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Microplastics (MPs) are environmental contaminants that are of increasing global concern. This study investigated the presence of MPs in four varieties of marine-derived commercial fish meal, followed by identification of their polymer composition using Fourier transform infrared (FTIR) spectroscopy. Exposure experiments were conducted on cultured common carp ( Cyprinus carpio ) by feeding four varieties of commercially available fish meal to determine relationships between abundance and properties of MPs found both in meal and in those transferred to cultured common carp. Mean particle sizes were 452 ± 161 μm (± SD). Fragments were the predominant shape of MP found in fish meal (67%) and C. carpio gastrointestinal tract and gills (65%), and polypropylene and polystyrene were the most present plastic polymers found in fish meal (45% and 24%, respectively) and C. carpio (37% and 33%, respectively). Positive relationships were found between MP levels in fish meal and C. carpio . This study highlights that marine-derived fish meal may be a source of MPs which can be transferred to cultured fish, thus posing a concern for aquaculture.

The present study aimed to investigate the effect of Foeniculum vulgare essential oil on the health of the common carp (Cyprinus carpio). A total of 120 healthy fish were provided with feed containing 200, 400, and 600 mg/kg of F. vulgare oil for 60 days. Findings revealed that the oral administration of 200 mg/kg of F. vulgare oil significantly increased final weight, weight gain, and specific growth rate compared to the control group (p < 0.05). Plasma biochemical enzyme levels remained unchanged in 200 and 400 mg/kg treatments, whereas they were found to be significantly increased in treatments with 600 mg/kg. Although no significant alterations in glucose, triglyceride, and cholesterol levels were observed, the treated groups exhibited significant increases in total protein, albumin, globulin, and creatinine levels. Results also indicated significantly decreasing levels of glutathione peroxidase, whereas superoxide dismutase activity increased. The gonadosomatic index showed notable improvement in the 200 and 400 mg/kg groups. Furthermore, plasma concentrations of estradiol and testosterone were significantly affected by doses of 400 and 200 mg/kg. Findings suggest that, following the administration of F. vulgare extract, the reproductive and general health of the fish appears to be improved. Nevertheless, it is recommended to supplement fish diets with up to 200 mg/kg of F. vulgare extract to improve their reproductive and general health. Concentrations above this limit can potentially cause harm.

The mucosa of vertebrates is a particularly complex but dynamic environment in which the host constantly interacts with trillions of commensal microorganisms and pathogens. Although the internal and external mucosal microbiomes with immune defense of mammals have been well investigated, the relationship between mucosal microbes and their host’s immune responses has not been systematically understood in the early vertebrates. In this study, we compared the composition and distribution of mucosal microbiota in common carp ( Cyprinus carpio ), and found that there were significant differences of microbiota between in the internal (gut) and external mucosal (buccal mucosa, gills and skin) tissues. Next, we successfully constructed an infection model with spring viremia of carp virus (SVCV). Specifically, following viral infection, the immune and antiviral related genes showed different up-regulation in all selected mucosal tissues while significant morphological changes were only found in external tissues including buccal mucosa, gills and skin. Using 16S rRNA gene sequence, we revealed that the abundance of Proteobacteria in mucosal tissues including buccal mucosa, gills and gut showed increased trend after viral infection, whereas the abundance of Fusobacteria significantly decreased in gut. In addition, the loss of dominant commensal microorganisms and increased colonization of opportunistic bacteria were discovered in the mucosal surfaces indicating that a secondary bacterial infection might occur in these mucosal tissues after viral infection. Overall, our results firstly point out the distribution of internal and external mucosal microbiota and analyze the changes of mucosal microbiota in common carp after SVCV infection, which may indicated that the potential role of mucosal microbiota in the antiviral process in early vertebrates.

