Exposure to nitrate and nitrite disrupts cardiovascular development through estrogen receptor in zebrafish embryos and larvae.

Increasing nitrate concentration on surface and groundwater due to anthropogenic activities is an environmental concern. In this study, Tg(fli1: EGFP) zebrafish embryos were exposed to nitrate (NO3-) and nitrite (NO2-), and their cardiovascular development were investigated. Exposure to 10 mg/L NO3-N and 1 and 10 mg/L NO2-N decreased heart rate at 48-96-h post-fertilization (hpf), ventricular volume, and red blood cell flow rate at 96 hpf. Similar concentrations increased the number of embryos and larvae with pericardial edema and missing intersegmental and parachordal vessels in the caudal region at 48-96 hpf. Addition of ICI 182,720 (ICI) reversed the effects of nitrate and nitrite, suggesting estrogen receptors (ER) are involved. 10 mg/L NO3-N and 1 mg/L NO2-N decreased cardiovascular-related genes, gata4,5,6, hand2, nkx2.5, nkx2.7, tbx2a, tbx2b, and fgf1a. Gene expressions of ovarian aromatase and brain aromatase (cyp19a1a and cyp19a1b, respectively) decreased in the exposed groups, whereas ERs (esr1, esr2a, and esr2b) and nitric oxide synthase 2a (nos2a) increased. The effects on gene expression were also reversed by addition of ICI. Taken together, nitrate and nitrite disrupt cardiovascular system through ER in developing zebrafish, implying that environmental nitrate and nitrite contamination may be harmful to aquatic organisms.

A synbiotic containing prebiotic prepared from a by-product of king oyster mushroom, Pleurotus eryngii and probiotic, Lactobacillus plantarum incorporated in diet to improve the growth performance and health status of white shrimp, Litopenaeus vannamei.

This study aimed to evaluate the effects of a synbiotic composite an extract from a by-product of king oyster mushroom, Pleurotus eryngii (KOME), and probiotic Lactobacillus plantarum 7-40 on the growth performance and health status of white shrimp, Litopenaeus vannamei. The KOME was able to stimulate the growth of probiotic, but not the growth of Vibrio pathogens, including V. alginolyticus, V. parahaemolyticus, and V. harveyi. Four diets were formulated, including a control diet supplemented without prebiotic and probiotic, a basal diet supplemented with KOME (5 g kg-1) (ME), a basal diet supplemented with probiotic (1 × 108 CFU kg-1) (LP), and a basal diet supplemented with KOME (5 g kg-1) and probiotic (1 × 108 CFU kg-1) (SYN). Shrimp fed the ME, LP, and SYN diets had significantly higher survival than that of shrimp fed with the control diet for 8 weeks. Shrimp in the SYN group also had a significantly higher weight gain and total final weight in comparison with the control and other treatments. In the intestinal tract, lactic acid bacteria count was significantly higher in the SYN group, whereas the Vibrio-like bacteria count was significantly higher in the ME group than in the control group. For the health status assessment, the disease resistance of shrimp against V. alginolyticus was improved in all treatments compared to the shrimp in control. Shrimps in the SYN group had significantly lower cumulative mortality due to the significant increase in immune responses, including phenoloxidase, respiratory burst, and lysozyme activity, and the gene expression of pexn and pen4 in the haemocytes, and lgbp, sp, propoii, pexn, pen3a, pen4, and gpx in the haepatopancreas of shrimp as compared to the control. Therefore, it is suggested that a combination of KOME and probiotics can be used as a synbiotic to improve the growth performance and reduce the risk of infectious diseases caused by Vibrio and at the same time significantly contribute to the circular economy.

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.

Formation and Cytotoxicity of Halophenylacetamides: A New Group of Nitrogenous Aromatic Halogenated Disinfection Byproducts in Drinking Water.

