DC-SIGN of Largemouth Bass (Micropterus salmoides) Mediates Immune Functions against Aeromonas hydrophila through Collaboration with the TLR Signaling Pathway.

C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of Micropterus salmoides was identified. MsDC-SIGN is classified as a type II transmembrane protein. The extracellular segment of MsDC-SIGN possesses a coiled-coil region and a carbohydrate recognition domain (CRD). The key amino acid motifs of the extracellular CRD of MsDC-SIGN in Ca2+-binding site 2 were EPN (Glu-Pro-Asn) and WYD (Trp-Tyr-Asp). MsDC-SIGN-CRD can bind to four pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), glucan, peptidoglycan (PGN), and mannan. Moreover, it can also bind to Gram-positive, Gram-negative bacteria, and fungi. Its CRD can agglutinate microbes and displays D-mannose and D-galactose binding specificity. MsDC-SIGN was distributed in seven tissues of the largemouth bass, among which the highest expression was observed in the liver, followed by the spleen and intestine. Additionally, MsDC-SIGN was present on the membrane of M. salmoides leukocytes, thereby augmenting the phagocytic activity against bacteria. In a subsequent investigation, the expression patterns of the MsDC-SIGN gene and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) exhibited an up-regulated expression response to the stimulation of Aeromonas hydrophila. Furthermore, through RNA interference of MsDC-SIGN, the expression level of the DC-SIGN signaling pathway-related gene (RAF1) and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) was decreased. Therefore, MsDC-SIGN plays a pivotal role in the immune defense against A. hydrophila by modulating the TLR signaling pathway.

Integrated multi-omics analysis reveals liver metabolic reprogramming by fish iridovirus and antiviral function of alpha-linolenic acid.

Iridovirus poses a substantial threat to global aquaculture due to its high mortality rate; however, the molecular mechanisms underpinning its pathogenesis are not well elucidated. Here, a multi-omics approach was applied to groupers infected with Singapore grouper iridovirus (SGIV), focusing on the roles of key metabolites. Results showed that SGIV induced obvious histopathological damage and changes in metabolic enzymes within the liver. Furthermore, SGIV significantly reduced the contents of lipid droplets, triglycerides, cholesterol, and lipoproteins. Metabolomic analysis indicated that the altered metabolites were enriched in 19 pathways, with a notable down-regulation of lipid metabolites such as glycerophosphates and alpha-linolenic acid (ALA), consistent with disturbed lipid homeostasis in the liver. Integration of transcriptomic and metabolomic data revealed that the top enriched pathways were related to cell growth and death and nucleotide, carbohydrate, amino acid, and lipid metabolism, supporting the conclusion that SGIV infection induced liver metabolic reprogramming. Further integrative transcriptomic and proteomic analysis indicated that SGIV infection activated crucial molecular events in a phagosome-immune depression-metabolism dysregulation-necrosis signaling cascade. Of note, integrative multi-omics analysis demonstrated the consumption of ALA and linoleic acid (LA) metabolites, and the accumulation of L-glutamic acid (GA), accompanied by alterations in immune, inflammation, and cell death-related genes. Further experimental data showed that ALA, but not GA, suppressed SGIV replication by activating antioxidant and anti-inflammatory responses in the host. Collectively, these findings provide a comprehensive resource for understanding host response dynamics during fish iridovirus infection and highlight the antiviral potential of ALA in the prevention and treatment of iridoviral diseases.

Participation of Hepcidins in the Inflammatory Response Triggered by λ-Carrageenin in Gilthead Seabream (Sparus aurata).

The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. While the expression of most of the genes studied was upregulated, the expression of ferroportin gene (slc40a) was downregulated. In the second part of the study, seabream specimens were injected intramuscularly with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b, and ferroportin genes (hamp1, fthb, and slc40a) was upregulated in the head kidney of fish from the λ-carrageenin-injected group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was downregulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J, and Hamp2D (hamp2.5/6) genes was downregulated in the λ-carrageenin-injected group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was upregulated in the λ-carrageenin-injected group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. The primary sequence of hepcidin was conserved among the different mature peptides, although changes in specific amino acid residues were identified. These changes affected the charge, hydrophobicity, and probability of hepcidins being antimicrobial peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.

GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination.

Grass carp reovirus (GCRV), particularly the highly prevalent type II GCRV (GCRV-II), causes huge losses in the aquaculture industry. However, little is known about the mechanisms by which GCRV-II invades grass carp and further disseminates among tissues. In the present study, monocytes/macrophages (Mo/Mφs) were isolated from the peripheral blood of grass carp and infected with GCRV-II. The results of indirect immunofluorescent microscopy, transmission electron microscopy, real-time quantitative RT-PCR (qRT-PCR), western blot (WB), and flow cytometry analysis collectively demonstrated that GCRV-II invaded Mo/Mφs and replicated in them. Additionally, we observed that GCRV-II induced different types (M1 and M2) of polarization of Mo/Mφs in multiple tissues, especially in the brain, head kidney, and intestine. To assess the impact of different types of polarization on GCRV-II replication, we recombinantly expressed and purified the intact cytokines CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B and successfully induced M1 and M2 type polarization of macrophages using these cytokines through in vitro experiments. qRT-PCR, WB, and flow cytometry analyses showed that M2 macrophages had higher susceptibility to GCRV-II infection than other types of Mo/Mφs. In addition, we found GCRV-II induced apoptosis of Mo/Mφs to facilitate virus replication and dissemination and also detected the presence of GCRV-II virus in plasma. Collectively, our findings indicated that GCRV-II could invade immune cells Mo/Mφs and induce apoptosis and polarization of Mo/Mφs for efficient infection and dissemination, emphasizing the crucial role of Mo/Mφs as a vector for GCRV-II infection.IMPORTANCEType II grass carp reovirus (GCRV) is a prevalent viral strain and causes huge losses in aquaculture. However, the related dissemination pathway and mechanism remain largely unclear. Here, our study focused on phagocytic immune cells, monocytes/macrophages (Mo/Mφs) in blood and tissues, and explored whether GCRV-II can invade Mo/Mφs and replicate and disseminate via Mo/Mφs with their differentiated type M1 and M2 macrophages. Our findings demonstrated that GCRV-II infected Mo/Mφs and replicated in them. Furthermore, GCRV-II infection induces an increased number of M1 and M2 macrophages in grass carp tissues and a higher viral load in M2 macrophages. Furthermore, GCRV-II induced Mo/Mφs apoptosis to release viruses, eventually infecting more cells. Our study identified Mo/Mφs as crucial components in the pathway of GCRV-II dissemination and provides a solid foundation for the development of treatment strategies for GCRV-II infection.

Stimulatory effect of dietary alpha-lipoic acid on growth performance, antioxidant capacity, liver enzymes, immunity and protection of African catfish, Clarias gariepinus (B.), Edwardsiella tarda infection.

