Immune Response In Grass Carp Research Articles

Evaluation of virulence of Aeromonas veronii strain GZ21-2 and development of a highly effective vaccine for grass carp with the potential for industrial application

Bacterial septicemia represents a significant disease affecting cultured grass carp culture, with the primary etiological agent identified as the Gram-negative bacterium Aeromonas veronii. In response to an outbreak of septicemia in Guangzhou, we developed a formaldehyde-inactivated vaccine against an A. veronii strain designated AV-GZ21-2. This strain exhibited high pathogenicity in experimental infections across at all developmental stages of grass carp. Mortality rates for grass carp weighing 15 ± 5 g ranged from 16 % to 92 % at exposure temperatures of 19 °C–34 °C, respectively. The median lethal dose (LD50) for grass carp groups weighing 15 ± 5 g, 60 ± 10 g, 150 ± 30 g and 500 ± 50 g were determined to be 1.43, 2.52, 4.65 and 7.12 × 107(CFU/mL), respectively. We investigated the inactivated vaccine in conbination with aluminum hydroxide gel (AV-AHG), Montanide ISA201VG (AV-201VG), and white oil (AV-WO) adjuvants. This study aimed to optimize inactivation conditions and identify the adjuvant that elicits the most robust immune response. The AV-GZ21-2 inactivated bacterial solution (AV),when combined with various adjuvants, was capable of inducing a strong specific immune response in grass carp. The relative percent survival (RPS) following a lethal challenge with AV-GZ21-2 were 94 % for AV-AHG, 88 % for AV-201VG, 84 % for AV-WO and 78 % for AV alone. The minimum immunization dose of the AV-AHG vaccine was determined to be 6.0 × 107 CFU per fish, providing immunity for a duration of six months with an immune protection level exceeding 75 %. Furthermore, the AV-AHG vaccine demonstrated significant protective efficacy against various epidemic isolates of A. veronii. Consequently, we developed an inactivated vaccine targeting a highly pathogenic strain of A. veronii, incorporating an aluminum hydroxide gel adjuvant, which resulted in high immune protection and a duration of immunity exceeding six months. These findings suggest that the AV-AHG vaccine holds substantial potential for industrial application.

ANAX4 is a downstream molecule of LGP2 and promotes GCRV proliferation

This study explored the key molecules and signal pathways in the pathogenesis of grass carp reovirus (GCRV). Using immunoprecipitation mass spectrometry and Co-IP validation, the protein CiANXA4 was identified which interacts indirectly with CiLGP2. CiANXA4 encodes 321 amino acids, including 4 ANX domains. To explore the role of CiANXA4 in the anti-GCRV immune response, we used overexpression and siRNA knockdown in cells. The results showed that overexpression of the CiANXA4 gene significantly increased the mRNA content of vp2 and vp7 in GCRV-infected cells, and the virus titer greatly increased. Knockdown of CiANXA4 significantly inhibited the mRNA levels of vp2 and vp7, and the protein levels of viral protein VP7 also significantly decreased. This suggests that CiANXA4 promotes viral proliferation. Further, we demonstrate that the ANX3 and ANX4 domains are key domains that limit CiANXA4 function by constructing domain-deletion mutants. Finally, we investigated the relationship between CiLGP2 and CiANXA4. RT-PCR and Western blot results showed that CiLGP2 mRNA and protein expression levels were not affected by CiANXA4 overexpression. In contrast, overexpression of CiLGP2 resulted in significant reductions in CiANXA4 mRNA and protein levels. This suggests that the function of CiANXA4 is restricted by CiLGP2, and CiANXA4 is a downstream molecule of CiLGP2. These results reveal that CiANXA4 plays a critical role in the anti-GCRV innate immune response of grass carp, and provides new targets and strategies to develop antiviral drugs and improve disease resistance in grass carp.

