Cyhexatin causes developmental toxic effects by disrupting endocrine system and inducing behavioral inhibition, apoptosis and DNA hypomethylation in zebrafish (Danio rerio) larvae.

Cyhexatin (CYT), an organotin acaricide, is extensively utilized in developing countries to mitigate plant diseases caused by mites and minimize agricultural crop losses. However, the comprehensive mechanisms underlying the developmental stage of non-target organisms remain largely unexplored. In this study, zebrafish embryos were firstly exposed to CYT (0.06, 0.12, and 0.20 ng/mL, referred to as CYTL, CYTM, and CYTH, respectively) from 2 hpf (hours post fertilization) to 30 dpf (days post fertilization). No developmental toxicity was observed in the CYTL and CYTM groups, except for induced deformed phenotypes in the CYTM group at 120 hpf. However, exposure to CYTH resulted in significant reductions in spontaneous movement (24 hpf), heart rate (48 hpf), hatching rate (48 and 72 hpf), body weight (30 dpf), whole body length (30 dpf), and locomotion (30 dpf). Additionally, CYTH exposure induced morphological malformations, including spinal curvature, pericardial edema, and tail curvature in zebrafish larvae. Moreover, CYTH treatment induced apoptosis, increased reactive oxygen species (ROS) production, and resulted in significant reductions in free T3, cholesterol, estradiol, and testosterone levels in zebrafish larvae, while free T4 levels were increased. RNA-Seq analysis indicated that CYTH exposure led to significant alterations in the genome-wide gene expression profiles of zebrafish, particularly in the thyroid hormone and steroid biosynthesis signaling pathways, indicating endocrine disruption. Furthermore, CYTH exposure induced global DNA hypomethylation, reduced S-adenosylmethionine (SAM) levels and the SAM/S-adenosylhomocysteine (SAH) ratio, elevated SAH levels, and suppressed the mRNA expression of DNA methyltransferases (DNMTs) while also downregulating DNMT1 at both the gene and protein levels in zebrafish larvae. Overall, this study partially elucidated the developmental toxicity and endocrine disruption caused by CYT in zebrafish, providing evidence of the environmental hazards associated with this acaricide.

Combined effects of dietary carbohydrate levels and ammonia stress on growth, antioxidant capacity and glucose metabolism in juvenile oriental river prawn (Macrobrachium nipponense).

Ammonia is a common environmental stress factor that constrains aquaculture industry development. This study evaluated the effect of carbohydrate levels and ammonia stress in oriental river prawn (Macrobrachium nipponense). The experiment had six treatments containing two water ammonia levels (0 and 5 mg/L) and three dietary carbohydrate levels (low carbohydrate diet (LCD, 10%), medium carbohydrate diet [MCD, 20%], and high carbohydrate diet [HCD, 30%]), and lasted six weeks. The results showed that the prawns fed on MCD had higher weight gain than those fed on LCD and HCD during ammonia stress. Moreover, the prawns fed on MCD had significantly lower acid phosphatase and alkaline phosphatase activities during ammonia stress. Feeding the prawns on the MCD increased B cells in the hepatopancreas during ammonia stress. Interestingly, the prawns fed on MCD had significantly lower superoxide dismutase activity compared to LCD and HCD during ammonia stress. Moreover, the prawns fed on MCD had significantly lower pyruvate kinase activity and pyruvate and lactic acid contents, while those fed on LCD had significantly higher succinic dehydrogenase, 6-phosphogluconic dehydrogenase, and phosphoenol pyruvate carboxykinase activities during ammonia stress. The prawns fed on the MCD increased significantly glutaminase activity and decreased the ammonia content in the serum during ammonia exposure. In addition, feeding the prawns on MCD decreased significantly the expression of apoptosis and inflammation-related genes. Taken together, the MCD supplied energy required to counteract ammonia stress, which increased growth, improved antioxidant capacity, facilitated ammonia excretion, and alleviated inflammation and apoptosis of the oriental river prawn.

Dietary thiamine modulates carbohydrate metabolism, antioxidant status, and alleviates hypoxia stress in oriental river prawn Macrobrachium nipponense (de Haan).

