Physiological adaptation in flagellar architecture improves Vibrio alginolyticus chemotaxis in complex environments.

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.

Application of nZnO supported with nanoclay for improving shrimp immunity.

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.

Transcriptome analysis reveals tissue-specific responses of Mytilus unguiculatus to Vibrio alginolyticus infection.

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.

Functional characterization of NOD1 from golden pompano Trachinotus ovatus.

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.

Comparative proteome analysis revealed potential biomarkers and the underlying immune mechanisms in Vibrio-resistant hybrid grouper, Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂.

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.

Tyramine beta hydroxylase-mediated octopamine synthesis pathway in Litopenaeus vannamei under thermal, salinity, and Vibrio alginolyticus infection stress.

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.

Adenosine 5'-monophosphate-activated protein kinase (AMPK) negatively regulates the immunity and resistance to Vibrio alginolyticus of white shrimp, Penaeus vannamei.

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.

Effect of nanoclay supported nanosilver on the growth inhibition of aquatic pathogens and immunomodulatory effect in Penaeusvannamei.

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.

Lactobacillus plantarum isolated from kefir enhances immune responses and survival of white shrimp (Penaeus vannamei) challenged with Vibrio alginolyticus.

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.

Effect of Clostridium butyricum on intestinal microbiota and resistance to Vibrio alginolyticus of Penaeus vannamei.

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.

Molecular characterization of a cation-dependent mannose-6-phosphate receptor gene in Crassostrea hongkongensis and its responsiveness in Vibrio alginolyticus infection.

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.

In vivo study of a novel protein kinase C that mediates immunocompetence and catecholamine biosynthesis in hemocytes of Litopenaeus vannamei by using its potential competitive inhibitor, bisindolylmaleimide I.

This study applied bisindolylmaleimide I (BSM), a pharmacological competitive inhibitor of protein kinase C (PKC) enzymatic activity, at 1.25 pmol shrimp-1 for 60 min to investigate the potential involvement of PKC in signal transduction pathways in the hemocytes of Litopenaeus vannamei. A novel PKC in L. vannamei (LvnPKC) was identified and characterized and was determined to be involved in mediating the neuroendocrine-immune regulatory network. The hemocytes of L. vannamei that receive BSM exhibit significantly decreased PKC activity and LvnPKC gene and protein expression levels. Furthermore, the total hemocyte count, hyaline cells, and semigranular cells increased significantly along with significant decreases in granular cells, and meanwhile, the significantly increased phenoloxidase activity, respiratory bursts, superoxide dismutase (SOD) activity, phagocytic activity, and neutrophil extracellular trap were observed; however, phagocytic activity decreased significantly. In a molecular model, the gene expressions of lipopolysaccharide- and ß-1,3-glucan-binding protein, peroxinectin, cytosolic manganese SOD, mitochondrial manganese SOD, and copper/zinc SOD in the hemocytes of L. vannamei that had received BSM decreased significantly, but prophenoloxidase I increased significantly. In catecholamine biosynthesis, tyrosine, dopamine, and norepinephrine decreased significantly in the hemocytes of L. vannamei that had received BSM, and l-dihydroxyphenylalanine increased. Moreover, tyrosine hydroxylase (TH) activity increased significantly, whereas TH and dihydroxyphenylalanine decarboxylase gene expression decreased significantly. These findings suggest that BSM inhibits PKC activity in hemocytes in which LvnPKC gene and protein expression are also inhibited. Additionally, the hemocytes' immunocompetence, including their prophenoloxidase and antioxidant systems, phagocytic activity, and catecholamine biosynthesis, was disrupted, confirming the roles of LvnPKC in mediating the neuroendocrine-immune regulatory network in hemocytes.

Sarcodia suae modulates the immunity and disease resistance of white shrimp Litopenaeus vannamei against Vibrio alginolyticus via the purine metabolism and phenylalanine metabolism.

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 significantly 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.

Molecular characterization, expression and functional analysis of large yellow croaker (Larimichthys crocea) peroxisome proliferator-activated receptor gamma.

