Evaluation of Salipiger thiooxidans and Exiguobacterium aestuarii from the Saemangeum Reservoir as Potential Probiotics for Pacific White Shrimp (Litopenaeus vannamei).

In this study, two bacterial species, Salipiger thiooxidans and Exiguobacterium aestuarii, were extracted and screened from the Saemangeum Reservoir. This study examined these species' suitability as a probiotic by confirming the effects of S. thiooxidans and E. aestuarii added to rearing water for L. vannamei. Three experimental groups were evaluated for 6 weeks: (1) a control group reared in natural (i.e., untreated) water (CON), (2) an experimental group in which S. thiooxidans was added to natural water (SMG-A), and (3) natural water inoculated with E. aestuarii (SMG-B). The SMG-B group inoculated with E. aestuarii showed significantly higher final body weight, weight gain, specific growth rates, and feed efficiency than the control group. The SMG-B group inoculated with E. aestuarii exhibited significantly higher levels of serum lysozyme, and ACP and ALP activity than the control and SMG-A groups. The SMG-A and SMG-B groups inoculated with probiotics showed significantly lower total ammonia nitrogen and nitrite than the control group. Our findings suggest that S. thiooxidans and E. aestuarii extracted from the Saemangeum Reservoir can improve the water quality of aquaculture water, and, in particular, E. aestuarii is a potential probiotic for L. vannamei.

Probiotic Potential of the Farmed Olive Flounder, Paralichthys olivaceus, Autochthonous Gut Microbiota.

In recent years, considerable and growing attention has been given to the application of host-associated microorganisms as a more suitable source of probiotics in aquaculture sector. Herein, we isolated and screened the olive flounder gut microbiota for beneficial bacterial strains that might serve as potential probiotics in a low fishmeal extruded aquafeed. Among the ten identified isolates, Bacillus amyloliquefaciens SK4079 and B. subtilis SK4082 were screened out based on their heat-resistant ability as well as enzymatic and non-hemolytic activities. Although both strains were well able to utilize carboxymethyl cellulose (CMC), xylan, and soybean meal (SBM) as a single carbon source in the minimal nutrient M9 medium, B. subtilis exhibited significantly higher cellulase, xylanase, and protease activities than B. amyloliquefaciens. The two selected strains were well able to degrade the undesirable anti-nutritional component of the SBM, which would limit its utilization as protein source in aquafeed industry. Significantly higher biofilm formation capacity and notably stronger adhesive interactions with the flounder's skin mucus were detected in B. subtilis than B. amyloliquefaciens. Immobilization of the spores from the selected strains, in a SBM complex carrier, remarkably enhances their thermal resistance at 120 °C for 5 min and different drying conditions. It was also interesting to learn that the B. subtilis spores could survive and remain viable after being sprayed onto extruded low-fish meal feed pellets for as long as 6 months. Overall, the findings of the present study could help the food/feed industries achieve their goal of developing cost-effective yet efficient products.

Dietary Supplementation of Bacillus sp. SJ-10 and Lactobacillus plantarum KCCM 11322 Combinations Enhance Growth and Cellular and Humoral Immunity in Olive Flounder (Paralichthys olivaceus).

