Characterization of TLR5 and TLR9 from silver pomfret (Pampus argenteus) and expression profiling in response to bacterial components.

Toll like receptor (TLR) 5 and 9 are important members of the TLR family that play key roles in innate immunity in all vertebrates. In this study, paTLR5 and paTLR9 were identified in silver pomfret (Pampus argenteus), a marine teleost of great economic value. Open reading frames (ORFs) of paTLR5 and paTLR9 are 2646 and 3225 bp, encoding polypeptides of 881 and 1074 amino acids, respectively. Sequence analysis revealed several conserved characteristic features, including signal peptides, leucine-rich repeat (LRR) motifs, and a Toll/interleukin-I receptor (TIR) domain. Sequence, phylogenetic and synteny analysis revealed high sequence identity with counterparts in other teleosts, confirming their correct nomenclature and conservation during evolution. Quantitative real-time PCR revealed that the that both TLRs were ubiquitously expressed in all investigated tissues, most abundantly in liver, kidney, spleen, intestine and gill, but lower in muscle and skin. In vitro immunostimulation experiments revealed that Aeromonas hydrophila lipopolysaccharide (LPS) and Vibrio anguillarum flagellin induced higher levels of paTLR9 and paTLR5 mRNA expression in isolated fish intestinal epithelial cells (FIECs) than Lactobacillus plantarum lipoteichoic acid (LTA), but all increased the secretion of IL-6 and TNF-α and induced cell apoptosis and necrosis. Together, these results indicate that paTLR5 and paTLR9 may function in the response to bacterial pathogens. Our findings enhance our understanding of the function of TLRs in the innate immune system of silver pomfret and other teleosts.

Ability of Lactobacillus plantarum lipoteichoic acid to inhibit Vibrio anguillarum-induced inflammation and apoptosis in silvery pomfret (Pampus argenteus) intestinal epithelial cells.

Lipoteichoic acid (LTA) is a major constituent of the cell wall of Gram-positive bacteria. The structure and immunomodulation of LTA vary greatly between different species. LTA from Lactobacillus plantarum has been shown to exert anti-pathogenic effects. Vibrio anguillarum is a major causative agent of vibriosis, one of the most prevalent fish diseases. The purpose of this study was to examine the effects of L. plantarum LTA on V. anguillarum growth, adhesion, and induced inflammation and apoptosis in intestinal epithelial cells of silvery pomfret (Pampus argenteus). Our results showed that L. plantarum LTA was unable to inhibit V. anguillarum growth; however, it significantly inhibited adhesion of V. anguillarum. It also showed significant inhibitory effects on EHEC-induced inflammation and apoptosis by modulating the expression of NF-κB (nuclear factor kappa B), IκB (inhibitor of NF-κB), Bcl2 (B-cell leukemia/lymphoma-2), BAX (Bcl-2-associated X protein), IL-8 (interleukin 8) and TNF-α (tumor necrosis factor-α), and via inhibition of caspase-9 and caspase-3 activation. These data extend our understanding of the beneficial effects of L. plantarum LTA, which is related to the inhibition of V. anguillarum, and suggest that L. plantarum LTA has potential as a new therapeutic agent against V. anguillarum-caused vibriosis in fish.

Transcriptome and analysis on the complement and coagulation cascades pathway of large yellow croaker (Larimichthys crocea) to ciliate ectoparasite Cryptocaryon irritans infection.

