Preventive and reparative potentials of heat-inactivated and viable commensal Bacillus pumilus SE5 in ameliorating the adverse impacts of high soybean meal in grouper (Epinephelus coioides).

Probiotic Bacillus pumilus SE5, heat-inactivated (HSE5) or active (ASE5), were supplemented to high soybean meal (HSM) (36 %) diet at whole term (0-56 days) and middle term (29-56 days) to investigate the preventing and repairing effects of B. pumilus SE5 in ameliorating the adverse effects of HSM in Epinephelus coioides. The results suggested that the HSM significantly decreased the weight gain rate (WGR), specific growth rate (SGR), and increased the feed conversion rate (FCR) at day 56 (P < 0.05), while HSE5 and ASE5 promoted the growth performance. The HSE5 and ASE5 showed preventive and reparative functions on the antioxidant capacity and serum immunity, with significantly increased the total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-PX) activities, and reduced malondialdehyde (MDA) level, and increased acid phosphatase (ACP), alkaline phosphatase (AKP), immunoglobulin M (IgM) and complement 3 (C3). The HSM impaired the intestinal health (destroyed the intestinal structure, significantly increased the contents of serum D-lactic acid and diamine oxidase, and reduced the expressions of claudin-3 and occludin), while HSE5 and ASE5 improved them at whole term and middle term. The HSM impaired the intestinal microbiota and reduced its diversity, and the HSE5 or ASE5 improved the intestinal microbiota (especially at whole term). HSE5 and ASE5 improved the intestinal mRNA expressions of anti-inflammatory genes (il-10 and tgf-ß1) and reduced the expressions of pro-inflammatory genes (il-1ß, il-8, il-12), and promoted the expressions of humoral immune factor-related genes (cd4, igm, mhcII-α) and antimicrobial peptide genes (ß-defensin, epinecidin-1 and hepcidin-1), and decreased the expressions of NF-κB/MAPK signaling pathway-related genes (ikk-α, nf-κb, erk-1), and improved the expressions of MAPK signaling pathway-related gene p38-α (P < 0.05). In conclusion, the heat-inactivated and active B. pumilus SE5 effectively prevented and repaired the suppressive effects of soybean meal in E. coioides, which underscored the potential of B. pumilus SE5 as a nutritional intervention agent in HSM diet in aquaculture.

LTA and PGN from Bacillus siamensis can alleviate soybean meal-induced enteritis and microbiota dysbiosis in Lateolabrax maculatus.

An eight-week feeding trial was designed to assess which component of commensal Bacillus siamensis LF4 can mitigate SBM-induced enteritis and microbiota dysbiosis in spotted seabass (Lateolabrax maculatus) based on TLRs-MAPKs/NF-кB signaling pathways. Fish continuously fed low SBM (containing 16 % SBM) and high SBM (containing 40 % SBM) diets were used as positive (FM group) and negative (SBM group) control, respectively. After feeding high SBM diet for 28 days, fish were supplemented with B. siamensis LF4-derived whole cell wall (CW), cell wall protein (CWP), lipoteichoic acid (LTA) or peptidoglycan (PGN) until 56 days. The results showed that a high inclusion of SBM in the diet caused enteritis, characterized with significantly (P < 0.05) decreased muscular thickness, villus height, villus width, atrophied and loosely arranged microvillus. Moreover, high SBM inclusion induced an up-regulation of pro-inflammatory cytokines and a down-regulation of occludin, E-cadherin, anti-inflammatory cytokines, apoptosis related genes and antimicrobial peptides. However, dietary supplementation with CW, LTA, and PGN of B. siamensis LF4 could effectively alleviate enteritis caused by a high level of dietary SBM. Additionally, CWP and PGN administration increased beneficial Cetobacterium and decreased pathogenic Plesiomonas and Brevinema, while dietary LTA decreased Plesiomonas and Brevinema, suggesting that CWP, LTA and PGN positively modulated intestinal microbiota in spotted seabass. Furthermore, CW, LTA, and PGN application significantly stimulated TLR2, TLR5 and MyD88 expressions, and inhibited the downstream p38 and NF-κB signaling. Taken together, these results suggest that LTA and PGN from B. siamensis LF4 could alleviate soybean meal-induced enteritis and microbiota dysbiosis in L. maculatus, and p38 MAPK/NF-κB pathways might be involved in those processes.

Pleurotus eryngii root waste and soybean meal co-fermented protein improved the growth, immunity, liver and intestinal health of largemouth bass (Micropterus salmoides).