Experiments were conducted to identify different ratios of Bacillus sp. SJ-10 and Lactobacillus plantarum KCCM 11322 mixtures at a concentration of 1 × 108 CFU/g diet; the effects on growth and cellular and humoral immune responses and the characteristics of disease protection in olive flounder (Paralichthys olivaceus). Flounder were divided into six groups and fed control diet D-1 (without Bacillus sp. SJ-10 and L. plantarum KCCM 11322), positive control diets D-2 (Bacillus sp. SJ-10 at 1 × 108 CFU/g feed) and D-3 (L. plantarum KCCM 11322 at 1 × 108 CFU/g feed); or treatment diets D-4 (3:1 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.75 + 0.25 × 108 CFU/g feed), D-5 (1:1 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.50 + 0.50 × 108 CFU/g feed), or D-6 (1:3 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.25 + 0.75 × 108 CFU/g feed) for 8 weeks. Group D-4 demonstrated better growth and feed utilization (P < 0.05) compared with the controls and positive controls. Similar modulation was also observed in respiratory burst for all treatments and in the expression levels of TNF-α, IL-1ß, IL-6, and IL-10 in different organs in D-4. D-4 and D-5 increased respiratory burst, superoxide dismutase, lysozyme, and myeloperoxidase activities compared with the controls, and only D-4 increased microvilli length. When challenged with 1 × 108 CFU/mL Streptococcus iniae, the fish in the D-4 and D-5 groups survived up to 14 days, whereas the fish in the other groups reached 100% mortality at 11.50 days. Collectively, a ratio-specific Bacillus sp. SJ-10 and L. plantarum KCCM 11322 mixture (3:1) was associated with elevated growth, innate immunity, and streptococcosis resistance (3:1 and 1:1) compared with the control and single probiotic diets.

A comparative study of effects of dietary mercuric chloride and methylmercury chloride on growth performance, tissue accumulation, stress and immune responses, and plasma measurements in Korean rockfish, Sebastes schlegeli.

Mercury (Hg) is known as a highly toxic heavy metal, and its toxicity varies depending on its form due to different toxicokinetics between inorganic and organic Hg. Limited information on comparison of Hg toxicity concerning its chemical form by oral exposure is currently available in cultured fishes. Therefore, we conducted a comparative study to have a better understanding of distinct toxic effects between mercuric chloride (HgCl2) and methylmercury chloride (CH3HgCl) in Korean rockfish. The 12-weeks dietary exposure of HgCl2 and CH3HgCl with its graded levels (0.4-6.4 ppm) (2 × 5 factorial design) in the young-of-the-year rockfish (initial weight: 82 ± 0.3 g) resulted in neither interactive nor main effects on whole-organism responses, including growth, feed utilization, and survival. However, the distinct pattern of Hg accumulation between the two forms in dorsal muscle, brain, liver and kidney tissues was observed, showing that the rockfish fed the CH3HgCl-contained diets exhibited the dose-dependent accumulation throughout the sampling points (1, 2, 4, 8, and 12 weeks post feeding), whereas those fed the HgCl2-contained diets did not show such response. The CH3HgCl exposure induced higher oxidative stress and immunotoxicity, reflected by the elevated plasma superoxide dismutase and lysozyme activities, respectively. In addition, the CH3HgCl-induced alteration in plasma measurements, including the plasma aspartate transaminase activity and total protein level was found. Taken together, the dietary exposure of methylmercury chloride had more pronounced toxic effects than mercuric chloride in the young-of-year rockfish, needed to be taken into consideration for regulation of maximum allowed levels for Hg by its chemical form.

Evaluation of floc-harvesting technologies in biofloc technology (BFT) system for aquaculture.

This study was conducted to examine floc-harvesting performance by three separation technologies, namely sedimentation, centrifugation, and membrane filtration, for biofloc generated from a BFT system in aquaculture. According to the experimental results, sedimentation demonstrated the poorest harvesting performance with the lowest energy consumption; centrifugation showed the highest harvesting performance with the highest energy burden; membrane filtration achieved better harvesting performance than sedimentation and better energy efficiency than centrifugation. In terms of large-scale floc recovery, a two-step harvesting process utilizing centrifugation with membrane filtration was found to be a reliable way to overcome the limitation of sedimentation and obtain moderate energy-efficiency. Overall, the energy-consuming aspects of the floc-recovery process on an industrial scale should be concerned, even though the use of biofloc as an aquaculture feed would be a positive in terms of an environment-friendly approach to recycling of aquaculture wastewater.

Biological wastewater treatment: Comparison of heterotrophs (BFT) with autotrophs (ABFT) in aquaculture systems.

