Effects of supplementing coated methionine in a high plant-protein diet on growth, antioxidant capacity, digestive enzymes activity and expression of TOR signaling pathway associated genes in gibel carp, Carassius auratus gibelio.

This study explored the impacts of supplementation of different levels of coated methionine (Met) in a high-plant protein diet on growth, blood biochemistry, antioxidant capacity, digestive enzymes activity and expression of genes related to TOR signaling pathway in gibel carp (Carassius auratus gibeilo). A high-plant protein diet was formulated and used as a basal diet and supplemented with five different levels of coated Met at 0.15, 0.30, 0.45, 0.60 and 0.75%, corresponding to final analyzed Met levels of 0.34, 0.49, 0.64, 0.76, 0.92 and 1.06%. Three replicate groups of fish (initial mean weight, 11.37 ± 0.02 g) (20 fish per replicate) were fed the test diets over a 10-week feeding period. The results indicated that with the increase of coated Met level, the final weight, weight gain (WG) and specific growth rate initially boosted and then suppressed, peaking at 0.76% Met level (P< 0.05). Increasing dietary Met level led to significantly increased muscle crude protein content (P< 0.05) and reduced serum alanine aminotransferase activity (P< 0.05). Using appropriate dietary Met level led to reduced malondialdehyde concentration in hepatopancreas (P< 0.05), improved superoxide dismutase activity (P< 0.05), and enhanced intestinal amylase and protease activities (P< 0.05). The expression levels of genes associated with muscle protein synthesis such as insulin-like growth factor-1, protein kinase B, target of rapamycin and eukaryotic initiation factor 4E binding protein-1 mRNA were significantly regulated, peaking at Met level of 0.76% (P< 0.05). In conclusion, supplementing optimal level of coated Met improved on fish growth, antioxidant capacity, and the expression of TOR pathway related genes in muscle. The optimal dietary Met level was determined to be 0.71% of the diet based on quadratic regression analysis of WG.

Roles of ß-catenin in innate immune process and regulating intestinal flora in Qi river crucian carp (Carassius auratus).

In mammals, ß-catenin participates in innate immune process through interaction with NF-κB signaling pathway. However, its role in teleost immune processes remains largely unknown. We aimed to clarify the function of ß-catenin in the natural defense mechanism of Qi river crucian carp (Carassius auratus). ß-catenin exhibited a ubiquitous expression pattern in adult fish, as indicated by real-time PCR analysis. Following lipopolysaccharide (LPS), Polyinosinic-polycytidylic acid (polyI: C) and Aeromonas hydrophila (A. hydrophila) challenges, ß-catenin increased in gill, intestine, liver and kidney, indicating that ß-catenin likely plays a pivotal role in the immune response against pathogen infiltration. Inhibition of the ß-catenin pathway using FH535, an inhibitor of Wnt/ß-catenin pathway, resulting in pathological damage of the gill, intestine, liver and kidney, significant decrease of innate immune factors (C3, defb3, LYZ-C, INF-γ), upregulation of inflammatory factors (NF-κB, TNF-α, IL-1, IL-8), and downregulation of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, increase of Malondialdehyde (MDA) content. Following A. hydrophila invasion, the mortality rate in the FH535 treatment group exceeded that of the control group. In addition, the diversity of intestinal microflora decreased and the community structure was uneven after FH535 treatment. In summary, our findings strongly suggest that ß-catenin plays a vital role in combating pathogen invasion and regulating intestinal flora in Qi river crucian carp.

Ginger polysaccharide alleviates the effects of acute exposure to carbonate in crucian carp (Carassius auratus) by regulating immunity, intestinal microbiota, and intestinal metabolism.