New insights into the role of myo-inositol in regulating the skin immune response of grass carp (Ctenopharyngodon idella)

Myo-inositol (MI) is a crucial constituent of the cell membrane and has a pivotal role to play in transmembrane signal transduction, ion channel regulation, and cell transport. Fish skin is a vital mucosal immune organ, and there are few studies that have examined the effect of MI on fish skin.Therefore, the purpose of this study was to investigate the role of dietary MI in regulating the skin immune response of grass carp (Ctenopharyngodon idella). A total of 540 fish (221.33 ± 0.84 g) were fed six diets containing graded levels of MI (27.0, 137.9, 286.8, 438.6, 587.7, and 737.3 mg/kg) for 10 weeks. When the growth test was complete, a 14-day challenge was conducted with Aeromonas hydrophila in each group. Our results indicated that the most effective MI concentrations (approximately 438.6–737.3 mg/kg diet) resulted in the following outcomes: (1) MI reduced the morbidity of grass carp skin and increased the thickness of the dense dermis layer. (2) MI enhanced the physical barrier function of the skin, maybe via increasing antioxidant capacity, inhibiting apoptosis, and improving tight junction-related signaling pathways (nuclear factor erythroid 2-related factor 2, p38 mitogen-activated protein kinase, and myosin light chain kinase). (3) MI enhanced skin immune barrier function, possibly via regulating anti-inflammatory and pro-inflammatory cytokines, as well as the target of rapamycin (TOR) and nuclear factor kappa B (NFκB) pathways. This study led to the following conclusion based on its findings: the MI supplementation strengthened the fish skin barrier function and protected it against Aeromonas hydrophila. The dietary MI requirement in on-growing grass carp was estimated to be 370.90 mg/kg by a broken-line method analysis of skin morbidity.

TRIM103 activates the RLRs pathway to enhance antiviral response by targeting VP5 and VP7

Grass carp (Ctenopharyngodon idella), crucial to global inland aquaculture with a production of 5.8 million tones in 2020, faces significant challenges from hemorrhagic disease caused by grass carp reovirus (GCRV). Rapid mutations compromise current vaccines, underscoring the need for a deeper understanding of antiviral mechanisms to enhance molecular marker-assisted selection. This study investigates the role of Tripartite Motif (TRIM) family in the innate immune response of grass carp, focusing on TRIM103 from Ctenopharyngodon Idella (CiTRIM103), a member of the TRIM-B30.2 family, which includes proteins with the B30.2 domain at the N-terminus, known for antiviral properties in teleosts. CiTRIM103 bind to the outer coat proteins VP5 and VP7 of GCRV. This binding is theorized to strengthen the function of the RIG-I-like Receptor (RLR) signaling pathway, crucial for antiviral responses. Demonstrations using overexpression and RNA interference (RNAi) techniques have shown that CiTRIM103 effectively inhibits GCRV replication. Moreover, molecular docking and pulldown assays suggest potential binding interactions of CiTRIM103's B30.2 domain with GCRV outer coat proteins VP5 and VP7. These interactions impede viral replication, enhance RLR receptor expression, and activate key transcription factors to induce type I interferons (IFNs). These findings elucidate the antiviral mechanisms of CiTRIM103, provide a foundation for future Molecular genetic breeding in grass carp.

Preliminary study of BF/C2 on immune mechanism of grass carp against GCRV infection

