Response signatures of intestinal microbiota and gene transcription of yellow catfish (Pelteobagrus fulvidraco) to Aeromonas hydrophila infection.

Bacterial intestinal inflammation is a common disease of yellow catfish (Pelteobagrus fulvidraco) in high-density aquaculture. Understanding the interactions between host and intestinal bacteria is helpful to intestinal inflammatory disease control. Here, we constructed a model of intestinal inflammation after Aeromonas hydrophila infection in yellow catfish, and characterized variations in gene expression and microbiome in the gut through high-throughput sequencing. Furthermore, host gene-microbiome interactions were identified. Histology observation showed disordered distribution of columnar epithelial cells and decrease of goblet cells in intestine. A total of 4741 genes showed differentially expression, mostly in comparisons between 12 hpi group with each other groups respectively, including control, 24 hpi and 48 hpi groups. These genes were enriched in immune-related pathways including the IL-17 signaling pathway, triggering strong inflammatory response at the invading stage within 12 h. Subsequently, the host strengthened energy consumption by activating carbohydrate and lipid metabolism pathways to repair the intestinal mucosal immune defense line. In addition, fish with A. hydrophila infection show decreased richness of gut microbial, reduced relative abundance of probiotics including Akkermansia, and elevated pathogenic bacteria such as Plesimonas. An integrative analysis identified A. hydrophila-related genes, such as il22 and stat3, for which expression level is close associated with the shift of A. hydrophila-related bacteria relative abundance, such as Akkermansia and Cetobacterium. Aside from picturing the variations of intestine gene expression and mucosal microbiome of yellow catfish coping with A. hydrophila infection, our study probed the underlying host-microbe interactions in A. hydrophila infection induced intestinal inflammatory, providing new insights for disease control in aquaculture.

Integrated Application of Multiomics Strategies Provides Insights Into the Environmental Hypoxia Response in Pelteobagrus vachelli Muscle.

Increasing pressures on aquatic ecosystems because of pollutants, nutrient enrichment, and global warming have severely depleted oxygen concentrations. This sudden and significant lack of oxygen has resulted in persistent increases in fish mortality rates. Revealing the molecular mechanism of fish hypoxia adaptation will help researchers to find markers for hypoxia induced by environmental stress. Here, we used a multiomics approach to identify several hypoxia-associated miRNAs, mRNAs, proteins, and metabolites involved in diverse biological pathways in the muscles of Pelteobagrus vachelli. Our findings revealed significant hypoxia-associated changes in muscles over 4 h of hypoxia exposure and discrete tissue-specific patterns. We have previously reported that P. vachelli livers exhibit increased anaerobic glycolysis, heme synthesis, erythropoiesis, and inhibit apoptosis when exposed to hypoxia for 4 h. However, the opposite was observed in muscles. According to our comprehensive analysis, fishes show an acute response to hypoxia, including activation of catabolic pathways to generate more energy, reduction of biosynthesis to decrease energy consumption, and shifting from aerobic to anaerobic metabolic contributions. Also, we found that hypoxia induced muscle dysfunction by impairing mitochondrial function, activating inflammasomes, and apoptosis. The hypoxia-induced mitochondrial dysfunction enhanced oxidative stress, apoptosis, and further triggered interleukin-1ß production via inflammasome activation. In turn, interleukin-1ß further impaired mitochondrial function or apoptosis by suppressing downstream mitochondrial biosynthesis-related proteins, thus resulting in a vicious cycle of inflammasome activation and mitochondrial dysfunction. Our findings contribute meaningful insights into the molecular mechanisms of hypoxia, and the methods and study design can be utilized across different fish species.

Assessing the ecotoxicity of florfenicol exposure at environmental levels: A case study of histology, apoptosis and microbiota in hepatopancreas of Eriocheir sinensis.

