Vibrio Alginolyticus Research Articles

Effect of dietary supplementation with Moringa oleifera leaf extract and Lactobacillus acidophilus on growth performance, intestinal microbiota, immune response, and disease resistance in whiteleg shrimp (Penaeus vannamei)

This study investigated the effect of the moringa (Moringa oleifera) leaf extract and Lactobacillus acidophilus individually or combined on growth performance, enzyme activity, intestinal and hepatopancreatic histology, intestinal microbiota, immune response, and resistance against Vibrio alginolyticus and Vibrio parahaemolyticus in whiteleg shrimp (Penaeus vannamei). Six diets were formulated: three diets without L. acidophilus containining 0 (control, ME0), 2.5 (ME2.5), and 5.0 g/kg of moringa (ME5.0) and the same three diets containing L. acidophilus at 1 × 107 CFU/g of diet (ME0+P, ME2.5 + P, and ME5.0 + P, respectively). Growth performance was measured after 60 days of the rearing period. On the final day, the shrimp were sampled to assess enzyme activity, intestinal and hepatopancreatic histology, and gut microbiota. Shrimp hemocytes were examined on Days 0, 1, 2, 4, 7, 14, 21, and 28 to measure the immune response in terms of the total hemocyte count, phenoloxidase activity, phagocytosis, and superoxide anion production. Furthermore, the shrimp were challenged with V. alginolyticus and V. parahaemolyticus. The results revealed that ME2.5 + P significantly increased (P < 0.05) final weight, weight gain, specific growth rate, enzyme activities, and villi height compared with ME2.5 and control. Wall thickness was increased in the shrimp fed diet supplemented with moringa and L. acidophilus compared with the control shrimp. Hepatopancreatic histology revealed that R cells were more abundant in the shrimp fed diet containing moringa and L. acidophilus compared with those fed diet containing moringa alone (P < 0.05) at the same concentration. High-throughput sequencing analysis indicated that the dietary supplementation with moringa and L. acidophilus affected the gut microbiota composition. All gene functions, members of KEGG level 2, related to metabolism were increased in diet supplemented with moringa with or without L. acidophilus compared with the control group. The immune assay revealed that the total hemocyte count, phenoloxidase activity, phagocytic rate, superoxide anion production, and immune-related gene expression (including those of prophenoloxidase II, alpha-2-macroglobulin, penaeidin2, antilipopolysaccharide factor, crustin, lysozyme, glutathione peroxidase, and superoxide dismutase) were higher in the experimental groups than in the control group on several observed days; however, the increases were observed more often in the ME2.5 + P group than in the other treatment groups. Furthermore, the ME2.5 + P group exhibited a significantly higher survival rate (P < 0.05) in the challenge test against V. alginolyticus and V. parahaemolyticus. In conclusion, supplementation with dietary moringa and L. acidophilus at ME2.5 + P improved growth performance, immune system, and resistance against Vibrio in the shrimp.

A New Crustin Gene Homolog SpCrus8 Identified in Scylla paramamosain Exerting In Vivo Protection Through Opsonization and Immunomodulation.