Alkaline stress poses a significant challenge to the healthy growth of fish. Ginger polysaccharide (GP) is one of the main active substances in ginger and has pharmacological effects, such as anti-oxidation and immune regulation. However, the physiological regulatory mechanism of GP addition to diet on alkalinity stress in crucian carp remains unclear. This study aimed to investigate the potential protective effects of dietary GP on antioxidant capacity, gene expression levels, intestinal microbiome, and metabolomics of crucian carp exposed to carbonate (NaHCO3). The CK group (no GP supplementation) and COG group (NaHCO3 stress and no GP supplementation) were set up. The GPCS group (NaHCO3 stress and 0.4% GP supplementation) was stressed for seven days. Based on these data, GP significantly increased the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), acid phosphatase (ACP), and alkaline phosphatase (AKP) in carp under alkalinity stress (p < 0.05) and decreased the activity of malon dialdehyde (MDA) (p < 0.05). GP restored the activity of GSH-PX, ACP, and AKP to CK levels. The expression levels of tumor necrosis factor ß (TGF-ß), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin 8 (IL-8) genes were decreased, and the expression levels of determination factor kappa-B (NF-κB) and interleukin 10 (IL-10) genes were increased (p < 0.05). Based on 16 S rRNA high-throughput sequencing, GP improved the changes in the intestinal microbial diversity and structural composition of crucian carp caused by NaHCO3 exposure. In particular, GP increased the relative abundance of Proteobacteria and Bacteroidetes and decreased the relative abundance of Actinobacteria. The metabolic response of GP to NaHCO3 exposed crucian carp guts was studied using LC/MS. Compared to the COG group, the GPCS group had 64 different metabolites and enriched 10 metabolic pathways, including lipid metabolism, nucleotide metabolism, and carbohydrate metabolism. The addition of GP to feed can promote galactose metabolism and provide an energy supply to crucian carp, thus alleviating the damage induced by alkalinity stress. In conclusion, GP can mitigate the effects of NaHCO3 alkalinity stress by regulating immune function, intestinal flora, and intestinal metabolism in crucian carp. These findings provide a novel idea for studying the mechanism of salt-alkali tolerance in crucian carp by adding GP to feed.

α-lipoic acid ameliorates hepatotoxicity induced by chronic ammonia toxicity in crucian carp (Carassius auratus gibelio) by alleviating oxidative stress, inflammation and inhibiting ERS pathway.

High environment ammonia (HEA) poses a deadly threat to aquatic animals and indirectly impacts human healthy life, while nutritional regulation can alleviate chronic ammonia toxicity. α-lipoic acid exhibits antioxidative effects in both aqueous and lipid environments, mitigating cellular and tissue damage caused by oxidative stress by aiding in the neutralization of free radicals (reactive oxygen species). Hence, investigating its potential as an effective antioxidant and its protective mechanisms against chronic ammonia stress in crucian carp is highly valuable. Experimental fish (initial weight 20.47 ± 1.68 g) were fed diets supplemented with or without 0.1% α-lipoic acid followed by a chronic ammonia exposure (10 mg/L) for 42 days. The results revealed that chronic ammonia stress affected growth (weight gain rate, specific growth rate, and feed conversion rate), leading to oxidative stress (decreased the activities of antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase; decreased total antioxidant capacity), increased lipid peroxidation (accumulation of malondialdehyde), immune suppression (decreased contents of nonspecific immune enzymes AKP and ACP, 50% hemolytic complement, and decrease of immunoglobulin M), impaired ammonia metabolism (reduced contents of Glu, GS, GSH, and Gln), imbalance of expression of induced antioxidant-related genes (downregulation of Cu/Zu SOD, CAT, Nrf2, and HO-1; upregulation of GST and Keap1), induction of pro-apoptotic molecules (transcription of BAX, Caspase3, and Caspase9), downregulation of anti-apoptotic gene Bcl-2 expression, and induction of endoplasmic reticulum stress (upregulation of IRE1, PERK, and ATF6 expression). The results suggested that the supplementation of α-lipoic acid could effectively induce humoral immunity, alleviate oxidative stress injury and endoplasmic reticulum stress, and ultimately alleviate liver injury induced by ammonia poisoning (50-60% reduction). This provides theoretical basis for revealing the toxicity of long-term ammonia stress and provides new insights into the anti-ammonia toxicity mechanism of α-lipoic acid.

Roundup® disrupts tissue architecture, attenuates Na+/K+-ATPase expression, and induces protein oxidation/nitration, cellular apoptosis, and antioxidant enzyme expressions in the gills of goldfish, Carassius auratus.