Carbonate alkalinity is a major environmental stress factor affecting aquatic feed configuration, which easily causes oxidative stress and hypoimmunity for fish. Hence, the purpose of the study is to assess the potential effect of phosphorus on growth, intestinal oxidation resistance, physical barrier function, and microflora for Songpu mirror carp ( Cyprinus carpio Songpu) (initial average weight of 2.95 ± 0.21 g) reared at the high-concentration carbonate alkalinity environment. A two-factor, three-level (2 × 3) design was applied, in which diets with three different phosphorus levels (3.6, 7.0, and 10.5 g/kg dry matter) were randomly assigned to 0 and 15 mmol/L carbonate alkalinity groups with three replicate aquariums. After the 8-week trial, we found that weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER), and lipase and amylase activities in the intestine significantly ( p < 0.05) declined with increasing carbonate alkalinity. Carbonate alkalinity of 15 mmol/L significantly reduced glutathione peroxidase (GSHPx) activities in the intestine ( p < 0.05). The relative expressions of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), glutathione peroxidase 1a (GPX1a), Clautin3, Clautin11, and tumor necrosis factor β (TNF-β) in the intestine were markedly downregulated by increasing carbonate alkalinity levels ( p < 0.05), whilst the relative expressions of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in the intestine were markedly upregulated ( p < 0.05). At the 15 mmol/L carbonate alkalinity treatment, Songpu mirror carp suffer from hypoimmunity status with failed digestion, antioxidant, inflammation, and immune response, thereby inducing impaired growth. Additionally, significant increments in the abundance of Proteobacteria and a significant decrease in the abundance of Fusobacteria and the Firmicutes/Bacteroidetes ratio were caused due to excessively high carbonate alkalinity (15 mmol/L) and excessively low dietary phosphorus supply (3.6 g/kg). Collectively, 7.0 g/kg dietary phosphorus supplementation was effective in promoting intestinal antioxidant enzyme activities and the corresponding gene expression via the Keap1-Nrf2 signaling pathway and in enhancing intestinal immunity by upregulating anti-inflammatory and downregulating pro-inflammatory genes. Appropriate dietary phosphorus supply could promote the formation of beneficial microflora in freshwater, and it has the potential ability to transfer the adverse effect of carbonate alkalinity stress to the structural composition of intestinal microflora. Hence, consideration should be given to suitable phosphorus supply for fish under the chronic carbonate alkalinity stress.

GSDME is the only direct executor of caspase-dependent pyroptosis in both canonical and non-canonical inflammasomes known to date in fish, and plays an important role in anti-bacterial infection and inflammatory response. In order to determine the regulation of GSDMEa on antibacterial infection in innate immune response, the CcGSDMEa gene in common carp ( Cyprinus carpio haematopterus ) was first identified and characterized, and then its function related to immune defense was investigated. Our results showed that the expressions of Cc GSDMEa at the mRNA and protein levels were both significantly increased after Aeromonas hydrophila intraperitoneal infection at the early stage than that in the control group. We found that Cc GSDMEa could be cleaved by inflammatory caspase ( Cc Caspase-1b) and apoptotic caspases ( Cc Caspase-3a/b and Cc Caspase-7a/b). Interestingly, only the Cc GSDMEa-NT (1-252 aa) displayed bactericidal activity to Escherichia coli and could punch holes in the membrane of HEK293T cells, whereas Cc GSDMEa-FL (1-532 aa) and Cc GSDMEa-CT (257-532 aa) showed no above activity and pore-forming ability. Overexpression of Cc GSDMEa increased the secretion of Cc IL-1β and the release of LDH, and could reduce the A. hydrophila burdens in fish. On the contrary, knockdown of Cc GSDMEa reduced the secretion of Cc IL-1β and the release of LDH, and could increase the A. hydrophila burdens in fish. Taken together, the elevated expression of Cc GSDMEa was a positive immune response to A. hydrophila challenge in fish. Cc GSDMEa could perform the pore-formation in cell membrane and the regulation on the secretion of IL-lβ, and further regulate the bacterial clearance in vivo . These results suggested that Cc GSDMEa played an important role in immune defense against A. hydrophila and could provide a new insight into understanding the immune mechanism to resist pathogen invasion in teleost.