BF/C2 is a crucial molecule in the coagulation complement cascade pathway and plays a significant role in the immune response of grass carp through the classical, alternative, and lectin pathways during GCRV infection. In vivo experiments demonstrated that the mRNA expression levels of BF/C2 (A, B) in grass carp positively correlated with GCRV viral replication at various stages of infection. Excessive inflammation leading to death coincided with peak levels of BF/C2 (A, B) mRNA expression and GCRV viral replication. Correspondingly, BF/C2 (A, B) recombinant protein, CIK cells and GCRV co-incubation experiments yielded similar findings. Therefore, 3 h (incubation period) and 9 h (death period) were selected as critical points for this study. Transcriptome sequencing analysis revealed significant differences in the expression of BF/C2A and BF/C2B during different stages of CIK infection with GCRV and compared to the blank control group (PBS). Specifically, the BF/C2A_3 and BF/C2A_9 groups exhibited 2729 and 2228 differentially expressed genes (DEGs), respectively, with 1436 upregulated and 1293 downregulated in the former, and 1324 upregulated and 904 downregulated in the latter. The BF/C2B_3 and BF/C2B_9 groups showed 2303 and 1547 DEGs, respectively, with 1368 upregulated and 935 downregulated in the former, and 818 upregulated and 729 downregulated in the latter. KEGG functional enrichment analysis of these DEGs identified shared pathways between BF/C2A and PBS groups at 3 and 9 h, including the C-type lectin receptor signaling pathway, protein processing in the endoplasmic reticulum, Toll-like receptor signaling pathway, Salmonella infection, apoptosis, tight junction, and adipocytokine signaling pathway. Additionally, the BF/C2B groups at 3 and 9 h shared pathways related to protein processing in the endoplasmic reticulum, glycolysis/gluconeogenesis, and biosynthesis of amino acids. The mRNA levels of these DEGs were validated in cellular models, confirming consistency with the sequencing results. In addition, the mRNA expression levels of these candidate genes (mapk1, il1b, rela, nfkbiab, akt3a, hyou1, hsp90b1, dnajc3a et al.) in the head kidney, kidney, liver and spleen of grass carp immune tissue were significantly different from those of the control group by BF/C2 (A, B) protein injection in vivo. These candidate genes play an important role in the response of BF/C2 (A, B) to GCRV infection and it also further confirmed that BF/C2 (A, B) of grass carp plays an important role in coping with GCRV infection.

Exploration of the immune response of grass carp (Ctenopharyngodon idellus) erythrocytes during bacterial infection

In teleost blood, red blood cells (RBCs) are the most common type of cell, and they differ from mammalian RBCs in having a nucleus and other organelles. As nucleated cells, teleost RBCs contribute to the immune response against pathogens, but their antibacterial mechanism remains unclear. Here, we utilized RNA-Seq to analyze gene expression patterns of grass carp (Ctenopharyngodon idellus) RBCs (GcRBCs) stimulated by Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus. Our transcriptomic data showed that bacterial stimulation generated many differentially expressed genes (DEGs). Furthermore, several inflammatory pathways responded to bacterial activation, and the TLR, IL-17, and tumor necrosis factor (TNF) signaling pathways were significantly activated based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Furthermore, the findings of qRT-PCR showed markedly elevated expression of various cytokines, including IL-1β, IL4, IL6, IL8, IL12, and TNFα, in GcRBCs after incubation with bacteria. Reactive oxygen species (ROS) production in GcRBCs was markedly increased after the cells were stimulated with the three bacteria, and the expression of superoxide dismutase, glutathione peroxidase, and antioxidant enzymes, including catalase, was altered. Flow cytometry analysis showed that the apoptosis rate of GcRBCs was enhanced after stimulation with the three bacteria for different times. In summary, our findings reveal that bacterial stimulation activates the immune response of GcRBCs by regulating ROS release, cytokine expression, and the antioxidant system, leading to apoptosis of GcRBCs.

Effects of biological flocculation technology (BFT) on water quality dynamics and immune response of grass carp (Ctenopharyngodon idella)

The biofloc technology (BFT) offers benefits in improving feed utilization and reducing the feed conversion ratio compared to the recirculating aquaculture system (RAS). In this study, high-throughput 16S rRNA gene sequencing was employed to investigate and compare the bacterial communities in these aquaculture systems and the gut microbiota of grass carp reared in them. We observed a significant distinction between the microbial communities of the aquaculture systems and the intestinal microbiota of grass carp. The genera cetobacterium and bacillus were more abundant in the BFT system, accounting for only 0.05% and 0.25% in the RAS. Our study also demonstrated that BFT could influence the intestinal microorganisms of grass carp (Ctenopharyngodon Idella) by reducing the relative abundance of burkholderia-caballeronia-paraburkholderia and increasing that of cetobacterium. Based on bugBase community predictions, the potential pathogenicity in BFT and grass carp intestine was lower than in RAS. Regarding gene expression in the grass carp intestine, immunosuppressive genes showed higher expression, while pro-inflammatory genes exhibited lower expression in grass carp reared in BFT. Furthermore, the final average length and weight of grass carp in the BFT system were significantly higher than those in the RAS. This study provides valuable insights into gut microbiome ecology in relation to two grass carp aquaculture systems, which could be effectively applied in grass carp management to promote health and improve water quality.

