotoxicimpacts on zebrafish by disrupting HPT and gut-liver axes atenvironmentally realistic levels.

Doublesex (DSX) proteins are members of the Doublesex/mab-3-related (DMRT) protein family and play crucial roles in sex determination and differentiation among the animal kingdom. In the present study, we identified two Doublesex (Dsx)-like mRNA isoforms in the brine shrimp Artemia franciscana (Kellogg 1906), which are generated by the combin-ation of alternative promoters, alternative splicing and alternative polyadenylation. The two transcripts exhibited sex-biased enrichment, which we termed AfrDsx(M) and AfrDsx(F). They share a common region which encodes an identical N-terminal DNA-binding (DM) domain. RT-qPCR analyses showed that AfrDsxM is dominantly expressed in male Artemia while AfrDsx(F) is specifically expressed in females. Expression levels of both iso-forms increased along with the developmental stages of their respective sexes. RNA interference with dsRNA showed that the knockdown of AfrDsx(M) in male larvae led to the appearance of female traits including an ovary-like structure in the original male reproductive system and an elevated expression of vitellogenin. However, silencing of AfrDsx(F) induced no clear phenotypic change in female Artemia. These results indicated that the male AfrDSX(M) may act as inhibiting regulator upon the default female developmental mode in Artemia. Furthermore, electrophoretic mobility shift assay analyses revealed that the unique DM domain of AfrDSXs can specifically bind to promoter segments of poten-tial downstream target genes like AfrVtg. These data show that AfrDSXs play crucial roles in regulating sexual development in Artemia, and further provide insight into the evolution of sex determination/differentiation in sexual organisms.

To survive under harsh environments, embryonic development of Artemia was arrested at the gastrula stage and released as the diapause embryo. Cell cycle and metabolism were highly suppressed in this state of quiescence. However, cellular mechanisms underlying diapause remain largely unclear. In this study, we found that the expression level of a CT10 regulator of kinase-encoding gene (Ar-Crk) in diapause embryos was significantly lower than non-diapause embryos at the early embryogenetic stage of Artemia. Knockdown of Ar-Crk by RNA interference induced formation of diapause embryos, while the control group produced nauplii. Western blot analysis and metabolic assays revealed that the diapause embryos produced by Ar-Crk-knocked-down Artemia had similar characteristics of diapause markers, arrested cell cycle, and suppressed metabolism with those diapause embryos produced by natural oviparous Artemia. Transcriptomic analysis of Artemia embryos revealed knockdown of Ar-Crk induced downregulation of the aurora kinase A (AURKA) signaling pathway, as well as energetic and biomolecular metabolisms. Taken together, we proposed that Ar-Crk is a crucial factor in deter-mining the process of diapause in Artemia. Our results provide insight into the functions of Crk in fundamental regulations such as cellular quiescence.

The ubiquitous presence of antibiotic residues in aqueous environments poses a great potential threat to aquatic organ-isms. Nevertheless, the behavioral effects of environmentally realistic levels of antibiotics remain poorly understood in fish species. In this study, the behavioral impacts of enrofloxacin, one of typical fluoroquinolone antibiotics that is fre-quently detected in aquatic environments, were evaluated by the classic light-dark test (LDT) and novel tank task (NTT) in zebrafish. Furthermore, the effects of enrofloxacin exposure on the microbiota-gut-brain axis were also assessed to reveal potential affecting mechanisms underlying the behavioral abnormality observed. Our results dem-onstrated that zebrafish exposed to 60 mu g/L enrofloxacin for 28 days took significantly longer to enter the stressful area of the testing tank and spent significantly less time there in both the LDT and NTT, indicating abnormal anxiety-like behaviors induced by the exposure. In addition, exposure to enrofloxacin at 6 and 60 mu g/L resulted in a significant elevation in Bacteroidetes and a marked decline in the Firmicutes/Bacteroidetes ratio of the gut microbiota. Moreover, the intestinal contents of interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), glucagon-like peptide 1 (GLP-1), and 5-hydroxytryptamine (5-HT) in zebrafish were significantly upregulated, whereas those of plasma adre-nocorticotropic hormone (ACTH) and cortisol (COR) were markedly downregulated upon enrofloxacin exposure. Incubation of zebrafish with a high dose of enrofloxacin (60 mu g/L) also resulted in evident increases in the contents of corticotropin-releasing hormone (CRH), brain-derived neurotrophic factor (BDNF), and neuropeptide Y (NPY) in the brain. Fortunately, no significant alteration in the expression of glial fibrillary acidic protein (GFAP) was detected in the brain after enrofloxacin exposure. Our findings suggest that the disruption of the microbiota-gut-brain axis may account for enrofloxacin-induced anxiety-like behaviors in zebrafish. Since the disruption of microbiota-gut-brain axis may give rise to various clinical symptoms, the health risk of antibiotic exposure deserves more attention.