Nitrogenous aromatic halogenated disinfection byproducts (DBPs) in drinking water have received considerable attention recently owing to their relatively high toxicity. In this study, a new group of nitrogenous aromatic halogenated disinfection byproducts, halophenylacetamides (HPAcAms), were successfully identified for the first time in both the laboratory experiments and realistic drinking water. The formation mechanism of HPAcAms during chlorination of phenylalanine in the presence of Br- and I-, occurrence frequencies, and concentrations in authentic drinking water were investigated, and a quantitative structure-activity relationship (QSAR) model was developed based on the acquired cytotoxicity data. The results demonstrated that HPAcAms could be formed from phenylalanine in chlorination via electrophilic substitution, decarboxylation, hydrochloric acid elimination, and hydrolysis. The HPAcAm yields from phenylalanine were significantly affected by contact time, pH, chlorine dose, and temperature. Nine HPAcAms with concentrations in the range of 0.02-1.54 ng/L were detected in authentic drinking water samples. Most tested HPAcAms showed significantly higher cytotoxicity compared with dichloroacetamide, which is the most abundant aliphatic haloacetamide DBP. The QSAR model demonstrated that the cellular uptake efficiency and the polarized distributions of electrons of HPAcAms play essential roles in their cytotoxicity mechanisms.

Occurrence and Cytotoxicity of Aliphatic and Aromatic Halogenated Disinfection Byproducts in Indoor Swimming Pool Water and Their Incoming Tap Water.

Disinfection byproducts (DBPs) in swimming pool water are of wide concern for public health. In this study, the occurrence of five categories of aliphatic halogenated DBPs, i.e., trihalomethanes (THMs), haloacetic acids (HAAs), haloacetonitriles (HANs), halonitromethanes (HNMs), and haloketones (HKs), and six categories of aromatic halogenated DBPs, i.e., halophenols (HPs), halonitrophenols (HNPs), halohydroxy-benzaldehydes (HBALs), halohydroxybenzoic acids (HBAs), halobenzoquinones (HBQs), and haloanilines (HAs), was examined in seven indoor swimming pool water and their incoming tap water. The correlations between the DBP concentrations and water quality parameters were explored. Moreover, the cytotoxicity of the aliphatic and aromatic halogenated DBPs was tested with human hepatoma (HepG2) cells, and the concentration-cytotoxicity contributions of different DBP categories were calculated. The results demonstrate that 24 aliphatic (5 THMs, 8 HAAs, 5 HANs, 4 HNMs, and 2 HKs) and 50 aromatic halogenated DBPs (9 HPs, 8 HNPs, 9 HBALs, 8 HBAs, 11 HBQs, and 5 HAs) were present in the swimming pool water, among which 41 aromatic halogenated DBPs were detected in swimming pool water for the first time. The average concentrations of the five categories of aliphatic halogenated DBPs in the swimming pool water were in the order of HAAs > HANs > HKs > THMs > HNMs, while those in their incoming tap water were in the order of THMs > HAAs > HKs > HANs > HNMs. The average concentrations of the aromatic halogenated DBPs in the swimming pool water were significantly lower than those of the aliphatic halogenated DBPs, following the order of HBQs > HPs > HBAs > HBALs > HAs > HNPs, while those in their incoming tap water were in the order of HBALs > HBQs > HPs > HBAs > HAs > HNPs. The average concentration-cytotoxicity contributions of different DBP categories in the swimming pool water followed the order of HAAs > HANs > HNMs > HKs > HBQs > THMs > HPs > HNPs > HBAs > HBALs > HAs, with HAAs, HANs, and HNMs possessing the main concentration-cytotoxicity contributions (93.2% in total) among all DBP categories.

Research on Trajectory Recognition and Control Technology of Real-Time Tracking Welding.