The intensification of production practices in the aquaculture industry has led to the indiscriminate use of antibiotics to combat diseases and reduce costs, which has resulted in environmental pollution, posing serious threats to aquaculture sustainability and food safety. However, the toxic effect of florfenicol (FF) exposure on the hepatopancreas of crustaceans remains unclear. Herein, by employing Chinese mitten crab (Eriocheir sinensis) as subjects to investigate the toxic effects on histopathology, oxidative stress, apoptosis and microbiota of hepatopancreas under environment-relevant (0.5 and 5 µg/L), and extreme concentrations (50 µg/L) of FF. Our results revealed that the damage of hepatopancreas tissue structure caused by FF exposure in a dose-and time-dependent manner. Combined with the increased expression of apoptosis-related genes (Caspase 3, Caspase 8, p53, Bax and Bcl-2) at mRNA and protein levels, activation of catalase (CAT) and superoxide dismutase (SOD), and malondialdehyde (MDA) accumulation, FF exposure also induced oxidative stress, and apoptosis in hepatopancreas. Interestingly, 7 days exposure triggered more pronounced toxic effect in crabs than 14 days under environment-relevant FF concentration. Integrated biomarker response version 2 (IBRv2) index indicated that 14 days FF exposure under extreme concentration has serious toxicity effect on crabs. Furthermore, 14 days exposure to FF changed the diversity and composition of hepatopancreas microbiota leading remarkable increase of pathogenic microorganism Spirochaetes following exposure to 50 µg/L of FF. Taken together, our study explained potential mechanism of FF toxicity on hepatopancreas of crustaceans, and provided a reference for the concentration of FF to be used in culture of Chinese mitten crab.

Construction of a high-density genetic linkage map and QTL mapping of growth and cold tolerance traits in Takifugu fasciatus.

Takifugu fasciatus is an aquaculture species with high economic value. In recent years, problems such as environmental pollution and inbreeding have caused a serious decline in T. fasciatus germplasm resources. In this study, a high-density genetic linkage map was constructed by whole-genome resequencing. The map consists of 4891 bin markers distributed across 22 linkage groups (LGs), with a total genetic coverage of 2381.353 cM and a mean density of 0.535 cM. Quantitative trait locus (QTL) localization analysis showed that a total of 19 QTLs associated with growth traits of T. fasciatus in the genome-wide significance threshold range, distributed on 11 LGs. In addition, 11 QTLs associated with cold tolerance traits were identified, each scattered on a different LG. Furthermore, we used QTL localization analysis to screen out three candidate genes (IGF1, IGF2, ADGRB) related to growth in T. fasciatus. Meanwhile, we screened three candidate genes (HSP90, HSP70, and HMGB1) related to T. fasciatus cold tolerance. Our study can provide a theoretical basis for the selection and breeding of cold-tolerant or fast-growing T. fasciatus.

Multi-omics analysis revealed the brain dysfunction induced by energy metabolism in Pelteobagrus vachelli under hypoxia stress.

Hypoxia in water environment has become increasingly frequent and serious due to global warming and environmental pollution. Revealing the molecular mechanism of fish hypoxia adaptation will help to develop markers of environmental pollution caused by hypoxia. Here, we used a multi-omics method to identify the hypoxia-associated mRNA, miRNA, protein, and metabolite involved in various biological processes in Pelteobagrus vachelli brain. The results showed that hypoxia stress caused brain dysfunction by inhibiting energy metabolism. Specifically, the biological processes involved in energy synthesis and energy consumption are inhibited in P. vachelli brain under hypoxia, such as oxidative phosphorylation, carbohydrate metabolism and protein metabolism. Brain dysfunction is mainly manifested as blood-brain barrier injury accompanied by neurodegenerative diseases and autoimmune diseases. In addition, compared with previous studies, we found that P. vachelli has tissue specificity in response to hypoxia stress and the muscle suffers more damage than the brain. This is the first report to the integrated analysis of the transcriptome, miRNAome, proteome, and metabolome in fish brain. Our findings could provide insights into the molecular mechanisms of hypoxia, and the approach could also be applied to other fish species. DATA AVAILABILITY: The raw data of transcriptome has been uploaded to NCBI database (ID: SUB7714154 and SUB7765255). The raw data of proteome has been uploaded to ProteomeXchange database (PXD020425). The raw data of metabolome has been uploaded to Metabolight (ID: MTBLS1888).