Although accumulating data demonstrated that gamma-aminobutyric acid (GABA), an inhibitory neurotrans-mitter, plays an important regulatory role in immunity of vertebrates, its immunomodulatory function and mechanisms of action remain poorly understood in invertebrates such as bivalve mollusks. In this study, the effect of GABA on phagocytic activity of hemocytes was evaluated in a commercial bivalve species, Tegillarca granosa. Furthermore, the potential regulatory mechanism underpinning was investigated by assessing potential downstream targets. Data obtained demonstrated that in vitro GABA incubation significantly constrained the phagocytic activity of hemocytes. In addition, the GABA-induced suppression of phagocytosis was markedly relieved by blocking of GABAA and GABAB receptors using corresponding antagonists. Hemocytes incubated with lipopolysaccharides (LPS) and GABA had significant higher K+-Cl- cotransporter 2 (KCC2) content compared to the control. In addition, GABA treatment led to an elevation in intracellular Cl-, which was shown to be leveled down to normal by blocking the ionotropic GABAA receptor. Treatment with GABAA receptor antagonist also rescued the suppression of GABAA receptor-associated protein (GABARAP), KCC, TNF receptor associated factor 6 (TRAF6), inhibitor of nuclear factor kappa-B kinase subunit alpha (IKK alpha), and nuclear factor kappa B subunit 1 (NF kappa B) caused by GABA incubation. Furthermore, incubation of hemocytes with GABA resulted in a decrease in cAMP content, an increase in intracellular Ca2+, and downregulation of cAMP-dependent protein kinase (PKA), calmodulin kinase II (CAMK2), calmodulin (CaM), calcineurin (CaN), TRAF6, IKK alpha, and NF kappa B. All these above-mentioned changes were found to be evidently relieved by blocking the metabotropic G-protein-coupled GABAB receptor. Our results suggest GABA may play an inhibitory role on phagocytosis through binding to both GABAA and GABAB receptors, and subsequently regulating corresponding downstream pathways in bivalve invertebrates.

Nanoplastics (NPs) may pass through the blood-brain barrier, giving rise to serious concerns about their potential toxicity to the brain. In this study, the effects of NPs exposure on learning and memory, the primary cognitive functions of the brain, are assessed in zebrafish with classic T-maze exploration tasks. Additionally, to reveal potential affecting mechanisms, the impacts of NPs exposure on brain aging, oxidative damage, energy provision, and the cell cycle are evaluated. The results demonstrate that NP-exposed zebrafish takes significantly longer for their first entry and spends markedly less time in the reward zone in the T-maze task, indicating the occurrence of learning and memory deficits. Moreover, higher levels of aging markers (beta-galactosidase and lipofuscin) are detected in the brains of NP-exposed fish. Along with the accumulation of reactive free radicals, NP-exposed zebrafish suffer significant levels of brain oxidative damage. Furthermore, lower levels of Adenosine triphosphate (ATP) and cyclin-dependent kinase 2 and higher levels of p53 are observed in the brains of NP-exposed zebrafish, suggesting that NPs exposure also results in a shortage of energy supply and an arrestment of the cell cycle. These findings suggest that NPs exposure may pose a severe threat to brain health, which deserves closer attention. © 2023 Wiley-VCH GmbH.

Large yellow croaker (Larimichthys crocea) is an important aquaculture species in China. This study analysed whole-genome methylation differences in liver tissues of young fish under different hypoxic and acidification conditions. Differentially methylated regions (DMRs) and differentially methylated genes (DMGs) were identified. Gene ontology (GO) and Kyoto encyclopaedia of genes and genomes (KEGG) enrichment analyses of DMGs were conducted to explore the mechanism of coping with hypoxic acidification. The main methylation type was CG, accounting for>70% of total methylation, significantly higher than CHG and CHH methylation types. GO enrichment analysis of DMGs revealed strong enrichment of nervous system development, cell periphery, plasma membrane, cell junction organisation, cell junction, signalling receptor activity, molecular sensor activity, cell-linked tissue junction organisation, cell-cell adhesion and nervous system development. KEGG enrichment analysis of DMR-related genes identified cell adhesion molecules, cortisol synthesis and secretion and aldosterone synthesis and secretion as the three key pathways regulating the physiological responses to hypoxia and acidification. Long-term hypoxic and acidification stress affected the immune system, nervous system and stress responses of large yellow croaker. Whole-genome sequencing analysis of exposed tissues was used to investigate changes that occur in L. crocea in response to hypoxic and acidic conditions at the DNA methylation level. The findings contribute to our comprehensive understanding of functional methylation in large yellow croaker and will support future research on the response mechanisms of this species under different environmental pressures. © 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.