Real-time tracking welding with the assistance of structured light vision enhances the intelligence of robotic welding, which significantly shortens teaching time and guarantees accuracy for user-customized product welding. However, the robustness of most image processing algorithms is deficient during welding practice, and the security regime for tracking welding is not considered in most trajectory recognition and control algorithms. For these two problems, an adaptive feature extraction algorithm was proposed, which can accurately extract the seam center from the continuous, discontinuous or fluctuating laser stripes identified and located by the CNN model, while the prior model can quickly remove a large amount of noise and interference except the stripes, greatly improving the extraction accuracy and processing speed of the algorithm. Additionally, the embedded Pauta criterion was used to segmentally process the center point data stream and to cyclically eliminate outliers and further ensure the accuracy of the welding reference point. Experimental results showed that under the guarantee of the above-mentioned seam center point extraction and correction algorithms, the tracking average error was 0.1 mm, and even if abnormal trajectory points existed, they did not cause welding torch shaking, system interruption or other accidents.

Haloquinone Chloroimides as Toxic Disinfection Byproducts Identified in Drinking Water.

Haloquinone chloroimides (HQCs) are suspected to be highly toxic contaminants, and their production during drinking water disinfection is predicted. However, HQC disinfection byproducts (DBPs) have not been reported in drinking water to date because of analytical limitations. In this study, we developed an analytical method to detect five HQCs, including 2,6-dichloroquinone-4-chloroimide (2,6-DCQC), 2,6-dibromoquinone-4-chloroimide (2,6-DBQC), 2-chloroquinone-4-chloroimide (2-CQC), 3-chloroquinone-4-chloroimide (3-CQC), and 2,6-dichloroquinone-3-methyl-chloroimide (2,6-DCMQC). This method combined a derivatization reaction of HQCs with phenol in alkaline solutions to produce halogenated indophenols, a solid-phase extraction pretreatment using hydrophilic-lipophilic balanced (HLB) cartridges, and a multiple reaction monitoring (MRM) method for quantification. The method was demonstrated to be sensitive and accurate with recoveries of 71-85% and limits of detection of 0.1-0.2 ng/L for the five tested HQCs. Using this method, five tested HQCs were identified in drinking water samples from nine water treatment plants and water distribution systems as new DBPs at concentrations of up to 23.1 ng/L. The cytotoxicity of the five tested HQCs in HepG2 cells was higher than or comparable to that of 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), an emerging DBP that was hundreds to thousands of times more toxic than regulated DBPs. This study presents the first analytical method for HQC DBPs in drinking water and the first set of occurrence and cytotoxicity data of HQC DBPs.

Effect of Tempeh on Gut Microbiota and Anti-Stress Activity in Zebrafish.

Rhizopus oryzae is a fungus used to ferment tempeh in Indonesia and is generally recognized as safe (GRAS) for human consumption by the USA FDA. We previously assessed the effect of a tempeh extract on cortisol levels in zebrafish but did not include behavioral studies. Here, we measured the GABA content in three strains of Rhizopus oryzae, two isolated by us (MHU 001 and MHU 002) and one purchased. We then investigated the effect of tempeh on cortisol and the gut microbiota in a zebrafish experimental model. GABA concentration was the highest in MHU 002 (9.712 ± 0.404 g kg-1) followed by our MHU 001 strain and the purchased one. The fish were divided into one control group fed a normal diet and three experimental groups fed soybean tempeh fermented with one of the three strains of Rhizopus oryzae. After two weeks, individual fish were subjected to unpredicted chronic stress using the novel tank diving test and the tank light-dark test. Next-generation sequencing was used to analyze gut microbial communities and RT-PCR to analyze the expression of BDNF (brain-derived neurotrophic factor) gene and of other genes involved in serotonin signaling/metabolism in gut and brain. Tempeh-fed zebrafish exhibited increased exploratory behavior (less stress) in both tank tests. They also had significantly reduced gut Proteobacteria (include E. coli) (51.90% vs. 84.97%) and significantly increased gut Actinobacteria (include Bifidobacterium spp.) (1.80% vs. 0.79%). The content of Bifidobacteriumadolescentis, a "psychobiotic", increased ten-fold from 0.04% to 0.45%. Tempeh also increases BDNF levels in zebrafish brain. Rhizopus oryzae MHU 001 greatly improved the anti-stress effect of tempeh and microbiota composition in zebrafish gut.

Formation and Decomposition of New Iodinated Halobenzoquinones during Chloramination in Drinking Water.

