Comparison of gut microbial communities, free amino acids or fatty acids contents in the muscle of wild Aristichthys nobilis from Xinlicheng reservoir and Chagan lake.

BACKGROUND: Fish is favored by consumers, while amino acids and fatty acids are the main nutrients of muscle. At present, it has been found that the gut microbial community may be involved in the regulation of host material anabolism. Juvenile and adult bighead carp (A. nobilis) from Chagan lake and Xinlicheng reservoir were selected, and divided into four groups to compare the differences of gut microbial communities, free amino acid and fatty acids in muscle. RESULTS: The results showed that fish in different lakes or ages contained specific microbiota, the gut microbial structure was similar, but the microbial content was significantly different. Gut microbial abundance of juvenile fish in Chagan lake was significantly higher than that of other groups. Phylum level analysis Proteobacteria was the dominant gut bacteria of fish in both adult and juvenile fish from two separate lakes. Actinobacteria was another dominant bacterial phylum in juvenile fish in both lakes. Contents of free amino acids and fatty acids in muscle were detected, and the relationships between them and gut microbial communities were analyzed. Bighead carp grew from juvenile to adult, Actinobacteria abundance decreased (P < 0.05) and Proteobacteria increased (P < 0.05). Proteobacteria was positively correlated with the contents of Thr, Lys, Pro, Asp, Gly and Glu, Actinobacteria was negatively correlated with Met and His. Meanwhile, EPA and DHA were positively correlated with Proteobacteria, EPA and DHA were not significantly associated with Actinobacteria. CONCLUSION: It was speculated that the contents of free amino acids and fatty acids in muscle might be affected by the difference of gut microbiota, thus affecting the taste and nutritional quality.

Transcriptomics reveals the mechanism of selenium-enriched Lactobacillus plantarum alleviating brain oxidative stress under cadmium stress in Luciobarbus capito.

Cadmium (Cd) is one of toxic metal in environment and is thought to affect nervous system. There were an increasing number of studies on selenium (Se)-enriched probiotics which were believed to produce bioactive nanoselenium. The antagonism of Se on heavy metals can significantly affect biological toxicity of heavy metals. This study aimed to elucidate possible mechanism of brain injury in Luciobarbus capito after Cd exposure and the mitigation of Se-enriched probiotics through transcriptome analysis. The results revealed 465 differentially expressed genes in the Cd and the control brains (Cd vs C), including 320 genes with upregulated expression and 145 genes with downregulated expression. In addition, we found that there were 4117 differentially expressed genes in the Se-enriched L. plantarum plus Cd and the control brains (S1L1-Cd vs C), including 2552 genes with upregulated expression and 1565 genes with downregulated expression. There were 147 differentially expressed genes in the Se-enriched L. plantarum plus Cd and the control brains (S1L1-Cd vs Cd), including 40 genes with upregulated expression and 107 genes with downregulated expression. Moreover, GO enrichment analysis indicated that the differentially expressed genes were involved in biological processes cellular component, and molecular function. KEGG enrichment analysis indicated that MAPK signaling pathway, calcium signaling pathway, and PI3K-Akt signaling pathway were significantly enriched. Subsequently, qRT-PCR was performed, and we selected 15 related differentially expressed genes for verification. The qRT-PCR results revealed the same trend as the RNA-Seq results. In conclusion, this study elucidated relieving effect of Se-enriched probiotics on Cd exposure-induced brain oxidative stress. This study provided a theoretical basis for further research on genes related to Cd poisoning and the amelioration of Se-enriched probiotics on Cd poisoning.

Selenium-enriched Bacillus subtilis reduces the effects of mercury-induced on inflammation and intestinal microbes in carp (Cyprinus carpio var. specularis).

