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.

Heat-killed Bacillus sp. SJ-10 probiotic acts as a growth and humoral innate immunity response enhancer in olive flounder (Paralichthys olivaceus).

Investigations were carried out to evaluate and quantify the effects of dietary supplementation with heat-killed (HK) Bacillus sp. SJ-10 (BSJ-10) probiotic (1 × 108 CFU g-1) on the growth and immunity of olive flounder (Paralichthys olivaceus). Flounder (averagely 9.64 g) were divided into two groups, and fed control and HK BSJ-10 (HKBSJ-10)-inoculated diets for 8 weeks. Investigations were carried out on growth and feed utilizations, innate immunity, serum biochemical parameters, microvilli length, and pro- and anti-inflammatory cytokine gene (tumor necrosis factor [TNF]-α, interleukin [IL]-1ß, IL-6, and IL-10) transcriptions. Compared to control, HKBSJ-10 diet significantly (P < 0.05) enhanced weight gain and protein efficiency ratio, 1.17 and 1.11 folds respectively. Humoral innate immune parameters, lysozyme and superoxide dismutase in treatment group were also elevated by 1.34 and 1.16 folds. Similarly, an increased (P < 0.05) relative expressions of TNF-α, IL-1ß, IL-6 were recorded in liver (2.71, 3.38, and 4.12 folds respectively), and gill (2.08, 1.98, and 1.81 folds respectively) than that of controls. Moreover, after challenge with Streptococcus iniae (1 × 108 CFU mL-1), the HKBSJ-10-fed group exhibited significantly higher protection (P < 0.05) against streptococcosis compared to controls, validating the observed changes in immune parameters and induction on the cytokine-encoding genes. Therefore, HKBSJ-10 increases growth, modulates innate immune parameters, and protects olive flounders against streptococcosis.

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.

Interactive effects of dietary leucine and isoleucine on growth, blood parameters, and amino acid profile of Japanese flounder Paralichthys olivaceus.

A 60-day feeding trial was conducted to assess the interactions of dietary leucine (Leu) and isoleucine (Ile) on Japanese flounder. Fish of 2.69 ± 0.04 g were fed experimental diets containing two levels of Leu (2.58 and 5.08% of diet) combined with three levels of Ile (1.44, 2.21, and 4.44% of diet), respectively. After the feeding trial, growth, proximate composition, muscle total amino acid profile, blood parameters, mucus lysozyme activity, and stress tolerance to freshwater were measured. Statistically significant (P < 0.05) interactive effects of Leu and Ile were found on growth parameters (final body weight, body weight gain, and special growth rate) of Japanese flounder. Antagonism was discovered in high dietary Leu groups, while stimulatory effects were obtained for increased dietary Ile in low Leu groups. Interactive effects of these two branched-chain amino acids were also found on hepatosomatic index of test fish. In addition, crude lipid content of fish whole body was significantly altered by various diets, with antagonism observed in low dietary Leu groups. Interactive effects also existed in muscle amino acid profiles for low fish meal diets, but no interactive impacts were observed on blood parameters. Furthermore, lysozyme activities and freshwater stress were significantly affected by different diets. And antagonism was found on lysozyme activities in low Leu groups. Moreover, high Leu and high Ile levels of diet significantly altered freshwater stress tolerance of Japanese flounder. These findings suggested that dietary Leu and Ile can effect interactively, and fish fed with diets containing 2.58% Leu with 4.44% Ile and 5.08% Leu with 1.44% Ile showed better growth performance.

Responses of juvenile southern flounder exposed to Deepwater Horizon oil-contaminated sediments.

