ABSTRACT
DNA methylation, an essential epigenetic alteration, is tightly linked to a variety of biological processes, such as immune response. To identify the epigenetic regulatory mechanism in Pacific oyster (Crassostrea gigas), whole-genome bisulfite sequencing (WGBS) was conducted on C. gigas at 0 h, 6 h, and 48 h after infection with Vibrio alginolyticus. At 6 h and 48 h, a total of 11,502 and 14,196 differentially methylated regions (DMRs) were identified (p<0.05, FDR<0.001) compared to 0 h, respectively. Gene ontology (GO) analysis showed that differentially methylated genes (DMGs) were significantly enriched in various biological pathways including immunity, cytoskeleton, epigenetic modification, and metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that transcription machinery (ko03021) is one of the most important pathways. Integrated transcriptome and methylome analyses allowed the identification of 167 and 379 DMG-related DEGs at 6 h and 48 h, respectively. These genes were significantly enriched in immune-related pathways, including nuclear factor kappa B (NF-κB) signaling pathway (ko04064) and tumor necrosis factor (TNF) signaling pathway (ko04668). Interestingly, it's observed that the NF-κB pathway could be activated jointly by TNF Receptor Associated Factor 2 (TRAF2) and Baculoviral IAP Repeat Containing 3 (BIRC3, the homolog of human BIRC2) which were regulated by DNA methylation in response to the challenge posed by V. alginolyticus infection. Through this study, we provided insightful information about the epigenetic regulation of immunity-related genes in the C. gigas, which will be valuable for the understanding of the innate immune system modulation and defense mechanism against bacterial infection in invertebrates.
Subject(s)
Crassostrea , DNA Methylation , Epigenesis, Genetic , NF-kappa B , Signal Transduction , Vibrio alginolyticus , Animals , Crassostrea/genetics , Crassostrea/immunology , Crassostrea/microbiology , Vibrio alginolyticus/physiology , NF-kappa B/genetics , NF-kappa B/metabolism , NF-kappa B/immunology , Signal Transduction/genetics , Immunity, Innate/genetics , Vibrio Infections/immunology , Vibrio Infections/veterinary , Vibrio Infections/geneticsABSTRACT
Interleukin-10 (IL-10) is an immunosuppressive cytokine, which plays a vital role in regulating inflammation for inhibiting the generation and function of pro-inflammatory cytokines in vivo or in vitro. In the present study, the full length cDNA of IL-10 was characterized from Nibea albiflora (named as NaIL-10) of 1238 base pairs (bp), containing a 5'-UTR (untranslated region) of 350 bp, a 3'-UTR of 333 bp and an open reading frame (ORF) of 555 bp (Fig. 1A) to encode 184 amino acid residues with a signal peptide at the N-terminus. The sequence analysis showed that NaIL-10 possessed the typical IL-10 family symbolic motif and conversed cysteine residues, similar to its teleost orthologues. Real-time PCR indicated that NaIL-10 had wide distribution in different healthy tissues, with a relatively high expression in immune-related tissues (head kidney, spleen, kidney, liver and gill). Significantly, up-regulations of NaIL-10 after infection against Vibrio parahaemolyticus, Vibrio alginolyticus and Poly I:C were also observed. Subcellular localization manifested that NaIL-10 mainly distributed in the cytoplasm unevenly and aggregately, and there was also a small amount on the cell membrane, indicating that NaIL-10 was secreted to the extracellular space as the known IL-10 homologous molecules. It could co-locate with IL-10 Rα on the membrane of HEK293T cells for their potential interaction, and GST pull-down and Co-IP studies certified the specific and direct interaction between NaIL-10 and NaIL-10 Rα, confirming that an IL-10 ligand-receptor system existed in N.albiflora. The expression of pro-inflammatory cytokines, including TNF-α, IL-6, IL-1ß, were dramatically inhibited in LPS-stimulated RAW264.7 macrophages pre-incubated with recombinant NaIL-10 protein, demonstrating its anti-inflammatory roles. Taken together, the results demonstrated the existence of IL-10 ligand-receptor system in N.albiflora for the first time, and indicated the suppressive function of NaIL-10 on pro-inflammatory cytokine expression in inflammatory response, which would be conducive to better comprehending the role of IL-10 in the immunomodulatory mechanisms of teleost.