Large yellow croaker (Larimichthys crocea) is one of the most valuable marine fish in southern China. Given to the rapid development of aquaculture industry, the L. crocea was subjected to ciliate ectoparasite Cryptocaryon irritans. It therefore is indispensable and urgent to understand the mechanism of L. crocea host defense against C. irritans infection. In the present study, the extensively analysis at the transcriptome level for Cryptocaryoniasis in L. crocea was carried out. These results showed that 15,826,911, 16,462,921, and 15,625,433 paired-end clean reads were obtained from three cDNA libraries (A: 0 theronts/fish, B: 12,000 theronts/fish, and C: 24,000 theronts/fish) of the L. crocea immune-related tissues by Illumina paired-end sequencing technology. Totally, 30,509 unique transcript fragments (unigenes) were assembled, with an average length of 1715 bp. In B/A, C/A, and C/B pairwise comparison, 972, 900, and 1126 genes showed differential expression respectively. Differently expressed immune-related genes (DEIGs) were scrutinized, in B/A pairwise comparison, 48 genes showed differential expression, including 26 up-regulated genes and 22 down-regulated genes in B; in C/A pairwise comparison, there were 39 DEIGs, including 7 up-regulated genes and 32 down-regulated genes in C; in C/B pairwise comparison, 40 genes showed differential expression, including 11 up-regulated genes and 29 down-regulated genes in C. There were 16 DEIGs enriched KEGG pathways, in which the complement and coagulation cascades pathway was the top most DEIGs enriched pathway (B:A = 42; C:A = 28; C:B = 42). The coagulation and fibrinolytic system was in a highly active state after infected by C. irritans with non-lethal concentration; the alternative complement pathway may play an important role in the early stages of C. irritans infection. These results demonstrated that low-concentration infection can significantly induce the immunological response in fishes, however, when fishes were in fatal conditions, the immunity was suppressed.

Molecular characterization and expression analysis of toll-like receptor 2 in response to bacteria in silvery pomfret intestinal epithelial cells.

Toll-like receptor 2 (TLR2) has been shown to play a crucial role in the host defense of pathogenic microbes in innate immunity. In this study, the full-length cDNA of TLR2 in silvery pomfret (Pampus argenteus) was cloned by homology cloning and the rapid amplification of cDNA ends (RACE) technique. The complete cDNA sequence of TLR2 was 2932 bp, containing an open reading frame (ORF) of 2469 bp encoding 822 amino acids. A multiple alignment analysis of the silvery pomfret TLR2 protein-coding sequence with other known TLR2 sequences from Oplegnathus fasciatus, Epinephelus coioides, Larimichthys crocea, Miichthys miiuy, Oreochromis niloticus, Paralichthys olivaceus, Trematomus bernacchii, Sparus aurata, and Chionodraco hamatus exhibited a high degree of homology of 78.83%, 75.91%, 74.21%, 74.94%, 71.95%, 72.57%, 73.68%, 75%, and 72.52 respectively, between these fish. Analysis of the TLR2 domain structures indicated that TLR2 from the silvery pomfret has the typical structural features of proteins that belong to the TLR family, including one transmembrane domain, eleven leucine-rich repeats (LRRs), and one Toll/IL-1 receptor homology domain (TIR). In vitro immunostimulation experiments revealed that Lactobacillus plantarum and Clostridium butyricum induce high levels of TLR2 mRNA and protein expression, but they induce only moderate levels of IL-8 and TNF-α production compared to Vibrio anguillarum. This suggests that TLR2 might play a vital role in the L. plantarum and C. butyricum-mediated immune response. In contrast, V. anguillarum significantly increased the secretion of IL-8 and TNF-α and induced cell apoptosis and necrosis. Due to the lower expression of TLR2 and higher levels of IL-8 and TNF-α induced by V. anguillarum, we hypothesize that a V. anguillarum infection is independent of the TLR2-induced production of pro-inflammatory cytokines. These results indicate that TLR2 may be involved in molecular interactions between the host and commensal bacteria, that exist in the silvery pomfret intestinal tract.

Growth, feed intake and immune responses of orange-spotted grouper (Epinephelus coioides) exposed to low infectious doses of ectoparasite (Cryptocaryon irritans).