Crustins are the most abundant class of antimicrobial peptides in crustaceans and are essential for protecting animals from infection. Among them, type II crustins usually exhibit potent antimicrobial activity. Interestingly, in this study, a newly identified type II crustin gene homolog (named SpCrus8) from mud crab Scylla paramamosain, the recombinant proteins of which (rSpCrus8 and rTrx-SpCrus8) showed no obvious antibacterial effects, but could significantly reduce the bacterial load in crab hemolymph and improve the survival rate of crabs infected with Vibrio alginolyticus. The immune-related function of SpCrus8 and the underlying mechanism deserve further investigation. It was found that the SpCrus8 gene was widely distributed in various tissues of adult crabs. In the hepatopancreas of crabs infected with V. alginolyticus or Staphylococcus aureus, transcripts of the SpCrus8 gene were remarkably induced, indicating that the SpCrus8 gene was involved in the immune response to bacterial infection in vivo. In addition, rSpCrus8 and rTrx-SpCrus8 had strong binding activity not only to microbial surface components (lipopolysaccharide, lipoteichoic acid, peptidoglycan, and glucan), but also to the tested bacteria (S. aureus, Pseudomonas aeruginosa and V. alginolyticus). Notably, rSpCrus8 and rTrx-SpCrus8 could significantly promote hemocyte phagocytosis. After rSpCrus8 and rTrx-SpCrus8 treatment, a large number of fluorescent microspheres were observed to aggregate into clusters and be phagocytosed by multiple hemocytes, while hemocytes in the control group phagocytosed only individual microspheres, indicating that SpCrus8 played an important role in opsonization. When the SpCrus8 gene was knocked down, the expression levels of the key phagocytosis-related genes SpRab5 and SpRab7 were significantly downregulated, as well as the IMD signaling pathway genes SpIKKβ and SpRelish, and another crustin gene SpCrus5. Correspondingly, all the SpIKKβ, SpRelish and SpCrus5 genes were significantly upregulated after rSpCrus8 treatment, suggesting that SpCrus8 might be involved in the immunomodulation of S. paramamosain. Taken together, this study revealed the immune-related functions of the SpCrus8 gene in opsonization and regulation, which will help us further understand the role of the crustin gene family in the immune system of mud crabs and provide new insights into the function of type II crutins.

A T3SS Regulator Mutant HY9901 ΔaraC of Vibrio alginolyticus Decreased the Expression of HopPmaJ and Provided Protection to Danio rerio as a Live-Attenuated Vaccine

Vibrio alginolyticus, a zoonotic bacterial pathogen, expresses a type III secretion system (T3SS) that is critical for pathogen virulence and disease development. In this study, the mutant HY9901 ΔaraC was obtained from the laboratory and its biological characteristics were analyzed. The swimming ability of ΔaraC decreased and exhibited a 2,600 times reduction in virulence to zebrafish. However, ΔaraC showed no difference in growth and extracellular protease activity compared to wild type. Biofilm-forming ability was improved at 24 h, but no difference was observed at other time points. The results of drug sensitivity testing showed that compared with the wild-type HY9901 strain, ΔaraC was sensitive to amikacin, tetracycline, neomycin, minocycline, and gentamicin. The transcription levels of T3SS effector proteins HopPmaJ, VopS, VcrV, and VopN were analyzed by qRT-PCR. The results showed that ΔaraC had significantly upregulated the mRNA expression of VopS, VcrV, and VopN, but significantly downregulated the mRNA expression of HopPmaJ at each stage compared with HY9901. Western blotting and the β-galactosidase reporter gene experiment also showed that the deletion of araC gene significantly downregulated the expression of HopPmaJ. Finally, the relative percent survival (RPS) rate of grouper inoculated by intramuscular (IM) injection of HY9901 ΔaraC was 61.3% after being challenged with HY9901. Real-time qPCR analysis showed that vaccination of HY9901 ΔaraC could enhance the expression of immune-related genes, including gata-1, il6, IgM, il-1β, and lyz in liver and spleen, indicating that ΔaraC applied as a live-attenuated vaccine effectively induced an immune response in the zebrafish. This study provides evidence for the subsequent development of an effective live-attenuated V. alginolyticus vaccine.

Molecular characterization and expression analysis of TRIF, TRAF6, and TBK1 of golden pompano (Trachinotus ovatus)

Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF), tumor necrosis factor receptor-associated factor 6 (TRAF6) and TANK-binding kinase 1 (TBK1) are critical signal transducers in toll-like receptors (TLRs) signaling pathway. In the present study, TRIF, TRAF6 and TBK1 were characterized from golden pompano (Trachinotus ovatus), named as TroTRIF, TroTRAF6 and TroTBK1, respectively. The full cDNA length of TroTRIF, TroTRAF6 and TroTBK1 was 2297 bp, 2293 bp, and 2482 bp, which respectively encoded 589, 573 and 723 amino acids. The deduced amino acids sequences of TroTRIF, TroTRAF6 and TroTBK1 contained conserved motifs, similar to their counterparts in other vertebrates. Phylogenetic tree analysis revealed that TroTRIF, TroTRAF6 and TroTBK1 were well clustered with their counterparts in other fish species. Quantitative Real-Time PCR (qPCR) analysis showed that TroTRIF, TroTBK1 and TroTRAF6 were detected in all examined tissues of healthy fish, but shared distinct transcript levels. Moreover, the expressions of TroTRIF, TroTBK1 and TroTRAF6 were generally induced by polyriboinosinic-polyribocytidylic acid (polyI:C), lipopolysaccharide (LPS), and Vibrio alginolyticus stimulation in vivo, indicating their critical roles in the immune defense of golden pompano against pathogen invasion. Our results provide valuable information for understanding the functions of these genes in golden pompano.

Transcriptome profiling of the Pacific oyster (Crassostrea gigas) suggests distinct host immune strategy in response to Vibrio alginolyticus infection

Bacterial infection is a serious challenge for oysters, which brings various disease problems to oyster culture. To better understand the immune response to bacterial infection, we performed RNA-seq to profile the transcriptome of gill tissue in the Pacific oyster (Crassostrea gigas) at short-term (6 h) and long-term (48 h) stages after Vibrio alginolyticus infection. A total of 1534 and 2483 differentially expressed genes (DEGs) were identified at short- and long-term stages after infection, respectively. Gene ontology (GO) analysis showed that the identified DEGs were significantly enriched in GO terms of stress response, ion transport, signal transduction and other biological processes. Notably, we observed that the immune strategy was altered in the C. gigas in response to Vibrio infection. In the short-term stage, most of the immune-related genes in C. gigas were down-regulated, and the immune response was low and may be suppressed. While in the long-term stage, large-scale immune-related genes associated with ion channels were activated and immune system was boosted. Distinct sets of immune-related genes with different functions were identified at short-term and long-term stages. Differences in these genes suggested the alteration of immune strategy in the C. gigas in response to Vibrio infection. This work provides valuable resources for better understanding of immune response to bacterial infection and will be important for disease monitoring and disease resistance breeding in the oysters.

Mobile genetic elements used by competing coral microbial populations increase genomic plasticity.

Intraspecies diversification and niche adaptation by members of the Vibrio genus, one of the most diverse bacterial genera, is thought to be driven by horizontal gene transfer. However, the intrinsic driving force of Vibrio species diversification is much less explored. Here, by studying two dominant and competing cohabitants of the gastric cavity of corals, we found that a phenotype influencing island (named VPII) in Vibrio alginolyticus was eliminated upon coculturing with Pseudoalteromonas. The loss of VPII reduced the biofilm formation and phage resistance, but activated motility, which may allow V. alginolyticus to expand to other niches. Mechanistically, we discovered that the excision of this island is mediated by the cooperation of two unrelated mobile genetic elements harbored in Pseudoalteromonas spp., an integrative and conjugative element (ICE) and a mobilizable genomic island (MGI). More importantly, these mobile genetic elements are widespread in cohabitating Gram-negative bacteria. Altogether, we discovered a new strategy by which the mobilome is employed by competitors to increase the genomic plasticity of rivals.

Cloning and expression of three small heat shock proteins in Cherax quadricarinatus under Vibrio alginolyticus or Aeromonas hydrophila stress