Effects of dietary nucleotide and yeast cell wall on growth performance, feed utilization, anti-oxidative and immune response of grass carp (Ctenopharyngodon idella)

A 70-day feeding trial was conducted to study the effects of dietary nucleotide, yeast cell wall (containing 20% β -glucan) and their combination on growth performance, feed utilization and immune response of grass carp (Ctenopharyngodon idella) with 69.97 ± 0.05 g of initial body weight. Four isonitrogenous (about 38% crude protein) and isolipidic (about 5% crude lipid) diets were established. Based on the control diet (CD), the other three experimental diets were prepared by adding 0.01% of nucleotide (NT), 0.1% of yeast cell wall (YCW) and NT (0.01%) +YCW (0.1%), respectively. Results showed that no significant difference was found in survival of grass carp ranging from 94.44% to 97.78% among all the groups (P > 0.05). Compared with the control group, weight gain rate, muscle crude protein content, serum protein, trypsin and chymotrypsin activities in midgut, lysozyme and immunoglobulin M in serum significantly increased in fish fed the YCW diet (P < 0.05). The significantly highest weight gain rate, villus height and digestive enzyme activities in midgut and innate immune parameters in serum were found in fish fed the NT + YCW diet (P < 0.05). The gene expressions of β-defensin, hepcidin, il-10 and tgf-β1 in the midgut, and tor and s6k1 in liver significantly increased in fish fed the NT + YCW diet. Meanwhile, the gene expressions of il-1β and tnf-α in the midgut decreased significantly (P < 0.05). The liver histology showed the better development in dietary NT and/or YCW supplemented groups than those in the control group. In conclusion, combination of dietary NT and YCW had significantly synergetic improvements on the growth, feed utilization, digestive enzymes, innate immunity and histology of midgut and liver of grass carp.

Dietary lysine levels modulate the lipid metabolism, mitochondrial biogenesis and immune response of grass carp, Ctenopharyngodon idellus

A 10-week experiment was conducted to evaluate the effects of dietary lysine levels on growth performance, lipid metabolism, anti-oxidant capacity, mitochondrial biogenesis and immune response of grass carp, Ctenopharyngodon idella. Fish (mean initial weight 51.01 ± 0.17 g) were fed seven diets containing graded levels of lysine (1.69 %, 1.79 %, 1.91 %, 2.01 %, 2.11 %, 2.24 % and 2.31 %). Results showed that fish fed the control diet (1.69 %) showed lower weight gain (WG) and higher feed conversion rate FCR than those fed 2.01–2.31 % lysine (P < 0.05). Viscerosomatic index decreased when dietary lysine increasing from 1.69 % to 2.11 % (P < 0.05). Suitable dietary lysine levels enhanced the lipolysis and mitochondrial biogenesis of grass carp. Total antioxidant capacity (T-AOC) in the liver was higher in fish fed 2.24 % dietary lysine than those fed diets containing 1.69 % and 1.79 % lysine (P < 0.05). The malonaldehyde (MDA) contents in liver decreased when dietary lysine increased from 1.69 % to 2.01 % (P < 0.05). Relative mRNA levels of glutathione peroxidase and catalase in liver and intestine were affected by dietary lysine levels (P < 0.05). B-cell lymphoma 2 (Bcl-2) mRNA level in liver was up-regulated and inhibitor kappa B kinase β (IKKβ) mRNA level in the intestine was down-regulated by increasing dietary lysine levels (P < 0.05). Second-order regression based on WG indicated the suitable level of lysine in feed of grass carp was 2.24 % (6.19 % protein).