This study aimed to evaluate the effects of fish meal (FM) replacement by yeast hydrolysate (YH) on liver antioxidant capability, intestinal morphology, and inflammation-related genes of juvenile Jian carp (Cyprinus carpio var. Jian). A total of 600 fish (average initial weight 19.44 ± 0.06 g) were randomly selected and divided into five groups. Five isonitrogenous and isocaloric diets replacing FM by YH 0% (YH0), 1% (YH1), 3% (YH3), 5% (YH5), and 7% (YH7) were formulated. Each diet was tested in four replicates for 10 weeks. The results have shown that, compared to the control group (YH0), liver total superoxide dismutase (t-SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione (GSH) activities of fish fed YH1 and YH3 diets were significantly higher (P < 0.05). Liver malondialdehyde (MDA) concentration significantly increased as supplementation levels of YH increased from 1 to 7% (P < 0.05). Moreover, intestinal microvillus length of juvenile Jian carp fed YH diets were significantly higher than that of fish fed the control diet (P < 0.05). In proximal intestine, the relative expression levels of inflammation-related genes (ALP, IL-1β, and TNF-α) in YH7 were significantly higher than that in the control group (P < 0.05). However, in midintestine, the expression levels of these genes in YH3 were significantly lower compared to the control group (P < 0.05). The results of this study indicated that dietary replacement of FM by 3%YH could improve antioxidant capability and intestinal microvillus morphology, as well as enhance the non-specific immunity of juvenile Jian carp.

An investigation on the toxicological assessment of 10 choline chloride (ChCl)-based deep eutectic solvents (DESs) towards four fungi strains and Cyprinus carpio fish was conducted. ChCl was combined with materials from different chemical groups such as alcohols, sugars, acids and others to form DESs. The study was carried out on the individual DES components, their aqueous mixture before DES formation and their formed DESs. The agar disc diffusion method was followed to investigate their toxicity on four fungi strains selected as a model of eukaryotic microorganisms ( Phanerochaete chrysosporium , Aspergillus niger , Lentinus tigrinus and Candida cylindracea ). Among these DESs, ChCl:ZnCl 2 exhibited the highest inhibition zone diameter towards the tested fungi growth in vitro, followed by the acidic group (malonic acid and p -toluenesulfonic acid). Another study was conducted to test the acute toxicity and determine the lethal concentration at 50 % (LC 50 ) of the same DESs on C. carpio fish. The inhibition range and LC 50 of DESs were found to be different from their individual components. DESs were found to be less toxic than their mixture or individual components. The LC 50 of ChCl:MA DES is much higher than that of ChCl:MA Mix . Moreover, the DESs acidic group showed a lower inhibition zone on fungi growth. Thus, DESs should be considered as new components with different physicochemical properties and toxicological profiles, and not merely compositions of compounds.

The common carp ( Cyprinus carpio L.) is an important farmed species worldwide. Mucosal-associated lymphoid tissues play an essential role in the fight against pathogen infection. Spring viremia of carp virus (SVCV) poses a serious threat to the common carp aquaculture industry. Understanding the molecular mechanisms driving mucosal immune responses to SVCV infection is critical. In this study, the mucosal tissues (gills, foregut and hindgut) were collected from normal and infected fishes for transcriptome analysis. A total of 932,378,600 clean reads were obtained, of which approximately 80% were successfully mapped to the common carp genome. 577, 1,054 and 1,014 differential expressed genes (DEGs) were identified in the gills, foregut and hindgut, respectively. A quantitative polymerase chain reaction assay indicated that the DEGs expression in the foregut following SVCV infection was consistent with the transcriptome results. Among them, two key genes of the retinoic acid-inducible gene I (RIG-I)-like receptor family, melanoma-differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) (i.e., Cc MDA5 and Cc LGP2), underwent further analysis. Overexpression of Cc MDA5 or Cc LGP2 increased phosphorylation of TANK-binding kinase 1 and interferon regulatory factor 3 and the expression of interferon-1 ( ifn-1 ), myxovirus resistance ( mx ), viperin and interferon-stimulated gene 15 ( isg15 ), and inhibited SVCV replication in epithelioma papulosum cyprini cells. Furthermore, Cc LGP2 significantly upregulated the Cc MDA5-induced ifn-1 mRNA expression and the activation of the ifn-1 promoter. Finally, confocal microscopy and coimmunoprecipitation experiments revealed that Cc LGP2 colocalizes and interacts with Cc MDA5 via the C-terminal regulatory domain. This study provides essential gene resources for understanding the fish immune response to SVCV infection and sheds light on the potential role of fish LGP2 in the MDA5 regulation.