The peroxisome proliferator-activated receptor gamma (PPARγ) are nuclear receptors with distinct roles in energy metabolism and immunity. Although extensively studied in mammals, immunomodulatory roles of this molecule in teleost fish remain to be investigated. In this study, large yellow croaker (Larimichthys crocea) PPARγ (LcPPARγ) sequence was cloned, which encodes a polypeptide of 541 amino acids that include signature domains belonging to the nuclear receptor superfamily. Phylogenetically, LcPPARγ was most closely related to PPARγ derived from European sea bass (Dicentrarchus labrax). Quantitative analysis shown a ubiquitous expression of this molecule, with highest expression level detected in the intestine. The expression of LcPPARγ was decreased in the intestine, muscle, body kidney, spleen and head kidney-derived monocytes/macrophages (MO/MФs) over the course of Vibrio alginolyticus (V. alginolyticus) infection. In contrast, an up-regulation of LcPPARγ was observed in head kidney-derived MO/MФs following docosahexaenoic acid (DHA) treatment. This increase in LcPPARγ leads to an up-regulation of LcCD11b and LcCD18 and an enhancement of complement-mediated phagocytosis. Furthermore, cytokine secretions of V. alginolyticus-stimulated MO/MФs were modulated following LcPPARγ activations that up-regulated the expression of LcIL-10, while decreased the expression of LcIL-1ß, LcTNF-α and LcTGF-ß1. Overall, our results indicated that LcPPARγ plays a role in regulating functions of MO/MФs and likely contribute to MO/MФs polarization.

Astragalus polysaccharides protect against inactivated Vibrio alginolyticus-induced inflammatory injury in macrophages of large yellow croaker.

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.

Molecular characterization and functional roles for Vibrio alginolyticus resistance of an octopamine/tyramine receptor of the white shrimp, Litopenaeus vannamei.

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.

Novel Amiloride Derivatives That Inhibit Bacterial Motility across Multiple Strains and Stator Types.

The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomA/PomB stator complex of Vibrio spp., can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium motility inhibitors have been described since the discovery of phenamil. In this study, we characterized two possible motility inhibitors, HM2-16F and BB2-50F, from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free-swimming bacteria, and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na+ motors comparable to phenamil at matching concentrations and could also stop rotation of tethered cells driven by H+ motors. Bead measurements in the presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na+ and H+ stator types and in pathogenic and nonpathogenic strains. IMPORTANCE Here, we characterized two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. These compounds were shown to inhibit flagellar motility at 10 µM across multiple strains: from nonpathogenic Escherichia coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (enterohemorrhagic E. coli or uropathogenic E. coli [EHEC or UPEC, respectively]), and finally, sodium-powered Vibrio alginolyticus. Broad antimotility compounds such as these are important tools in our efforts to control virulence of pathogens in health and agricultural settings.

White shrimp Litopenaeus vannamei that have received mixtures of heat-killed and formalin-inactivated Vibrio alginolyticus and V. harveyi exhibit recall memory and show increased phagocytosis and resistance to Vibrio infection.

Heat-killed Vibrio alginolyticus (HVa), formalin-inactivated V. alginolyticus (FVa), heat-killed Vibrio harveyi (HVh), formalin-inactivated V. harveyi (FVh), live V. alginolyticus (LVa), and live V. harveyi (LVh) were used in this study. White shrimp Litopenaeus vannamei receiving two mixtures (HVa + FVa) or four mixtures (HVa + FVa + HVh + FVh) served as primary exposure, and shrimp receiving LVa or LVh afterward served as secondary exposure. Shrimp receiving marine saline and then receiving either LVa or LVh served as controls. Phagocytic activity and clearance efficiency were examined in shrimp that received two mixtures after 1-8 weeks and then received LVa. Both the phagocytic activity and clearance efficiency of shrimp receiving two mixtures were significantly higher than in control shrimp after 1-8 weeks. In another experiment, phagocytic activity and clearance efficiency were examined in shrimp that received four mixtures after 1-8 weeks and then received LVa and LVh, respectively. The phagocytic activity of shrimp receiving four mixtures was significantly higher than in control shrimp after 1-8 weeks post exposure to LVa and LVh. The clearance efficiency of shrimp receiving four mixtures was significantly higher than in control shrimp after 1-6 weeks post exposure to LVa, and 1-7 weeks post exposure to LVh. In the other experiment, the survival rate of shrimp that received four mixtures after five weeks were challenged with LVa at 6.4 × 107 colony-forming units (cfu) shrimp-1 and LVh at 4.4 × 106 cfu shrimp-1. Shrimp that received marine saline for five weeks and then challenged with LVa and LVh at a same dose served as challenged controls. The survival rate of shrimp that received four mixtures was significantly higher (90%) than that of control shrimp (67%), and significantly higher (73%) than that of control shrimp (53%) after 3-7 days post challenge with LVa and LVh. It is concluded that the mixtures have feature of adjuvant and antigen, and shrimp receiving mixtures of heat-killed and formalin-inactivated V. alginolyticus and V. harveyi even after 5-8 weeks exhibit memory recall and show increased phagocytosis and resistance to Vibrio infections.