Growth performance, intestinal microbiota and immune response of grass carp fed isonitrogenous and isoenergetic diets containing faba bean extracts

Functionally active components of faba bean (Vicia faba, FB) are known to improve the muscle texture and growth performance of grass carp (Ctenopharyngodon idellus), but this has not yet been proven by feeding trials using isonitrogenous and isoenergetic diets. Here, we formulated isonitrogenous and isoenergetic diets containing four types of FB extracts and examined their effects on the weight gain, textural quality, intestinal microbiota, metabolic enzymes and immune response of grass carp by a 100-day feeding trial. An isonitrogenous and isoenergetic diet with no FB extracts (control) and whole FB were also used. Compared to the control, the FB water extract diet increased weight gain, muscle hardness and chewiness, intestinal microbiota diversity, the activity of metabolic enzymes including lipase (LP), glucose-6-phosphatase (G-6-P), acetyl-CoA and succinate dehydrogenase (SDH) (P < 0.05). The FB alcohol extract diet also increased weight gain, intestinal microbiota diversity, but the total cholesterol (TC) and triglycerides (TG) contents were also increased (P < 0.05). The FB protein extract and FB residues diets had negative and no effects on weight gain, respectively, and caused intestinal microbiota disorder. FB water and alcohol extracts enhanced intestinal amino acids metabolism, whereas FB water extract and residues enhanced intestinal carbohydrate metabolism. These results further extend previous findings that FB water and alcohol extracts contain most functionally active components of FB, opening up the possibility that these extracts can be used as functional feed additives for freshwater fish.

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.

Effects of glucose, insulin, glucagon, LPS and poly (I:C) on adiponectin receptors expression in grass carp (Ctenophcuyngodon idellus)

Adiponectin plays critical roles in energy metabolism and immunity of mammals by binding to its receptors (AdipoR1 and AdipoR2). However, they have not been fully understood in fish. The present study examined the distribution of adiponectin receptors (AdipoRs) mRNA in grass carp, and the changes of their expressions under glucose load, insulin or glucagon stimulation, lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid [poly (I:C)] challenge. As for glucose load, fish were orally administrated with glucose (1.67 mg/g BW) for 1, 3, 6, 12 h. For insulin or glucagon stimulation, the primary hepatocytes of grass carp were incubated with 0, 10, 100, 1000 nM insulin or glucagon for 1, 3, 6, 12 h. In the immune challenge experiment, grass carp were intraperitoneally injected with LPS or poly (I:C) (5 μg/g BW) for 3, 6, 12, 24, 48 h. Finally, AdipoRs mRNA expression in all experiments was detected by Real-time quantitative PCR. The results showed that AdipoRs mRNA was widely expressed in various tissues of grass carp. The adipoR1a mRNA was highly expressed in the brain regions, and the highest level of adipoR2 mRNA was observed in the adipose tissue. Glucose load up-regulated adipoR1b and adipoR2 mRNA expressions in the liver at 6 h post administration and decreased adipoR1a expression in the muscle at 3 h. Insulin significantly enhanced adipoR1b and adipoR2 mRNA expressions in the hepatocytes at 1–12 h, and promoted adipoR1a mRNA expression at 1, 3 and 12 h, while glucagon had little effect on their expressions. LPS and poly (I:C) stimuli induced AdipoRs expression in the liver at 6 and 12 h, and their expressions in the muscle were decreased slightly or remained unchanged. In addition, LPS promoted AdipoRs expression in the kidney and spleen, whereas poly (I:C) only induced adipoR1a mRNA expression in the kidney, and increased adipoR2 mRNA level in the spleen. These results suggest that AdipoRs may act as one of the targets of insulin and immune response in grass carp, but whether they are direct target remains to be further studied.