Cherax quadricarinatus has been widely introduced and cultured around the world and has been seriously affected by pathogens, including viruses and bacteria. Small heat shock proteins (sHsps) have been shown to have a positive effect on immune responses in animals. In this study, three subunits of sHsps (CqsHsps, named CqHsp24.06, CqHsp41.97 and CqHsp40.17) from C. quadricarinatus were cloned. The coding sequence lengths of the CqHsp24.06, CqHsp41.97 and CqHsp40.17 genes were 654 bp, 1128 bp and 1074 bp, with a molecular weight of 24.06, 41.97 and 40.17 kDa, respectively. In normal tissues, the expression level of CqHsp24.06 was the highest in the intestinal tract and was 12 times higher than that in the hepatopancreas, while CqHsp41.97 was mostly expressed in the gills which was 8 times higher than that in the nerves. However, CqHsp40.17 was highest in the nerves and was 12 times higher than its expression level in the gonads. In gills of C. quadricarinatus stressed by Vibrio Alginolyticus, the relative expression levels of CqHsp24.06, CqHsp41.97 and CqHsp40.17 in gill first increased and then decreased, and the maximum expression increased by 25 times. Under Aeromonas hydrophila stress, the expression of CqHsp24.06 and CqHsp41.97 reached the highest level at the 3-h time point and at the 12-h time point for CqHsp40.17, and the maximum expression increased by 3.5 times. These results indicated that CqsHsps could induce the activation of the immune system, and they were more sensitive to Vibrio Alginolyticus than to Aeromonas hydrophila.

Transcriptome profiles of genes related to growth and virulence potential in Vibrio alginolyticus treated with modified clay

Vibrio alginolyticus is a globally distributed opportunistic pathogen that causes different degrees of disease in various marine organisms, such as fish, shrimp and shellfish. At present, vibriosis caused by V. alginolyticus has a wide epidemic range and causes frequent outbreaks, resulting in substantial losses in aquaculture. According to previous studies, modified clay (MC) could effectively flocculate and reduce the density of Vibrio in water, but it is still unknown whether MC inhibits growth and how it affects virulence in bottom flocs. Here, we studied the response mechanism of V. alginolyticus in flocs treated with MC at the transcriptome level and verified the transcriptomic data combined with relevant physiological experiments and reverse transcription quantitative real-time PCR (RT–qPCR) for the first time. It was found that the morphology of Vibrio in the MC flocs changed, the membrane function was damaged, the antioxidant system was activated, and the material and energy metabolism also changed. In addition, MC could inhibit the expression of virulence factors of V. alginolyticus; for example, flagella, pilus, siderophores, quorum sensing, and other related genes were significantly downregulated. In general, MC effectively inhibited the growth of Vibrio and reduced its virulence potential in flocs, which could provide theoretical support for a new model of healthy aquaculture.

Molecular mimicry among human proteinase 3 and bacterial antigens: implications for development of c-ANCA associated vasculitis.

Wegener's granulomatosis is an autoimmune disease where autoantibodies target human autoantigen PR3, a serine protease locates on the neutrophil membrane. This disease affects blood small vessels and could be deadly. The origin of these autoantibodies is unknown, but infections have been implicated with autoimmune disease. In this study, we explored potential molecular mimicry between human PR3 and homologous pathogens through in silico analysis. Thirteen serine proteases from human pathogens (Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella sp., Streptococcus suis, Vibrio parahaemolyticus, Bacteroides fragilis, Enterobacter ludwigii, Vibrio alginolyticus, Staphylococcus haemolyticus, Enterobacter cloacae, Escherichia coli and Pseudomonas aeruginosa) shared structural homology and amino acid sequence identity with human PR3. Epitope prediction found an only conserved epitope IVGG, located between residues 59-74. However, multiple alignments showed conserved regions that could be involved in cross-reactivity between human and pathogens serine proteases (90-98, 101-108, 162-169, 267 and 262 residues positions). In conclusion, this is the first report providing in silico evidence about the existence of molecular mimicry between human and pathogens serine proteases, that could explain the origins of autoantibodies found in patients suffering from Wegener's granulomatosis.

Role of Marine Bacterial Contaminants in Histamine Formation in Seafood Products: A Review.