Hypoxia is one of the challenges in prawns aquaculture. However, the role of thiamine, which is a coenzyme in carbohydrate metabolism with antioxidant properties, in reducing hypoxia in prawns aquaculture is currently unknown. We investigated the effects of thiamine on antioxidant status, carbohydrate metabolism and acute hypoxia in oriental river prawn, Macrobrachium nipponense. One thousand eight hundred prawns (0.123 ± 0.003 g) were fed five diets (60 prawns each tank, six replicates per diet) supplemented with graded thiamine levels (5.69, 70.70, 133.67, 268.33 and 532.00 mg/kg dry mater) for eight weeks and then exposed to hypoxia stress for 12 h followed by reoxyegnation for 12 h. The results showed that, under normoxia, prawns fed the 133.67 or 268.33 mg/kg thiamine diet had significantly lower glucose 6-phosphatedehydrogenase, succinate dehydrogenase and phosphoenolpyruvate carboxykinase activities than those fed the other diets. Moreover, total antioxidant capacity (T-AOC) increased significantly when prawns were fed the 133.67 mg/kg thiamine diet. Superoxide dismutase (SOD) activity and malonaldehyde (MDA) content also increased significantly when prawns were fed the 268.33 or 532.00 mg/kg thiamine diet under hypoxia. And the significantly increased SOD activity and MDA level also observed in prawns fed 532.00 mg/kg thiamine under reoxygenation. Under normoxia, prawns fed the 70.70 or 133.67 mg/kg thiamine diet decreased the mRNA expressions of AMP-activated protein kinase-alpha (AMPK-α), pyruvate dehydrogenase-E1-α subunit (PDH-E1-α) and hypoxia-inducible factor-1s (HIF-1α, HIF-1ß), but increased the mRNA expressions of phosphofructokinase (PFK) significantly. After 12 h of hypoxia, the energy metabolism related genes (AMPK-ß, AMPK-γ, PFK, PDH-E1-α), hypoxia-inducible factor related genes (HIF-1α, HIF-1ß) and thiamine transporter gene (SLC19A2) were up-regulated significantly in prawns fed the 133.67 or 268.33 mg/kg thiamine diets. After 12 h of reoxygenation, prawns fed the 133.67 or 268.33 mg/kg diet significantly decreased the SOD activity, MDA level and SLC19A2 mRNA expression compared with other diets. The optimum thiamine was 161.20 mg/kg for minimum MDA content and 143.17 mg/kg for maximum T-AOC activity based on cubic regression analysis. In summary, supplementing 143.17 to 161.20 mg/kg thiamine in the diets for M. nipponense improves the antioxidant capacity under normoxia and reduces the oxidative damage under hypoxia stress.

Cloning, tissue distribution and mRNA expression of type I collagen alpha 1 gene from Chu's croaker (Nibea coibor).

The demand for collagen has been increasing over years due to its wide application in food, cosmetics and biomedicine industries. The synthesis of collagen protein in fish depends on instructions provided by collagen, type I, alpha 1 (COL1A1) gene. However, cloning, tissue distribution and mRNA expression of COL1A1 gene in a gel-producing Chu's croaker (Nibea coibor) is currently unknown. This study cloned the cDNA of COL1A1 gene (GenBank accession number: MK641512) from six N. coibor fish. The distribution and mRNA expression pattern of COL1A1 was analyzed in eight tissues of N. coibor. The COL1A1 cDNA had a full length of 6130 bp and contained a 4344 bp open reading frame (ORF) encoding a polypeptide of 1448 amino acids. The homology of N. coibor COL1A1 amino acid had 98% similarity with Larimichthys crocea, indicating conservatism with other members in same family (Sciaenidae). The deduced polypeptide contained the same signal peptides, C-propeptide and N-propeptide domains, and triple helix domains, which are the characteristics of type I collagen in vertebrates. The mRNA of COL1A1 gene was expressed significantly higher in the spine of N. coibor than in all other tissues (P < 0.05), followed by swim bladder, skin and scales. The swim bladder had higher collagen and hydroxyproline contents than other tissues, followed by spine >, scales > and > skin (P < 0.05). Our study successfully cloned the COL1A1 gene from N. coibor for the first time. The COL1A1 gene contained all the features of collagen pro-α1(I) chain proteins, and shared high homology with other marine teleost. COL1A1 gene in N. coibor is highly expressed in spine and swim bladder, consistent with collagen distribution. Our study contributes to better understanding on collagen biosynthesis in N. coibor tissues for various industrial uses.