Mercury (Hg) is a global pollutant that affects the health of humans and ecosystems. Selenium (Se) is an essential trace element for many organisms including humans. Bacillus subtilis is one of the main probiotics used in aquaculture, and has a certain adsorption effect on heavy metals. The interaction between Hg and Se was rigorously studied, especially due to the observation of the protective effect of Se on Hg toxicity. The objective of this study was to research the effects of Hg, Se, and B. subtilis on inflammation and intestinal microbes in common carp. The common carp was exposed to Hg (0.03 mg/L), and 105 cfu/g Se-rich B. subtilis was added to the feed. After 30 days of feeding, samples were taken to evaluate the growth performance, serological response, inflammatory response, and intestinal microbial changes. In this study, when fish were exposed to Hg, the growth performance of the Se-rich B. subtilis plus 0.03 mg/L Hg fish group was lower than that of the control group and higher than 0.03 mg/L Hg; The levels of serum immunoglobulin M (IgM) and lysozyme (LZM) decreased, but after supplementation with Se-rich B. subtilis, the levels of LZM and IgM increased; Hg treatment significantly upregulated the mRNA expression of interleukin-1ß (IL-1ß), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and nuclear factor-kB (NF-κB P65), but downregulated the mRNA expression of interleukin-10 (IL-10), transforming growth factor-ß (TGF-ß) and NF-kappa-B inhibitor alpha (IkBα). However, compared with the Hg group, the Se-rich B. subtilis plus Hg group can significantly increase the mRNA expression levels of IL-1ß, IL-8, TNF-α, and NF-κB P65, but reduce the regulation of IL-10, TGF-ß, and IkBα expression. Through the analysis of the microbiological, we found that the Hg group was mainly composed of Aeromonas sobria and Aeromonas hydrophila. However, in the Se-rich B. subtilis treatment group, we found that Aeromonas sobria was significantly less than the Hg group. Se-rich B. subtilis improves Hg-induced intestinal microbial changes, alleviates the abundance of Aeromonas, and alleviates the inflammation of the fish. The results of this study show that Se-rich B. subtilis dietary supplements can effectively protect common carp against Hg toxicity.

Transcriptome analysis reveals the mechanism of alkalinity exposure on spleen oxidative stress, inflammation and immune function of Luciobarbus capito.

Saline-alkali land is distributed all over the world, and it affects the economic development of fisheries. The alkalinity in water is related to the accumulation of carbonate, so the is generally higher. To understand how alkalinity impacts the immune response in Luciobarbus capito, we performed transcriptomic profiles for spleen, the immune organ of Luciobarbus capito which were underwent alkalinity exposure. Totally there are 47,727,954, 53,987,820 and 51,398,546 high quality clean reads obtained from the control groups, and 46,996,982, 49,650,460 and 45,964,986 clean reads from the alkalinity exposure groups. Among them, 611 genes were differently expressed, including 534 upregulated and 77 down-regulated genes. The identified genes were enriched using databases of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). It was found that differentially expressed genes in Luciobarbus capito spleen tissue were enriched into 14 GO pathways, and differentially expressed genes in Luciobarbus capito spleen were enriched into 25 corresponding KEGG pathways under alkalinity stress. Inflammation and immune function genes and pathways were identified and validated by quantitative real-time RT-PCR. Our results showed that alkalinity exposure leads to inflammation and immunoregulation in spleen of Luciobarbus capito. These results provide new insights for unveiling the biological effects of alkalinity in Luciobarbus capito.

Brain Injury Induced by Mercury in Common Carp: Novel Insight from Transcriptome Analysis.

Mercury is a heavy metal which causes irreversible toxicity to fish and is detected in aquatic environment around the world. We aimed to explore the relative mechanism of mercury exposure on the brain injury. In this study, high-throughput sequencing RNA-Seq technology was carried out to analyze the changes of gene expression of brain tissues exposed to mercury. A large number of differentially expressed genes were identified. And 366 genes were up-regulated and 688 genes were down-regulated. Gene Ontology (GO) functional enrichment analysis showed that DNA-templated and transport were highly enriched in the biological process. Membrane, nucleus, and cytoplasm were highly enriched in the cellular component, and metal ion binding and DNA binding were highly enriched in molecular function. The differential genes were enriched in ferroptosis, necroptosis, calcium signaling pathway, and ion channels. Real-time quantitative reverse transcription PCR (qRT-PCR) results demonstrated the selected genes exhibited the same trends with the RNA-Seq results, which indicates the transcriptome sequencing data is reliable. Our results may provide an insightful view for the toxic effects of mercury on brain injury of common carp.

Effect of arsenic stress on the intestinal structural integrity and intestinal flora abundance of Cyprinus carpio.