Subject(s)
Amino Acid Sequence , Fish Diseases , Fish Proteins , Gene Expression Regulation , Immunity, Innate , Interleukin-10 , Phylogeny , Animals , Fish Proteins/genetics , Fish Proteins/immunology , Fish Proteins/chemistry , Fish Diseases/immunology , Interleukin-10/genetics , Interleukin-10/immunology , Immunity, Innate/genetics , Gene Expression Regulation/immunology , Gene Expression Regulation/drug effects , Vibrio parahaemolyticus/physiology , Sequence Alignment/veterinary , Gene Expression Profiling/veterinary , Poly I-C/pharmacology , Vibrio Infections/immunology , Vibrio Infections/veterinary , Cyprinidae/immunology , Cyprinidae/genetics , Vibrio alginolyticus/physiology , Base SequenceABSTRACT
Vibrio alginolyticus is one of the most common opportunistic pathogens in marine animals and humans. In this study, A transposon mutation library of the V. alginolyticus E110 was used to identify motility-related genes, and we found three flagellar and one capsular polysaccharide (CPS) synthesis-related genes were linked to swarming motility. Then, gene deletion and complementation further confirmed that CPS synthesis-related gene ugd is involved in the swarming motility of V. alginolyticus. Phenotype assays showed that the Δugd mutant reduced CPS production, decreased biofilm formation, impaired swimming ability, and increased cytotoxicity compared to the wild-type strain. Transcriptome analysis showed that 655 genes (15%) were upregulated and 914 genes (21%) were downregulated in the Δugd strain. KEGG pathway and heatmap analysis revealed that genes involved in two-component systems (TCSs), chemotaxis, and flagella assembly pathways were downregulated in the Δugd mutant. On the other hand, genes involved in pathways of human diseases, biosynthesis ABC transporters, and metabolism were upregulated in the Δugd mutant. The RT-qPCR further validated that ugd-regulated genes are associated with motility, biofilm formation, virulence, and TCSs. These findings imply that ugd may be an important player in the control of some physiological processes in V. alginolyticus, highlighting its potential as a target for future research and potential therapeutic interventions.
Subject(s)
Bacterial Capsules , Bacterial Proteins , Biofilms , Flagella , Gene Expression Regulation, Bacterial , Vibrio alginolyticus , Vibrio alginolyticus/genetics , Vibrio alginolyticus/physiology , Vibrio alginolyticus/metabolism , Biofilms/growth & development , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Flagella/genetics , Flagella/metabolism , Flagella/physiology , Bacterial Capsules/metabolism , Bacterial Capsules/genetics , Polysaccharides, Bacterial/biosynthesis , Polysaccharides, Bacterial/metabolism , Polysaccharides, Bacterial/genetics , Virulence , Animals , Gene Expression Profiling , Gene Deletion , Humans , Vibrio Infections/microbiologyABSTRACT
Fish rely on innate immune system for immunity, and nucleotide-binding oligomerization domain-like receptors (NLRs) are a vital group of receptor for recognition. In the present study, NOD1 gene was cloned and characterized from golden pompano Trachinotus ovatus, a commercially important aquaculture fish species. The ORF of T. ovatus NOD1 was 2820 bp long, encoding 939 amino acid residues with a highly conserved domains containing CARD-NACHT-LRRs. Phylogenetic analysis revealed that the T. ovatus NOD1 clustered with those of fish and separated from those of birds and mammals. T. ovatus NOD1 has wide tissue distribution with the highest expression in gills. Bacterial challenges (Streptococcus agalactiae and Vibrio alginolyticus) significantly up-regulated the expression of NOD1 with different response time. The results of T. ovatus NOD1 ligand recognition and signaling pathway analysis revealed that T. ovatus NOD1 could recognize iE-DAP at the concentration of ⧠100 ng/mL and able to activate NF-κB signaling pathway. This study confirmed that NOD1 play a crucial role in the innate immunity of T. ovatus. The findings of this study improve our understanding on the immune function of NOD1 in teleost, especially T. ovatus.
Subject(s)
Amino Acid Sequence , Fish Diseases , Fish Proteins , Immunity, Innate , Nod1 Signaling Adaptor Protein , Phylogeny , Sequence Alignment , Vibrio alginolyticus , Animals , Nod1 Signaling Adaptor Protein/genetics , Nod1 Signaling Adaptor Protein/immunology , Nod1 Signaling Adaptor Protein/chemistry , Fish Proteins/genetics , Fish Proteins/immunology , Fish Proteins/chemistry , Immunity, Innate/genetics , Fish Diseases/immunology , Sequence Alignment/veterinary , Vibrio alginolyticus/physiology , Streptococcal Infections/immunology , Streptococcal Infections/veterinary , Streptococcus agalactiae/physiology , Gene Expression Regulation/immunology , Gene Expression Profiling/veterinary , Vibrio Infections/immunology , Vibrio Infections/veterinary , Diaminopimelic Acid/chemistry , Diaminopimelic Acid/analogs & derivatives , Perciformes/immunology , Perciformes/genetics , Fishes/immunology , Fishes/geneticsABSTRACT
Vibrio alginolyticus is the causative agent of vibriosis, a common bacterial infection in grouper aquaculture that is associated with the development of haemorrhagic and non-haemorrhagic ulcerations on the fish. In the present study, comparative proteome analysis was performed on serum samples from Vibrio-resistant and Vibrio-susceptible grouper. Samples were analysed using high-throughput LC-MS/MS and identified 2770 unique peptides that corresponded to 344 proteins. Subsequent analysis identified 21 proteins that were significantly up-regulated in the resistant group compared to the control and the susceptible groups. Those proteins are associated with immunostimulatory effects, signalling and binding cascade, metabolism, and maintaining tissue integrity and physiological condition. Besides, potential protein biomarkers related to the immune system were identified, which could be associated with the disease-resistant phenotype. These data provide insights into the underlying immune mechanism of hybrid groupers upon Vibrio sp. infection.