The present study aimed to evaluate the effect of king oyster mushroom (Pleurotus eryngii) root waste and soybean meal co-fermented protein (CFP) on growth performance, feed utilization, immune status, hepatic and intestinal health of largemouth bass (Micropterus salmoides). Largemouth bass (12.33 ± 0.18 g) were divided into five groups, fed with diets containing 0 %, 5 %, 10 %, 15 % and 20 % CFP respectively for 7 weeks. The growth performance and dietary utilization were slightly improved by the supplementation of CFP. In addition, improved immunoglobulin M (IgM) content and lysozyme activity in treatments confirm the enhancement of immunity in fish by the addition of CFP, especially in fish fed 20 % CFP (P < 0.05). Furthermore, CFP significantly improved liver GSH (glutathione) content in groups D10 and D15 (P < 0.05), and slightly improved total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity while slightly reduced malondialdehyde (MDA) content. Simultaneously, the upregulation of lipolysis-related genes (PPARα, CPT1 and ACO) expression and downregulation of lipid synthesis-related genes (ACC and DGAT1) expression was recorded in the group D20 compared with the control (P < 0.05), which were consistent with the decreased liver lipid contents, suggests that lipid metabolism was improved by CFP. In terms of intestinal structural integrity, ameliorated intestinal morphology in treatments were consistent with the upregulated Occludin, Claudin-1 and ZO-1 genes expression. The intestinal pro-inflammatory cytokines (TNF-α and IL-8) expression were suppressed while the anti-inflammatory cytokines (IL-10 and TGF-ß) were activated in treatments. The expression of antimicrobial peptides (Hepcidin-1, Piscidin-2 and Piscidin-3) and intestinal immune effectors (IgM and LYZ) were slightly up-regulated in treatments. Additionally, the relative abundance of intestinal beneficial bacteria (Firmicutes) increased while the relative abundance of potential pathogenic bacteria (Fusobacterium and Proteobacteria) decreased, which indicated that the intestinal microbial community was well-reorganized by CFP. In conclusion, dietary CFP improves growth, immunity, hepatic and intestinal health of largemouth bass, these data provided a theoretical basis for the application of this novel functional protein ingredient in fish.

Paraprobiotic and postbiotic forms of Bacillus siamensis improved growth, immunity, liver and intestinal health in Lateolabrax maculatus fed soybean meal diet.

Live commensal Bacillus siamensis LF4 showed reparative potentials against high SM-induced negative effects, but whether its paraprobiotic (heat-killed B. siamensis, HKBS) and postbiotic (cell-free supernatant, CFS) forms had reparative functions and potential mechanisms are not yet known. In this study, the reparative functions of HKBS and CFS were investigated by establishing an injured model of spotted seabass (Lateolabrax maculatus) treated with dietary high soybean meal (SM). The results showed that HKBS and CFS effectively mitigated growth suppression, immune deficiency, and liver injury induced by dietary high SM. Simultaneously, HKBS and CFS application positively shaped intestinal microbiota by increased the abundance of beneficial bacteria (Fusobacteria, Firmicutes, Bacteroidota, and Cetobacterium) and decreased harmful bacteria (Proteobacteria and Plesiomonasare). Additionally, HKBS and CFS improved SM-induced intestinal injury by restoring intestinal morphology, upregulating the expression of tight junction proteins, anti-inflammatory cytokines, antimicrobial peptides, downregulating the expression of pro-inflammatory cytokines and apoptotic proteins. Furthermore, HKBS and CFS intervention significantly activated TLR2, TLR5 and MyD88 signaling, and eventually inhibited p38 and NF-κB pathways. In conclusion, paraprobiotic (HKBS) and postbiotic (CFS) from B. siamensis LF4 can improve growth, immunity, repair liver and intestinal injury, and shape intestinal microbiota in L. maculatus fed high soybean meal diet, and TLRs/p38 MAPK/NF-κB signal pathways might be involved in those processes. These results will serve as a base for future application of paraprobiotics and postbiotics to prevent and repair SM-induced adverse effects in fish aquaculture.

The interactions between the host immunity and intestinal microorganisms in fish.