Edwardsiella tarda is one of the most common causes of fish diseases that hinder aquaculture. Oxidative stress in farm animals can induce a number of pathological disorders, production and general animal welfare. The use of exogenous dietary nonenzymatic antioxidants such as alpha-lipoic acid (ALA) can stop a pro-oxidant state and thus appears to have the potential to modulate the immune system and protect fish from bacterial infection. Thus, this study investigates the stimulatory effect of dietary ALA on growth performance, antioxidant capacity, liver enzymes, immunity and protection of African catfish, Clarias gariepinus (B.), against an infection with E. tarda. Five isonitrogenous and isocaloric diets (400 g/kg of crude protein) containing ALA at doses of 0.0 (control), 500, 1000, 1500 or 2000 mg/kg diet were served to 300 juveniles of African catfish (mean weight = 8.2 ± 0.2 g) adequately thrice per day for 12 weeks. Thereafter, 0.1 mL of E. tarda (ATCC 15947; 1.0 × 108 CFU/mL) was intraperitoneally injected into 10 fish from each tank and was monitored for 14 days. The results showed that ALA-fortified diets significantly boosted the fish growth, feed consumption and utilization and feed conversion ratio but no did not affect fish survival rate. The highest final fish weight (g), weight growth (g) and weight gain (%) were all considerably higher in fish fed with ALA-fortified diets (p < 0.05), especially from 1000 to 200 mg/kg ALA than the control group. Also, an enhanced hemato-biochemical, antioxidant and immune indices were noticed in African catfish-fed ALA-enriched diets. In a dose-dependent order, the levels of haematological indices such Ht, Hb, RBCs, WBCs and platelets were markedly increased (p < 0.05). Additionally, fish fed with ALA-based diets showed substantial (p < 0.05) declines in aspartate and alanine aminotransferase values, with the lowest values being found in the 2000 mg/kg diet while control group had highest values. Further, African catfish fed the feed fortified with 2000 mg ALA/kg diet showed the highest levels of lysozyme, respiratory burst, proteases and esterase activities (p < 0.05). Following exposure of fish to E. tarda infection, a significant reduction in the mortality was obtained in African catfish fed with ALA-based diets, especially from 1500 to 2000 mg ALA/kg diet (3.3%); while fish fed with the control diet had highest mortality (86.7%). Therefore, diets supplemented with ALA evoked fish growth performance, antioxidants and nonspecific immunity of African catfish. Also, resistance of African catfish to E. Tarda infection were raised when fed ALA-fortified diets at optimum inclusion rate of 1300 mg ALA/kg diet.

EFHD2 cooperates with E3 ubiquitin ligase Smurf1 to facilitate virus infection by promoting the degradation of TRAF6 in teleost fish.

The ubiquitin-proteasome system is one of the most important protein stability regulation systems. It can precisely regulate host immune responses by targeting signaling proteins. TRAF6 is a crucial E3 ubiquitin ligase in host antiviral signaling pathway. Here, we discovered that EF-hand domain-containing protein D2 (EFHD2) collaborated with the E3 ubiquitin ligase Smurf1 to potentiate the degradation of TRAF6, hence facilitating RNA virus Siniperca chuatsi rhabdovirus infection. The mechanism analysis revealed that EFHD2 interacted with Smurf1 and enhanced its protein stability by impairing K48-linked polyubiquitination of Smurf1, thereby promoting Smurf1-catalyzed degradation of TRAF6. This study initially demonstrated a novel mechanism by which viruses utilize host EFHD2 to achieve immune escape and provided a new perspective on the exploration of mammalian innate immunity.IMPORTANCEViruses induce host cells to activate several antiviral signaling pathways. TNF receptor-associated factor 6 (TRAF6) plays an essential role in these pathways. Numerous studies have been done on the mechanisms of TRAF6-mediated resistance to viral invasion. However, little is known about the strategies that viruses employ to antagonize TRAF6-mediated antiviral signaling pathway. Here, we discovered that EFHD2 functions as a host factor to promote viral replication. Mechanistically, EFHD2 potentiates Smurf1 to catalyze the ubiquitin-proteasomal degradation of TRAF6 by promoting the deubiquitination and stability of Smurf1, which in turn inhibits the production of proinflammatory cytokines and interferons. Our study also provides a new perspective on mammalian resistance to viral invasion.

Dissecting the early and late endosomal pathways of Singapore grouper iridovirus by single-particle tracking in living cells.