To explore the effect of low-dose Cryptocaryon irritans infection on growth, feeding and antiparasitic immunity of orange-spotted grouper (Epinephelus coioides), this study utilized C. irritans at concentrations of 5500 theronts/fish (Group I, 1/10 of 96 h LC50) or 11,000 theronts/fish (Group II) to infect E. coioides weighing 38 g on average at week 0, 2 and 4, respectively. Food consumption was recorded daily; the fish were weighed weekly; serum immobilizing titer (SIT), and acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), lysozyme (LZM) activity were recorded every 2 weeks; the fish were treated with lethal dose (70,000 theronts/fish) of C. irritans in the 8th week and death number were recorded. The result shows that in the 1st week after the first infection, the fish's weight gain (WG), length gain (LG), and specific growth rate (SGR) dropped as parasite dose increased, and WG, SGR values were negative; while, after the 2nd and the 3rd infection, no significant differences were detected among the three groups. These results indicated that the 1st infection affected the fish most, while the following infections were protected by some immunity. In the 3rd, 7th, and 8th week, condition factor (CF) increased with the increased infectious dose, indicating that the parasite affected body length more than body weight. As the experiment went on, accumulated food consumption (AFC) of all three groups steadily grew (control > Group I > Group II). But on the 2nd day after the first infection, daily food consumption (DFC) of Group I and II significantly dropped, the decline of Group II was greater than that of Group I, DFC recovered in the following week, with Group I earlier than Group II. After the 2nd infection, DFC of Group I and II dropped again, Group II still dropped more than Group I, and both groups recovered on the 3rd day after infection. The 3rd infection caused no significant difference in week food consumption (WFC). These results indicated that a higher dose of infection causes a greater drop in FC and a slower recovery. Weekly feed conversion ratio (WFCR) values of Group I and II in the 1st week was negative; in the 2nd week, WFCR was lower in the group infected by a higher dose of parasite; while in the 3rd and following weeks, no significant pattern was observed. Accumulate feed conversion ratio (AFCR) dropped as the infectious dose increased (control > Group I > Group II), AFCR of Group I and II reached above 0 in the 2nd and 4th week, respectively. From the 4th week on, the inter-group AFCR of the 3 groups still took on a declining trend with the increased infectious dose but the gap became smaller. One week after the first infection, SIT of Group I and Group II were 0; one week after the 2nd infection, SIT reached up to 8 (Group I) and 16 (Group II) respectively; and after the 3rd infection, SIT further increased and peaked in the 7th week. When challenged by lethal dose of C. irritans, fish of all 3 groups began to die since the 3rd day after infection, and the final deaths were 14, 12 and 8 for the control group, Group I and Group II, respectively. ACP activity in the 1st, 5th, 7th but the 3rd week was higher in the experiment group than that in the control group, but no significant difference was detected between Group I and II throughout the experiment. AKP activity increased as the infectious dose increased, but the difference among the three groups gradually became less obvious in latter infections, and no significant difference can be detected in the end. SOD activity increased with infection dose at each time point, while both group I and group II had their SOD activities first increased and then decreased as times of infection increased. The LZM activity of the two infection groups increased as the infectious times increased. Combining the results on growth and feeding, we speculated that the fish's physiological condition stabilized after 3 rounds of infection. To sum up, low-dose infection by C. irritans can induce the fish's immunity, but at the cost of decreasing food intake, decreased food conversion, and lagged growth.

Effects of Cryptocaryon irritans infection on the survival, feeding, respiratory rate and ionic regulation of the marbled rockfish Sebastiscus marmoratus.

To clarify the effects of a Cryptocaryon irritans infection on the physiological functions of the marbled rockfish Sebastiscus marmoratus, this study utilized C. irritans at concentrations of 2500; 5000; 7500; 10,000; 20,000; and 30,000 theronts/fish to infect marbled rockfish weighing 45 ± 3 g. The survival rate, food intake, respiratory rate, serum ion concentrations and gill Na+/K+-ATPase activity were determined. With the increase of the infection concentration and the passage of time, the survival rate of the rockfish gradually decreased. The groups infected with more than 5000 theronts/fish had stopped feeding within 4 days. The respiratory rates of the fish in the groups infected with 2500 and 5000 theronts/fish initially increased and then decreased. In contrast, the respiratory rate of the fish in the groups infected with more than 7500 theronts/fish was elevated to levels significantly higher than the control group after 12 h. The Na+/K+-ATPase activity and serum Na+ and Cl- concentrations increased with increasing infection concentration. In conclusion, the physiological functions of the fish infected with low concentrations of C. irritans can be effectively restored, whereas a high concentration infection induced severe stress. The declined food intake and accelerated respiratory rate could be useful for an early warning system as important indicators.