The present study was conducted to compare wastewater purification capacities between heterotrophs (BFT) and autotrophs (ABFT) and to evaluate the effects on the growth of fish (Nile tilapia, Oreochromis niloticus) in aquaculture systems. The wastewater treatment capacity of heterotrophs is far superior to that of autotrophs, but the BFT system requires more energy for aquaculture than does the ABFT system. Regardless, both systems effected positive influences on fish growth performance, showing excellent water-purification capacities compared with the control group (CON). No significant differences were found between CON and ABFT or between ABFT and BFT, but there were significant differences between CON and BFT. Both systems BFT and ABFT were revealed to be cost effective in relation to CON, having reduced water replacement by 82%. Therefore, the BFT and ABFT systems could be economical aquaculture systems if due advantage is taken of what both have to offer.

Characterization of a Bacillus sp. isolated from fermented food and its synbiotic effect with barley ß-glucan as a biocontrol agent in the aquaculture industry.

The purpose of this study was to evaluate Bacillus sp. SJ-10, isolated from traditional Korean seafood, as a probiotic. Strain SJ-10 was demonstrated to be safe, on the basis of in vitro tests confirming the absence of cytotoxicity, hemolysis, and genes with toxigenic potential, and was susceptible to antibiotics. It met the probiotic prerequisites of a spore count that remained almost constant, acid and bile tolerance under simulated gastrointestinal conditions, and significant adhesion to Caco-2 cells. Moreover, SJ-10 demonstrated beneficial properties as a probiotic: broad-spectrum antimicrobial activity, hydroxyl radical, antioxidant activity, production of functional enzymes such as ß-galactosidase and phytase, and selective growth via ß-glucan fermentation. The fish-feeding trial demonstrated that olive flounder fed diets containing SJ-10 alone or in combination with ß-glucan exhibited significantly higher growth performance and pathogenic disease resistance compared with those fed diets containing ß-glucan alone, indicating that SJ-10 diets exerted a beneficial effect as an antibiotic replacer in terms of growth performance and disease resistance in olive flounder.

Autochthonous Bacillus licheniformis: Probiotic potential and survival ability in low-fishmeal extruded pellet aquafeed.

In recent years, Bacillus spp. have garnered attention as probiotic supplements in aquafeed owing to the production of heat stable and low pH resistant spores. Herein, we isolated and characterized an autochthonous Bacillus licheniformis KCCM 43270 from the intestine of olive flounder (Paralichthys olivaceus) for supplementation in low-fishmeal extruded aquafeeds. The KCCM 43270 was screened based on amylase, protease, cellulase, and lipase as well as non-hemolytic activities. The isolate was able to grow in carboxymethyl cellulose (CMC), xylan, and soybean meal (SBM) when used as a single carbon source in the minimal nutrient M9 medium. The KCCM 43270 spores displayed complete survival in acid (pH 2.5) and bile (0.3%, w/v) for 3 hr, strong biofilm formation, and nearly 50% adhesion with intestinal mucus. The spores of the isolate also showed significant survival ability at 80, 90, 100°C for 60, 30, and 1 min, respectively. In addition, the spores in a blend of SBM complex carrier showed significant heat stability at 120°C for 5 min and under different drying conditions. Furthermore, the spores also survived the extrusion process during low-fishmeal aquafeed manufacturing, implying the potential application of B. licheniformis KCCM 43270 in aquafeed industry.

Effects of Lactococcus lactis subsp. lactis I2 with ß-Glucooligosaccharides on Growth, Innate Immunity and Streptococcosis Resistance in Olive Flounder (Paralichthys olivaceus).