Iridoviruses are large DNA viruses that infect a wide range of invertebrates and lower vertebrates, causing serious threats to ecological security and aquaculture industry worldwide. However, the mechanisms underlying intracellular transport of iridovirus remain unknown. In this study, the transport of Singapore grouper iridovirus (SGIV) in early endosomes (EEs) and late endosomes (LEs) was explored by single-particle tracking technology. SGIV employs EEs to move rapidly from the cell membrane to the nucleus, and this long-range transport is divided into "slow-fast-slow" stages. SGIV within LEs mainly underwent oscillatory movements near the nucleus. Furthermore, SGIV entered newly formed EEs and LEs, respectively, possibly based on the interaction between the viral major capsid protein and Rab5/Rab7. Importantly, interruption of EEs and LEs by the dominant negative mutants of Rab5 and Rab7 significantly inhibited the movement of SGIV, suggesting the important roles of Rab5 and Rab7 in virus transport. In addition, it seems that SGIV needs to enter clathrin-coated vesicles to move from actin to microtubules before EEs carry the virus moving along microtubules. Together, our results for the first time provide a model whereby iridovirus transport depending on EEs and LEs, helping to clarify the mechanism underlying iridovirus infection, and provide a convenient tactic to investigate the dynamic infection of large DNA virus.

Eukaryotic translation elongation factor 1 alpha (eEF1A) inhibits Siniperca chuatsi rhabdovirus (SCRV) infection through two distinct mechanisms.

IMPORTANCE: Although a virus can regulate many cellular responses to facilitate its replication by interacting with host proteins, the host can also restrict virus infection through these interactions. In the present study, we showed that the host eukaryotic translation elongation factor 1 alpha (eEF1A), an essential protein in the translation machinery, interacted with two proteins of a fish rhabdovirus, Siniperca chuatsi rhabdovirus (SCRV), and inhibited virus infection via two different mechanisms: (i) inhibiting the formation of crucial viral protein complexes required for virus transcription and replication and (ii) promoting the ubiquitin-proteasome degradation of viral protein. We also revealed the functional regions of eEF1A that are involved in the two processes. Such a host protein inhibiting a rhabdovirus infection in two ways is rarely reported. These findings provided new information for the interactions between host and fish rhabdovirus.

A novel protein encoded by circVPS13D attenuates antiviral innate immunity by targeting MAVS in teleost fish.

IMPORTANCE: The expression of circVPS13D was upregulated with SCRV invasion, which proved that circVPS13D was involved in the regulation of the antiviral immune response. Our study revealed that the existence of circVPS13D promoted the replication of SCRV. Functionally, circVPS13D negatively regulates the antiviral responses of fish. Mechanistically, we confirmed that circVPS13D inhibited RLRs antiviral signaling pathway via the encoded protein VPS13D-170aa by targeting MAVS. Our study provided novel insights into the roles of protein-coding circRNAs and supported VPS13D-170aa as a negative regulator in the antiviral immune responses of teleost fish.

A C-type lectin-like receptor CD302 in yellow drum (Nibea albiflora) functioning in antibacterial activity and innate immune signaling.

Molecular dissection of disease resistance against Vibrio harveyi infection in yellow drum at the genome-wide level uncovered a C-type lectin-like receptor cluster of differentiation CD302 (named as YdCD302) in our previous study. Here, the gene expression pattern of YdCD302 and its function in mediating the defense response to V. harveyi attack were investigated. Gene expression analysis demonstrated that YdCD302 was ubiquitously distributed in various tissues with the highest transcript abundance in liver. The YdCD302 protein exhibited agglutination and antibacterial activity against V. harveyi cells. Binding assay indicated that YdCD302 can physically interact with V. harveyi cells in a Ca2+-independent manner, and the interaction can activate reactive oxygen species (ROS) production in the bacterial cells to induce RecA/LexA-mediated cell death. After infection with V. harveyi, the expression of YdCD302 can be up-regulated significantly in the main immune organs of yellow drum and potentially further trigger the cytokines involved innate immunity. These findings provide insight into the genetic basis of the disease resistance trait in yellow drum and shed light on the functioning of the CD302 C-type lectin-like receptor in host-pathogen interactions. The molecular and functional characterization of YdCD302 is a significant step towards a better understanding of disease resistance mechanisms and the development of new strategies for disease control.

Phosphoproteomic insight into the changes in structural proteins of muscle architecture associated with texture softening of grass carp (Ctenopharyngodon idella) fillets during chilling storage.