Subject(s)
Bass , Biomarkers , Disease Resistance , Fish Diseases , Fish Proteins , Proteome , Vibrio Infections , Vibrio alginolyticus , Animals , Fish Diseases/immunology , Fish Diseases/microbiology , Vibrio Infections/veterinary , Vibrio Infections/immunology , Bass/immunology , Fish Proteins/immunology , Fish Proteins/genetics , Vibrio alginolyticus/physiology , Tandem Mass Spectrometry/veterinary , Immunity, Innate , Vibrio/physiologyABSTRACT
This study discloses the nanoscale silicate platelet-supported nZnO (ZnONSP) applied as novel feed additives in aquaculture. The preparation of the nanohybrid (ZnO/NSP = 15/85, w/w) was characterized by UV-visible spectroscopy, powder X-ray diffraction and transmission electron microscope. The effects of ZnONSP on growth, zinc accumulation, stress response, immunity and resistance to Vibrio alginolyticus in white shrimp (Penaeus vannamei) were \demonstrated. To evaluate the safety of ZnONSP, shrimps (2.0 ± 0.3 g) were fed with ZnONSP containing diets (200, 400 and 800 mg/kg) for 56 days. Dietary ZnONSP did not affect the weight gain, specific growth rate, feed conversion ratio, survival rate, zinc accumulation, and the expression of heat shock protein 70 in tested shrimps. To examine the immunomodulatory effect of ZnONSP, shrimps (16.6 ± 2.4 g) were fed with the same experimental diets for 28 days. Dietary ZnONSP improved the immune responses of haemocyte in tested shrimps, including phagocytic rate, phagocytic index, respiratory burst, and phenoloxidase activity, and upregulated the expression of several genes, including lipopolysaccharide, ß-1,3-glucan binding protein, peroxinectin, penaeidin 2/3/4, lysozyme, crustin, anti-lipopolysaccharide factor, superoxide dismutase, glutathione peroxidase, clotting protein and α-2-macroglobulin. In the challenge experiment, shrimps (17.2 ± 1.8 g) were fed with ZnONSP containing diets (400 and 800 mg/kg) for 7 days and then infected with Vibrio alginolyticus. Notably, white shrimps that received ZnONSP (800 mg/kg) showed significantly improved Vibrio resistance, with a survival rate of 71.4 % at the end of 7-day observation. In conclusion, this study discovers that ZnONSP is a new type of immunomodulatory supplement that are effective on enhancing innate cellular and humoral immunities, and disease resistance in white shrimp.
Subject(s)
Immunity, Innate , Penaeidae , Animals , Dietary Supplements , Diet/veterinary , Disease Resistance , Vibrio alginolyticus/physiology , Zinc/pharmacologyABSTRACT
An increasing number of evidences have shown that invertebrate taxa can be primed to produce immune memory to resist the secondary infection of pathogens, which was considered as a viable option to protect invertebrates from pathogens. In this work, we compared the protective effect of several different immune priming methods on the Vibrio alginolyticus secondary infection of the Crassostrea gigas. The results showed that C. gigas primed with live V. alginolyticus had higher ROS level, which led to hemocytes necrosis and higher mortality rate in the later stage. Low-dose of formalin-inactivated V. alginolyticus (including 5 × 104 CFU/mL and 5 × 105 CFU/mL) elicited appropriate immune response in C. gigas, protecting C. gigas from V. alginolyticus infection. Immersion with 5 × 104 CFU/mL formalin-inactivated V. alginolyticus was performed to prime C. gigas immunity in the trans-generational immune priming. Trans-generational immune priming significantly increased the resistance of larvae to various Vibrio species. Overall, these results suggested that low-dose of formalin-inactivated V. alginolyticus can protect C. gigas from secondary infection and confer broad-spectrum Vibrio resistance on offspring. This work provided valuable information toward a new direction for the protection of C. gigas from Vibrio infection.