Effects of Florfenicol on Intestinal Histology, Apoptosis and Gut Microbiota of Chinese Mitten Crab (Eriocheir sinensis).

Excessive use of antibiotics in aquaculture causes residues in aquatic animal products and harms human health. However, knowledge of florfenicol (FF) toxicology on gut health and microbiota and their resulting relationships in economic freshwater crustaceans is scarce. Here, we first investigated the influence of FF on the intestinal health of Chinese mitten crabs, and then explored the role of bacterial community in FF-induced intestinal antioxidation system and intestinal homeostasis dysbiosis. A total of 120 male crabs (48.5 ± 4.5 g) were experimentally treated in four different concentrations of FF (0, 0.5, 5 and 50 µg/L) for 14 days. Responses of antioxidant defenses and changes of gut microbiota were assessed in the intestine. Results revealed that FF exposure induced significant histological morphology variation. FF exposure also enhanced immune and apoptosis characteristics in the intestine after 7 days. Moreover, antioxidant enzyme catalase activities showed a similar pattern. The intestinal microbiota community was analyzed based on full-length 16S rRNA sequencing. Only the high concentration group showed a marked decrease in microbial diversity and change in its composition after 14 days of exposure. Relative abundance of beneficial genera increased on day 14. These findings illustrate that exposure to FF could cause intestinal dysfunction and gut microbiota dysbiosis in Chinese mitten crabs, which provides new insights into the relationship between gut health and gut microbiota in invertebrates following exposure to persistent antibiotics pollutants.

Long-term hypoxia stress-induced oxidative stress, cell apoptosis, and immune response in the intestine of Pelteobagrus vachelli.

Hypoxia is a common phenomenon in aquaculture. With the dissolved oxygen (DO) 3.75 ± 0.25 mg O2 /L for hypoxia group and 7.25 ± 0.25 mg O2 /L for control group for 30, 60, and 90 days, long-term hypoxia stress was used to investigate the oxidative stress, apoptosis, and immunity in the intestine of Pelteobagrus vachelli. According to the results of measurement of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX), and catalase (CAT) activities and malondialdehyde (MDA) content, the oxidative stress ability of the intestine was activated at 30 days and impaired at 60 and 90 days. The upregulation of Bcl-2-associated x (Bax); downregulation of B cell lymphoma-2 (Bcl-2); increased activities of caspase-3, caspase-9, and Na+-K+-ATPase; decreased activities of succinate dehydrogenase (SDH); and the release of cytochrome c (Cyt-c) in mitochondria revealed that hypoxia induced the apoptosis. Moreover, heat shock protein 70 (HSP 70), heat shock protein 90 (HSP 90), immunoglobulin M (IgM), and C-lysozyme (C-LZM) were activated to inhibit apoptosis, but the immunoregulatory function might be damaged at 60 and 90 days. This study provides a theoretical foundation for understanding the mechanisms of hypoxia stress and aquaculture management of P. vachelli.

Integrated Analysis of Transcriptome and Metabolome Reveals Distinct Responses of Pelteobagrus fulvidraco against Aeromonas veronii Infection at Invaded and Recovering Stage.