MiR-148 targets CiGadd45ba and CiGadd45bb to modulate the inflammatory response to bacterial infection in grass carp

In grass carp (Ctenopharyngodon idella), septicemia is a systemic inflammatory response to bacterial infection. Once infected bacteria, a hyper-inflammatory state that could lead to septic shock and death. There is increasing evidence that microRNAs are involved in the regulation of the inflammatory response. Ctenopharyngodon idella growth arrest and DNA damage-inducible 45 ba and bb (CiGadd45ba and CiGadd45bb) are two subtypes of Gadd45b. In the present study, miR-148 was confirmed to be involved in the inflammatory response after infection with Aeromonas hydrophila. Dual-luciferase reporter assays and miRNA expression profiling confirmed that miR-148 targeted both CiGadd45ba and CiGadd45bb. Transfection with miR148 mimics and inhibitors altered the expression levels of proinflammatory genes, suggesting that miRNAs regulate the immune response in grass carp. Our results provide a theoretical basis for studying the molecular mechanism underlying the regulation of inflammation by miR-148 in grass carp.

The immunoprotective effect of whole-cell lysed inactivated vaccine with SWCNT as a carrier against Aeromonas hydrophila infection in grass carp

Aeromonas hydrophila is a strong gram-negative bacterium that can cause a mass death of grass carp, and result in the huge economic loss. Development of practical vaccines is the best way to control the outbreak of this bacterial disease. In this study, a whole-cell inactivated vaccine was obtained via sonication, and then single-walled carbon nanotubes (SWCNTs) was used to link to the bacterial lysate (BL) for a novel vaccine (SWCNTs-BL). A total of 400 fish were vaccinated with BL and SWCNTs-BL via immersion (5, 10 mg L−1) or injection (5, 10 μg/fish) before challenge with live A. hydrophila at the 28 days post immunization (d.p.i.). The results showed that the antibody titer, enzymatic activity, expression of some immune-related genes (especially IgM and TNF-α) and RPS of fish in the injection groups were significantly increased compared to the control group after 28 d.p.i. For the immersion groups, immunological parameters were increased compared to the control group. Furthermore, the immuno-protective effects of SWCNTs-BL were better than BL. The above results indicated that BL of A. hydrophila can effectively induce specific immune response of grass carp, and BL linked with functionalized SWCNTs could enhance the protective effect of immersion immunization. Our results may provide a practical vaccine, with a simple production, to fight against bacterial diseases in aquaculture industry.

Effects of microRNA-731 on inflammation and apoptosis by targeting CiGadd45aa in grass carp

Ctenopharyngodon idellagrowth arrest and DNA damage-inducible 45aa (CiGadd45aa) is a member of the Gadd45 family of immune-related proteins in grass carp. There is increasing evidence that microRNAs (miRNAs) are involved in the regulation of inflammatory responses and apoptosis. However, little is known about the regulatory effects of miRNAs on CiGadd45aa expression in grass carp. In the present study, CiGadd45aa was identified as a target gene of miR-731 based on miRNA expression profiling and dual-luciferase reporter assay. Our study revealed that miR-731 targets CiGadd45aa and regulates the expression of proinflammatory factors, thereby regulating immune response in grass carp. In addition, CiGadd45aa and miR-731 were both found induced apoptosis. Hence, our findings provide a theoretical basis for exploring the molecular mechanism by which miR-731 regulates inflammation and apoptosis in grass carp.