Aquatic organisms such as fish can accumulate high concentrations of arsenic (As), which has toxic effects on fish. However, whether the intestinal flora are involved in As damage to fish intestinal tissues and the underlying process are unclear. Common carp (Cyprinus carpio) were exposed to As (2.83 mg/L) in water for 30 days, and blood, muscle, intestine, and intestine samples were collected. Intestinal pathological sections were observed, and the lipopolysaccharide (LPS) levels in serum and the levels of As accumulation and tight junction-related factors in intestinal tissues were measured. The gut microbiota was analysed by 16S rRNA sequencing. The results showed that As treatment decreased the abundance of microbiota, increased the number of harmful bacteria, and decreased the number of beneficial bacteria in the intestine. In our experiment, the top 30 harmful and beneficial bacteria with the highest relative abundance were identified. Among the top 30 harmful and beneficial bacteria, As treatment resulted in a significant (P < 0.05) increase in harmful bacteria (such as Fusobacteriota, Bacteroidota (LPS-producing bacteria), Verrucomicrobiota, Bacteroides, Aeromonas, and Stenotrophomonas) and a significant (P < 0.05) decrease in beneficial bacteria (such as Actinobacteriota, Planctomycetota, Firmicutes, Reyranella, Akkermansia, and Pseudorhodobacter), which further demonstrated that As affects the abundance of intestinal flora. In addition, As exposure increased the LPS level in serum and the abundance of Bacteroidota (LPS-producing bacteria) in the intestine. Bacteroidota exhibits the six highest relative abundance at the phylum level, which indicates that LPS produced by Bacteroidota can increase the LPS level in serum. Additionally, the protein and gene levels of the tight junction markers ZO-1 and occludin in the intestine were reduced by As treatment, which further indicated that As exposure impaired the structural integrity of the intestine. In conclusion, the results obtained in our study indicate that the intestinal flora, LPS, and tight junctions participate in the impairment of the structural integrity of the common carp intestine resulting from As exposure.

Transcriptome analysis revealed the mechanism of Luciobarbus capito (L. capito) adapting high salinity: Antioxidant capacity, heat shock proteins, immunity.

Salinity has a significant influence on the physiology of freshwater aquatic organisms. However, there are few studies on the hematology and immunology of freshwater fish under high salinity. In the current study, we aimed to analyze the adaptive effect of salt stress on L. capito spleen immune function and hematology using transcriptomic analysis. We replicated a L. capito acute salinity stress model, and collected blood and spleens from freshwater and saltwater fish. It was found that salinity affected significantly the numbers of leukocytes, lymphocytes, neutrophils, and red blood cells, as well as the content of haemoglobin. Salt treatment resulted in a significant increase in the expression of HSP70, HSP90, CAT, SOD, and GPX1 genes in L. capito spleens. Transcriptomic analysis revealed a total of 546 differentially expressed genes (DEGs) in spleens, including 224 up-regulated DEGs and 322 down-regulated DEGs. In addition, GO enrichment analysis revealed immune system process, multicellular organismal process, and biological regulation of genes with the most differences in biological processes. KEGG enrichment analysis showed that the regulation of lipolysis in adipocyte, FoxO signaling pathway, Hematopoietic cell lineage signaling pathway, and HIF-1 signaling pathway were significantly enriched. L. capito adapted oxidative to high salinity through FoxO signaling pathway and immune to high salinity through Hematopoietic cell lineage signaling pathway. At the same time, we selected 10 DEGs for qRT-PCR detection, and the results showed that the qRT-PCR results were consistent with our RNA-Seq results, indicating that transcriptome sequencing was accurate and reliable. In conclusion, our results demonstrated that the improvement of antioxidant capacity, heat shock protein and immunity are involved in the molecular mechanism of L. capito adapting to high salinity. Our findings provided a rationale for further study on high salinity adaptation and related enrichment pathways.

Effects of exposure to cadmium (Cd) and selenium-enriched Lactobacillus plantarum in Luciobarbus capito: Bioaccumulation, antioxidant responses and intestinal microflora.

Cadmium (Cd) is a dangerous pollutant with multiple toxic effects on aquatic animals, and it exists widely in the environment. Selenium (Se) is a biologically essential trace element. Interactions between heavy metals and selenium can significantly affect their biological toxicity, although little is known about the mechanism of this antagonism. Lactobacillus is one of the dominant probiotics, given that a certain dose promotes host health. In this study, we evaluated the protective effect of a dietary probiotic supplementation, Se-enriched Lactobacillus plantarum (L. plantarum), on the bioaccumulation, oxidative stress and gut microflora of Luciobarbus capito exposed to waterborne Cd. Fish were exposed for 28 days to waterborne Cd at 0.05 mg/L and/or dietary Se-enriched L. plantarum. Exposure to Cd in water leads to Cd accumulation in tissues, oxidative stress and significant changes in gut microflora composition. Adding Se-enriched L. plantarum to the diet can reduce the accumulation of Cd in tissues, enhance the activity of antioxidant enzymes, and reverse changes in intestinal microbial composition after Cd exposure. The results obtained indicate that Se-enriched L. plantarum provides significant protection against the toxicity of Cd by inhibiting bioaccumulation. Selenium reduced oxidative stress by increasing the activity of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and malondialdehyde (MDA). Se-enriched L. plantarum can reduce the increase in the number of pathogenic Aeromonas caviae bacteria in the intestine caused by Cd stress and increase the number of Gemmobacter to regulate the microbial population. The results of this study show that Se-enriched L. plantarum dietary supplements can effectively protect Luciobarbus capito against Cd toxicity at subchronic levels.