There is a huge quantity of microorganisms in the gut of fish, which exert pivotal roles in maintaining host intestinal and general health. The fish immunity can sense and shape the intestinal microbiota and maintain the intestinal homeostasis. In the meantime, the intestinal commensal microbes regulate the fish immunity, control the extravagant proliferation of pathogenic microorganisms, and ensure the intestinal health of the host. This review summarizes developments and progress on the known interactions between host immunity and intestinal microorganisms in fish, focusing on the recent advances in zebrafish (Danio rerio) showing the host immunity senses and shapes intestinal microbiota, and intestinal microorganisms tune host immunity. This review will offer theoretical references for the development, application, and commercialization of intestinal functional microorganisms in fish. KEY POINTS: • The interactions between the intestinal microorganisms and host immunity in zebrafish • Fish immunity senses and shapes the microbiota • Intestinal microbes tune host immunity in fish.

High dietary wheat starch negatively regulated growth performance, glucose and lipid metabolisms, liver and intestinal health of juvenile largemouth bass, Micropterus salmoides.

Largemouth bass (Micropterus salmoides) were fed with three diets containing 6%, 12%, and 18% wheat starch for 70 days to examine their impacts on growth performance, glucose and lipid metabolisms, and liver and intestinal health. The results suggested that the 18% starch group inhibited the growth, and improved the hepatic glycogen content compared with the 6% and 12% starch groups (P < 0.05). High starch significantly improved the activities of glycolysis-related enzymes, hexokinase (HK), glucokinase (GK), phosphofructokinase (PFK), and pyruvate kinase (PK) (P < 0.05); promoted the mRNA expression of glycolysis-related phosphofructokinase (pfk); decreased the activities of gluconeogenesis-related enzymes, pyruvate carboxylase (PC), and phosphoenolpyruvate carboxykinase (PEPCK); and reduced the mRNA expression of gluconeogenesis-related fructose-1,6-bisphosphatase-1(fbp1) (P < 0.05). High starch reduced the hepatic mRNA expressions of bile acid metabolism-related cholesterol hydroxylase (cyp7a1) and small heterodimer partner (shp) (P < 0.05), increased the activity of hepatic fatty acid synthase (FAS) (P < 0.05), and reduced the hepatic mRNA expressions of lipid metabolism-related peroxisome proliferator-activated receptor α (ppar-α) and carnitine palmitoyltransferase 1α (cpt-1α) (P < 0.05). High starch promoted inflammation; significantly reduced the mRNA expressions of anti-inflammatory cytokines transforming growth factor-ß1 (tgf-ß1), interleukin-10 (il-10), and interleukin-11ß (il-11ß); and increased the mRNA expressions of pro-inflammatory cytokine tumor necrosis factor-α (tnf-α), interleukin-1ß (il-1ß), and interleukin-8 (il-8) in the liver and intestinal tract (P < 0.05). Additionally, high starch negatively influenced the intestinal microbiota, with the reduced relative abundance of Trichotes and Actinobacteria and the increased relative abundance of Firmicutes and Proteobacteria. In conclusion, low dietary wheat starch level (6%) was more profitable to the growth and health of M. salmoides, while high dietary starch level (12% and 18%) could regulate the glucose and lipid metabolisms, impair the liver and intestinal health, and thus decrease the growth performance of M. salmoides.

Commensal Bacillus siamensis LF4 induces antimicrobial peptides expression via TLRs and NLRs signaling pathways in intestinal epithelial cells of Lateolabrax maculatus.

Antimicrobial peptides (AMPs) play an important role in modulating intestinal microbiota, and our previous study showed that autochthonous Baccilus siamensis LF4 could shape the intestinal microbiota of spotted seabass (Lateolabrax maculatus). In the present study, a spotted seabass intestinal epithelial cells (IECs) model was used to investigate whether autochthonous B. siamensis LF4 could modulate the expression of AMPs in IECs. And then, the IECs were treated with active, heat-inactivated LF4 and its supernatant to illustrate their AMPs inducing effects and the possible signal transduction mechanisms. The results showed that after 3 h of incubation with 108 CFU/mL B. siamensis LF4, lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), glutamic propylic transaminase (GPT) activities in supernatant decreased significantly and obtained minimum values, while supernatant alkaline phosphatase (AKP) activity, ß-defensin protein level and IECs Na+/K+-ATPase activity, AMPs (ß-defensin, hepcidin-1, NK-lysin, piscidin-5) genes expression increased significantly and obtained maximum values (P < 0.05). Further study demonstrated that the active, heat-inactivated LF4 and its supernatant treatments could effectively decrease the LDH, GOT, and GPT activities in IECs supernatant, increase AKP activity and ß-defensin (except LF4 supernatant treatment) protein level in IECs supernatant and Na+/K+-ATPase and AMPs genes expression in IECs. Treatment with active and heat-inactivated B. siamensis LF4 resulted in significantly up-regulated the expressions of TLR1, TLR2, TLR3, TLR5, NOD1, NOD2, TIRAP, MyD88, IRAK1, IRAK4, TRAF6, TAB1, TAB2, ERK, JNK, p38, AP-1, IKKα, IKKß and NF-κB genes. Treatment with B. siamensis LF4 supernatant also resulted in up-regulated these genes, but not the genes (ERK, JNK, p38, and AP-1) in MAPKs pathway. In summary, active, heat-inactivated and supernatant of B. siamensis LF4 can efficiently induce AMPs expression through activating the TLRs/NLRs-MyD88-dependent signaling, active and heat-inactivated LF4 activated both the downstream MAPKs and NF-κB pathways, while LF4 supernatant only activated NF-κB pathway.