Black soldier fly (Hermetia illucens) larvae meal in diets of European seabass: Effects on antioxidative capacity, non-specific immunity, transcriptomic responses, and resistance to the challenge with Vibrio alginolyticus.

Black soldier fly larvae meal (BSFLM) has been successfully demonstrated as a promising fish meal (FM) replacer in diets of several fish species including European seabass (Dicentrarchus labrax). However, its impacts on antioxidant capacity, and immune responses of treated fish are still poorly understood. A 60-day feeding trial was carried out to evaluate the effects of partial substitution of FM with different levels of dry BSFLM on the antioxidative status, non-specific immunity, transcriptomic responses, and resistance of European seabass to the challenge with Vibrio alginolyticus. Four isoproteic (45%) and isolipidic diets were formulated by replacing 0.0%, 25%, 35%, and 50% of the dietary FM. Each diet was randomly assigned to four fish groups (in triplicates) (initial mean body weight, 12.1 ± 0.21 g) (20 fish per aquarium) (n = 240). Fish were fed three times daily to the apparent satiation. At the end of the feeding trial, serum antioxidant biomarkers such as malondialdehyde levels, and catalase, superoxide dismutase, and glutathione peroxidase enzyme activities were significantly increased in all BSFLM groups in a dose-dependent manner compared to the control group (P < 0.05). The non-specific immune indices, including phagocytic activity, phagocytic index, serum lysozyme and respiratory burst activities were significantly elevated in BSFLM groups compared to those in the control group (P < 0.05). Significant upregulation of the mRNA expression levels of hepatic heat shock protein 70, interleukin-1beta and interleukin-10 genes were observed in all BSFLM groups compared to the control group (P < 0.05). Additionally, after the challenge with V. alginolyticus, the relative percent of survival was significantly elevated in fish groups fed on diets containing graded levels of BSFLM over the control group (P < 0.05). Conclusively, the present study suggests the potential efficacy of partial replacement of dietary FM protein for up to 50% by BSFLM without negative effects on fish health with possible potentiation of the antioxidative status, and the immune responses of the European seabass.

Expression analysis and tissue localization of IgZ in the grouper Epinephelus coioides after Vibrio alginolyticus infection and vaccination.

The orange-spotted grouper (Epinephelus coioides) is an important marine farmed fish in China. It is affected by the bacterial pathogen Vibrio alginolyticus, which causes high mortality and substantial economic losses. We studied the transcriptional changes of the IgZ gene in E. coioides following V. alginolyticus stimulation and investigated the distribution of IgZ in different tissues. The highest expression level of IgZ occurred in the head kidney. When fish were stimulated with live and inactivated V. alginolyticus, the expression levels of IgZ in the head kidney, spleen, intestine, gills and blood cells were significantly upregulated. In an in situ hybridization study, IgZ mRNA-positive cells were detected in the head kidney, spleen and gill, but positive signals were not detected in the liver and intestine. IgZ-labelled cells increased in the head kidney, spleen and gills post-infection with V. alginolyticus for 21 days. The present study provides additional evidence that IgZ is involved in mucosal immune responses and helps explain the role of IgZ in E. coioides defence against V. alginolyticus infection.