Histamine is a toxic biogenic amine commonly found in seafood products or their derivatives. This metabolite is produced by histamine-producing bacteria (HPB) such as Proteus vulgaris, P. mirabilis, Enterobacter aerogenes, E. cloacae, Serratia fonticola, S. liquefaciens, Citrobacter freundii, C. braakii, Clostridium spp., Raoultella planticola, R. ornithinolytica, Vibrio alginolyticus, V. parahaemolyticus, V. olivaceus, Acinetobacter lowffi, Plesiomonas shigelloides, Pseudomonas putida, P. fluorescens, Aeromonas spp., Photobacterium damselae, P. phosphoreum, P. leiognathi, P. iliopiscarium, P. kishitanii, and P. aquimaris. In this review, the role of these bacteria in histamine production in fish and seafood products with consequences for human food poisoning following consumption are discussed. In addition, methods to control their activity in countering histamine production are proposed.

Sequence and functional features of a novel scavenger receptor homolog, SCARA5 from Yellow drum (Nibea albiflora)

As an important member in SR-As, member 5 (SCARA5) can swallow apoptotic cells and foreign bodies, and participate multiple signaling pathways to inhibit tumor occurrence, development growth and metastasis. To explore its immune function, SCARA5 was identified from the yellow drum (Nibea albiflora) according to its transcriptome data, and its full-length cDNA was 6968 bp (named as NaSCARA5, GenBank accession no: MW070211) encoding 497 amino acids with a calculated molecular weight of 55.12 kDa, which had the typical motifs of SR family, such as transmembrane helix region, coil region, Pfam collagens region and SR region. BLASTp and the phylogenetic relationship analysis illustrated that the sequences shared high similarity with known SCARA5 of teleosts. Quantitative real time RT-PCR analysis showed that NaSCARA5 was expressed in intestine, stomach, liver, kidney, gill, heart and spleen, with the highest in the spleen (24.42-fold compared with that in heart). After being infected with Polyinosinic:polycytidylic acid (PolyI:C), Vibrio alginolyticus and Vibrio parahaemolyticus, NaSCARA5 mRNA were up-regulated with time dependent mode in spleen, which suggested that NaSCARA5 might play an important role in the immune process of fish. The extracellular domain of NaSCARA5 was successfully expressed in BL21 (DE3), and yielded the target protein of the expected size with many active sites for their conferring protein-protein interaction functions. After being purified by Ni-NAT Superflow resin and renatured, it was found to bind all the tested bacteria (V.parahaemolyticus,V.alginolyticus and Vibrio harveyi). The eukaryotic expression vector of the NaSCARA5-EGFP fusion protein was constructed and transferred into epithelioma papulosum cyprini (EPC) cells, and it was mainly expressed on the cell membrane indicating that NaSCARA5 was a typical transmembrane protein. The aforementioned results indicated that NaSCARA5 played a significant role in the defense against pathogenic bacteria infection as PRRs, which may provide some further understandings of the regulatory mechanisms in the fish innate immune system for SR family.

Functional analysis of the N-terminal region of Vibrio FlhG, a MinD-type ATPase in flagellar number control.

GTPase FlhF and ATPase FlhG are two key factors involved in regulating the flagellum number in Vibrio alginolyticus. FlhG is a paralogue of the Escherichia coli cell division regulator MinD and has a longer N-terminal region than MinD with a conserved DQAxxLR motif. The deletion of this N-terminal region or a Q9A mutation in the DQAxxLR motif prevents FlhG from activating the GTPase activity of FlhF in vitro and causes a multi-flagellation phenotype. The mutant FlhG proteins, especially the N-terminally deleted variant, were remarkably reduced compared to that of the wild-type protein in vivo. When the mutant FlhG was expressed at the same level as the wild-type FlhG, the number of flagella was restored to the wild-type level. Once synthesized in Vibrio cells, the N-terminal region mutation in FlhG seems not to affect the protein stability. We speculated that the flhG translation efficiency is decreased by N-terminal mutation. Our results suggest that the N-terminal region of FlhG controls the number of flagella by adjusting the FlhF activity and the amount of FlhG in vivo. We speculate that the regulation by FlhG, achieved through transcription by the master regulator FlaK, is affected by the mutations, resulting in reduced flagellar formation by FlhF.

Ecological Impacts of Aged Freshwater Biofilms on Estuarine Microbial Communities Elucidated Through Microcosm Experiments: A Microbial Invasion Perspective.