Subject(s)
Coinfection , Crassostrea , Vibrio Infections , Vibrio , Animals , Vibrio alginolyticus/physiology , Formaldehyde , HemocytesABSTRACT
Mytilus unguiculatus is an important economic bivalve species with wide consumption and aquaculture value. Disease is one of the primary limiting factors in mussel aquaculture, thus understanding the response of different tissues of M. unguiculatus to pathogens is crucial for disease prevention and control. In this study, we investigated the physiological and transcriptomic responses of the gills, adductor muscle, and mantle of M. unguiculatus infected with Vibrio alginolyticus. The results showed that V. alginolyticus infection caused inflammation and tissue structure changes in the gill, adductor muscle and mantle of M. unguiculatus. Meanwhile, the activities of superoxide dismutase and catalase in the three tissues increased, while the total antioxidant capacity decreased, suggesting that M. unguiculatus have an activated defense mechanism against infection-induced oxidative stress, despite a compromised total antioxidant capacity. Transcriptomic studies reveal that infected M. unguiculatus exhibits upregulation of endocytosis, lysosome activity, cellular apoptosis, and immune-related signaling pathways, indicating that M. unguiculatus responds to pathogen invasion by upregulating efferocytosis. Compared with the gill and adductor muscle, the mantle had a higher level of mytimycin, mytilin and myticin, and the three tissues also increased the expression of mytimycin to cope with the invasion of pathogens. In addition, the analysis of genes related to taste transduction pathways and muscle contraction and relaxation found that after infection with V. alginolyticus, M. unguiculatus may reduce appetite by inhibiting taste transduction in the gill, while improving muscle contraction of the adductor muscle and keeping the shell closed, to resist further invasion of pathogens and reduce the risk of pathogen transmission in the population.
Subject(s)
Mytilus , Vibrio Infections , Vibrio , Animals , Mytilus/genetics , Vibrio alginolyticus/physiology , Antioxidants , Vibrio Infections/veterinary , Gene Expression Profiling/veterinary , Vibrio/physiologyABSTRACT
Stress responses impact the immune systems, growth, and reproduction of aquatic organisms. Neuroendocrine regulation involving biogenic amines, including octopamine (OA), plays a pivotal role in maintaining physiological balance during stress. This study focuses on the synthesis pathway of OA, particularly the role of tyramine beta hydroxylase (TBH), in Litopenaeus vannamei under stress. TBH catalyzes the conversion of tyramine to OA, a process critical for physiological responses. The present study demonstrated LvTBH at the protein level under different stress conditions during acute (0.5, 1, 2 h) and chronic stress (24, 72, 168 h) periods. LvTBH increased in thoracic ganglia within 2 h under hyperthermal stress, accompanied by elevated OA levels. Conversely, LvTBH decreased in the brain and circumesophageal connective tissues during acute and chronic hypothermal stress. Additionally, LvTBH increased in the brain and circumesophageal connective tissues under acute infection stress, coinciding with elevated OA levels. These findings collectively contribute to a more intricate understanding of the neuroendocrine dynamics within L. vannamei under stress, underscoring the role of TBH in orchestrating responses crucial for adaptation.
Subject(s)
Octopamine , Penaeidae , Animals , Octopamine/metabolism , Vibrio alginolyticus/physiology , Salinity , Mixed Function Oxygenases , TyramineABSTRACT
Bacteria navigate natural habitats with a wide range of mechanical properties, from the ocean to the digestive tract and soil, by rotating helical flagella like propellers. Species differ in the number, position, and shape of their flagella, but the adaptive value of these flagellar architectures is unclear. Many species traverse multiple types of environments, such as pathogens inside and outside a host. We investigate the hypothesis that flagellar architectures mediate environment-specific benefits in the marine pathogen Vibrio alginolyticus which exhibits physiological adaptation to the mechanical environment. In addition to its single polar flagellum, the bacterium produces lateral flagella in environments that differ mechanically from water. These are known to facilitate surface motility and attachment. We use high-throughput 3D bacterial tracking to quantify chemotactic performance of both flagellar architectures in three archetypes of mechanical environments relevant to the bacterium's native habitats: water, polymer solutions, and hydrogels. We reveal that lateral flagella impede chemotaxis in water by lowering the swimming speed but improve chemotaxis in both types of complex environments. Statistical trajectory analysis reveals two distinct underlying behavioral mechanisms: In viscous solutions of the polymer PVP K90, lateral flagella increase the swimming speed. In agar hydrogels, lateral flagella improve overall chemotactic performance, despite lowering the swimming speed, by preventing trapping in pores. Our findings show that lateral flagella are multipurpose tools with a wide range of applications beyond surfaces. They implicate flagellar architecture as a mediator of environment-specific benefits and point to a rich space of bacterial navigation behaviors in complex environments.