Previously four chlorinated and brominated halo-benzoquinones were reported as new disinfection byproducts (DBPs) in drinking water, which have drawn great concern due to their high toxicity. In this study, three new iodinated halobenzoquinones, including 2-chloro-6-iodo-1,4-benzoquinone (2,6-CIBQ), 2-bromo-6-iodo-1,4-benzoquinone (2,6-BIBQ), and 2,6-diiodo-1,4-benzoquinone (2,6-DIBQ), were detected and identified in drinking water for the first time. Their cytotoxicity was evaluated, and their formation under various conditions was examined. Since they were not stable during chloramination, their further decomposition during chloramination was also explored. The results indicated that the concentrations of 2,6-CIBQ, 2,6-BIBQ, and 2,6-DIBQ in drinking water were in the ranges of 0.7-1.3, 1.8-8.0, and 0.4-15.9 ng/L, respectively. Compared with 2,6-dibromo-1,4-benzoquinone, the iodinated halobenzoquinones were generally more cytotoxic. The formation of 2,6-DIBQ during chloramination was significantly affected by the iodide concentration, pH, and natural organic matter. The five tested iodinated halobenzoquinones decomposed during chloramination following pseudo-first-order decay, with the decomposition rate constants in the rank order of 2,6-CIBQ > 2,6-BIBQ > 2,6-DIBQ > 2,3-diiodo-1,4-benzoquinone >2-iodo-1,4-benzoquinone. Nine polar halogenated intermediates as well as ten aliphatic halogenated DBPs were detected as the decomposition products of 2,6-DIBQ during chloramination, based on which the decomposition pathways of 2,6-DIBQ during chloramination were proposed and verified.

Dietary administration of Bacillus amyloliquefaciens R8 reduces hepatic oxidative stress and enhances nutrient metabolism and immunity against Aeromonas hydrophila and Streptococcus agalactiae in zebrafish (Danio rerio).

Zebrafish (Danio rerio) are an excellent model for assessing the beneficial effects of probiotics before applying them in aquaculture. This study evaluated the effects on zebrafish of dietary supplementation with the probiotic Bacillus amyloliquefaciens R8, which heterologously expresses xylanase from rumen fungi. Nutrient metabolism, hepatic oxidative stress, and innate immunity against pathogen infections were investigated. Treated zebrafish received feed supplemented with B. amyloliquefaciens R8 for 30 days and then were compared to zebrafish that were fed a control diet. The treated fish showed significant increases in xylanase activity in the intestines. The livers of the treated fish showed increased mRNA expressions of glycolysis-related genes of hexokinase, glucokinase, glucose-6-phosphatase, and pyruvate kinase; and higher enzyme activities of 3-hydroxyacyl-coenzyme A dehydrogenase and citrate synthase which are associated with fatty acid ß-oxidation and mitochondrial integrity. The livers of treated fish also showed decreased mRNA expressions of oxidative stress-related genes (SOD, Gpx, NOS2, and Hsp70) and an apoptotic gene (tp53), as well as increased expression of an anti-apoptotic gene (bcl-2). The probiotics-treated fish had increased expression of innate immune-related genes (IL-1ß, IL-6, IL-21, TNF-α, and TLR-1, -3, and -4). Following challenge with Aeromonas hydrophila and Streptococcus agalactiae, treated fish showed increased a higher survival rate than control fish. Overall, results showed that the administration of xylanase-expressing B. amyloliquefaciens R8 can potentially improve nutrient metabolism and hepatic stress tolerance, and enhance immunity and disease resistance against A. hydrophila and S. agalactiae in zebrafish.

Dietary administration of probiotic Paenibacillus ehimensis NPUST1 with bacteriocin-like activity improves growth performance and immunity against Aeromonas hydrophila and Streptococcus iniae in Nile tilapia (Oreochromis niloticus).