Effects of diet α-ketoglutarate (AKG) supplementation on the growth performance, antioxidant defense system, intestinal digestive enzymes, and immune response of grass carp (Ctenopharyngodon idellus)

This study was conducted to investigate the effects of diet 7.5 g/kg α-ketoglutarate (AKG) on the growth performance, antioxidant defense system, digestive enzymes, and immune response of grass carp (Ctenopharyngodon idellus). A total of 400 grass carp with an average body weight 10.81 ± 0.68 g was randomly allocated into 2 groups with 4 replicates of 50 fish respectively. The experiment was conducted in net cages (1.5 m × 1.5 m × 1.5 m) suspended in an indoor cement pool. Fish were fed a basic diet containing either 0 (control) or 7.5 g/kg AKG (supplemented diet). The experiment lasted for 8 weeks (56 days). Results indicated that compared with the control group, the final weight (FW), weight gain rate (WGR), specific growth rate (SGR), and protein efficiency ratio (PER) in the AKG group were increased significantly (P < 0.05). However, the feed conversion ratio (FCR) was decreased significantly (P < 0.05). The 7.5 g/kg AKG supplementation significantly increased the activities of glutamine synthetase (GS), glutathione peroxidase (GSH-Px), catalase (CAT), total superoxide dismutase (T-SOD), and hexokinase (HK), as well as the concentrations of glutathione (GSH), total antioxidant capacity (T-AOC), and complement 3 (C3) in blood (P < 0.05), while significantly decreased the concentrations of malondialdehyde (MDA) and hemoglobin (Hb) (P < 0.05). The GS activity and GSH concentration in hepatopancreas were increased significantly (P < 0.05), whereas the glycogen concentration in hepatopancreas, and the glycogen concentration and GS activity in the muscle were significantly decreased (P < 0.05). In addition, 7.5 g/kg AKG supplementation significantly increased the concentration of GSH and the activities of amylase, protease, and lipase in fore-gut, alkaline phosphates (ALP) in the mid-gut, and Na-ATP and Ca-ATP in the gill (P < 0.05), as well as γ-glutamyl transpeptidase (γ-GT) both in fore-gut and mid-gut (P < 0.05), whereas the activity of acid phosphatase (ACP) in the mid-gut was decreased significantly (P < 0.05). In conclusion, diet 7.5 g/kg AKG supplementation in grass carp may improve the growth performance and immune response and play crucial roles in regulating the activities of GS, antioxidant defense system, and digestive enzymes.

Complement component 3 (C3): An important role in grass carp (Ctenopharyngodon idella) experimentally exposed to Aeromonas hydrophila

Complement is traditionally recognized as part of the innate immune system, defending the host against the invasion of foreign pathogens. In complement system, C3 (complement component 3) is a central component. Therefore, research into C3 can help us better understand the functions of fish complement system. In this study, we detected the grass carp C3 (gcC3) mRNA expression in all sample tissues from healthy grass carp, which was highest in the liver, followed by the heart and the spleen, and lowest in the muscle, head kidney, trunk kidney, blood and intestine. After infection with Aeromonas hydrophila, gcC3 mRNA expression levels were significantly upregulated in the gill, liver, spleen, intestine, trunk kidney and head kidney. Interestingly, C3 protein levels were downregulated and subsequently upregulated in the liver and serum. Histologically, C3 protein at 24 h pi was over expressed in necrotic liver sites, and the liver index (LI) at this point was significantly higher than that of the control. These findings are indicated that C3 plays an important role in the immune response of grass carp after A. hydrophila infection, and C3 protein may play an assistant role in repairing liver tissues from A. hydrophila injury.

MDA5 and LGP2 acts as a key regulator though activating NF-κB and IRF3 in RLRs signaling of mandarinfish