The Deepwater Horizon oil spill released millions of barrels of crude oil into the northern Gulf of Mexico, much of which remains associated with sediments and can have continuing impacts on biota. Juvenile southern flounder (Paralichthys lethostigma) were exposed for 28 d in the laboratory under controlled conditions to reference and Deepwater Horizon oil-contaminated sediments collected from coastal Louisiana to assess the impacts on an ecologically and commercially important benthic fish. The measured polycyclic aromatic hydrocarbon (PAH) concentrations in the sediments ranged from 0.25 mg/kg to 3940 mg/kg suite of 50 PAH analytes (tPAH50). Mortality increased with both concentration and duration of exposure. Exposed flounder length and weight was lower compared to controls after 28 d of exposure to the sediments with the highest PAH concentration, but condition factor was significantly higher in these fish compared with all other treatments. Histopathological analyses showed increased occurrence of gill abnormalities, including telangiectasis, epithelial proliferation, and fused lamellae in flounder exposed to sediments with the highest tPAH50 concentrations. In addition, hepatic vascular congestion and macrovesicular vacuolation were observed in flounder exposed to the more contaminated sediments. These data suggest that chronic exposure to field collected oil-contaminated sediments results in a variety of sublethal impacts to a benthic fish, with implications for long-term recovery from oil spills. Environ Toxicol Chem 2017;36:1067-1076. © 2016 SETAC.

Comparison of the effects of dietary single and multi-probiotics on growth, non-specific immune responses and disease resistance in starry flounder, Platichthys stellatus.

An 8-week feeding trial was conducted to evaluate the effects of dietary probiotics on growth performance and non-specific immune responses in starry flounder, Platichthys stellatus. Fish averaging 46.5 ± 0.65 g (mean ± SD) were fed one of the six experimental diets; one control (Cont), and five other diets were prepared by supplementing single-probiotics 1 (Bacillus subtilis; SP1, 2 × 109 CFU kg-1 diet), single-probiotics 2 (Bacillus licheniformis; SP2, 2 × 109 CFU kg-1 diet), multi-probiotics 1 (Bacillus subtilis + Bacillus licheniformis; MP1, 2 × 109 CFU kg-1 diet), multi-probiotics 2 (commercial probiotics; Bacillus subtills + Bacillus licheniformis + Paenibacillus polymyxa + Aspergillus oryzae + Saccharomyces cerevisiae; MP2, 2 × 109 CFU kg-1 diet) and oxytetracycline (OTC) at 5 g OTC kg-1 diet. At the end of 8 weeks feeding trial, weight gain (WG) and specific growth rate (SGR) of fish fed SP1, MP1 and MP2 diets were significantly higher than those of fish fed control diet (P < 0.05). Superoxide dismutase (SOD) activity of fish fed MP2 diet was significantly higher than those of fish fed OTC diet (P < 0.05). Nitro blue tetrazolium (NBT) activity and lysozyme activity of fish fed SP1, MP1 and MP2 diets were significantly higher than those of fish fed OTC diet (P < 0.05). However, there was no significant difference among fish fed SP1, SP2, MP1 and MP2 diets. During the Edwardsiella tarda challenge test, the first mortality occurred on day 2. After the 14 days challenge test, cumulative survival rate of fish fed MP1 and MP2 diets were significantly higher than those of fish fed control diet (P < 0.05). However, there was no significant difference among fish fed SP1, SP2, MP1, MP2 and OTC diets in survival rate at the termination of the challenge test. Although there was little advantage in immunological parameters with fish fed MP diets, single and multi-probiotics were equally effective statistically. These results demonstrated that single or multi-probiotics had equal beneficial effect as an antibiotic replacer in terms of growth performance, non-specific immune responses and disease resistance in starry flounder.

Effects of resveratrol on growth and skeletal muscle physiology of juvenile southern flounder.