Texture is an important sensory attribute of fish affected by modifications of structural proteins in muscle architecture. To investigate the changes in protein phosphorylation during texture softening of fish, the proteins of grass carp muscle after chilling storage of 0 day and 6 days were compared by phosphoproteomics, and their association with texture was analyzed. Totally 1026 unique phosphopeptides on 656 phosphoproteins were identified as differential. They were mainly classified as intracellular myofibril and cytoskeleton, and extracellular matrix, of which the molecular function and biological process were binding into supramolecular assembly and myofilament contraction. The concomitant dephosphorylation of kinases and assembly regulators indicated dephosphorylation and disassembly tendency of sarcomeric architecture. Correlation analysis defined the relation between texture and dephosphorylation of myosin light chain, actin, collagen and cytoskeleton. This study revealed that protein phosphorylation may affect the texture of fish muscle through regulating sarcomeric assembly of structural proteins in muscle architecture.

Treatments of orange-spotted grouper (Epinephelus coioides) against Cryptocaryon irritans with •OH, ClO2 or HCHO: Survival, physiological and histological response.

Cryptocaryon irritans causes one of the most serious diseases in various wild and cultured marine fish, leading to mass mortality and economic loss. In this study, hydroxyl radical (•OH) solution produced by strong ionization discharge combined with water jet cavitation effect was injected into orange-spotted grouper (Epinephelus coioides) aquaculture tanks for C. irritans control. The results showed that all C. irritans theronts were inactivated by •OH solution at concentrations of 0.5 mg/L within 2 min. •OH could induce alteration of shape, the absence of motility and macronucleus dispersion in theronts. A possible explanation was that the macronucleus of C. irritans might be damaged by •OH; as a result, its metabolism and life activities were disturbed. The •OH treatment increased the survival rate of E. coioides challenged with C. irritans from 64.7 ± 8.0% (mean ± SD) to 100% and reduced their infection intensity significantly. Stress response biomarkers such as malonaldehyde, glutathione, glutathione peroxidase, superoxide dismutase (SOD) and catalase levels in the gills of E. coioides at different time points were analysed. The SOD activity in the •OH group first decreased and then recovered to the initial level at the end of the experiment. The other stress response biomarkers had no significant difference from that in the uninfected control group after •OH treatment. Additionally, the gill of E. coioides in the •OH group exhibited slight and reversible transformation compared with the uninfected control group. Compared with •OH treatment, chlorine dioxide and formalin treatment reduced the survival rate, induced oxidative damage and changed the histological gill structure in E. coioides. In conclusion, •OH could be applied effectively to control C. irritans infection without affecting the normal physiological condition of E. coioides.

Transcriptome analysis of the spleen provides insight into the immunoregulation of Scortum barcoo under Streptococcus agalactiae infection.

Jade perch (Scortum barcoo) is a freshwater fish with substantial economic value, which has been widely cultivated all over the world. However, with the intensification and expansion of farming, several bacterial and viral diseases have occurred in jade perch. To understand the immune response of jade perch against Streptococcus agalactiae (Group B Streptococcus, GBS), we performed a histopathological examination and transcriptome sequencing of jade perch spleen after artificial bacterial infection. GBS infection can cause structural changes and even necrosis of the jade perch spleen, which may affect the survival of infected individuals. A total of 144,458 unigenes were obtained through de novo assembly of spleen transcriptome. Among them, 1821 unigenes were identified as DEGs, including 1415 up-regulated and 406 down-regulated unigenes in the infection group. Moreover, the analysis of GO and KEGG revealed that many GO terms and pathways were involved in the host immune response, such as Toll-like receptor signaling pathway, Cytokine-cytokine receptor interaction, and TNF signaling pathway. In addition, according to transcriptome data and qRT-PCR analysis, the expression levels of many cytokines that participate in the inflammatory response changed a lot after GBS infection. Overall, this transcriptomic analysis provided valuable information for studying the immune response of jade perch against bacterial infection.

Functional Characterization of Serum Amyloid P Component (SAP) in Host Defense against Bacterial Infection in a Primary Vertebrate.