To identify and quantify the effects of a combination of dietary 1 × 108 CFU/g Lactococcus lactis subsp. lactis I2 (LI2) and 0.1% ß-glucooligosaccharides (BGO) on the growth and immunity of olive flounder (Paralichthys olivaceus), a feeding experiment was conducted. Flounder (14 ± 0.5 g) were divided into two groups and fed control and synbiotic feeds for 8 weeks. Investigations were carried out on growth and feed utilization, innate immunity, serum biochemical parameters, intestinal lactic acid bacterial (LAB) viability, microvillus length, and changes in the expression levels of genes encoding pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1ß, and IL-6). Results demonstrated the synbiotic diet had significantly better (p < 0.05) responses in terms of weight gain and specific growth rate, three innate immune parameters (respiratory burst, serum lysozyme, and superoxide dismutase), intestinal LAB viability, and the relative TNF-α expression level (p < 0.05). Moreover, after challenge with Streptococcus iniae (1 × 108 CFU/ml), the synbiotically fed group exhibited significantly higher (p < 0.05) protection against streptococcosis, validating the observed changes in immune parameters and induction of the cytokine-encoding gene. Therefore, according to the results of the present study, synbiotic feed (LI2 + BGO) increased growth, modulated innate immune parameters and protected olive flounder against streptococcosis.

Immobilization of ß-1,3-1,4-glucanase from Bacillus sp. on porous silica for production of ß-glucooligosaccharides.

(1,3)(1,4)-ß-d-glucan has been determined to have various beneficial effects due to its unique structure. ß-glucooligosaccharides (ß-GOS), which are hydrolysates of barley (1,3)(1,4)-ß-d-glucan, provide a useful prebiotic material for selective growth of probiotic bacteria. In this study, recombinant ß-1,3-1,4-glucanase (Bg1314) from Bacillus sp. SJ-10 (KCCM 90078) was immobilized on porous silica using glutaraldehyde as a crosslinking reagent to achieve efficient production of ß-GOS. We investigated the effects of factors such as the amounts of enzyme and glutaraldehyde, reaction temperature, and pH on catalytic activity. Enzyme activity decreased sharply at high concentrations of glutaraldehyde, likely due to the reaction of glutaraldehyde with lysine residues at the catalytic site of Bg1314, because lysine-substituted Bg1314 retained its activity under the same conditions. Immobilized Bg1314 protein (ImBg1314) was stable over a wide range of pH and could be stored long term at 4 °C. The optimal conditions of ImBg1314 were similar to those of Bg1314. However, the optimal temperature of ImBg1314 differed from that of Bg1314. The products were ß-GOS composed of 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-cellotriosyl-d-glucose. After ImBg1314 was reused for 10 cycles, it retained 42% of its initial catalytic activity. This study showed that the Imbg1314 applied economical production of ß-GOS.

Dietary Hizikia fusiformis glycoprotein-induced IGF-I and IGFBP-3 associated to somatic growth, polyunsaturated fatty acid metabolism, and immunity in juvenile olive flounder Paralichthys olivaceus.

This study was aimed to examine the effect of dietary glycoprotein extracted from the sea mustard Hizikia fusiformis (Phaeophyceae: Sargassaceae) as a dietary supplement on growth performance in association with somatotropin level, proximate compositions, and immunity in juvenile olive flounder Paralichthys olivaceus. Water-ethanol extracted glycoprotein from H. fusiformis was supplemented to three fishmeal-based diets at the concentration of 0, 5, and 10gkg(-1) diet (designated as H0, H5, and H10, respectively). After a 12week-long feeding trial, growth performance and biochemical responses were analyzed including proximate composition, and whole body amino acids and fatty acids. We also measured plasma insulin like growth factor (IGF), IGF-binding protein (IGFBP) and interleukin (IL). The fish fed H5 showed the greatest weight gain among the dietary treatments. In parallel with the growth, the fish fed the diets containing H. fusiformis glycoprotein showed an increased plasma IGF-I activity and increased expression of 43-kDa IGFBP-3 compared to that in the control, whereas an opposite trend was observed for 34-kDa IGFBP-1. Although no differences were found in the level of whole body linoleic acid (C18:2n-6) and linolenic acid (C18:3n-3) among treatments, increases in arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) were observed in fish fed H5 compared to control. IL-2 and -6 levels increased significantly in fish fed H10 compared to those in the control indicating increased immunity. These results suggest that supplementation of H. fusiformis glycoprotein in fish diet may be beneficial for fish growth and immunity in juvenile olive flounder.