Subject(s)
Chemotaxis , Vibrio alginolyticus , Vibrio alginolyticus/physiology , Adaptation, Physiological , Flagella , Hydrogels , PolymersABSTRACT
Shrimp immunology is vital in establishing prophylactic and therapeutic strategies for controlling pathological problems that threaten shrimp production. Apart from dietary treatments, the adenosine 5'-monophosphate-activated protein kinase (AMPK), an important regulatory enzyme that restores cellular energy balance during metabolic and physiological stress, is known to have therapeutic potential to improve shrimp's defense mechanism. Despite this, studies targeting the AMPK pathway in shrimp exposed to stressful conditions are vastly limited. In this study, AMPK was knocked down to assess the immunological changes and white shrimp, Penaeus vannamei resistance to Vibrio alginolyticus infection. Shrimps were injected individually and simultaneously with dsRNA targeting specific genes such as AMPK, Rheb, and TOR, after which the hepatopancreas was analyzed for the different gene expressions. The gene expressions of AMPK, Rheb, and TOR were effectively suppressed after being treated with dsRNAs. The Western blot analysis further confirmed a reduction in the protein concentration of AMPK and Rheb in the hepatopancreas. The suppression of AMPK gene led to a robust increase in the shrimp's resistance to V. alginolyticus, whereas the activation of AMPK by metformin decreased the shrimp's disease resistance. Among the mTOR downstream targets, the HIF-1α expression in shrimp treated with dsAMPK significantly increased at 48 h but returned to normal levels when shrimp were treated with dsAMPK and either dsRheb or dsTOR. Immune responses such as respiratory burst, lysozyme activity, and phagocytic activity increased, while superoxide dismutase activity decreased following the knockdown of the AMPK gene compared to the control group. However, co-injection with dsAMPK and dsTOR or dsRheb restored immune responses to normal levels. Collectively, these results demonstrate that the inactivation of AMPK may ameliorate shrimp's innate immune response to recognize and defend against pathogens via the AMPK/mTOR1 pathway.
Subject(s)
Penaeidae , Vibrio Infections , Animals , Vibrio alginolyticus/physiology , AMP-Activated Protein Kinases/genetics , Immunity, Innate/genetics , AdenosineABSTRACT
Mitogen-activated protein kinase kinase 4 (MKK4), serves as a critical component of the mitogen-activated protein kinase signaling pathway, facilitating the direct phosphorylation and activation of the c-Jun N-terminal kinase (JNK) and p38 families of MAP kinases in response to environmental stresses. In the current research, we identified two MKK4 subtypes, namely SpMKK4-1 and SpMKK4-2, from Scylla paramamosain, followed by the analysis of their molecular characteristics and tissue distributions. The expression of SpMKK4s was induced upon WSSV and Vibrio alginolyticus challenges, and the bacteria clearance capacity and antimicrobial peptide (AMP) genes' expression upon bacterial infection were significantly decreased after knocking down SpMKK4s. Additionally, the overexpression of both SpMKK4s remarkably activated NF-κB reporter plasmid in HEK293T cells, suggesting the activation of the NF-κB signaling pathway. These results indicated the participation of SpMKK4s in the innate immunity of crabs, which shed light on a better understanding of the mechanisms through which MKK4s regulate innate immunity.
Subject(s)
Brachyura , White spot syndrome virus 1 , Humans , Animals , NF-kappa B , Vibrio alginolyticus/physiology , White spot syndrome virus 1/physiology , MAP Kinase Kinase 4/genetics , Phylogeny , HEK293 Cells , Gene Expression Profiling , Immunity, Innate , Mitogen-Activated Protein Kinases , Arthropod Proteins/metabolismABSTRACT
In order to evaluate the effect of Clostridium butyricum (C. butyricum) feeding on intestinal microorganisms and protection against infection by Vibrio alginolyticus (V. alginolyticus) in Penaeus vannamei (P. vannamei). We set up two groups, CG30 (fed normal feed) and CB30 (fed feed supplemented with C. butyricum), for the 30d C. butyricum feeding test, and four groups, CG (CG30 group injected with PBS), CB (CB30 group injected with PBS), VACG (CG30 group injected with V. alginolyticus), and VACB (CB30 group injected with V. alginolyticus), for the 24 h infection test. The protective effect of C. butyricum against acute V. alginolyticus infection in P. vannamei was explained in terms of survival, histopathology, changes in enzyme activity, transcriptome analysis, and immune-related genes. We found that feeding C. butyricum significantly altered intestinal microbial populations' abundance and significantly reduced Vibrio spp. In the V. alginolyticus stress test, C. butyricum improved the survival rate and alleviated pathological changes in hepatopancreatic tissues, alleviated the reduction of superoxide dismutase (SOD) and phenoloxidase (PO) activity caused by infection, and increased the lysozyme content in P. vannamei. VACB group compared with the VACG group, 1730 up-regulated differentially expressed genes (DEGs) and 2029 down-regulated DEGs were screened. Quantitative real-time PCR (qRT-PCR) showed that dietary supplementation with C. butyricum suppressed the upregulation of alkaline phosphatase (AKP) transcription factors and the downregulation of prophenoloxidase (proPO), alpha-2-macroglobulin (A2M), and anti-lipopolysaccharide factor (ALF) induced by V. alginolyticus infection. In conclusion, feed supplementation with C. butyricum changed P. vannamei's population ratio of intestinal microorganisms. Moreover, C. butyricum has the potential to act as an inhibitor of V. alginolyticus infection and enhance the resistance of P. vannamei to V. alginolyticus infection.