Resveratrol is a naturally occurring antioxidant that has been widely studied in mammals due to its potential to extend lifespan. However, antioxidants may also limit protein damage and therefore reduce rates of protein degradation, providing a potential avenue for enhancing growth in an aquaculture setting. The present study tested the hypotheses that in Southern flounder, Paralichthys lethostigma, resveratrol would decrease protein carbonylation and 4-HNE (indicators of protein and lipid oxidative damage, respectively), levels of ubiquitinylation and LC3 (indicators of non-lysosomal and lysosomal protein degradation, respectively), while having no effect on S6K activation (indicator of protein synthesis). These effects were predicted to increase growth rate. Mitochondrial volume density was also examined since resveratrol may lead to the proliferation of mitochondria, which are the principal source of reactive oxygen species (ROS) that cause oxidative damage. Juvenile fish (n=142) were fed a control diet or a diet supplemented with 600 µg resveratrol per g of food for 16 weeks. Fish treated with resveratrol had a 9% greater length and 33% greater body mass than control fish after 16 weeks. Additionally, there was lower protein carbonylation and lipid 4-HNE within the muscle tissues of treated fish, indicating decreased oxidative damage, and reduced protein ubiquitinylation in the resveratrol fed flounder, indicating less protein degradation. However, there was not a significant difference in LC3, S6K activation, or mitochondrial volume density. These results suggest that resveratrol has positive effects on growth due to its antioxidant properties that reduce non-lysosomal protein degradation.

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.

Effects of dietary supplementation of citrus by-products fermented with a probiotic microbe on growth performance, innate immunity and disease resistance against Edwardsiella tarda in juvenile olive flounder, Paralichthys olivaceus (Temminck & Schlegel).

Two consecutive studies were conducted to evaluate the dietary supplementation of citrus by-products (CB) fermented with probiotic bacteria on growth performance, feed utilization, innate immune responses and disease resistance of juvenile olive flounder. In Experiment I, five diets were formulated to contain 0% (control) or 3% four different CB fermented with Bacillus subtilis (BS), Enterococcus faecium (EF), Lactobacillus rhamnosus (LR) and L. plantarum (LP) (designated as CON, CBF-BS, CBF-EF, CBF-LR and CBF-LP, respectively). During 10 weeks of a feeding trial, growth performance and feed efficiency were not significantly different among all the fish groups. However, fish fed CBF containing diets had significantly higher survivals than the CON group. Disease resistance of fish against Edwardsiella tarda was increased by the fermentation of CB. In Experiment II, we chose the BS as a promising probiotic and formulated five diets to contain 0%, 2%, 4%, 6% and 8% CBF-BS. Growth performance was not significantly affected by the CBF-BS supplementation during 6 weeks of a feeding trial. Innate immunity of fish was significantly enhanced by CBF-BS supplementation. Myeloperoxidase and lysozyme activities were increased in a dose-dependent manner by dietary CBF-BS inclusions. In a consecutive challenge test against E. tarda, an increased disease resistance was found by CBF-BS supplementation. These studies indicate that the fermentation process of CB with probiotic has beneficial effects on innate immunity and thereby increases disease resistance of olive flounder against E. tarda. Bacillus subtilis can be used as a promising probiotic microbe for by-product fermentation in fish feeds.

Effects of dietary supplementation of inosine monophosphate on growth performance, innate immunity and disease resistance of olive flounder (Paralichthys olivaceus).

This study was investigated to examine the effects of dietary inosine monophosphate (IMP) supplementation on growth performance, feed utilization, innate immunity, hematological parameters and disease resistance of juvenile olive flounder. Five experimental diets were formulated to contain IMP at levels of 0, 0.1, 0.2, 0.4 and 1.0%. All diets were maintained isonitrogenous (48% crude protein) and isocaloric (20.7 MJ/kg diet). Triplicate groups of olive flounder (initial body weight, 7.5 ± 0.02 g) were fed one of the experimental diets to apparent satiation (twice a day) for 14 weeks. Final body weight of fish fed 0.1-0.2% IMP were significantly higher than that of fish fed the 1.0% IMP. Groups of fish fed 0.2 or 0.4% IMP diet had significantly higher myeloperoxidase and lysozyme activities than fish fed the control diet. However, nitro-blue-tetrazolium and superoxide dismutase activities were not significantly different among all treatments. In the challenge test against Streptococcus iniae, cumulative mortality of fish fed IMP supplemented diets was significantly lower (15%, 4%, 4% and 9% for 0.1%, 0.2%, 0.4% and 1.0% IMP, respectively) than that of fish fed the control group (87%). The results suggest that IMP supplementation of 0.46-1.84 g into a kg of fish meal based diet (0.1-0.4% IMP product) can enhance innate immunity and disease resistance of olive flounder.