Serum amyloid P component (SAP), an ancient short pentraxin of the pentraxin family, plays an essential role in resistance to bacterial infection. In this study, the expression and functional characterization of SAP (OnSAP) in Nile tilapia (Oreochromis niloticus), a primary vertebrate, are investigated. The open reading frame of OnSAP is 645 bp of a nucleotide sequence encoding a polypeptide of 214 amino acids. As a calcium-binding protein, the structure and relative motif of OnSAP is highly similar to those of humans, containing amino acid residues Asn, Glu, Gln and Asp. In healthy fish, OnSAP mRNA is extensively distributed in all eleven tissues examined, with the highest level in spleen. The mRNA expression of OnSAP was significantly up-regulated after being challenged with gram-positive bacterium Streptococcus agalactiae and gram-negative bacterium Aeromonas hydrophila in vivo. In addition, recombinant OnSAP ((r)OnSAP) protein had capacities of binding S. agalactiae or A. hydrophila in the presence of Ca2+. Further, (r)OnSAP helped monocytes/macrophages to efficiently phagocytize bacteria. Moreover, the (r)OnSAP was able to enhance the complement-mediated lysis of the chicken red blood cells. Collectively, the evidence of SAP in tilapia, based on the results including its evolutionary conserved protein structure, bacterial binding and agglutination, opsonophagocytosis of macrophage and hemolysis enhancement, enriches a better understanding of the biological functions of the pentraxin family.

Acute nitrite exposure-induced oxidative damage, endoplasmic reticulum stress, autophagy and apoptosis caused gill tissue damage of grass carp (Ctenopharyngodon idella): Relieved by dietary protein.

Nitrite poses a serious threat to intensive aquaculture. Protein, as a major nutrient in animals, is vital for protecting animal tissues from damage. In this study, we investigated the protective effect of dietary protein on gill tissue structure and the underlying mechanisms in sub-adult grass carp (Ctenopharyngodon idella) exposed to nitrite stress. Six iso-energetic semi-purified diets containing different protein levels (16-31 %) were formulated, and fed to fish for 60 d. The fish were then exposed to a nitrite solution for 4 d. Histopathological observation and determination of related indices (serum glucose, serum cortisol, nitric oxide, peroxynitrite, reactive oxygen species, malondialdehyde, and protein carbonyl) showed that 22-25 % dietary protein significantly alleviated the nitrite-induced stress response, gill tissue damage and oxidative damage. Further research found that a suitable dietary protein suppressed the nitrite-induced endoplasmic reticulum stress (ERS) 78 kDa glucose-regulated protein (GRP78) related signaling pathway which possibly activated autophagy and apoptosis. Interestingly, we discovered that proper dietary protein reduced autophagy, probably through unc-51-like kinase 1 (Ulk1), BCL-2-interacting myosin-like coiled-coil protein (Beclin1), autophagy-related gene 5 (Atg5), Atg12, microtubule-associated protein1 light chain 3 (LC3), BCL-2 interacting protein 3 (BNIP3) and autophagy receptor P62 (p62). We also found that an appropriate dietary protein inhibited nitrite-induced apoptosis via mitochondrial and death receptor pathways. In summary, our findings are the first to demonstrate that 22-25 % of dietary protein levels can play a protective role against nitrite-induced gill injury.

Gill remodeling increases the respiratory surface area of grass carp (Ctenopharyngodon idella) under hypoxic stress.