Extensive agricultural activities to feed the growing population are one major driving force behind aquatic pollution. Different types of pesticides are used in farmlands to increase crop production and wash up into water bodies. Glyphosate-based herbicide Roundup® is one of the most used pesticides in the United States; however, its effects on teleost species are still poorly understood. This study focused on the effects of environmentally relevant concentrations of Roundup exposure (low- and high-dose: 0.5 and 5 µg/L for 2-week) on Na+/K+-ATPase (NKA, a biomarker for sodium­potassium ion pump efficacy), cytochrome P450-1A (CYP1A, a monooxygenase enzyme), 2,4-dinitrophenyl protein (DNP, a biomarker for protein oxidation), 3-nitrotyrosine protein (NTP, a biomarker for protein nitration), superoxidase dismutase (SOD, an antioxidant enzyme), catalase (CAT, an antioxidant enzyme) expressions, and cellular apoptosis in the gills of goldfish. Histopathological and in situ TUNEL analyses showed widespread tissue damage, including lamellar fusion, loss of gill architecture, club shape of primary lamellae, mucous formation, and distortion in the epithelium layer, as well as apoptotic nuclei in gills. Immunohistochemical and qRT-PCR analyses provided insights into the expressions of molecular indicators in gills. Fish exposed to Roundup exhibited a significant (P < 0.05) downregulation of NKA expression in gills. Additionally, we observed upregulation of CYP1A, DNP, NTP, SOD, and CAT expressions in the gills of goldfish. Overall, our results suggest that exposure to Roundup causes disruption of gill architecture, induces protein oxidation/nitration and cellular apoptosis, and alters prooxidant-antioxidant homeostasis in tissues, which may lead to reduced fitness and survivability of teleost species.

Oral administration of wild plant-derived minerals and red ginseng ameliorates insulin resistance in fish through different pathways.

Many kinds of fish are characterized by a limited efficiency to use carbohydrates. For this reason, raw fish and mixed feed containing a lot of fish meal have been used as feed for fish farming. However, continuing to use high-protein diets not only increases the cost of fish farming, but may also fuel animal protein shortages. Furthermore, carbohydrates are added to improve the texture of the feed and act as a binding agent and are usually contained at 20% in the feed. It makes sense, therefore, to find ways to make good use of carbohydrates rather than wasting them. The physiological mechanisms of glucose intolerance in fish are not yet well understood. Therefore, we investigated the glucose utilization of fish, omnivorous goldfish Carassius auratus and carnivorous rainbow trout Oncorhynchus mykiss. Furthermore, the effects of oral administration of wild plant-derived minerals and red ginseng on the glucose utilization in these fish muscle cells were investigated. As a result, we found the following. (1) An extremely high insulin resistance in fish muscle and the symptom was more pronounced in carnivorous rainbow trout. (2) Administration of wild plant-derived minerals promotes the translocation of the insulin-responsive glucose transporter GLUT4 to the cell surface of white muscle via activation of the PI3 kinase axis, whereas administration of red ginseng not only promotes GLUT4 transfer and translocation to the cell surface of white muscle via AMPK activation as well as promoting glucose uptake into muscle cells via a pathway separate from the insulin signaling system. (3) In fish, at least goldfish and rainbow trout, both PI3K/Akt and AMPK signaling cascades exist to promote glucose uptake into muscle cells, as in mammals.

The role of visfatin/ NAMPT in the regulation of feeding in goldfish (Carassius auratus).

The protein NAMPT (nicotinamide phosphoribosyltransferase, encoded by the NAPMT gene) is present in two forms. The intracellular form of NAMPT (iNAMPT) is the rate-limiting enzyme in a major nicotinamide adenine dinucleotide (NAD) biosynthetic pathway and regulates cellular metabolism. NAMPT is also secreted by cells in the extracellular milieu, and referred to as extracellular NAMPT (eNAMPT or visfatin). In mammals, visfatin has been linked to various metabolic disorders. However, the role of visfatin in regulating energy homeostasis in fish is not known. In this study, we assessed the effects of nutritional status on NAMPT mRNA expression and the effects of visfatin peripheral injections on food intake and the expression of appetite regulators in goldfish. Our results show that NAMPT is widely expressed in peripheral tissues and brain. Fasting induced increases in NAMPT expression in liver but had no effect on either brain or intestine NAMPT expression levels. Intraperitoneal injections of visfatin (400 ng/g) induced an increase in food intake and in expression levels of hepatic leptin and sirtuin1. Visfatin injections decreased intestine CCK and PYY, and telencephalon (but not hypothalamic) orexin and NPY expression levels. Visfatin did not affect plasma glucose levels, intestine ghrelin or brain CART, POMC and AgRP expressions. These data suggest that visfatin/NAMPT might be involved in the regulation of feeding and energy homeostasis in goldfish.