Exposure of lead on intestinal structural integrity and the diversity of gut microbiota of common carp.

Lead is an environmental toxicant that has toxicity effect to the health of aquatic organisms. Gut microbiota has been reported to be closely related to human health. The aim of this study was to investigate the effects of lead exposure on the composition of gut microbiota. The composition of gut microbiota alteration was detected by 16S rRNA gene sequencing, following a 42-day exposure of lead (1 mg/L). The results showed that compared with the normal control group, the carp of lead group showed severe intestinal tissues injury and decreased Zona Occludens 1 (ZO-1) and occludin expression. The production of LPS in serum was increased by the treatment of lead exposure. Our results showed gut bacterial diversity in lead-treated common carp was lower than the control group. At the phylum level, the abundance of Bacteroidetes (LPS producing bacteria) and Fusobacteria in lead-treated carp were much higher than the control carp. And the abundance of Actinobacteria decreased by lead exposure. At the genus level, we found the abundance of Bacteroides (LPS producing bacteria) and Plesiomonas (an important pathogenic bacteria), increased significantly by lead exposure. And the abundance of Akkermansia, a critical probiotics, was markedly inhibited by lead exposure. In conclusion, this study indicated exposure of carp to lead causes gut microbiota alterations and intestinal structural integrity destruction.

Effect of selenium-rich Bacillus subtilis against mercury-induced intestinal damage repair and oxidative stress in common carp.

Mercury (Hg) poisoning in humans and fish represents a significant global problem. Hg is one of the most dangerous threats to the aquatic ecosystem due to its high toxicity. Mercury has a high oxidative stress-inducing potential, and can compounds exert toxic effects by interacting with many important enzymes involved in the regulation of antioxidants. Selenium (Se) supplementation can reactivate the mercury-inhibited enzymes viability. The probiotic Bacillus subtilis is widely used in aquaculture, and it has a certain adsorption effect on heavy metals. The interactions between Hg and Se have been rigorously investigated, particularly due to the observed protective effects of Se against Hg toxicity. The objective of this study was to evaluate whether Se-rich B. subtilis ameliorated Hg-induced toxicity in C. carpio var. specularis. Fish were exposed to waterborne Hg (0.03 mg/L) and fed a diet supplemented with 105 cfu/g Se-rich B. subtilis for 30 days. Fish were sampled, antioxidant activity, and Intestinal damage repair were assessed. Our results indicated that Se-rich B. subtilis protected the Intestinal from Hg-induced morphological changes. Hg treatment significantly decreased the activity levels of SOD, CAT and GSH-PX while increasing the activity levels of MDA, GST, and GSH. Hg treatment also upregulated the mRNA expression of Nrf2, CAT, GSH-PX and HO-1, and reduced expression of keap1. Se-rich B. subtilis had a significant protective effect against Hg-induced oxidative stress.

Effects of Fluorine on Intestinal Structural Integrity and Microbiota Composition of Common Carp.

Fluorine is an environmental toxicant and exposure of fluorine could induce various health disorders. Gut microbiota has been known to be involved in maintaining animal or human health. Therefore, in the present study, we aimed to evaluate the relationship between fluorine exposure and gut microbiota in common carp. Gut microbiota composition was detected by 16S rRNA gene sequencing. Intestinal structural integrity was assessed by hematoxylin-eosin staining and tight junction protection detection. The results showed that exposure of carp to fluorine led to the injury of intestinal tissues. And compared to the control group, the expression of tight junction protein ZO-1 and occludin was decreased. Meanwhile, the gut microbial diversity and composition were changed by fluorine exposure. At the phylum level, the abundance of Fusobacteria and Firmicutes increased significantly, and the abundance of Actinobacteria decreased markedly after treatment of fluorine. At the genus level, interestingly, we found the abundance of Plesiomonas, an important pathogenic bacteria, increased significantly by the treatment of fluorine. And the abundance of Akkermansia, a critical probiotics, was markedly inhibited by the treatment of fluorine. In conclusion, the results suggested fluorine exposure changed the gut microbiome composition and led to the damage of intestinal structural integrity.