Yellow catfish (Pelteobagrus fulvidraco) is an important aquaculture fish susceptible to Aeromonas veronii infection, which causes acute death resulting in huge economic losses. Understanding the molecular processes of host immune defense is indispensable to disease control. Here, we conducted the integrated and comparative analyses of the transcriptome and metabolome of yellow catfish in response to A. veronii infection at the invaded stage and recovering stage. The crosstalk between A. veronii-induced genes and metabolites uncovered the key biomarkers and pathways that strongest contribute to different response strategies used by yellow catfish at corresponding defense stages. We found that at the A. veronii invading stage, the immune defense was strengthened by synthesizing lipids with energy consumption to repair the skin defense line and accumulate lipid droplets promoting intracellular defense line; triggering an inflammatory response by elevating cytokine IL-6, IL-10 and IL-1ß following PAMP-elicited mitochondrial signaling, which was enhanced by ROS produced by impaired mitochondria; and activating apoptosis by up-regulating caspase 3, 7 and 8 and Prostaglandin F1α, meanwhile down-regulating FoxO3 and BCL6. Apoptosis was further potentiated via oxidative stress caused by mitochondrial dysfunction and exceeding inflammatory response. Additionally, cell cycle arrest was observed. At the fish recovering stage, survival strategies including sugar catabolism with D-mannose decreasing; energy generation through the TCA cycle and Oxidative phosphorylation pathways; antioxidant protection by enhancing Glutathione (oxidized), Anserine, and α-ketoglutarate; cell proliferation by inducing Cyclin G2 and CDKN1B; and autophagy initiated by FoxO3, ATG8 and ATP6V1A were highlighted. This study provides a comprehensive picture of yellow catfish coping with A. veronii infection, which adds new insights for deciphering molecular mechanisms underlying fish immunity and developing stage-specific disease control techniques in aquaculture.

Integrated Analysis of Transcriptomic, miRNA and Proteomic Changes of a Novel Hybrid Yellow Catfish Uncovers Key Roles for miRNAs in Heterosis.

Heterosis is a complex biological phenomenon in which hybridization produces offspring that exhibit superior phenotypic characteristics compared with the parents. Heterosis is widely utilized in agriculture, for example in fish farming; however, its underlying molecular basis remains elusive. To gain a comprehensive and unbiased molecular understanding of fish heterosis, we analyzed the mRNA, miRNA, and proteomes of the livers of three catfish species, Pelteobagrus fulvidraco, P. vachelli, and their hybrid, the hybrid yellow catfish "Huangyou-1" (P. fulvidraco ♀ × P. vachelli ♂). Using next-generation sequencing and mass spectrometry, we show that the nonadditive, homoeolog expression bias and expression level dominance pattern were readily identified at the transcriptional, post-transcriptional, or protein levels, providing the evidence for the widespread presence of dominant models during hybridization. A number of predicted miRNA-mRNA-protein pairs were found and validated by qRT-PCR and PRM assays. Furthermore, several diverse key pathways were identified, including immune defense, metabolism, digestion and absorption, and cell proliferation and development, suggesting the vital mechanisms involved in the generation of the heterosis phenotype in progenies. We propose that the high parental expression of genes/proteins (growth, nutrition, feeding, and disease resistance) coupled with low parental miRNAs of the offspring, are inherited from the mother or father, thus indicating that the offspring were enriched with the advantages of the father or mother. We provide new and important information about the molecular mechanisms of heterosis, which represents a significant step toward a more complete elucidation of this phenomenon.

Effects of acute hypoxia and reoxygenation on oxygen sensors, respiratory metabolism, oxidative stress, and apoptosis in hybrid yellow catfish "Huangyou-1".

The regulation mechanism of the hybrid yellow catfish "Huangyou-1" was assessed under conditions of hypoxia and reoxygenation by examination of oxygen sensors and by monitoring respiratory metabolism, oxidative stress, and apoptosis. The expressions of genes related to oxygen sensors (HIF-1α, HIF-2α, VHL, HIF-1ß, PHD2, and FIH-1) were upregulated in the brain and liver during hypoxia, and recovered compared with control upon reoxygenation. The expressions of genes related to glycolysis (HK1, PGK1, PGAM2, PFK, and LDH) were increased during hypoxia and then recovered compared with control upon reoxygenation. The mRNA levels of CS did not change during hypoxia in the brain and liver, but increased during reoxygenation. The mRNA levels of SDH decreased significantly only in the liver during hypoxia, but later increased compared with control upon reoxygenation in both tissues. Under hypoxic conditions, the expressions of genes related to oxidative stress (SOD1, SOD2, GSH-Px, and CAT) and the activity of antioxidant enzymes (SOD, CAT, and GSH-Px) and MDA were upregulated compared with control. The expressions of genes related to apoptosis (Apaf-1, Bax, Caspase 3, Caspase 9, and p53) were higher than those in control during hypoxic exposure, while the expressions of Bcl-2 and Cyt C were decreased. The findings of the transcriptional analyses will provide insights into the molecular mechanisms of hybrid yellow catfish "Huangyou-1" under conditions of hypoxia and reoxygenation. Overall, these findings showed that oxygen sensors of "Huangyou-1" are potentially useful biomarkers of environmental hypoxic exposure. Together with genes related to respiratory metabolism, oxidative stress and apoptosis occupy a quite high position in enhancing hypoxia tolerance. Our findings provided new insights into the molecular regulatory mechanism of hypoxia in "Huangyou-1."