Mitochondrial Fatty Acid ß-Oxidation Inhibition Promotes Glucose Utilization and Protein Deposition through Energy Homeostasis Remodeling in Fish.

BACKGROUND: Fish cannot use carbohydrate efficiently and instead utilize protein for energy supply, thus limiting dietary protein storage. Protein deposition is dependent on protein turnover balance, which correlates tightly with cellular energy homeostasis. Mitochondrial fatty acid ß-oxidation (FAO) plays a crucial role in energy metabolism. However, the effect of remodeled energy homeostasis caused by inhibited mitochondrial FAO on protein deposition in fish has not been intensively studied. OBJECTIVES: This study aimed to identify the regulatory role of mitochondrial FAO in energy homeostasis maintenance and protein deposition by studying lipid, glucose, and protein metabolism in fish. METHODS: Carnitine-depleted male Nile tilapia (initial weight: 4.29 ± 0.12 g; 3 mo old) were established by feeding them with mildronate diets (1000 mg/kg/d) for 6 wk. Zebrafish deficient in the carnitine palmitoyltransferase 1b gene (cpt1b) were produced by using CRISPR/Cas9 gene-editing technology, and their males (154 ± 3.52 mg; 3 mo old) were used for experiments. Normal Nile tilapia and wildtype zebrafish were used as controls. We assessed nutrient metabolism and energy homeostasis-related biochemical and molecular parameters, and performed 14C-labeled nutrient tracking and transcriptomic analyses. RESULTS: The mitochondrial FAO decreased by 33.1-88.9% (liver) and 55.6-68.8% (muscle) in carnitine-depleted Nile tilapia and cpt1b-deficient zebrafish compared with their controls (P < 0.05). Notably, glucose oxidation and muscle protein deposition increased by 20.5-24.4% and 6.40-8.54%, respectively, in the 2 fish models compared with their corresponding controls (P < 0.05). Accordingly, the adenosine 5'-monophosphate-activated protein kinase/protein kinase B-mechanistic target of rapamycin (AMPK/AKT-mTOR) signaling was significantly activated in the 2 fish models with inhibited mitochondrial FAO (P < 0.05). CONCLUSIONS: These data show that inhibited mitochondrial FAO in fish induces energy homeostasis remodeling and enhances glucose utilization and protein deposition. Therefore, fish with inhibited mitochondrial FAO could have high potential to utilize carbohydrate. Our results demonstrate a potentially new approach for increasing protein deposition through energy homeostasis regulation in cultured animals.

Environmental estrogen exposure converts lipid metabolism in male fish to a female pattern mediated by AMPK and mTOR signaling pathways.

Environmental estrogens, including bisphenol A (BPA) and 17ß-estradiol (E2), which are widely used in industries and medicine, pose a severe ecological threat to fish due to feminization induction. However, the related metabolic basis for reproductive feminization in male fish has not been well addressed. We first found that female zebrafish exhibited higher lipid accumulation and lipogenesis activity than males. Next, we exposed male and female zebrafish to E2 (200 ng/L) or BPA (100 µg/L) for six weeks, and observed an early-phase reproductive feminization in males, accompanied with reduced spermatids, significant fat deposition and lipogenic gene expressions that mimicked female patterns. Cellular signaling assays revealed that, E2 or BPA modulated lipid metabolism in males mainly through lowering 5' AMP-activated protein kinase (AMPK) and upregulating the lipogenic mechanistic target of rapamycin (mTOR) pathways. For the first time, we show that environmental estrogens could alter lipid metabolism in male fish to a female pattern (metabolic feminization) prior to gonad feminization in male fish, to allows males to accumulate efficiently lipids to harmonize with the feminized gonads. This study suggests that negative effects of environmental estrogens, as hazardous materials, on vertebrate health are more complicated than originally thought.