Seasonal changes, diurnal variations, and eutrophication result in periodic hypoxia in fish habitats, thus affecting the success of commercial aquaculture. In this study, the grass carp (Ctenopharyngodon idella) presented moderate hypoxia tolerance; they showed a medium critical oxygen tension during the loss of equilibrium. In response to 7 d of hypoxic exposure, the erythrocyte count and hemoglobin (Hb) concentration significantly increased (p < 0.01). To cope with the hypoxic environment, the grass carp underwent gill remodeling marked by reduction in the interlamellar cell mass (ILCM) and an increase in respiratory surface area. The gill remodeling under hypoxia was enabled by apoptosis induction. Although apoptotic signals were not found on ILCM cells, transferase dUTP nick end labeling (TUNEL) assay results indicated that after 1 d of hypoxic exposure, the number of TUNEL-positive cells per lamella increased until 4 d and then began to decrease. Consistent with the results of the TUNEL assay, the mRNA expression of apoptosis-related genes, caspase-3, Bax, and Bcl-2, increased at 1, 4, and 7 d of the hypoxia treatment. In addition, gill remodeling significantly (p < 0.01) decreased the concentration of sodium and chloride ions in the fish serum. These findings provide evidence that grass carps increase their respiratory surface area through gill remodeling by apoptosis in the gill filaments to acclimate to a hypoxic environment. This study expands our understanding of the morphological and physiological changes in grass carp in response to a hypoxic environment; therefore, it could be useful for maintaining grass carp production.

The circulating plasma metabolome of Neoparamoeba perurans-infected Atlantic salmon (Salmo salar).

Metabolomics can provide insights into the dynamic small-molecule fluctuations occurring in response to infection and has become a valuable tool in studying the pathophysiology of diseases in recent years. However, its application in fish disease research is limited. Here, we report the circulating plasma metabolome of Atlantic salmon (Salmo salar) experimentally infected with Neoparamoeba perurans-the causative agent of amoebic gill disease (AGD). Plasma samples were collected from fish with varying degrees of infection inferred from an external gross morphological score of gill pathology (i.e., gill score [GS] 1 -- GS3), where a higher GS indicates advanced infection stage. Uninfected fish (GS0) served as the control. Typical pathologies associated with AGD infection, such as hyperplastic lesions and lamellar fusion, were evident in infected gill samples. Plasma metabolites were identified by ultra-performance liquid chromatography coupled with a high-resolution quadrupole-orbitrap mass spectrometer. Identification of compounds were performed at four levels of certainty, where level 1 provided the most accurate compound identity. A total of 900 compounds were detected in the samples of which 143 were annotated at level 3, 68 on level 2b, 74 on level 2a, and 66 on level 1. Versus GS0, GS1 showed the highest number of significantly affected metabolites (104), which decreased with a higher GS. Adrenaline and adenosine were the two Level 1 compounds significantly affected by AGD regardless of GS, with the former increasing and the latter decreasing in infected fish. Hippuric acid significantly increased in GS1 and GS2, while the tryptophan metabolite indole-3-lactic acid decreased in response to the initial stage of infection but returned to basal levels at a higher GS. There were ten significantly affected metabolic pathways: Eight of which were significantly downregulated while two were downregulated in GS1 relative to GS0. The super-pathway of purine nucleotide salvage was enriched both within the upregulated metabolites in GS1vsGS0 and the down-regulated metabolites in GS3vsGS1. This is the first report on the circulating plasma metabolome of AGD infected salmon, and the results show that low infection levels resulted in a more dramatic metabolomic dysregulation than advanced infection stages. The metabolites identified are potential biological markers for the systemic physiological impact of AGD.

The mTOR/PGC-1α/SIRT3 Pathway Drives Reductive Glutamine Metabolism to Reduce Oxidative Stress Caused by ISKNV in CPB Cells.