A high-density SNP-based genetic map and several economic traits-related loci in Pelteobagrus vachelli.

BACKGROUND: A high-density genetic linkage map is essential for QTL fine mapping, comparative genome analysis, identification of candidate genes and marker-assisted selection in aquaculture species. Pelteobagrus vachelli is a very popular commercial species in Asia. However, some specific characters hindered achievement of the traditional selective breeding based on phenotypes, such as lack of large-scale genomic resource and short of markers tightly associated with growth, sex determination and hypoxia tolerance related traits. RESULTS: By making use of 5059 ddRAD markers in P. vachelli, a high-resolution genetic linkage map was successfully constructed. The map' length was 4047.01 cM by using an interval of 0.11 cm, which is an average marker standard. Comparative genome mapping revealed that a high proportion (83.2%) of markers with a one-to-one correspondence were observed between P. vachelli and P. fulvidraco. Based on the genetic map, 8 significant genome-wide QTLs for 4 weight, 1 body proportion, 2 sex determination, and 1 hypoxia tolerance related traits were detected on 4 LGs. Some SNPs from these significant genome-wide QTLs were observably associated with these phenotypic traits in other individuals by Kompetitive Allele Specific PCR. In addition, two candidate genes for weight, Sipa1 and HSD11B2, were differentially expressed between fast-, medium- and slow-growing P. vachelli. Sema7a, associated with hypoxia tolerance, was induced after hypoxia exposure and reoxygenation. CONCLUSIONS: We mapped a set of suggestive and significant QTLs as well as candidate genes for 12 growth, 1 sex determination and 1 hypoxia tolerance related traits based on a high-density genetic linkage map by making use of SNP markers for P. fulvidraco. Our results have offered a valuable method about the much more efficient production of all-male, fast growth and hypoxia tolerance P. vachelli for the aquaculture industry.

Integrated application of multi-omics provides insights into cold stress responses in pufferfish Takifugu fasciatus.

BACKGROUND: T. fasciatus (Takifugu fasciatus) faces the same problem as most warm water fish: the water temperature falls far below the optimal growth temperature in winter, causing a massive death of T. fasciatus and large economic losses. Understanding of the cold-tolerance mechanisms of this species is still limited. Integrated application of multi-omics research can provide a wealth of information to help us improve our understanding of low-temperature tolerance in fish. RESULTS: To gain a comprehensive and unbiased molecular understanding of cold-tolerance in T. fasciatus, we characterized mRNA-seq and metabolomics of T. fasciatus livers using Illumina HiSeq 2500 and UHPLC-Q-TOF MS. We identified 2544 up-regulated and 2622 down-regulated genes in the liver of T. fasciatus. A total of 40 differential metabolites were identified, including 9 down-regulated and 31 up-regulated metabolites. In combination with previous studies on proteomics, we have established an mRNA-protein-metabolite interaction network. There are 17 DEMs (differentially-expressed metabolites) and 14 DEGs-DEPs (differentially co-expressed genes and proteins) in the interaction network that are mainly involved in fatty acids metabolism, membrane transport, signal transduction, and DNA damage and defense. We then validated a number of genes in the interaction network by qRT-PCR. Additionally, a number of SNPs (single nucleotide polymorphisms) were revealed through the transcriptome data. These results provide key information for further understanding of the molecular mechanisms of T. fasciatus under cold stress. CONCLUSION: The data generated by integrated application of multi-omics can facilitate our understanding of the molecular mechanisms of fish response to low temperature stress. We have not only identified potential genes and SNPs involved in cold tolerance, but also show that some nutrient metabolites may be added to the diet to help the overwintering of T. fasciatus.

iTRAQ analysis of liver immune-related proteins from darkbarbel catfish (Pelteobagrus vachelli) infected with Edwardsiella ictaluri.

Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), a major disease occurring in these siluriform fish. As the liver is an important organ for defending against bacterial pathogens in fish, this study aimed to determine the liver immune response at the protein level. The differential proteomes of the darkbarbel catfish liver in response to E. ictaluri infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using a 1.2-fold change in expression as a physiologically significant benchmark, a total of 819 differentially expressed proteins were reliably quantified using iTRAQ analysis, including 6 up-regulated proteins and 813 down-regulated proteins. GO enrichment analysis indicated that the "complement activation, alternative pathway" and "complement activation, classical pathway" were significantly enriched. KEGG enrichment analysis indicated the "antigen processing and presentation" and "bacterial secretion system" were significantly enriched. We selected the 6 up-regulated proteins and 10 immune-related down-regulated proteins for validation using real-time PCR. The 10 immune-related proteins included complement component C1r, C3, C5, C7, and C9 and plasma protease C1 inhibitor (C1-INH), signal recognition particle 54 kDa protein (SRP54), SRP receptor, proteasome activator complex subunit 1 (PSME1) and major histocompatibility complex class I (MHC class I) were selected from the GO clusters and KEGG pathways. The variations in mRNA expression for these genes were similar to the results of iTRAQ. This is the first report detailing the proteome response in the darkbarbel catfish liver during E. ictaluri infection and markedly contributes to our understanding of the defense mechanisms in the livers of darkbarbel catfish.

Copper nanoparticles induced oxidation stress, cell apoptosis and immune response in the liver of juvenile Takifugu fasciatus.

Copper nanoparticles (Cu NPs) are a new pollutant in aquaculture, representing a hazard to aquatic organisms. We investigated the effects of Cu NPs exposure on oxidative stress, apoptosis and immune response in an economically important model species, Takifugu fasciatus. The juvenile fish were exposed to control, 20 or 100 µg Cu NPs/L for 30 days. The growth of T. fasciatus was inhibited after Cu NPs exposure. Copper accumulation in liver increased with increasing Cu NPs dose. Oxidative stress indicators [malondialdehyde (MDA), total superoxide dismutase (T-SOD), catalase (CAT) and glutathione (GSH)], apoptosis index and activities of caspases (caspase-3, caspase-9) were all increased with the increase of Cu NPs concentration in liver. With an increase in Cu NPs dose, the activities of succinate dehydrogenase (SDH) and Na+-K+-ATPase as well as cytochrome c (Cyt-c) concentration in mitochondria decreased, accompanied by increased Cyt-c concentration in cytosol. Apoptosis-related gene expressions of p53, caspase-3, caspase-9 and Bax were increased with the increase of Cu NPs dose. However, the opposite result was found in Bcl2 expression. The physiological indicators of immune response [heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), immunoglobulin M (IgM) and lysozyme (LZM)] as well as the mRNA levels of HSP70, HSP90, IgM and C-LZM were all increased after Cu NPs exposure. Our results will be helpful in understanding the mechanism of Cu NPs toxicity in T. fasciatus.

Studies of the mechanism of fatty liver formation in Takifugu fasciatus following copper exposure.