Inadvertent introductions of alien species via biofilms as a vector released through ballast water are of environmental importance, yet their consequences are not much known. In the present study, biofilm communities developed in an inland freshwater port under in situ and dark conditions were subjected to long-term dark incubations. Subsequently, the impact of these aged biofilms as vectors on estuarine water column communities were evaluated using microcosm experiments in the laboratory. Variations in biofilm and planktonic microbial communities were quantified using quantitative PCR.Upon prolonged dark incubation, a shift in bacterial diversity with an increase in tolerant bacterial communities better adapted to stress was observed. Actinobacteria were the dominant taxa in both aged biofilms upon dark incubations. The laboratory studies indicated that on exposure of these biofilms to estuarine water, resuscitation of Vibrio alginolyticus, V. parahaemolyticus, and V. cholerae from a dormant state existing in these biofilms to culturable form was observed. Moreover, the results revealed that both the biofilm types can pose a threat to the environment, but the degree of risk can be attributed to the imbalance caused by significant changes in the surrounding estuarine microbial communities. Consequently, this may result in either proliferation or decline of some genera with different metabolic potential and resuscitation of pathogenic forms not present earlier, thereby influencing the ecology of the environment. Quantifying these effects in the field using biofilm metagenomes with an emphasis on virulent species and understanding traits that enable them to adapt to changing environments is a way forward.

Characterizing the Effect of the Lysine Deacetylation Modification on Enzyme Activity of Pyruvate Kinase I and Pathogenicity of Vibrio alginolyticus.

Pyruvate kinase I (PykF) is one of the key enzymes of glycolysis and plays a crucial role in bacterial metabolism. Several acetylation sites of Vibrio alginolyticus PykF were reported in previous studies and then 11 sites were first verified in this study, however, the specific roles of PykF acetylation remains unclear. Overlap-PCR and homologous recombination were implied to delete V. alginolyticus pykF gene and constructed complementary strains of site-directed mutagenesis for the further research focus on the deacetylation regulation on PykF. The results showed that the pyruvate kinase activity was sharply suppressed in the deacetylation status of K52, K68, and K317 of PykF, as well as the extracellular protease activity was significantly decreased in the deacetylation status of K52 and K68, but not induced with K317. Moreover, the growth rates of V. alginolyticus were not influenced with these three deacetylation sites. The ΔpykF mutant exhibited a 6-fold reduction in virulence to zebrafish. Site-directed mutations of K52R and K68R also showed reduced virulence while mutations of K317R didn't. The in vitro experiments showed that PykF was acetylated by acetyl phosphate (AcP), with the increase of incubation time by AcP, the acetylation level of PykF increased while the enzyme activity of PykF decreased correspondingly. Besides, PykF was deacetylated by CobB deacetylase and in result that the deacetylation was significantly down-regulated while the pyruvate kinase activity of PykF increased. Moreover, deletion of cobB gene had no significant difference in pyruvate kinase activity. These results confirm that CobB can regulate the acetylation level and pyruvate kinase activity of PykF. In summary, the results of this study provide a theoretical basis for further understanding of the deacetylation modification of PykF. It provides a new idea for the prevention and cure of vibriosis.

Prevalence of Histamine-Forming Bacteria in Two Kinds of Salted Fish at Town Markets of Guangdong Province of South China

Histamine-forming bacteria (HFB) were isolated from the 70 salted fish samples bought from town markets of Guangdong Province of south China. In addition, the histamine-forming ability of HFB was analyzed. There were 31 strains of HFB isolated from 36 salted fish pickled overnight. They were identified as six bacteria species: Vibrio alginolyticus, Vibrio rumoiensis, Staphylococcus saprophyticus, Staphylococcus xylosus, Lactococcus lactis, and Morganella morganii. The rate of confirmation of V. alginolyticus was highest (23 of 31), exceeding 200 mg/kg histamine. In particular, M. morganii produced a histamine amount that exceeded 2,000 mg/kg, although it was only one isolate in this study. In addition, five bacteria species of HFB were isolated from 34 dried salted fish. Among them, S. saprophyticus was dominant in the dried salted fish but produced histamines below 200 mg/kg. However, Enterobacter aerogenes from dried salted fish formed a histamine amount exceeding 200 mg/kg. The study showed that the dominant strain of HFB was different in two kinds of salted fish. Both kinds of salted fish contained HFB whose histamine-forming capacity exceeded 200 mg/kg. As a result, the safety of salted fish should be of concern, especially salted fish pickled overnight.