Bacteria-induced diseases are a major cause of mortality in aquaculture. Probiotics have commonly been used to replace antibiotics for prophylactic biocontrol in aquaculture. In the present study, Paenibacillus ehimensis NPUST1 was isolated from a tilapia culture pond. This probiotic has bacteriocin-like activities against Aeromonas hydrophila and was characterized by biochemical analysis and 16S rDNA sequencing. The physiochemical properties of a crude extract of the bacteriocin-like substance revealed low pH and high thermal tolerance. The substance exhibited broad-spectrum antimicrobial activity against diverse aquatic pathogens, food spoilage, clinical pathogens, and plant pathogens. The effect of dietary supplementation with P. ehimensis NPUST1 was evaluated in regard to the growth of Nile tilapia (Oreochromis niloticus) and immunity against pathogenic infection. The results showed significantly increased weight gain (WG), feed conversion ratio (FCR), and feed efficiency (FE) in Nile tilapia fed P. ehimensis NPUST1 for 2 months compared with fish fed a control diet. When challenged with A. hydrophila and S. iniae, the fish fed P. ehimensis NPUST1 also exhibited a higher survival rate than fish fed the control diet. The immune parameters revealed that the P. ehimensis NPUST1-fed fish had significantly higher phagocytic activity, respiratory burst, and superoxide dismutase (SOD) of the head kidney leukocytes, as well as higher serum lysozyme activity and expression of cytokines TNF-α and IL-1ß than the fish fed the control diet. These results indicate that dietary supplementation with P. ehimensis NPUST1 improved the growth performance, immunity, and disease resistance in Nile tilapia.

Improvements in the growth performance, immunity, disease resistance, and gut microbiota by the probiotic Rummeliibacillus stabekisii in Nile tilapia (Oreochromis niloticus).

The application of probiotics as an eco-friendly alternative to antibiotics is an emerging strategy for sustainable aquaculture. In the present study, Rummeliibacillus stabekisii was isolated from the gut of Nile tilapia, and the effects of R. stabekisii on the growth, innate immunity, disease resistance, and gut microbiota of Nile tilapia (Oreochromis niloticus) were investigated. The results showed significantly increased weight gain (WG), feed conversion ratio (FCR), and feed efficiency (FE) in Nile tilapia fed R. stabekisii for 8 weeks compared to those in fish fed a control diet. Intestinal digestive enzymes such as protease, cellulase, and xylanase were also significantly increased in the R. stabekisii-administered groups. Enhanced cumulative survival was exhibited in fish fed R. stabekisii after challenge with Aeromonas hydrophila and Streptococcus iniae. Immune parameters such as the phagocytic activity, respiratory bursts, and superoxide dismutase of head kidney leukocytes; serum lysozyme activity; and expression of the cytokine genes interleukin-1ß, tumor necrosis factor-α, transforming growth factor-ß, and heat shock protein 70 were significantly elevated in fish fed R. stabekisii. Administration of R. stabekisii considerably increased the abundance of potential probiotics (Bacillus and Lactobacillus spp.) and reduced abundances of potential pathogenic bacteria (Streptococcus and Staphylococcus spp.) in fish intestines. The present study indicated that dietary supplementation with R. stabekisii improved the growth, immunity, disease resistance, and gut microflora of Nile tilapia. This research is the first study reporting that the genus Rummeliibacillus is a potential probiotic in animals, suggesting that R. stabekisii can be used as a feed additive to enhance the growth and health status in tilapia.

A potential probiotic Chromobacterium aquaticum with bacteriocin-like activity enhances the expression of indicator genes associated with nutrient metabolism, growth performance and innate immunity against pathogen infections in zebrafish (Danio rerio).