Boosted growth performance, immunity, antioxidant capacity and disease resistance of crucian carp (Carassius auratus) by single or in combination dietary Bacillus subtilis and xylo-oligosaccharides.

In this study, a total of 420 healthy crucian carp (9.77 ± 0.04 g) were randomly divided into CK, B·S, XOS and B·S + XOS group, and cultured for 8 weeks. Results showed that the dietary Bacillus subtilis (B. subtilis) and xylo-oligosaccharides (XOS) can significantly increased the final weight, weight gain, specific growth rate, feed efficiency, protein efficiency and survival rate of crucian carp. Dietary B. subtilis and XOS can significantly increased the activities of catalase, glutathione, superoxide dismutase and total antioxidant capacity, significantly decreased the contents of malondialdehyde, and significantly increased the activities of alkaline phosphatase, acid phosphatase, lysozyme and the contents of complement component 3,4 and immunoglobulin M in crucian carp serum. In addition, compared with CK group, the expression levels of TGF-ß and IL-10 in B·S, XOS and B·S + XOS group were significantly increased, and the expression levels of TNF-α, HSP90, IL-1ß, TLR4 and MyD88 were significantly decreased. Supplementation of B. subtilis and XOS can also improve the intestinal tissue morphology of crucian carp. After injection of 1 × 107 CFU/mL Aeromonas hydrophila (A. hydrophila), compared with CK group, the survival rates of the B·S group, the XOS group and the B·S + XOS group were increased by 13.98%, 10.56% and 30.74%, respectively. These results show that dietary B. subtilis and XOS can significantly improve the growth performance, antioxidant capacity, immunity and resistance to A. hydrophila of crucian carp, and the combined effect is better than that of single addition.

Nesfatin-1 stimulates the hypothalamus-pituitary-interrenal axis hormones in goldfish.

Stress in vertebrates is mediated by the hypothalamus-pituitary-adrenal (in mammals)/interrenal (in fish) (HPA/I) axis, which produces the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and corticosteroids, respectively. Nesfatin-1, a novel anorexigenic peptide encoded in the precursor nucleobindin-2 (NUCB2), is increasingly acknowledged as a peptide that influences the stress axis in mammals. The primary aim of this study was to characterize the putative effects of nesfatin-1 on the fish HPI axis, using goldfish (Carassius auratus) as an animal model. Our results demonstrated that nucb2/nesfatin-1 transcript abundance was detected in the HPI tissues of goldfish, with most abundant expression in the pituitary. NUCB2/nesfatin-1-like immunoreactivity was found in the goldfish hypothalamus, pituitary, and interrenal cells of the head kidney. GPCR12, a putative receptor for nesfatin-1, was also detected in the pituitary and interrenal cells. NUCB2/nesfatin-1-like immunoreactivity was observed in ACTH-expressing pituitary corticotrophs. Acute netting and restraint stress upregulated nucb2/nesfatin-1 mRNA levels in the forebrain, hypothalamus, and pituitary, as well as crf and crf-r1 expression in the forebrain and hypothalamus. Intraperitoneal and intracerebroventricular administration of nesfatin-1 increased cortisol release and hypothalamic crf mRNA levels, respectively. Finally, we found that nesfatin-1 significantly stimulated ACTH secretion from dispersed pituitary cells in vitro. Collectively, our data provide the first evidence showing that nesfatin-1 is a stress responsive peptide, which modulates the stress axis hormones in fish.

A potential role for hyperpolarization-activated cyclic nucleotide-gated sodium/potassium channels (HCNs) in teleost acid-base and ammonia regulation.