Concentration-dependent effects of 17ß-estradiol and bisphenol A on lipid deposition, inflammation and antioxidant response in male zebrafish (Danio rerio).

Environmental estrogenic compounds are important pollutants, which are widely distributed in natural water bodies. They produce various adverse effects on fish, but their concentration-dependent toxicities in fish metabolism and health are not fully understood. This study investigated the effects of 17ß-estradiol (E2) and bisphenol A (BPA) at low and high concentrations on lipid deposition, inflammation and antioxidant response in male zebrafish. We measured fish growth parameters, gonad development, lipid contents and the activities of inflammatory and antioxidant enzymes, as well as their mRNA expressions. All E2 and BPA concentrations used increased body weight, damaged gonad structure and induced feminization in male zebrafish. The exposure of zebrafish to E2 and BPA promoted lipid accumulation by increasing total fat, liver triglycerides and free fatty acid contents, and also upregulated lipogenic genes expression, although they decreased total cholesterol content. Notably, zebrafish exposed to low concentrations of E2 (200 ng/L) and BPA (100 µg/L) had higher lipid synthesis and deposition compared to high concentrations (2000 ng/L and 2000 µg/L, respectively). However, the high concentrations of E2 and BPA increased inflammation and antioxidant response. Furthermore, BPA caused greater damage to fish gonad development and more severe lipid peroxidation compared to E2. Overall, the results suggest that the toxic effects of E2 and BPA on zebrafish are concentration-dependent such that, the relative low concentrations used induced lipid deposition, whereas the high ones caused adverse effects on inflammation and antioxidant response.

Molecular cloning and functional characterization of elongase (elovl5) and fatty acyl desaturase (fads2) in sciaenid, Nibea diacanthus (Lacepède, 1802).

In the present paper, we investigated the molecular cloning and functional characterization of elongase of very long chain fatty acid (elovl) and fatty acyl desaturase (fads) genes in a marine teleost, Nibea diacanthus. The elongase cDNA sequence encoded a polypeptide of 294 amino acids exhibiting Elovl5 activity, which effectively elongated both C18 (18:2n-6, 18:3n-3 and 18:3n-6) and C20 (20:4n-6 and 20:5n-3) polyunsaturated fatty acids. The desaturase cDNA sequence specified a polypeptide of 445 amino acids indicating Δ6 desaturation activity, which coul converted C18:2n-6 and C18:3n-3 to C18:3n-6 and C18:4n-3, respectively. Tissue distribution analysis by quantitative real-time PCR revealed that the elovl5 was primarily expressed in intestine and liver, while the fads2 was mainly expressed in liver and brain. These results increase our knowledge of the ability of endogenous highly unsaturated fatty acids (HUFA) biosynthesis in marine carnivorous fish.

Tracking pollutants in dietary fish oil: From ocean to table.

Dietary fish oil used in aquafeed transfers marine pollutants to farmed fish. However, the entire transfer route of marine pollutants in dietary fish oil from ocean to table fish has not been tracked quantitatively. To track the entire transfer route of marine pollutants from wild fish to farmed fish through dietary fish oil and evaluate the related human health risks, we obtained crude and refined fish oils originating from the same batch of wild ocean anchovy and prepared fish oil-containing purified aquafeeds to feed omnivorous lean Nile tilapia and carnivorous fatty yellow catfish for eight weeks. The potential human health risk of consumption of these fish was evaluated. Marine persistent organic pollutants (POPs) were concentrated in fish oil, but were largely removed by the refining process, particularly dioxins and polychlorinated biphenyls (PCBs). The differences in the POP concentrations between crude and refined fish oils were retained in the fillets of the farmed fish. Fillets fat content and fish growth were positively and negatively correlated to the final POPs deposition in fillets, respectively. The retention rates of marine POPs in the final fillets through fish oil-contained aquafeeds were 1.3%-5.2%, and were correlated with the POPs concentrations in feeds and fillets, feed utilization and carcass ratios. The dietary crude fish oil-contained aquafeeds are a higher hazard ratio to consumers. Prohibiting the use of crude fish oil in aquafeed and improving growth and feed efficiency in farmed fish are promising strategies to reduce health risks originating from marine POPs.