Construction of the flagellin F mutant of Vibrio parahaemolyticus and its toxic effects on silver pomfret (Pampus argenteus) cells.

Vibrio parahaemolyticus causes diseases in aquatic organisms, leading to substantial financial losses to the aquaculture industry; its flagellin F (flaF) protein triggers severe inflammation in host cells. To enhance the understanding of the function of flaF in V. parahaemolyticus infection, in this study, a flaF-deficient mutant was constructed by employing two-step homologous recombination. The flaF-deficient mutant induced a significantly lower toll-like receptor 5 (TLR5) expression and apoptosis in fish intestinal epithelial cells than the wild-type V. parahaemolyticus. Furthermore, fluorescence labelling and microscopy analysis of TLR5 showed that V. parahaemolyticus and its mutant strain significantly enhanced TLR5 expression. Additionally, the findings suggest that flaF deletion did not significantly affect the expression of myeloid differentiation factor 88 (MyD88) and interleukin-8 (IL-8) induced by V.parahaemolyticus. In summary, V. parahaemolyticus induced a TLR5-dependent inflammatory response and apoptosis through MyD88, which was observed to be influenced by flaF deletion. In this study, we obtained stable mutants of V. parahaemolyticus via target gene deletion-which is a rapid and effective approach-and compared the induction of inflammatory response and apoptosis by V. parahaemolyticus and its mutant strain, providing novel perspectives for functional gene research in V. parahaemolyticus.

In Vitro Protective Efficacy of Clostridium butyricum Against Fish Pathogen Infections.

Most pathogens in intestine are opportunist, called "opportunistic pathogens" that usually do not cause disease in a healthy host. Only when the host's resistance is lowered or the intestinal microecological balance is destroyed, the opportunistic pathogens are capable of causing disease. Here, two opportunistic pathogens, Salmonella enteritidis and Vibrio parahaemolyticus were chosen to test the possible antagonistic effect of the probiotic agent Clostridium butyricum on these pathogens infections in vitro using fish intestinal epithelial cells (FIECs). The C. butyricum and its spent culture supernatants exhibited significant inhibitory activity on S. enteritidis and V. parahaemolyticus growth and adherence to FIECs. The C. butyricum also showed significant inhibitory effects on S. enteritidis and V. parahaemolyticus induced apoptosis, which may due to its growth and adhesion inhibitory effects. These results indicated that the probiotic bacterium C. butyricum has preventive and therapeutic effects on S. enteritidis and V. parahaemolyticus infections in fish.

Alterations of the Gut Microbiota and Metabolomics Associated with the Different Growth Performances of Macrobrachium rosenbergii Families.

To investigate the key gut microbiota and metabolites associated with the growth performance of Macrobrachium rosenbergii families, 16S rRNA sequencing and LC-MS metabolomic methods were used. In this study, 90 M. rosenbergii families were bred to evaluate growth performance. After 92 days of culture, high (H), medium (M), and low (L) experimental groups representing three levels of growth performance, respectively, were collected according to the weight gain and specific growth rate of families. The composition of gut microbiota showed that the relative abundance of Firmicutes, Lachnospiraceae, Lactobacillus, and Blautia were much higher in Group H than those in M and L groups. Meanwhile, compared to the M and L groups, Group H had significantly higher levels of spermidine, adenosine, and creatinine, and lower levels of L-citrulline. Correlation analysis showed that the abundances of Lactobacillus and Blautia were positively correlated with the levels of alpha-ketoglutaric acid and L-arginine. The abundance of Blautia was also positively correlated with the levels of adenosine, taurine, and spermidine. Notably, lots of metabolites related to the metabolism and biosynthesis of arginine, taurine, hypotaurine, and fatty acid were upregulated in Group H. This study contributes to figuring out the landscape of the gut microbiota and metabolites associated with prawn growth performance and provides a basis for selective breeding.