Characterization of two new Scygonadin homologous SCY4 and SCY5 in Scylla paramamosain

Two new peptides homologous to the reported antimicrobial peptide Scygonadin were characterized in Scylla paramamosain with 48.51% and 49.23% identity in the amino acid sequences, named as SCY4 and SCY5, respectively. The mRNA transcripts and proteins of the two peptides were abundant in the ejaculatory duct of male crabs, and they were transferred to the spermatheca of female crabs along with semen during mating. Immunofluorescence analysis showed that both peptides were localized in sperm cells at different developmental stages and in the acrosomal tubes of acrosomal reactions, suggesting their potential roles in protecting sperm and in the acrosomal reactions. The analysis of progesterone stimulation in vivo showed that the expression patterns of SCY4 and SCY5 were different, with SCY5 up-regulated and SCY4 down-regulated. The recombinant proteins (rSCY4 and rSCY5) were more active against Gram-positive bacteria than the Gram-negative bacteria we tested. It is noteworthy that although rSCY4 and rSCY5 had no bactericidal effect on the pathogen Vibrio alginolyticus in vitro, both proteins could obviously enhance the survival rate of crabs challenged with V. alginolyticus. In summary, SCY4 and SCY5 may play a role similar to Scygonadin in the reproductive process, thereby maintaining internal sterility during sperm transfer or in the spermatheca of female S. paramamosain.

Natural Feed Supplements Improve Growth, Non-Specific Immune Responses and Resistance against Vibrio alginolyticus in Lates calcarifer

This study aimed to investigate the effects of dietary natural feed supplement on the growth performance, non-specific responses, and disease resistance in Lates calcarifer. Three commercial products (A, B, and C) containing a basal probiotic mixture were tested. Product A contained a basal mixture of Paenibacillus sp., Bacillus subtilis, Bacillus amyloliquefaciens, and Lactobacillus rhamnosus (107 cfu/g for each probiotic); product B contained additional Lactobacillus plantarum (1010 cfu/g); and product C contained additional soybean peptides (500 g/kg) and garlic powder (1 g/kg). Each product was supplemented into subject diets at dosages of 1 or 2 g/kg (designated as the A1, A2, B1, B2, C1, and C2 groups, respectively). Following an eight-week trial, growth parameters (specific growth rate and feed conversion ratio), non-specific immune responses (O2− production, phagocytic rate, and phagocytic index), and the results of a challenge test against Vibrio alginolyticus were evaluated. The results show that all probiotic supplement groups exhibited an improvement in growth performance compared to the control group (non-probiotic diet). In terms of non-specific immunity parameters, a significant improvement in O2− production was found in the C2 group, whereas significant improvements in phagocytic activity were found in all the B and C groups. The C2 group displayed optimal O2− production, phagocytic rate, and phagocytic index results. For the challenge test, the C groups showed higher Vibrio resistance than the other experimental groups and the control group. These results suggest that product C, given at dosages of 2 g/kg, may serve as a growth-promoting and immunostimulatory additive for the cultivation of Asian seabass.

Synergistic Interaction of Low Salinity Stress With Vibrio Infection Causes Mass Mortalities in the Oyster by Inducing Host Microflora Imbalance and Immune Dysregulation.