Under oxidative stress, viruses prefer glycolysis as an ATP source, and glutamine is required as an anaplerotic substrate to replenish the TCA cycle. Infectious spleen and kidney necrosis virus (ISKNV) induces reductive glutamine metabolism in the host cells. Here we report that ISKNV infection the increased NAD+/NADH ratio and the gene expression of glutaminase 1 (GLS1), glutamate dehydrogenase (GDH), and isocitrate dehydrogenase (IDH2) resulted in the phosphorylation and activation of mammalian target of rapamycin (mTOR) in CPB cells. Inhibition of mTOR signaling attenuates ISKNV-induced the upregulation of GLS1, GDH, and IDH2 genes expression, and exhibits significant antiviral activity. Moreover, the expression of silent information regulation 2 homolog 3 (SIRT3) in mRNA level is increased to enhance the reductive glutamine metabolism in ISKNV-infected cells. And those were verified by the expression levels of metabolic genes and the activities of metabolic enzymes in SIRT3-overexpressed or SIRT3-knocked down cells. Remarkably, activation of mTOR signaling upregulates the expression of the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) gene, leading to increased expression of SIRT3 and metabolic genes. These results indicate that mTOR signaling manipulates reductive glutamine metabolism in ISKNV-infected cells through PGC-1α-dependent regulation of SIRT3. Our findings reveal new insights on ISKNV-host interactions and will contribute new cellular targets to antiviral therapy. IMPORTANCE Infectious spleen and kidney necrosis virus (ISKNV) is the causative agent of farmed fish disease that has caused huge economic losses in fresh and marine fish aquaculture. The redox state of cells is shaped by virus into a favorable microenvironment for virus replication and proliferation. Our previous study demonstrated that ISKNV replication induced glutamine metabolism reprogramming, and it is necessary for the ISKNV multiplication. In this study, the mechanistic link between the mTOR/PGC-1α/SIRT3 pathway and reductive glutamine metabolism in the ISKNV-infected cells was provided, which will contribute new insights into the pathogenesis of ISKNV and antiviral treatment strategies.

Engagement of gcFKBP5/TRAF2 by spring viremia of carp virus to promote host cell apoptosis for supporting viral replication in grass carp.

Spring viremia of carp virus (SVCV) causes severe morbidity and mortality in grass carp (Ctenopharyngodon idellus) in Europe, America and several Asian countries. We found that FKBP5 (FK506-binding protein 5) is an SVCV infection response factor; however, its role in the innate immune mechanism caused by SVCV infection remains unknown. This study cloned gcFKBP5 (grass carp FKBP5) and made its mimic protein structure for function discussion. We found that gcFKBP5 expression in the primary innate immune organs of grass carp, including intestine, liver and spleen, was highly upregulated by SVCV in 24 h, with a similar result in fish cells by poly(I:C) treatment. gcFKBP overexpression aggravates viral damage to cells and increases viral replication. Furthermore, SVCV engages gcFKBP5 interacting with TRAF2 (tumour necrosis factor receptor-associated factor 2) to promote host cell apoptosis for supporting viral replication. The enhanced viral replication seems not to be due to the repression of IFN and other antiviral factors as expected. For the first time, these data show the pivotal role of gcFKBP5 in the innate immune response of grass carp to SVCV infection.

Amoebic gill disease increases energy requirements and decreases hypoxia tolerance in Atlantic salmon (Salmo salar) smolts.

Globally, Atlantic salmon (Salmo salar Linnaeus) aquaculture is now routinely affected by amoebic gill disease (AGD; Neoparamoeba perurans). The disease proliferates throughout the summer and is implicated in decreasing tolerance of salmon to environmental perturbations, yet little empirical evidence exists to support these observations. Using salmon acclimated to 15 or 19 °C, our aim was to determine the effects of clinically light-moderate (industry-relevant) AGD on metabolism (MO2rest and MO2max), aerobic scope (MO2max - MO2rest), excess post-exercise oxygen consumption (EPOC), and hypoxia tolerance. An increase in MO2rest (~8% and ~ 13% increase within the 15 and 19 °C acclimation groups, respectively) with increasing disease signs demonstrated an increase in baseline energy requirements as the disease progressed. Conversely, MO2max remained stable at both temperatures (~364 mg O2 kg-1 h-1), resulting in a decline in aerobic scope by 13 and 19% in the 15 and 19 °C groups, respectively. There was evidence of a decrease in hypoxia tolerance as the dissolved oxygen concentrations at loss of equilibrium increased by ~8% with more severe lesion coverage of the gills. These results suggest an increase in basal energy requirements and reduction in hypoxia tolerance as AGD proliferates, lending support to the idea that AGD reduces environmental tolerance. However, the lack of an effect of acclimation temperature indicates that the temperature-disease interaction may be more complicated than currently thought.