The use of probiotics as alternatives to antibiotics for disease control is a relatively eco-friendly approach in aquaculture; hence, studies isolating and assessing the benefit of potential probiotics to fish farming are common. The zebrafish is an excellent model system for validating beneficial functions of potential probiotics before their practical application in aquaculture. Here, a potentially probiotic Chromobacterium aquaticum was isolated from lake water samples and characterized by biochemical analysis and 16S rDNA sequencing. The probiotic produced extracellular enzymes (protease and xylanase) and a bacteriocin-like substance, which exhibited tolerance to extreme pH and high-temperature conditions and broad-spectrum bactericidal activity against diverse pathogens, including aquatic, foodborne, clinical and plant pathogens. The effects of C. aquaticum on zebrafish nutrient metabolism, growth performance and innate immunity were evaluated by measuring the expression of indicator genes after C. aquaticum feeding for 8 weeks. Fish administered the probiotic exhibited significantly increased hepatic mRNA expression of carbohydrate metabolism-related genes, including glucokinase (GK), hexokinase (HK), glucose-6-phosphatase (G6Pase), and pyruvate kinase (PK-L), and growth-related genes, including the growth hormone receptor (GHR) and insulin-like growth factor-1 (IGF-1). Innate immune-related genes (IL-1ß, IL-6, TNF-α, IL-10, IL-21, NF-κb, lysozyme and complement C3b) were induced in fish with probiotic supplementation. Probiotic-treated fish exhibited a higher survival rate than control fish after challenge with Aeromonas hydrophila and Streptococcus iniae. Together, these data suggest that C. aquaticum, as a probiotic feed supplement, could enhance nutrient metabolism and growth performance and could modulate innate immunity against A. hydrophila and S. iniae in zebrafish.

Multiple-strain probiotics appear to be more effective in improving the growth performance and health status of white shrimp, Litopenaeus vannamei, than single probiotic strains.

The probiotic efficiencies of the mixed probiotics containing Lactobacillus pentosus BD6, Lac. fermentum LW2, Bacillus subtilis E20, and Saccharomyces cerevisiae P13 for shrimp growth and health status improvement were better than those when using single probiotics. The probiotic mixture at a level of 108 colony-forming units (cfu) (kg diet)-1 and the diets containing BD6 and E20 at 109 cfu (kg diet)-1 significantly improved the growth and health status of shrimp, whereas the diets containing P13 or LW2 did not significantly affect the growth of shrimp. No significant difference in the carcass composition was recorded among the control and treatments. After 56 days of feeding, shrimp fed the diet containing the probiotic mixture (107∼109 cfu (kg diet)-1) had higher survival after injection with the V. alginolyticus, but 109 cfu (kg diet)-1 of single probiotics (except for S. cerevisiae P13) had to be administered to improve shrimp survival. The better disease resistance of shrimp in groups fed the probiotic mixture might have been due to increased phenoloxidase activity, respiratory bursts, and lysozyme activity of hemocytes. Therefore, we considered that the probiotic mixture could adequately provide probiotic efficiency for white shrimp, and a diet containing 108 cfu (kg diet)-1 probiotic mixture is recommended.

Molecular Characterization and Heterologous Production of the Bacteriocin Peocin, a DNA Starvation/Stationary Phase Protection Protein, from Paenibacillus ehimensis NPUST1.

The production of a bacteriocin-like substance with antimicrobial activity, named peocin, by the probiotic Paenibacillus ehimensis NPUST1 was previously reported by our laboratory. The present study aimed to identify peocin and increase the peocin yield by heterologous expression in Escherichia coli BL21(DE3). Peocin was identified as a DNA starvation/stationary phase protection protein, also called DNA-binding protein from starved cells (Dps), by gel overlay and LC-MS/MS analysis. For mass production of peocin, fed-batch cultivation of E. coli was performed using a pH-stat control system. Purification by simple nickel affinity chromatography and dialysis yielded 45.3 mg of purified peocin from a 20-mL fed-batch culture (49.3% recovery). The biological activity of the purified peocin was confirmed by determination of the MIC and MBC against diverse pathogens. Purified peocin exhibited antimicrobial activity against aquatic, food spoilage, clinical and antibiotic-resistant pathogens. In an in vivo challenge test, zebrafish treated with purified peocin exhibited significantly increased survival rates after A. hydrophila challenge. The present study is the first to show the antimicrobial activity of Dps and provides an efficient strategy for production of bioactive peocin, which will aid the development of peocin as a novel antimicrobial agent with potential applications in diverse industries.