Fatty liver is widely observed during Takifugu fasciatus production, but the mechanisms underlying fatty liver formation remain unknown. The present study was conducted to determine the potential effects of copper (Cu) on hepatic lipid deposition and metabolism in T. fasciatus after 21 days of exposure to Cu (levels: 0, 20 and 100 µg/L). Copper exposure decreased the weight gain rate (WG) in T. fasciatus, but increased the values of the viscerosomatic index (VSI) and hepatosomatic index (HSI) compared with the control. The time-dependent Cu accumulation in tissues increased as the Cu concentration increased. The order of Cu accumulation was liver > intestine > muscle. The lipid content, triglyceride (TG) content and lipoprotein lipase (LPL) activity increased after Cu exposure compared with the control. In addition, more lipid droplets and greater vacuolization were observed in the liver after exposure to 20 µg/L Cu than after 100 µg/L Cu. The expression of genes involved in lipogenesis (g6pd, 6pgd, lpl, fas and acc), lipolysis (hsl and cpt 1) and transcription (ppar α and ppar ©) was dependent on Cu. An analysis of the intestinal microbiome community showed that the highest values of the Chao 1 index, ACE, Shannon index and Simpson index were obtained in fish exposed to 20 µg/L Cu, whereas the lowest values were obtained after the 100 µg/L Cu treatment. The Principal Coordinates Analysis (PCoA) plots of the data revealed structural differences in the groups treated with Cu compared with the control group. At the phylum level, the intestinal microbiota in the Cu-treated and control fish were dominated by Proteobacteria and Bacteroidetes. The higher Firmicutes to Bacteroidetes ratio was observed in fish treated with 20 µg/L Cu compared with other groups, while the lowest ratio was observed in fish exposed to 100 µg/L Cu. Our study revealed the mechanisms by which Cu exposure altered (i) lipid deposition in the body and (ii) the intestinal microbiome, which may contribute to maintain the health status of T. fasciatus for the aquaculture.

Individual and combined effects of salinity and lipopolysaccharides on the immune response of juvenile Takifugu fasciatus.

Lipopolysaccharides (LPS) and salinity are important variables in aquatic environments. High concentration of LPS and large changes in salinity seriously threat the survival of a variety of organisms, including fish. To reveal the effects of salinity and LPS on a fish immune response, we measured the immune-related parameters (total leukocyte count, total serum protein, albumin and globulin concentrations, complement C3 concentration, and lysozyme activity) and genes (the expressions of TNF-α, IL-1ß, and SOCS1-3 at the mRNA and protein levels) of juvenile Takifugu fasciatus exposed to phosphate buffered saline (PBS) or LPS (25 µg mL-1) under different salinities (0, 15, and 30 ppt) for 24 h. Changes in key immunological indicators suggested that the LPS challenge induced considerable damage to T. fasciatus, whereas an increase in salinity mitigated the harmful effects. Moreover, although the immune responses in blood and other selected tissues (gill and kidney) were suppressed with an increase in salinity, the increased response in liver in saltwater enabled T. fasciatus to conquer large salinity variation during migration. The appropriate addition of salts appeared to be a sensible strategy to mitigate LPS-induced toxicity in the aquaculture of T. fasciatus.

The improved energy metabolism and blood oxygen-carrying capacity for pufferfish, Takifugu fasciatus, against acute hypoxia under the regulation of oxygen sensors.

Hypoxia frequently occurs in aquatic ecosystem, which is influenced by salinity, water temperature, weather, and surface water runoff. In order to shed further light on the evolutionary and adaptive mechanisms in fish under hypoxic condition, the impact of acute hypoxia (1.63 ± 0.2 mg/L) and reoxygenation (7.0 ± 0.3 mg/L) on oxygen sensors, energy metabolism, and hematological indices was evaluated in Takifugu fasciatus. Data from transcriptional level analysis show that the expressions of genes related to oxygen sensors (HIF-1α, PHD2, and VHL) were upregulated in the brain and liver under hypoxia and recovered under reoxygenation. The upregulation of GLUT2, VEGF-A, and EPO in conjugation with VEGF-A protein and hematological indices conferred the rapid adjustments of cellular glucose uptake and blood oxygen-carrying capacities in pufferfish. Higher levels of glycolysis-related mRNAs (HK, PGK1, and PGAM2), HK activity, and proteins (PGK1 and PGAM2) were detected in the brain and liver under hypoxic condition compared with control. Interestingly, the expression of MDH1 at the mRNA, enzyme activity, and protein levels was significantly increased in the brain at 0 or 2 h and in the liver at 8 h under hypoxic condition. In addition, although the enzyme activity and mRNA expression of LDH in the brain were not significantly changed, a persistent upregulation was observed in the liver during hypoxia exposure. This study demonstrated that pufferfish could counterpoise the energetic demands and hematological functional properties evoked by oxygen sensors after hypoxia. Our findings provided new insights into the molecular regulatory mechanism of hypoxia in pufferfish.