Subject(s)
Clostridium butyricum , Gastrointestinal Microbiome , Penaeidae , Animals , Vibrio alginolyticus/physiology , Penaeidae/genetics , Dietary Supplements , Immunity, Innate/geneticsABSTRACT
The cation-dependent mannose-6-phosphate receptor (CD-M6PR) is a P-type lectin that plays a crucial role in lysosomal enzyme transport, bacterial resistance, and viral entry. In this study, we cloned and analyzed the ORF of the CD-M6PR gene from Crassostrea hongkongensis and named it ChCD-M6PR. We analyzed the nucleotide and amino acid sequence of ChCD-M6PR, its tissue expression pattern and immune response to Vibrio alginolyticus. Our results showed that the ORF of ChCD-M6PR was 801 bp long and encoded a protein of 266 amino acids with a signal peptide at the N-terminus, as well as Man-6-P_recep, ATG27 and transmembrane structural domains. Phylogenetic analysis indicated that Crassostrea hongkongensis shared the highest similarity with Crassostrea gigas in the terms of CD-M6PR. The ChCD-M6PR gene was found to be expressed in various tissues, with the highest expression observed in the hepatopancreas and the lowest in the hemocytes by the fluorescence quantitative PCR. Furthermore, the expression of ChCD-M6PR gene was significantly up-regulated for a short time in response to Vibrio alginolyticus infection in the gill and hemocytes, while it was down-regulated in the gonads. The expression patterns of ChCD-M6PR also varied in the other tissues. The 96 h cumulative mortality rate of Crassostrea hongkongensis infected with Vibrio alginolyticus after knockdown the ChCD-M6PR gene was significantly higher. Overall, our findings suggests that ChCD-M6PR plays a crucial role in the immune response of Crassostrea hongkongensis to Vibrio alginolyticus infection, and its tissue-specific expression patterns may be indicatitive of varied immune responses across tissues.
Subject(s)
Crassostrea , Vibrio Infections , Humans , Animals , Vibrio alginolyticus/physiology , Base Sequence , Crassostrea/metabolism , Phylogeny , Immunity, Innate/genetics , HemocytesABSTRACT
Lactobacillus plantarum is known for its probiotics benefit to host, although the effects vary among strains. This study conducted a feeding experiment of three Lactobacillus strains, MRS8, MRS18 and MRS20, which were isolated from kefir and incorporated into the diets of shrimp to evaluate the effects of non-specific immunity, immune-related gene expression, and disease resistance of white shrimp (Penaeus vannamei) against Vibrio alginolyticus. To prepare the experimental feed groups, the basic feed was mixed with different concentrations of L. plantarum strains MRS8, MRS18, and MRS 20, which were incorporated at 0 CFU (control), 1 × 106 CFU (groups 8-6, 18-6, and 20-6), and 1 × 109 CFU (groups 8-9, 18-9, and 20-9) per gram of diet for an in vivo assay. During the rearing period for 28 days of feeding each group, immune responses, namely the total hemocyte count (THC), phagocytic rate (PR), phenoloxidase activity, and respiratory burst were examined on days 0, 1, 4, 7, 14, and 28. The results showed that groups 20-6, 18-9 and 20-9 improved THC, and groups 18-9 and 20-9 improved phenoloxidase activity and respiratory burst as well. The expression of immunity-related genes was also examined. Group 8-9 increased the expression of LGBP, penaeidin 2 (PEN2) and CP, group 18-9 increased the expression of proPO1, ALF, Lysozyme, penaeidin 3 (PEN3) and SOD, and group 20-9 increased the expression of LGBP, ALF, crustin, PEN2, PEN3, penaeidin 4 (PEN4) and CP (p < 0.05). Groups 18-6, 18-9, 2-6, and 20-9 were further used in the challenge test. After feeding for 7 days and 14 days, Vibrio alginolyticus was injected into white shrimp and observed the shrimp survival for 168 h. The results showed that compared to the control, all groups improved the survival rate. Especially, feeding group 18-9 for 14 days improved the survival rate of white shrimp (p < 0.05). After the challenge test for 14 days, the midgut DNA of survival white shrimps was extracted to analyze the colonization of L. plantarum. Among the groups, (6.61 ± 3.58) × 105 CFU/pre shrimp of L. plantarum in feeding group 18-9 and (5.86 ± 2.27) × 105 CFU/pre shrimp in group 20-9 were evaluated by qPCR. Taken together, group 18-9 had the best effects on the non-specific immunity, the immune-related gene expression, and the disease resistance, which might be due to the benefit of the probiotic colonization.