Transformation among Aromatic Iodinated Disinfection Byproducts in the Presence of Monochloramine: From Monoiodophenol to Triiodophenol and Diiodonitrophenol.

Aromatic iodinated disinfection byproducts (DBPs) are a newly identified category of highly toxic DBPs. Among the identified aromatic iodinated DBPs, 2,4,6-triiodophenol and 2,6-diiodo-4-nitrophenol have shown relatively widespread occurrence and high toxicity. In this study, we found that 4-iodophenol underwent transformation to form 2,4,6-triiodophenol and 2,6-diiodo-4-nitrophenol in the presence of monochloramine. The transformation pathways were investigated, the decomposition kinetics of 4-iodophenol and the formation of 2,4,6-triiodophenol and 2,6-diiodo-4-nitrophenol were studied, the factors affecting the transformation were examined, the toxicity change during the transformation was evaluated, and the occurrence of the proposed transformation pathways during chloramination of source water was verified. The results revealed that 2,4,6-triiodophenol and 2,6-diiodo-4-nitrophenol, which could account for 71.0% of iodine in the transformed 4-iodophenol, were important iodinated transformation products of 4-iodophenol in the presence of monochloramine. The transformation pathways of 4-iodophenol in the presence of monochloramine were proposed and verified. The decomposition of 4-iodophenol in the presence of monochloramine followed a pseudo-second-order decay. Various factors including monochloramine dose, pH, temperature, nitrite concentration, and free chlorine contact time (before chloramination) affected the transformation. The cytotoxicity of the chloraminated 4-iodophenol samples increased continuously with contact time. The proposed transformation pathways occurred during chloramination of source water.

Dietary supplementation with xylanase-expressing B. amyloliquefaciens R8 improves growth performance and enhances immunity against Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus).

Bacillus amyloliquefaciens has attracted attention as a probiotic in aquaculture due to its immunostimulatory activity against pathogenic infection. Xylanases are extensively used in animal feed to degrade plant ingredients, enhancing nutrient utilization and increasing the growth rate of various animals. In the present study, the effects of dietary supplementation with B. amyloliquefaciens and xylanase-expressing B. amyloliquefaciens R8 on the growth of Nile tilapia (Oreochromis niloticus) and immunity against Aeromonas hydrophila were evaluated. The results showed that the xylanase activity in the intestine, weight gain (WG), feed efficiency (FE) and condition factor (CF) of Nile tilapia fed B. amyloliquefaciens R8 for 2 months were significantly increased compared with those of the fish fed the control diet and B. amyloliquefaciens. Moreover, the mRNA expression of growth- and metabolism-related genes, such as insulin-like growth factor-1 (igf-1), glucokinase (GK), glucose-6-phosphate 1-dehydrogenase (G6PD), and glucose-6-phosphatase (G6Pase), was significantly induced in Nile tilapia fed administered B. amyloliquefaciens R8, and this group also exhibited a higher survival rate than the control fish following a challenge with A. hydrophila. The phagocytic activity and respiratory burst activity of head kidney leukocytes as well as the serum lysozyme activity of B. amyloliquefaciens R8-fed Nile tilapia were significantly higher than those of fish fed the control diet for 2 months. Superoxide dismutase (SOD) levels in the head kidney leukocytes of Nile tilapia fed B. amyloliquefaciens R8 differed from those of fish fed the control diet, but this was not significant. These results indicate that dietary supplementation with xylanase-expressing B. amyloliquefaciens R8 improves growth performance and enhances immunity and disease resistance against A. hydrophila in Nile tilapia.

Transgenic expression of omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection.