Identification of immune-related genes in gill cells of Japanese eels (Anguilla japonica) in adaptation to water salinity changes.

The changes in ambient salinity influence ion and water homeostasis, hormones secretion, and immune response in fish gills. The physiological functions of hormones and ion transporters in the regulation of gill-osmoregulation have been widely studied, however the modulation of immune response under salinity changes is not determined. Using transcriptome sequencing, we obtained a comprehensive profile of osmo-responsive genes in gill cells of Japanese eel (Anguilla japonica). Herein, we applied bioinformatics analysis to identify the immune-related genes that were significantly higher expressed in gill pavement cells (PVCs) and mitochondrial-rich cells (MRCs) in freshwater (FW) than seawater (SW) adapted fish. We validated the data using the real-time qPCR, which showed a high correlation between the RNA-seq and real-time qPCR data. In addition, the immunohistochemistry results confirmed the changes of the expression of selected immune-related genes, including C-reactive protein (CRP) in PVCs, toll-like receptor 2 (TLR2) in MRCs and interleukin-1 receptor type 2 (IL-1R2) in both PVCs and MRCs. Collectively our results demonstrated that those immune-related genes respond to salinity changes, and might trigger related special signaling pathways and network. This study provides new insights into the impacts of ambient salinity changes on adaptive immune response in fish gill cells.

Physiological mechanism of osmoregulatory adaptation in anguillid eels.

In recent years, the production of eel larvae has dramatic declines due to reductions in spawning stocks, overfishing, growth habitat destruction and access reductions, and pollution. Therefore, it is particularly important and urgent for artificial production of glass eels. However, the technique of artificial hatching and rearing larvae is still immature, which has long been regarded as an extremely difficult task. One of the huge gaps is artificial condition which is far from the natural condition to develop their capability of osmoregulation. Thus, understanding their osmoregulatory mechanisms will help to improve the breed and adapt to the changes in the environment. In this paper, we give a general review for a study progress of osmoregulatory mechanisms in eels from five aspects including tissues and organs, ion transporters, hormones, proteins, and high throughput sequencing methods.

Comparative iTRAQ-Based Quantitative Proteomic Analysis of Pelteobagrus vachelli Liver under Acute Hypoxia: Implications in Metabolic Responses.

More and more frequently these days, aquatic ecosystems are being stressed by nutrient enrichment, pollutants, and global warming, leading to a serious depletion in oxygen concentrations. Although a sudden, significant lack of oxygen will result in mortality, fishes can have an acute behavior (e.g., an increase in breathing rate, reduction in swimming frequency) and physiology responses (e.g., increase in oxygen delivery, and reduction in oxygen consumption) to hypoxia, which allows them to maintain normal physical activity. Therefore, in order to shed further light on the molecular mechanisms of hypoxia adaptation in fishes, the authors conduct comparative quantitative proteomics on Pelteobagrus vachelli livers using iTRAQ. The research identifies 511 acute hypoxia-responsive proteins in P. vachelli. Furthermore, comparison of several of the diverse key pathways studied (e.g., peroxisome pathway, PPAR signaling pathway, lipid metabolism, glycolysis/gluco-neogenesis, and amino acid metabolism) help to articulate the different mechanisms involved in the hypoxia response of P. vachelli. Data from proteome analysis shows that P. vachelli can have an acute reaction to hypoxia, including detoxification of metabolic by-products and oxidative stress in light of continued metabolic activity (e.g., peroxisomes), an activation in the capacity of catabolism to get more energy (e.g., lipolysis and amino acid catabolism), a depression in the capacity of biosynthesis to reduce energy consumption (e.g., biosynthesis of amino acids and lipids), and a shift in the aerobic and anaerobic contributions to total metabolism. The observed hypoxia-related changes in the liver proteome of the fish can help to understand or can be related to the hypoxia-related response that takes place in similar conditions in the liver or other proteomes of mammals.