Subject(s)
Kefir , Lactobacillus plantarum , Penaeidae , Animals , Vibrio alginolyticus/physiology , Immunity, Innate , Monophenol Monooxygenase/metabolism , Disease ResistanceABSTRACT
Hybrid of nanosilver and nanoscale silicate platelet (AgNSP) is a safe, non-toxic nanomaterial which has been applied in medical use due to its strong antibacterial activity. The application of AgNSP in aquaculture was first proposed in the present study by evaluating the in vitro antibacterial activities against four aquatic pathogens, in vitro effects toward shrimp haemocytes as well as the immune responses and disease resistance in Penaeus vannamei fed with AgNSP for 7 days. For evaluating the antibacterial activities of AgNSP in culture medium, the minimum bactericidal concentration (MBC) values against Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus and Vibrio parahaemolyticus were 100, 15, 625 and 625 mg/L, respectively. Moreover, the inhibition of pathogen growth over a period of 48 h could be achieved by the appropriate treatment of AgNSP in culturing water. In freshwater containing bacterial size of 103 and 106 CFU/mL, the effective doses of AgNSP against A. hydrophila were 12.5 and 450 mg/L, respectively while the effective doses against E. tarda were 0.2 and 50 mg/L, respectively. In seawater with same bacterial size, the effective doses against V. alginolyticus were 150 and 2000 mg/L, respectively while the effective doses against V. parahaemolyticus were 40 and 1500 mg/L, respectively. For the in vitro immune tests, the superoxide anion production and phenoloxidase activity in haemocytes were elevated after in vitro incubation with 0.5-10 mg/L of AgNSP. In the assessment of dietary supplemental effects of AgNSP (2 g/kg), no negative effect on the survival was found at the end of 7 day feeding trail. In addition, the gene expression of superoxide dismutase, lysozyme and glutathione peroxidase were up-regulated in haemocytes taken from shrimps received AgNSP. The following challenge test against Vibrio alginolyticus showed that the survival of shrimp fed with AgNSP was higher than that of shrimp fed with control diet (p = 0.083). Dietary AgNSP improved the Vibrio resistance of shrimp by increasing 22.7% of survival rate. Therefore, AgNSP could potentially be used as a feed additive in shrimp culture.
Subject(s)
Immunity, Innate , Penaeidae , Animals , Dietary Supplements , Diet , Disease Resistance , Superoxides , Superoxide Dismutase/metabolism , Vibrio alginolyticus/physiologyABSTRACT
As an effective immunostimulant, Astragalus polysaccharides (APS) have been widely used in fish aquaculture, however, their action mechanisms remain poorly understood. In the present paper, the inflammatory macrophage model of large yellow croaker (Larimichthys crocea) was constructed by using formalin-inactivated Vibrio alginolyticus. Inactivated V. alginolyticus could cause cellular damage of primary head kidney macrophages (PKM) by decreasing cell activity and inducing reactive oxygen species (ROS) production and cell apoptosis. When PKM were pretreated with APS, the depressed cell activity induced by inactivated V. alginolyticus was significantly improved, and ROS overproduction and cell apoptosis were inhibited. Then the protection mechanism of APS was investigated by transcriptome analysis. After treated with inactivated V. alginolyticus, the expression of immune-related genes (TLR5s, TLR13, Clec4e, IKK, IκB, BCL-3, NF-κB2, REL, IL-1ß, and IL-6) and pyroptosis-related genes (caspase-1, NLRP3, and NLRC3) in PKM were significantly up-regulated. However, APS pretreatment reversed the up-regulation of most of the above-mentioned genes, where TLR5s, BCL-3, REL, caspase-1, NLRP12, IL-1ß, and IL-6 were significantly down-regulated compared with inactivated V. alginolyticus-treated group. These results suggested that APS could protect large yellow croaker PKM against inactivated V. alginolyticus-induced inflammatory injury, and may exert their protection effects by activating NF-κB and pyroptosis signaling pathways. These findings therefore advance our understanding of the immune regulation mechanism of APS in fish, and facilitate the application of APS in prevention and control of fish bacteriosis.