BACKGROUND: Highly desaturated n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are synthesized by desaturases and elongase. They exert hepatoprotective effects to prevent alcoholic fatty liver syndrome or cholestatic liver injury. However, it is unclear how n-3 PUFAs improve immune function in liver. Vibrio vulnificus, a gram-negative bacterial pathogen, causes high mortality of aquaculture fishes upon infection. Humans can become infected with V. vulnificus through open wounds or by eating raw seafood, and such infections may result in systemic septicemia. Moreover, patients with liver diseases are vulnerable to infection, and are more likely than healthy persons to present with liver inflammation following infection. This study quantified n-3 PUFAs and their anti-bacterial effects in Fadsd6 and Elvol5a transgenic zebrafish. RESULTS: Two transgenic zebrafish strains with strong liver specific expression of Fadsd6 and Elvol5a (driven by the zebrafish Fabp10 promoter) were established using the Tol2 system. Synthesis of n-3 PUFAs in these strains were increased by 2.5-fold as compared to wild type (Wt) fish. The survival rate in 24 h following challenge with V. vulnificus was 20 % in Wt, but 70 % in the transgenic strains. In addition, the bacteria counts in transgenic fish strains were significantly decreased. The expression levels of pro-inflammatory genes, such as TNF-α, IL-1ß, and NF-κB, were suppressed between 9 and 12 h after challenge. This study confirms the anti-bacterial function of n-3 PUFAs in a transgenic zebrafish model. CONCLUSIONS: Fadsd6 and Elvol5a transgenic zebrafish are more resistant to V. vulnificus infection, and enhance survival by diminishing the attendant inflammatory response.

Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture.

Growth hormone (GH) performs important roles in regulating somatic growth, reproduction, osmoregulation, metabolism and immunity in teleosts, and thus, it has attracted substantial attention in the field of aquaculture application. Herein, giant grouper GH (ggGH) cDNA was cloned into the pET28a vector and expressed in Shuffle® T7 Competent Escherichia coli. Recombinant N-terminal 6× His-tagged ggGH was produced mainly in insoluble inclusion bodies; the recombinant ggGH content reached 20% of total protein. For large-scale ggGH production, high-cell density E. coli culture was achieved via fed-batch culture with pH-stat. After 30h of cultivation, a cell concentration of 41.1g/l dry cell weight with over 95% plasmid stability was reached. Maximal ggGH production (4.0g/l; 22% total protein) was achieved via mid-log phase induction. Various centrifugal forces, buffer pHs and urea concentrations were optimized for isolation and solubilization of ggGH from inclusion bodies. Hydrophobic interactions and ionic interactions were the major forces in ggGH inclusion body formation. Complete ggGH inclusion body solubilization was obtained in PBS buffer at pH 12 containing 3M urea. Through a simple purification process including Ni-NTA affinity chromatography and refolding, 5.7mg of ggGH was obtained from 10ml of fed-batch culture (45% recovery). The sequence and secondary structure of the purified ggGH were confirmed by LC-MS/MS mass spectrometry and circular dichroism analysis. The cell proliferation-promoting activity was confirmed in HepG2, ZFL and GF-1 cells with the WST-1 colorimetric bioassay.

Molecular cloning and functional characterization of the hepcidin gene from the convict cichlid (Amatitlania nigrofasciata) and its expression pattern in response to lipopolysaccharide challenge.

The hepcidin gene is widely expressed in many fish species and functions as an antimicrobial peptide, suggesting that it plays an important role in the innate immune system of fish. In the present study, the Amatitlania nigrofasciata hepcidin gene (AN-hepc) was cloned from the liver and its expression during an immune response was characterized. The results of quantitative PCR and RT-PCR showed that the AN-hepc transcript was most abundant in the liver. The expression of AN-hepc mRNA was significantly increased in the liver, stomach, heart, intestine, gill and muscle but was not significantly altered in the spleen, kidney, brain or skin after lipopolysaccharide challenge. The synthetic AN-hepc peptide showed a wide spectrum of antimicrobial activity in vitro toward gram-positive and gram-negative bacteria. In particular, this peptide demonstrated potent antimicrobial activity against the aquatic pathogens Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus, Aeromonas hydrophila and Streptococcus agalactiae. The in vivo bacterial challenge results demonstrated that the synthetic AN-hepc peptide significantly improved the survival rate of S. agalactiae- and V. vulnificus-infected zebrafish. Taken together, these data indicate an important role for AN-hepc in the innate immunity of A. nigrofasciata and suggest its potential application in aquaculture for increasing resistance to disease.