A sudden drop in salinity following extreme precipitation events usually causes mass mortality of oysters exposed to pathogens in ocean environment. While how low salinity stress interacts with pathogens to cause mass mortality remains obscure. In this study, we performed an experiment by low salinity stress and pathogen infection with Vibrio alginolyticus to investigate their synergistic effect on the mortality of the Pacific oyster toward understanding of the interaction among environment, host, and pathogen. We showed that low salinity stress did not significantly affect proliferation and virulence of V. alginolyticus, but significantly altered microbial composition and immune response of infected oysters. Microbial community profiling by 16S rRNA amplicon sequencing revealed disrupted homeostasis of digestive bacterial microbiota with the abundance of several pathogenic bacteria being increased, which may affect the pathogenesis in infected oysters. Transcriptome profiling of infected oysters revealed that a large number of genes associated with apoptosis and inflammation were significantly upregulated under low salinity, suggesting that low salinity stress may have triggered immune dysregulation in infected oysters. Our results suggest that host-pathogen interactions are strongly affected by low salinity stress, which is of great significance for assessing future environmental risk of pathogenic diseases, decoding the interaction among environment, host genetics and commensal microbes, and disease surveillance in the oyster.

Prediction and characterization of a novel hemoglobin-derived mutant peptide (mTgHbP7) from Tegillarca granosa

The hemoglobin (Hb) is identified in Tegillarca granosa and its derived peptides have been proved to possess antibacterial activity against gram-positive and gram-negative bacteria. In this study, we identified a series of novel antimicrobial peptides (AMPs) and artificially mutated AMPs derived from subunits of T. granosa Hbs, among which, a mutant T. granosa hemoglobin peptide (mTgHbP) mTgHbP7, was proved to possess predominant antibacterial activity against three bacteria strains (Vibrio alginolyticus, V. parahaemolyticus and Escherichia coli). Besides, mTgHbP7 was predicted to form α-helical structure, which was known to be an important feature of bactericidal AMPs. Furthermore, upon contact with HEK293 cell line, we confirmed that mTgHbP7 had no cytotoxicity to mammalian cell even at a high concentration of 160 μM. Therefore, the findings reported here provide a rationalization for antimicrobial peptide prediction and optimization from mollusk hemoglobin, which will be useful for future development of antimicrobial agents.

Improvement in the probiotic efficacy of Bacillus subtilis E20-stimulates growth and health status of white shrimp, Litopenaeus vannamei via encapsulation in alginate and coated with chitosan

The aim of this study was to increase the efficacy of probiotic Bacillus subtilis E20 by encapsulating the probiotic in alginate and coating it with chitosan. The protective effect was evaluated by firstly ensuring the viability of encapsulated probiotics in simulated gastrointestinal fluid (SGF) and simulated intestinal fluid (SIF) conditions and then at different storage temperatures. In addition, the encapsulated probiotic was incorporated into the diet to improve the growth performance and health status of white shrimp, Litopenaeus vannamei. B. subtilis E20 has the ability to survive in SGF when encapsulated in 1.5–2% alginate and coated with 0.4% chitosan. Furthermore, viability increased significantly in SIF compared to the probiotic encapsulated in 1% alginate and coated with 0.4% chitosan and the non-encapsulated probiotic. Longer storage time and adverse conditions affected probiotics' survival, which was improved by the encapsulation with significantly higher viability than the non-encapsulated probiotic at different temperatures and storage duration. Encapsulation of B. subtilis E20 and dietary administration at 107 CFU kg−1 decreased shrimp mortality after a Vibrio infection, thereby improving shrimp's disease resistance, while the non-encapsulated probiotic required 109 CFU kg−1 to achieve better resistance. Although the best results of growth performance, immune response, and disease resistance against Vibrio alginolyticus were found in the shrimp fed with the diets supplemented with encapsulated probiotic at >108 CFU kg−1, shrimp's growth performance and health status improved after being fed 107 CFU kg−1 encapsulated probiotic for 56 days. Together, the results of this study prove that encapsulation could improve the viability of probiotic in different gastrointestinal conditions and adverse storage temperatures. Overall, lower concentrations of encapsulated probiotic B. subtilis E20 (107 CFU kg−1) was able to increase the growth performance and health status of shrimp.