Increasing evidence suggests the involvement of hyperpolarization-activated cyclic nucleotide-gated sodium/potassium channels (HCNs) not only in cardiac and neural function, but also in more general physiological processes including acid-base and ammonia regulation. We have identified four different HCN paralogs/isoforms in the goldfish Carassius auratus (CaHCN1, CaHCN2b, CaHCN4a and CaHCN4b) as likely candidates to contribute to renal, branchial and intestinal acid-base and ammonia regulation in this teleost. Quantitative real-time PCR showed not only high mRNA abundance of all isoforms in heart and brain, but also detectable levels (particularly of CaHCN2b and CaHCN4b) in non-excitable tissues, including gills and kidneys. In response to an internal or external acid-base and/or ammonia disturbance caused by feeding or high environmental ammonia, respectively, we observed differential and tissue-specific changes in mRNA abundance of all isoforms except CaHCN4b. Furthermore, our data suggest that the functions of specific HCN channels are supplemented by certain Rhesus glycoprotein functions to help in the protection of tissues from elevated ammonia levels, or as potential direct routes for ammonia transport in gills, kidney, and gut. The present results indicate important individual roles for each HCN isoform in response to acid-base and ammonia disturbances.

First evidence on the role of palmitoylethanolamide in energy homeostasis in fish.

The objective of this study was to investigate the role of palmitoylethanolamide (PEA) in the regulation of energy homeostasis in goldfish (Carassius auratus). We examined the effects of acute or chronic intraperitoneal treatment with PEA (20 µg·g-1 body weight) on parameters related to food intake and its regulatory mechanisms, locomotor activity, glucose and lipid metabolism, and the possible involvement of transcription factors and clock genes on metabolic changes in the liver. Acute PEA treatment induced a decrease in food intake at 6 and 8 h post-injection, comparable to that observed in mammals. This PEA anorectic effect in goldfish could be mediated through interactions with leptin and NPY, as PEA increased hepatic expression of leptin aI and reduced hypothalamic expression of npy. The PEA chronic treatment reduced weight gain, growth rate, and locomotor activity. The rise in glycolytic potential together with the increased potential of glucose to be transported into liver suggests an enhanced use of glucose in the liver after PEA treatment. In addition, part of glucose may be exported to be used in other tissues. The activity of fatty acid synthase (FAS) increased after chronic PEA treatment, suggesting an increase in the hepatic lipogenic capacity, in contrast with the mammalian model. Such lipogenic increment could be linked with the PEA-induction of REV-ERBα and BMAL1 found after the chronic treatment. As a whole, the present study shows the actions of PEA in several compartments related to energy homeostasis and feeding behavior, supporting a regulatory role for this N-acylethanolamine in fish.

Effects of short-term exercise on food intake and the expression of appetite-regulating factors in goldfish.

In mammals, growing evidence indicates that exercise affects food intake, metabolism and the expression and blood levels of appetite regulators. In this study, we examined the effects of short-term (30 min, at low and high water flow) exercise on food intake, glucose levels and the expressions of appetite regulators in goldfish hypothalamus (irisin, orexin, CART, leptin), intestine (CCK, PYY, proglucagon/GLP-1), muscle (irisin) and liver (leptin), of brain-derived neurotrophic factor (BDNF) in brain, interleukin-6 (IL6) in muscle and hypothalamus, and major metabolic enzymes, the glycolytic enzyme glucokinase (GCK) and its regulatory protein (GCKR) in liver, the lipolytic enzyme lipoprotein lipase in intestine and muscle, and trypsin in intestine. Fish submitted to high flow exercise had a lower post-exercise food intake compared to control fish but no differences were seen in glucose levels between groups. Exercise induced an increase in hypothalamic expression levels of CART, IL6 and BDNF, but not orexin, irisin, CRF, leptin and NPY. High flow exercise induced an increase in intestine CCK, PYY and GLP-1, and muscle irisin and IL-6 expression levels. Exercise had no effects on expression levels of hepatic leptin or any of the metabolic enzymes examined. Our results suggest that, in goldfish, short-term exercise might decrease feeding in part by affecting the expressions of myokines and peripheral, but not central appetite regulators or metabolic enzyme/hormones.