The role of flagellin F in Vibrio Parahaemolyticus-induced intestinal immunity and functional domain identification.

The marine pathogen Vibrio parahaemolyticus has caused huge economic losses to aquaculture. Flagellin is a key bacterial virulence factor that induces an inflammatory response via activation of Toll-like receptor 5 (TLR5) signaling. Herein, to explore the inflammatory activity of V. parahaemolyticus flagellins (flaA, flaB, flaC, flaD, flaE, and flaF), we investigated their ability to induce apoptosis in a fish cell line. All six flagellins induced severe apoptosis. Moreover, treatment with V. parahaemolyticus flagellins increased TLR5 and myeloid differentiation factor 88 (MyD88) expression and the production of TNF-α and IL-8 significantly. This indicated that flagellins might induce a TLR5-meditated immune response via an MyD88-dependent pathway. FlaF exhibited the strongest immunostimulatory effect; therefore, the interaction between TLR5 and flaF was screened using the yeast two-hybrid system. A significant interaction between the two proteins was observed, indicating that flaF binds directly to TLR5. Finally, the amino acids that participate in the TLR5-flaF interaction were identified using molecular simulation, which indicated three binding sites. These results deepen our understanding of the immunogenic properties of flagellins from V. parahaemolyticus, which could be used for vaccine development in the future.

Integrated comparative transcriptome and weighted gene co-expression network analysis provide valuable insights into the response mechanisms of crayfish (Procambarus clarkii) to copper stress.

One of the significant limitations of aquaculture worldwide is the prevalence of divalent copper (Cu). Crayfish (Procambarus clarkii) are economically important freshwater species adapted to a variety of environmental stimuli, including heavy metal stresses; however, large-scale transcriptomic data of the hepatopancreas of crayfish in response to Cu stress are still scarce. Here, integrated comparative transcriptome and weighted gene co-expression network analyses were initially applied to investigate gene expression profiles of the hepatopancreas of crayfish subjected to Cu stress for different periods. As a result, 4662 significant differentially expressed genes (DEGs) were identified following Cu stress. Bioinformatics analyses revealed that the "focal adhesion" pathway was one of the most significantly upregulated response pathways following Cu stress, and seven DEGs mapped to this pathway were identified as hub genes. Furthermore, the seven hub genes were examined by quantitative PCR, and each was found to have a substantial increase in transcript abundance, suggesting a critical role of the "focal adhesion" pathway in the response of crayfish to Cu stress. Our transcriptomic data can be a good resource for the functional transcriptomics of crayfish, and these results may provide valuable insights into the molecular response mechanisms underlying crayfish to Cu stress.

An important role of interleukin-10 in counteracting excessive immune response in HT-29 cells exposed to Clostridium butyricum.

BACKGROUND: Clostridium butyricum has become increasingly important in preventing and treating intestinal inflammation. In the intestine it may increase the resistance of the gut to pathogen invasion via inducing the secretion of anti-inflammatory cytokines. Interleukin 10 (IL-10) plays a central role in preventing certain inflammatory diseases by down-regulating inflammatory cascades. In a previous study, we observed that the level of IL-10 mRNA was modulated by C. butyricum. The aim of this study was to investigate whether C. butyricum achieves its beneficial effects through IL-10. RESULTS: We treated HT-29 cells with anti-IL-10 (IL-10 antibody) or siIL-10 (IL-10 small interfering RNA) to disrupt IL-10. In both cases, the effects of C. butyricum-induced NF-κB activation and IL-8 expression were enhanced. We also found that neutralization or knockdown of IL-10 could induce apoptosis and necrosis of HT-29 cells treated with C. butyricum compared with control cells. CONCLUSIONS: These findings show that IL-10 serves an important role in C. butyricum-mediated immune protection, and in host recognition of C. butyricum.

Clostridium butyricum activates TLR2-mediated MyD88-independent signaling pathway in HT-29 cells.