RIG-I-like receptors (RLRs), as key cytoplasmic sensors of viral pathogen-associated molecular patterns, can recognise viral RNA and enhance the antiviral response. Some investigations have focused on the roles of RLRs in the innate immune response in grass carp, large yellow croaker, and rainbow trout. However, little is known about the function of RLRs in mandarinfish (Siniperca chuatsi), an important economic fish in Perciformes. Here, we functionally characterized the RLRs involved in the immune responses of mandarinfish (Siniperca chuatsi), by evaluating three RLRs, namely, RIG-I, MDA5, and LGP2. The results revealed that MDA5 and LGP2 were present in mandarinfish, whereas RIG-I was absent. The MDA5 and LGP2 cDNA sequences contained 2976 and 2046 bp and encoded 991 and 681 amino acids, respectively. Multiple sequence alignments showed that MDA5 and LGP2 of mandarinfish were clustered together with their homologs from other teleost fishes and shared high similarities with those from other vertebrates, and RIG-I of mandarinfish was absent. Moreover, quantitative real-time PCR (qPCR) analysis suggested that MDA5 and LGP2 were constitutively expressed in all tissues tested, and MDA5 mRNA expression was relatively high in the gill, and spleen, whereas LGP2 mRNA expression was high in the liver, gill, and head kidney. After stimulation with lipopolysaccharide or poly I:C, the expression of MDA5 and LGP2 was upregulated in spleen, gill and head kidney, but the pattern was not exactly the same, MDA5 transcripts generally increased and then declined with the prolonged infection, while LGP2 transcripts went up continuously, which showed that mandarinfish MDA5 and LGP2 may play independent roles in antiviral response. Besides, it is further revealed that the MDA5 could activate NF-κB and IRF3 to inducing the production of IFN-β by constructing tet-on stable strain of 293T cell, however over-expression of LGP2 resulted in decreased NF-κB, IRF3 and IFN-β production in cells challenged with LPS and polyI:C Taken together, our results demonstrated that MDA5 and LGP2, as a positive and negative regulator, respectively, played an important role in modulating antibacterial andantiviral immune responses though activating NF-κB and IRF3 in RLRs signaling of mandarinfish.

Effect of high temperature on immune response of grass carp (Ctenopharyngodon idellus) by transcriptome analysis

Exposure to high temperature is an inherent feature of grass carp culture in southern China and juvenile grass carps are predisposed to infectious disease in this condition. To understand how high temperature impacts the immune response to pathogens in grass carp, the transcriptomic profiles of the spleens from immune injected grass carp groups undergoing heat stress and normal temperature were investigated. An average of 72 million clean reads per library was obtained, and approximate 80% of these genes were successfully mapped to the reference genome. A total of 2287 up-regulated and 1068 down-regulated genes were identified. 10 immune-related categories involving 90 differently expressed genes were scrutinized. Expression patterns of 18 differentially expressed genes involving in immune response were validated by quantitative real-time RT-PCR. These results provide further significant insights into the influence mechanism of high temperature to immune response in grass carp.

Effect of a new recombinant Aeromonas hydrophila vaccine on the grass carp intestinal microbiota and correlations with immunological responses

Intestinal microbiota has become an integral component of the fish, and plays a key role in host metabolism, immunity and health maintenance. However, information on the immune responses after vaccine administration in relation to the intestinal microbiota is absent in fish. The present study focused on the effect of a new recombinant Aeromonas hydrophila vaccine (Aera) by using a novel functionalized, single-walled carbon nanotubes (SWCNTs) as a delivery vehicle on the intestinal microbiota of grass carp (Ctenopharyngodon idella) through the bath immunization, and further explored the immunological responses in intestine, kidney and spleen. By performing deep sequencing, a total of 81,979 valid reads and 609 OTUs obtained from 4 intestine samples were analyzed. We detected 141 genuses, most of which belonged to Firmicutes, Fusobacteria and Proteobacteria. Of note, the quantity of Aeromonas in library Aera (after 6 h Aera vaccine pretreatment, fish were transferred to tanks without SWCNTs-Aera for 28 d) and Aera-GD (6 h Aera vaccine pretreated prior to the group injected by A. hydrophila) was declined 6.5% and 14.6% compared with the control, respectively. Moreover, the expression of seven immune-related genes (IFN-I, TNF-α, CRP, IL-8, IgM, MHC I and CD8α) in the intestine, kidney and spleen of Aera treated fish was significantly enhanced, which indicated that a better tissue immune response in grass carp was induced by the SWCNTs-Aera vaccine. Therefore, a new recombinant SWCNT-Aera vaccine may represent potentially efficient and immunological role in grass carp intestine to resist A. hydrophila infection.