Molecular characterization and taurine regulation of two novel CDOs (CDO1 and CDO2) from Carassius auratus.

Cysteine oxygenase (CDO) is a mononuclear nonhemoglobin enzyme that catalyzes the production of taurine through the cysteine (Cys) pathway and plays a key role in the biosynthesis of taurine in mammals. However, the function of CDOs in bony fish remains poorly understood. In this study, we cloned CDO genes (CaCDO1 and CaCDO2) from Carassius auratus. The cDNA sequences of both CaCDO1 and CaCDO2 encoded putative proteins with 201 amino acids, which included structural features typical of the CDO protein family. Multiple sequence alignment and phylogenetic analysis showed that CaCDO1 and CaCDO2 shared high sequence identities and similarities with C. carpio homologs. Quantitative real-time polymerase chain reaction (qRT-PCR) results revealed that CaCDO1 and CaCDO2 were both broadly expressed in all selected tissues and developmental stages in C. auratus but had differing mRNA levels. In addition, compared to those of the taurine-free group, the in vivo mRNA expression levels of both CaCDO1 and CaCDO2 significantly decreased with increasing dietary taurine levels from 1.0 to 9.0 g/kg. Furthermore, in vitro taurine treatments showed similar inhibitory effects on the expression of CaCDO1 and CaCDO2 in the intestines of C. auratus. Our results also showed that the mRNA expression of CaCDO2 in the intestines was higher than that of CaCDO1 in response to in vivo and in vitro taurine supplementation. Overall, these data may provide new insights into the regulation of fish CDO expression and provide valuable knowledge for improving dietary formulas in aquaculture.

Effect on Serum Parameters and Immune Responses of Carassius auratus gibelio Exposed to Dietary Lead and Bacillus subtilis.

Lead (Pb), a heavy metal and an environmental stressor, may affect many physiological processes, including the serum index and the immune response. The aim of this study was to explore the toxic effects of Pb on the serum index and the immune response of Carassius auratus gibelio (C. gibelio) fed 0, 120, or 240 mg/kg Pb, and 109 cfu/g Bacillus subtilis (B. subtilis). After 15 and 30 days of dietary exposure, the serum indices and the immune responses of the fish were assessed. Dietary Pb exposure significantly affected various components of the serum index, including calcium, magnesium, glucose, cholesterol, total protein, glutamic-pyruvic transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH). However, sIgA activity in the gut increased significantly following B. subtilis supplementation. Notable changes were also observed in the expression levels of immune-related genes, including HSP70, IgM, HSP90, IL-1ß, IL-6, and TNF-α. B. subtilis supplementation effectively attenuated the effects of dietary Pb exposure.

Effect of Dietary Probiotic Lactobacillus helveticus on Growth Performance, Antioxidant Levels, and Absorption of Essential Trace Elements in Goldfish (Carassius auratus).

In this study, we have investigated the effect of probiotic Lactobacillus helveticus CD6 supplemented into simple laboratory fish feed (LFF) and complex, multi-ingredient market fish feed (MFF) on growth performance, antioxidant levels, and essential trace element absorption in goldfish (Carassius auratus). Twenty-four healthy goldfish (average weight 3-4 g) were acclimatized and divided into four experimental diets supplemented with 3 × 107 CFU/g of probiotic (LFF + Pro, MFF + Pro) and without probiotic (LFF, MFF) for 14 weeks. Fish fed with probiotic were healthy, active, and intense orange-gold as compared to control (without probiotic). At 14 weeks, fish fed with MFF + Pro/LFF + Pro showed 91/47% weight gain as compared to 34/- 12% weight observed with MFF/LFF. The average weight gain differences recorded between probiotic and control diets were not significant. No mortality to report when fish fed with probiotic. In contrast, fish fed without probiotic showed mortalities (LFF, two fish; MFF, one fish) during the trial. DPPH activity revealed high levels of antioxidants into the intestine of probiotic-fed fish. Trace element analysis showed that probiotic colonization enhanced diet-dependent absorption of Fe and Zn. The in vitro antimicrobial activity was exhibited by probiotic L. helveticus CD6 against infected fish isolate Aeromonas spp. JA showed an ability to protect fish from infections. Moreover, complex, multi-ingredient feed had a highest impact on viability of probiotic during storage. In conclusion, L. helveticus CD6 did not significantly enhance growth performance; however, it improved health and reduced mortalities in goldfish (C. auratus) regardless of the composition of the diet.