Oral administration of Clostridium butyricum as probiotic is increasingly gaining importance in the treatment of diarrhea and the improvement of animal performance. However, the mechanisms of host cell receptor recognition of C. butyricum and the downstream immune signaling pathways leading to these benefits remain unclear. The objective of this study was to analyze the mechanisms involved in C. butyricum induction of the toll-like receptor (TLR) signaling. Knockdown of myeloid differentiation primary response protein 88 (MyD88) expression using small interfering RNA in this manner did not affect C. butyricum-induced elevated levels of nuclear factor κB (NF-κB), interleukin-8 (IL-8), IL-6, and tumor necrosis factor alpha (TNF-α), suggesting a MyD88-independent route to TLR signaling transduction. However, a significant reduction in the levels of NF-κB, IL-8, IL-6, and TNF-α was evident in the absence of TLR2 expression, implying the need for TLR2 in C. butyricum recognition. Hence, C. butyricum activates TLR2-mediated MyD88-independent signaling pathway in human epithelial cells, which adds to our understanding of the molecular mechanisms of this probiotic action on gut epithelium.

Transcriptomic Analysis of Large Yellow Croaker (Larimichthys crocea) during Early Development under Hypoxia and Acidification Stress.

Fishes live in aquatic environments and several aquatic environmental factors have undergone recent alterations. The molecular mechanisms underlying fish responses to hypoxia and acidification stress have become a serious concern in recent years. This study revealed that hypoxia and acidification stress suppressed the growth of body length and height of the large yellow croaker (Larimichthys crocea). Subsequent transcriptome analyses of L. crocea juveniles under hypoxia, acidification, and hypoxia-acidification stress led to the identification of 5897 differentially expressed genes (DEGs) in the five groups. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that several DEGs were enriched in the 'protein digestion and absorption' pathway. Enrichment analysis revealed that this pathway was closely related to hypoxia and acidification stress in the five groups, and we found that genes of the collagen family may play a key role in this pathway. The zf-C2H2 transcription factor may play an important role in the hypoxia and acidification stress response, and novel genes were additionally identified. The results provide new clues for further research on the molecular mechanisms underlying hypoxia-acidification tolerance in L. crocea and provides a basic understanding of the potential combined effects of reduced pH and dissolved oxygen on Sciaenidae fishes.

Dietary jellyfish affect digestive enzyme activities and gut microbiota of Pampus argenteus.

For many years, jellyfish were described as 'dead ends' in marine food webs, due to their high-water content and low nutritional value. However, it has been confirmed that silver pomfret (Pampus argenteus) has a particular preference for preying on jellyfish. In this study, we determined the effect of consuming jellyfish on the intestinal microbes of silver pomfret. Analysis of bacterial 16S rRNA gene amplicons showed that jellyfish had a dramatic impact on the composition of the gut microbiota. The content of Proteobacteria was reduced from 99% to 51%, while Firmicutes, Bacteroidetes and Actinobacteria increased, accounting for 35%, 9% and 2% of the total flora, respectively. At the genus level, the content of Photobacterium decreased sharply to <1% of the total flora. By contrast, Lactobacillus, Burkholderia and Sphingomonas increased to 12%, 9% and 7% of the total flora, respectively. After feeding jellyfish, the functions of intestinal microbes and the activity of digestive enzymes also changed, resulting in better digestion and absorption of jellyfish. The results provide insights into the specific bacterial taxa within the silver pomfret intestinal microbiome that are impacted by jellyfish. Silver pomfret can better digest and absorb jellyfish by adjusting the intestinal microbial composition. The findings provide a theoretical basis for the digestive mechanism by which silver pomfret consume jellyfish.

Structure and Properties of Polyoxymethylene/Silver/Maleic Anhydride-Grafted Polyolefin Elastomer Ternary Nanocomposites.