Probiotics and herbal mixtures enhance the growth, blood constituents, and nonspecific immune response in Paralichthys olivaceus against Streptococcus parauberis.

The present study was reported the effect of probiotics and herbals mixture supplementation diet on growth, blood constituents, and nonspecific immune response in olive flounder Paralichthys olivaceus against Streptococcus parauberis on weeks 1, 2, 4, 6, 8, 10, and 12 after injected intraperitoneally (i.p.) with 50 µl of PBS (phosphate buffer saline) containing S. parauberis (2.1 × 107 CFU ml⁻¹). The initial weight did not significantly increased in supplementation diet group from 1 to 4 weeks, whereas it was significantly increased from weeks 6 to 12 as compared to fish fed without supplementation diet. The serum aspartate aminotransferase (SGOT) and alanine aminotransferase (SGPT) activities significantly increased from weeks 4 to 12 in infected fish fed with supplementation diet compared to fish fed without supplementation diet. However, the total protein (TP) and glucose (GLU) levels were significantly increased in infected fish fed with supplementation diet after 6 weeks. The phagocytic, respiratory burst, complement, and lysozyme activities significantly enhanced in infected fish fed with supplementation diet from weeks 4 to 12 as compared to fish fed without supplementation diet. These results suggested that different probiotics and herbals mixture supplementation diet enhanced the growth, blood biochemical constituents, and nonspecific immunity in olive flounder against S. parauberis.

Effects of heavy oil in the developing spotted halibut, Verasper variegatus.

It is well known that heavy oil (HO) on the sea surface causes serious problems in the aquatic environment. In particular, some species of teleosts which develop on the sea surface are thought to be affected by the HO which flows out from tankers or coastal industry. However, the toxicological effects of HO are not fully understood. We performed exposure experiments using the Pleuronectiformean fish, spotted halibut (Verasper variegatus), which is an important fishery resource in Japan. In course of the development, HO-exposed embryos showed remarkable delay in developmental processes including somite formation. We further observed abnormal development of the head morphology. Notably, treated embryos had relatively small eyes and craniofacial structures. These findings strongly suggest that HO seriously affects the cell proliferation and differentiation of the embryo. In addition, HO-exposed embryos showed abnormal neuronal development. We also performed the exposure in the larval stage. Treatment of post-hatching larvae with HO resulted in significantly greater mortality compared with controls. Through these observations, we finally conclude that HO is strongly toxic to halibut in their early life stages.

Comparison of taurine biosynthesis ability between juveniles of Japanese flounder and common carp.

This study was conducted to investigate taurine deficiency and the ability of taurine biosynthesis in both juvenile Japanese flounder (JF) and juvenile common carp (CC) in vivo using low taurine level diets. Three different taurine level diets were prepared by the supplementation of taurine to the basal composition (JF--0, 0.5 and 1.5% in JF; CC--0, 1, 3% in CC). The final average body weight and feed efficiency of JF fed the JF - 1.5% was significantly higher than those of fish fed on the JF--0%. On the other hand, no significant difference was observed in CC fed with CC--0, 1, and 3% diets. The taurine retention rate was negative in the case of JF-fed with the taurine-free supplement (JF--0%). On the other hand, the taurine retention rate was about 280% in the case of CC-fed with the taurine-free supplement (CC--0%). These findings indicate that while taurine is essential for growth of JF, it is not essential for the growth of CC.