Xenin is a novel anorexigen in goldfish (Carassius auratus).

Xenin, a highly conserved 25 amino acid peptide cleaved from the N-terminus of the coatomer protein alpha (COPA), is emerging as a food intake regulator in mammals and birds. To date, no research has been conducted on xenin biology in fish. This study aims to identify the copa mRNA encoding xenin in goldfish (Carassius auratus) as a model, to elucidate its regulation by feeding, and to describe the role of xenin on appetite. First, a partial sequence of copa cDNA, a region encoding xenin, was identified from goldfish brain. This sequence is highly conserved among both vertebrates and invertebrates. RT-qPCR revealed that copa mRNAs are widely distributed in goldfish tissues, with the highest levels detected in the brain, gill, pituitary and J-loop. Immunohistochemistry confirmed also the presence of COPA peptide in the hypothalamus and enteroendocrine cells on the J-loop mucosa. In line with its anorexigenic effects, we found important periprandial fluctuations in copa mRNA expression in the hypothalamus, which were mainly characterized by a gradually decrease in copa mRNA levels as the feeding time was approached, and a gradual increase after feeding. Additionally, fasting differently modulated the expression of copa mRNA in a tissue-dependent manner. Peripheral and central injections of xenin reduce food intake in goldfish. This research provides the first report of xenin in fish, and shows that this peptide is a novel anorexigen in goldfish.

α-Melanocyte-stimulating hormone promotes bone resorption resulting from increased osteoblastic and osteoclastic activities in goldfish.

We examined the effects of α-melanocyte-stimulating hormone (α-MSH) on bone metabolism using regenerating goldfish scales. Normally developed scales on the bodies of goldfish were removed to allow the regeneration of scales under anesthesia. Thereafter, the influence of α-MSH on the regeneration of goldfish scales was investigated in vivo. In brief, α-MSH was injected at a low dose (0.1 µg/g body weight) or a high dose (1 µg/g body weight) into goldfish every other day. Ten days after removing the scales, we collected regenerating scales and analyzed osteoblastic and osteoclastic activities as respective marker enzyme (alkaline phosphatase for osteoblasts, tartrate-resistant acid phosphatase for osteoclasts) activity in the regenerating scales as well as plasma calcium levels. At both doses, osteoblastic and osteoclastic activities in the regenerating scales increased significantly. Plasma calcium concentrations in the α-MSH-treated group (high doses) were significantly higher than those in the control group. Next, in vitro experiments were performed to confirm the results of in vivo experiments. In the cultured regenerating scales, osteoblastic and osteoclastic activities significantly increased with α-MSH (10-7 and 10-6 M) treatment. In addition, real-time PCR analysis indicated that osteoclastogenesis in α-MSH-treated scales was induced by the receptor activator of the NF-κB/receptor activator of the NF-κB ligand/osteoprotegerin pathway. Furthermore, we found that α-MSH receptors (melanocortin receptors 4 and 5) were detected in the regenerating scales. Thus, in teleosts, we are the first to demonstrate that α-MSH functions in bone metabolism and promotes bone resorption via melatonin receptors 4 and/or 5.

The pH-dependent toxicity of triclosan to five aquatic organisms (Daphnia magna, Photobacterium phosphoreum, Danio rerio, Limnodrilus hoffmeisteri, and Carassius auratus).

Triclosan (TCS) is an antibacterial and antifungal agent widely used in personal care products, and it has been frequently detected in the aquatic environment. In the present study, the acute toxicity of TCS to Daphnia magna, Photobacterium phosphoreum, Danio rerio, and Limnodrilus hoffmeisteri was assessed under different pH conditions. Generally, TCS was more toxic to the four aquatic organisms in acidic medium. The LC50 values for D. magna and D. rerio were smaller among the selected species, suggesting that D. magna and D. rerio were more sensitive to TCS. In addition, the oxidative stress-inducing potential of TCS was evaluated in Carassius auratus at three pH values. Changes of superoxide dismutase (SOD) and catalase (CAT) activity, glutathione (GSH) level, and malondialdehyde (MDA) content were commonly observed in all TCS exposure groups, indicating the occurrence of oxidative stress in the liver of C. auratus. The integrated biomarker response (IBR) index revealed that a high concentration of TCS induced great oxidative stress in goldfish under acidic condition. This work supplements the presently available data on the toxicity data of TCS, which would provide some useful information for the environmental risk assessment of this compound.