In the present study, silver (Ag) nanoparticles and maleic anhydride-grafted polyolefin elastomer (MAH-g-POE) were used as enhancement additives to improve the performance of the polyoxymethylene (POM) homopolymer. Specifically, the POM/Ag/MAH-g-POE ternary nanocomposites with varying Ag nanoparticles and MAH-g-POE contents were prepared by a melt mixing method. The effects of the additives on the microstructure, thermal stability, crystallization behavior, mechanical properties, and dynamic mechanical thermal properties of the ternary nanocomposites were studied. It was found that the MAH-g-POE played a role in the bridging of the Ag nanoparticles and POM matrix and improved the interfacial adhesion between the Ag nanoparticles and POM matrix, owing to the good compatibility between Ag/MAH-g-POE and the POM matrix. Moreover, it was found that the combined addition of Ag nanoparticles and MAH-g-POE significantly enhanced the thermal stability, crystallization properties, and mechanical properties of the POM/Ag/MAH-g-POE ternary nanocomposites. When the Ag/MAH-g-POE content was 1 wt.%, the tensile strength reached the maximum value of 54.78 MPa. In addition, when the Ag/MAH-g-POE content increased to 15wt.%, the elongation at break reached the maximum value of 64.02%. However, when the Ag/MAH-g-POE content further increased to 20 wt.%, the elongation at break decreased again, which could be attributed to the aggregation of excessive Ag nanoparticles forming local defects in the POM/Ag/MAH-g-POE ternary nanocomposites. Furthermore, when the Ag/MAH-g-POE content was 20 wt.%, the maximum decomposition temperature of POM/Ag/MAH-g-POE ternary nanocomposites was 398.22 °C, which was 71.39 °C higher than that of pure POM. However, compared with POM, the storage modulus of POM/Ag/MAH-g-POE ternary nanocomposites decreased with the Ag/MAH-g-POE content, because the MAH-g-POE elastomer could reduce the rigidity of POM.

Relationships between the Gut Microbiota of Juvenile Black Sea Bream (Acanthopagrus schlegelii) and Associated Environment Compartments in Different Habitats.

The fish-gut microbiota play a key role in the physiology, development, and fitness of its host. An understanding of fish-gut microbial communities and the factors influencing community composition is crucial for improving fish performance. In this study, we compared the gut microbiota of juvenile black sea bream Acanthopagrus schlegelii among habitats: (1) wild, (2) offshore cage-culture, and (3) pond-culture. We also explored the relationships between the gut microbiota and host-associated environmental factors. Gut samples and associated environmental compartments were investigated using 16S rRNA gene sequencing. Our results revealed significant habitat-specific differences among the gut microbiota of juvenile A. schlegelii. Wild populations of juvenile A. schlegelii had more diverse gut microbiota than populations cultured in pond habitats due to their omnivorous feeding habits and the corresponding abundance of natural food resources. Significant variations in the composition, core taxa, and diversity of the microbiota were also found between the gut and the environmental compartments. However, no significant differences were observed among the microbiota of the environmental compartments in the relatively isolated pond habitat. Source tracking analysis recovered connections between the fish-gut microbiota and the diet, water and sediment environmental compartments. This connection was especially strong between the microbiota of the fish gut and that of the diet in the pond habitat: the diet microbiota accounted for 33.48 ± 0.21% of the gut microbiota. Results suggested that all A. schlegelii shared a core gut microbiota, regardless of differences in diet and habitat. However, environmental factors associated with both diet and habitat contributed to the significant differences between the gut microbiota of fish living in different habitats. To the authors' knowledge, this study presents the first comparison of gut microbiota among juvenile A. schlegelii with different diets and habitats. These findings enrich our understanding of the gut microbiota of A. schlegelii and help to clarify the interaction between gut microbiota and environmental factors. Our results may also help to guide and improve fish ecological fitness via the regulation of gut microbiota, thereby increasing the efficacy of stock enhancement programs for this species.

Construction of a Vibrio anguillarum flagellin B mutant and analysis of its immuno-stimulation effects on Macrobrachium rosenbergii.