Subject(s)
Fish Diseases , Perciformes , Animals , Vibrio alginolyticus/physiology , Fish Proteins , Reactive Oxygen Species/metabolism , Interleukin-6/metabolism , Macrophages , Polysaccharides/metabolism , Caspases/metabolismABSTRACT
Octopamine and Tyramine are biogenic amines that have been demonstrated to play an important immunological role in white shrimp, Litopenaeus vannamei. G protein-coupled receptors, known as seven-transmembrane domain receptors, are a variety of neurotransmitter receptors which are sensitive to biogenic amines for initiating the cell signaling pathway. In present study, we cloned and characterized an octopamine/tyramine receptor (LvOA/TA-R) from the hemocytes of L. vannamei, with a 1194 b.p. open reading frame that encodes 398 amino acids. Several bioinformatics analyses indicated that LvOA/TA-R had seven conserved hydrophobic transmembrane domains. The phylogenetic analysis and multiple sequence alignment indicated that LvOA/TA-R was orthologous to the OA/TA receptor of tiger shrimp, P. monodon. LvOA/TA-R was expressed in hemocytes and nervous tissue including circumoesphageal connective tissue and the thoracic and abdominal ganglia. Significant increases in LvOA/TA-R occurred in hemocytes of L. vannamei under Vibrio alginolyticus infection within 30-60 min of infection. Here, we demonstrated that LvOA/TA-R expression is upregulated in response to Vibrio alginolyticus infection and appears to be functionally responsible for the observed immune response. These results suggest that LvOA/TA-R mediates regulation of immunity, which promotes the resistance of L. vannamei to V. alginolyticus.
Subject(s)
Penaeidae , Vibrio Infections , Amino Acids/metabolism , Animals , Hemocytes , Immunity, Innate/genetics , Octopamine/metabolism , Phylogeny , Receptors, Biogenic Amine , Receptors, Neurotransmitter/metabolism , Tyramine , Vibrio alginolyticus/physiologyABSTRACT
Red seaweeds have several biofunctional properties, including immunomodulatory, antitumor, antioxidant, and antibacterial activities. In this study, we examined the effects of diets containing Sarcodia suae on the immune response, immune-related gene expressions, and disease resistance against Vibrio alginolyticus in white shrimp Litopenaeus vannamei. In addition, 1H NMR metabolomics was applied to analyze the metabolites extracted from shrimp fed with S. suae and their functions in regulating immunity. A diet containing only fish meal was used as the control diet (S0), and three diets containing different concentrations of S. suae powder, 2.5% (S2.5), 5% (S5), and 7.5% (S7.5) were used as experimental diets. Shrimp were fed diets for 20 days. Compared to the control group (S0), results showed that (1) shrimp fed diets supplemented with 5-7.5% of S. suae powder signiï¬cantly increased anti-V. alginolyticus activity; (2) phagocytic activity (PA) increased in all shrimp fed with S. suae, but total haemocyte count (THC) only increased in S7.5 group; and (3) the expression of glutathione peroxidase (GPx) in haemocyte were significantly higher in S7.5 groups. Results from the 1H NMR analysis revealed that 19 heapatopancreatic metabolites were matched and identified among groups. Based on the KEGG enrichment analysis, the up-regulated metabolites in the shrimp fed S5 and S7.5 diets were primarily due to the metabolism of purine and phenylalanine and their respective pathways. Results from these trials reveal that diets containing S. suae can increase immune response, thereby increasing shrimp resistance to V. alginolyticus. The purine and phenylalanine metabolic pathways may be considered as the relevant pathways for optimizing immunomodulatory responses.
Subject(s)
Penaeidae , Rhodophyta , Animals , Disease Resistance , Immunity, Innate , Metabolic Networks and Pathways , Phenylalanine , Powders/pharmacology , Purines/pharmacology , Vibrio alginolyticus/physiologyABSTRACT
Penaeus vannamei is an important cultured shrimp that has high commercial value in the worldwide. However, the industry suffers heavy economic losses each year due to disease outbreaks caused by pathogenic bacteria. In the present study, after Vibrio alginolyticus infection, DNA damage in the hemocytes of the shrimp markedly increased, and autophagy and apoptosis increased significantly. Subsequently, hemocytes were sampled from the control and infected shrimp and sequenced for mRNA and microRNA (miRNA) 24 h after V. alginolyticus infection to better understand the response mechanism to bacterial infection in P. vannamei. We identified 1,874 and 263 differentially expressed mRNAs (DEGs) and miRNAs (DEMs) respectively, and predicted that 997 DEGs were targeted by DEMs. These DEGs were involved in the regulation of multiple signalling pathways, such as Toll and IMD signalling, TGF-beta signalling, MAPK signalling, and cell apoptosis, during Vibrio alginolyticus infection of the shrimp. We identified numerous mRNA-miRNA interactions, which provide insight into the defense mechanism that occur during the antimicrobial process of P. vannamei.