Significant improvement of intestinal microbiota of gibel carp (Carassius auratus gibelio) after traditional Chinese medicine feeding.

AIMS: Increasing attention has been attracted to intestinal microbiota, due to interactions with nutrition, metabolism and immune defence of the host. Traditional Chinese medicine (TCM) feed additives have been applied in aquaculture to improve fish health, but the interaction with fish gut microbiota is still poorly understood. This study aimed to explore the effect of adding TCM in feed on the intestinal microbiota of gibel carp (Carassius auratus gibelio). METHODS AND RESULTS: Bacterial communities of 16 fish intestinal contents and one water sample were characterized by high-throughput sequencing and analysis of the V4-V5 region of the 16S rRNA gene. The results showed that the composition and structure of the bacterial community were significantly altered by the TCM feeding. Some phyla increased markedly (Proteobacteria, Actinobacteria, Acidobacteria, etc.), while Fusobacteria were significantly reduced. Concurrently, the richness and diversity of the taxonomic units increased, and the microbiota composition of TCM-treated fish was more homogeneous among individuals. At the genus level, the addition of TCM tended to reduce the incidence of potential pathogens (Aeromonas, Acinetobacter and Shewanella), while stimulating the emergence of some potential probiotics (Lactobacillus, Lactococcus, Bacillus and Pseudomonas). CONCLUSIONS: These data suggested that the feed additive could regulate the fish intestinal microbiota by reinforcing the microbial balance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study may provide useful information for further application of TCM for diseases prevention and stress management in aquaculture.

Single and combined effects of aluminum (Al2O3) and zinc (ZnO) oxide nanoparticles in a freshwater fish, Carassius auratus.

The increasing use of nanoparticles (NPs) worldwide has raised some concerns about their impact on the environment. The aim of the study was to assess the toxicity of metal oxide nanoparticles, singly or combined, in a freshwater fish (Carassius auratus). The fish were exposed for 7, 14, and 21 days to different concentrations of NPs (10 µg Al2O3.L-1, 10 µg ZnO.L-1, 10 µg Al2O3.L-1 plus 10 µg ZnO.L-1, 100 µg Al2O3.L-1, 100 µg ZnO.L-1, and 100 µg Al2O3.L-1 plus 100 µg ZnO.L-1). At the end of each exposure period, antioxidant enzyme activity (catalase, glutathione-S-transferase, and superoxide dismutase), lipid peroxidation, and histopathology were assessed in the gills and livers of C. auratus. The results show an increase in catalase (CAT) and superoxide dismutase (SOD) activity in the gills and livers of fish, especially after 14 days of exposure to single and combined NPs, followed by a reduction at 21 days. An increase in glutathione S-transferase (GST) was observed in gills after 7 days for all tested NP concentrations (single and combined); while in livers, a significant increase was determined after 14 days of exposure to 100 µg.L-1 of both single ZnO and Al2O3 NPs. Lipid peroxidation (LPO) significantly increased in gills after 7 days of exposure to 100 µg.L-1 Al2O3 NPs (single or combined). In livers, LPO increased significantly after 7 days of exposure to all tested concentrations of both single ZnO and Al2O3 (except for 10 µg Al2O3.L-1), and after 14 days of exposure to ZnO (10 and 100 µg.L-1) and Al2O3 (100 µg.L-1). The results from histological observations suggest that exposure to metal oxide NPs affected both livers and gills, presenting alterations such as gill hyperplasia and liver degeneration. However, the most pronounced effects were found in gills. In general, this study shows that the tested NPs, single or combined, are capable of causing sub-lethal effects on C. auratus, but when combined, NPs seem to be slightly more toxic than when added alone.