Vibrio anguillarum is a globally distributed aquatic pathogen, and its flagellin B (FlaB) protein can evoke innate immune responses in hosts. In order to explore the role of FlaB in V. anguillarum infection, we constructed a FlaB-deficient mutant using overlapping PCR and two-step homologous recombination, and gene sequencing confirmed successful knockout of the FlaB gene. Scanning electron microscopy showed no significant differences in the morphological structure of the flagellum between wild-type and FlaB-deficient strains. The mutant was subsequently injected into the freshwater prawn (Macrobrachium rosenbergii) to explore its pathogenicity in the host, and expression of myeloid differentiation factor 88, prophenoloxidase, catalase, superoxide dismutase and glutathione peroxidase was investigated by real-time PCR. The results showed that deletion of FlaB had little effect on V. anguillarum-induced expression of these immune-related genes (p > 0.05). In general, the FlaB mutant displayed similar flagella morphology and immune characteristics to the wild-type strain, hence we speculated that knockout of FlaB might promote the expression and function of other flagellin proteins. Furthermore, this study provides a rapid and simple method for obtaining stable mutants of V. anguillarum free from foreign plasmid DNA.

Molecular identification and functional analysis of MyD88 in giant freshwater prawn (Macrobrachium rosenbergii) and expression changes in response to bacterial challenge.

Myeloid differentiation factor 88 (MyD88) is a crucial adaptor protein for Toll-like receptor (TLR)-mediated signaling pathways and plays an important role in immune response. In this study, the full-length cDNA of MyD88 from Macrobrachium rosenbergii (MRMyD88) was cloned. The MRMyD88 cDNA is 1758 bp long and contains a 1398-bp open reading frame. Multiple sequence alignment and phylogenetic analysis revealed that the amino acid sequence of MRMyD88 shared high identity with the known MyD88 proteins. The MRMyD88 mRNA was widely expressed in all examined tissues, with highest level in intestine, followed by gonad and pleopod. Furthermore, the MRMyD88 promoter region, spanning 1622 bp, contains several transcription factor-binding sites, including nine GATA-1 box motifs. Electrophoretic mobility shift assay showed that Gfi-1, SRF, and Oct-1 bind to the upstream region of MRMyD88. Additionally, the results showed that the expression levels of TLR1, TLR2 and TLR3 were different in response to Vibrio anguillarum, Lactobacillus plantarum and Aeromonas hydrophila infections. However, these bacteria significantly increased the expression levels of MyD88 and prophenoloxidase. These data suggest that the TLR-mediated signaling pathway is MyD88-dependent in response to pathogenic and probiotic bacteria in M. rosenbergii.

The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification.

Vibrio anguillarum, an opportunistic pathogen of aquatic animals, moves using a filament comprised of polymerised flagellin proteins. Flagellins are essential virulence factors for V. anguillarum infection. Herein, we investigated the effects of flagellins (flaA, flaB, flaC, flaD and flaE) on cell apoptosis, TLR5 expression, and production of IL-8 and TNF-α. FlaB exhibited the strongest immunostimulation effects. To explore the functions of flaB in infection, we constructed a flaB deletion mutant using a two-step recombination method, and in vitro experiments showed a significant decrease in the expression of TLR5 and inflammatory cytokines compared with wild-type cells. However in the in vivo study, expression of inflammatory cytokines and intestinal mucosal structure showed no significant differences between groups. Additionally, flaB induced a significant increase in TLR5 expression based on microscopy analysis of fluorescently labelled TLR5, indicating interactions between the two proteins, which was confirmed by native PAGE and yeast two-hybrid assay. Molecular simulation of interactions between flaB and TLR5 was performed to identify the residues involved in binding, revealing two binding sites. Then, based on molecular dynamics simulations, we carried out thirteen site-directed mutations occurring at the amino acid sites of Q57, N83, N87, R91, D94, E122, D152, N312, R313, N320, L97, H316, I324 in binding regions of flaB protein by TLR5, respectively. Surface plasmon resonance (SPR) was employed to compare the affinities of flaB mutants for TLR5, and D152, D94, I324, N87, R313, N320 and H316 were found to mediate interactions between flaB and TLR5. Our comprehensive and systematic analysis of V. anguillarum flagellins establishes the groundwork for future design of flagellin-based vaccines.