Molecular cloning of Kazal-type proteinase inhibitor of the shrimp Fenneropenaeus chinensis.

Proteinase inhibitors play important roles in host defence systems involving blood coagulation and pathogen digestion. We isolated and characterized a cDNA clone for a Kazal-type proteinase inhibitor (KPI) from a hemocyte cDNA library of the oriental white shrimp Fenneropenaeus chinensis. The KPI gene consists of three exons and two introns. KPI cDNA contains an open reading frame of 396 bp, a polyadenylation signal sequence AATAAA, and a poly (A) tail. KPI cDNA encodes a polypeptide of 131 amino acids with a putative signal peptide of 21 amino acids. The deduced amino acid sequence of KPI contains two homologous Kazal domains, each with six conserved cysteine residues. The mRNA of KPI is expressed in the hemocytes of healthy shrimp, and the higher expression of KPI transcript is observed in shrimp infected with the white spot syndrome virus (WSSV), suggesting a potential role for KPI in host defence mechanisms.

Isolation and characterization of polymorphic microsatellite DNA markers in the sea squirt Halocynthia roretzi.

The sea squirt Halocynthia roretzi is an important marine food resource species that is found in the waters around Korea. We describe the isolation and characterization of 13 new polymorphic microsatellite loci in 96 sea squirt samples that were collected from the marine environment of Samcheok on the east coast of Korea. The number of alleles that were observed for each locus ranged from six to 32, and the value of expected and observed heterozygosities was 0.504-0.922 and 0.396-0.813, respectively. These markers will be useful tools for future population studies.

Development of 52 new polymorphic microsatellite markers for the olive flounder, Paralichthys olivaceus.

We characterized 52 new microsatellite markers isolated from (GT)(n) and (CT)(n) microsatellite-enriched genomic libraries of the olive flounder (Paralichthys olivaceus). All markers were polymorphic, with eight to 30 (mean 15.1) alleles detected in 30 individuals from a single natural population. Observed heterozygosity ranged from 0.20 to 1.0. Segregation analysis within a mapping family revealed non-amplifying null alleles at six loci. These results indicate that these new microsatellite markers will be useful for population genetic, parentage, and genome mapping studies.

Identification of softness syndrome-associated candidate genes and DNA sequence variation in the sea squirt, Halocynthia roretzi.

The mortality of sea squirts, Halocynthia roretzi, with softness syndrome threatens the sea squirt aquaculture industry in Asian countries. The molecular approach to understanding the pathogenesis of softness syndrome began with differential gene expression analysis of tissues from normal and dying organisms. In the present study, we show that the expression of Halocynthia roretzi metalloproteinase (HrMMP) was significantly upregulated in the tissues of dying organisms through screening of differentially expressed genes, reverse transcription-polymerase chain reaction (RT-PCR), and real-time PCR. HrMMP is composed of 482 amino acids, contains a conserved domain found in the astacin family, and has typical metalloproteinase activity. To discriminate between the differential expression of the HrMMP gene in normal and dying organisms, we cloned the HrMMP gene promoter and identified a polymorphism in the HrMMP promoter region that resulted in distinct polymorphisms (G/T) at position - 308 bp. These results suggest that organisms with the GT genotype may have more resistance to softness syndrome than those with the TT genotype. These findings suggest that the HrMMP promoter polymorphism may be associated with an increased risk of softness syndrome in cultivated sea squirts and should be evaluated as a candidate molecular marker for the selective breeding of softness syndrome-resistant sea squirts.