Effects of Fluorine on Neutrophil Extracellular Trap Formation through Regulating AMPK/p38 Signaling Pathway.

Fluorine is an important trace element that is widely dispersed, and studies showed that fluorine could cause severe toxicity to fish. The aim of this study was to investigate the effects of fluorine on neutrophil extracellular trap (NET) formation in common carp and clarify the possible mechanism. The neutrophils were isolated and exposed to 0.25, 0.5, or 1 mM sodium fluoride (NaF). The results showed that NaF could induce the formation of NETs which exhibited a DNA-based network structure modified with histones and myeloperoxidase (MPO). Furthermore, NaF led to the production of reactive oxygen species (ROS) in neutrophils. Western blot results showed that NaF significantly increased the phosphorylation of AMPK and p38. In addition, our results showed that NaF-induced NET formation could be inhibited by an AMPK or p38 inhibitor. In conclusion, our results showed that NaF induced NET formation in neutrophils through regulation of the AMPK/p38 signaling pathway.

Transcriptome analysis reveals the mechanism of common carp brain injury after exposure to lead.

Lead, a widespread industrial pollutant, has been known as a powerful neurotoxin that could affect the central nervous system. Accumulating evidences demonstrated that lead exposure could result in the damage of brain tissues both in fish and human. However, the mechanism of lead induced brain injury has not been fully elucidated. The purpose of this study was to clarify the possible mechanism of common carp brain injury after exposure to lead through transcriptome analysis. Transcriptome analysis showed that 2141 differentially expressed genes were identified. Among these, 502 genes were up-regulated and 1639 genes were down-regulated. Meanwhile, GO enrichment analysis showed Transport, biological_process, DNA-templated (regulation of transcription) and signal transduction contained the most differential genes in the biological process. Furthermore, KEGG pathway enrichment analysis showed Ion channels, GnRH signaling pathway, cell adhesion molecules, Wnt signaling pathway, and calcium signaling pathway were significantly enriched. In addition, 10 differentially expressed genes were selected for qRT-PCR detection, and the results demonstrated that the selected genes exhibited the same trends with the RNA-Seq results, which indicates the transcriptome sequencing data is reliable. In conclusion, the above results provide a theoretical basis for clarifying the relationship between lead exposure and brain injury in common carp and for further studying of the genes related to lead poisoning.

Transcriptome analysis reveals the mechanism of fluorine exposure on memory loss of common carp.

Fluorine, an environmental toxicant in our daily life, has been reported to have adverse effects on nervous system. Previous studies demonstrated that fluorine exposure could induce brain injury in fish and human. However, the possible mechanism remains unclear. In the present study, we aimed to reveal the mechanism of fluorine exposure on brain injury of common carp through transcriptome analysis. In the fluorine-exposed carp, 444 brain genes were up-regulated, whereas 742 genes were down-regulated. DNA-templated (regulation of transcription) and multicellular organism development in the GO function annotation accounted for the most biological processes. Nucleus and membrane accounted for the most cellular components and DNA binding and metal ion binding accounted for the most molecular function. Meanwhile, 196 metabolic pathways were identified in Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway significant enrichment analysis, including long-term depression, Cushing syndrome, nuclear receptors, vascular smooth muscle contraction, Ion channels, and other pathways. Furthermore, we found that the up-regulated and down-regulated trends were similar between the quantitative real-time-PCR and RNA-Seq results, which indicate the transcriptome sequencing data is reliable. In conclusion, our data may provide insights into the mechanisms underlying brain injury induced by fluorine exposure.

Amelioration of Cd-Induced Oxidative Stress, MT Gene Expression, and Immune Damage by Vitamin C in Grass Carp Kidney Cells.

Cadmium (Cd) is the most common heavy metal and is easily detected in aquatic environments on a global scale. Vitamin C was a widely used vitamin in aquaculture. The aim of this study was to explore the potential of vitamin C in ameliorating Cd-induced toxicity in grass carp kidney (CIK) cells. Cell viability, oxidative response, metallothionein (MT), and immune-related gene expression was analyzed in the present study. The results show that cell viability was significantly reduced following Cd exposure, but the vitamin C supplementation attenuated the increased in cell viability. In addition, vitamin C supplementation can increase the antioxidation response and MT and immune-related gene expression. These results indicate that vitamin C has the potential to alleviate the effects of Cd toxicity in CIK cells.