•An effective live attenuated vaccine for A. hydrophila was screened using rifampicin.•The vaccine candidate can stir specific immunity against A. hydrophila.•The vaccine can induce sensitive expression of immune-related genes. Aeromonas hydrophila, as a strong Gram-negative bacterium, can infect a wide range of freshwater fish, including common carp Cyprinus carpio, and cause the huge economic loss. To create the effective vaccine is the best way to control the outbreak of the disease caused by A. hydrophila. In this study, a live attenuated A. hydrophila strain, XX1LA, was screened from the pathogenic A. hydrophila strain XX1 cultured on medium containing the antibiotic rifampicin, which was used as a live attenuated vaccine candidate. The immune protection of XX1LA against the pathogen A. hydrophila in common carp was evaluated by the relative percent survival (RPS), the specific IgM antibody titers, serum lysozyme activity and the expression profiles of multiple immune-related genes at the different time points following immunization. The results showed that the variable up-regulations of the immune-related genes, such as the pro-inflammatory cytokine IL-1β, the chemokine IL-10 and IgM, were observed in spleen and liver of common carp injected in the vaccines with the formalin-killed A. hydrophila (FKA) and the live attenuated XX1LA. Specific antibody to A. hydrophila was found to gradually increase during 28 days post-vaccination (dpv), and the RPS (83.7%) in fish vaccinated with XX1LA, was significant higher than that (37.2%) in fish vaccinated with FKA (P<0.05) on Day 28 after challenged by pathogen. It was demonstrated that the remarkable immune protection presented in the group vaccinated with XX1LA. During the late stage of 4-week immunization phase, compared with FKA and the control, specific IgM antibody titers significantly increased (P<0.05) in the XX1LA group. The activity of the lysozyme in serum indicated no significant change among three groups. In summary, the live attenuated bacterial vaccine XX1LA, screened in this study, indicates the better protect effect on common carp against A. hydrophila, which can be applied in aquaculture of common carp to prevent from the disease outbreak in the future.

Toll-like receptor 19 (Tlr19) is a fish-specific TLR that plays a critical role in innate immunity. In the present study, we aimed to identify tlr19 from common carp ( Cyprinus carpio L.) and explored its expression profile, localization, adaptor, and signaling pathways. A novel tlr19 cDNA sequence ( Cctlr19 ) was identified in common carp. Phylogenetic analysis revealed that Cc Tlr19 was most closely related to Danio rerio Tlr19. Subcellular localization analysis indicates that Cc Tlr19 was synthesized in the free ribosome and then transported to early endosomes. Cctlr19 was constitutively expressed in all the examined tissues, with the highest expression in the brain. After poly(I:C) and Aeromonas hydrophila injection, the expression of Cctlr19 was significantly upregulated in immune-related organs. In addition, the expression of Cctlr19 was upregulated in head kidney leukocytes (HKL) upon stimulation with different ligands. Immunofluorescence and luciferase analyses indicate that Cc Tlr19 recruited TRIF as an adaptor. Furthermore, Cc Tlr19 can activate the expression of ifn-1 and viperin . Taken together, these findings lay the foundation for future research to investigate the mechanisms underlying fish tlr19 .