Probiotic components of Bacillus siamensis LF4 mitigated ß-conglycinin caused cell injury via modulating TLR2/MAPKs/NF-κB signaling in Lateolabrax maculatus.

ß-conglycinin is a recognized factor in leading to intestinal inflammation and limiting application of soybean meal in aquaculture. Our previous study reported that heat-killed B. siamensis LF4 could effectively mitigate inflammatory response and apoptosis caused by ß-conglycinin in spotted seabass (Lateolabrax maculatus) enterocytes, but the mechanisms involved are not fully understood. In the present study, therefore, whole cell wall (CW), peptidoglycan (PG) and lipoteichoic acid (LTA) and cell-free supernatant (CFS) have been collected from B. siamensis LF4 and their mitigative function on ß-conglycinin-induced adverse impacts and mechanisms underlying were evaluated. The results showed that ß-conglycinin-induced cell injury, characterized with significantly decreased cell viability and increased activities of lactate dehydrogenase, glutamic oxaloacetic transaminase, glutamic propylic transaminase (P < 0.05), were reversed by subsequent heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS treatment. Enterocytes co-cultured with heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS (especially PG) significantly increased expressions of anti-inflammatory genes (IL-2, IL-4, IL-10 and TGF-ß1), tight junction proteins (ZO-1, occludin and claudin-b) and antimicrobial peptides (ß-defensin, hepcidin-1, NK-lysin and piscidin-5), and decreased expressions of pro-inflammatory genes (IL-1ß, IL-8 and TNF-α) and apoptosis-related genes (caspase 3, caspase 8 and caspase 9) (P < 0.05), indicating their excellent mitigation effects on ß-conglycinin-induced cell damages. In addition, heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS significantly increased TLR2 mRNA level (especially in PG treatment), and decreased MAPKs (JNK, ERK, p38 and AP-1) and NF-κB related genes expressions. In conclusion, heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS could modulating TLR2/MAPKs/NF-κB signaling and alleviating ß-conglycinin-induced enterocytes injury in spotted seabass (L. maculatus), and PG presented the best potential.

Commensal Bacillus siamensis LF4 ameliorates ß-conglycinin induced inflammation in intestinal epithelial cells of Lateolabrax maculatus.

ß-conglycinin and glycinin, two major heat-stable anti-nutritional factors in soybean meal (SM), have been suggested as the key inducers of intestinal inflammation in aquatic animals. In the present study, a spotted seabass intestinal epithelial cells (IECs) were used to compare the inflammation-inducing effects of ß-conglycinin and glycinin. The results showed that IECs co-cultured with 1.0 mg/mL ß-conglycinin for 12 h or 1.5 mg/mL glycinin for 24 h significantly decreased the cell viability (P < 0.05), and overstimulated inflammation and apoptosis response by significantly down-regulating anti-inflammatory genes (IL-2, IL-4, IL-10 and TGF-ß1) expressions and significantly up-regulated pro-inflammatory genes (IL-1ß, IL-8 and TNF-α) and apoptosis genes (caspase 3, caspase 8 and caspase 9) expressions (P < 0.05). Subsequently, a ß-conglycinin based inflammation IECs model was established and used for demonstrating whether commensal probiotic B. siamensis LF4 can ameliorate the adverse effects of ß-conglycinin. The results showed ß-conglycinin-induced cell viability damage was completely repaired by treated with 109 cells/mL heat-killed B. siamensis LF4 for ≥12 h. At the same time, IECs co-cultured with 109 cells/mL heat-killed B. siamensis LF4 for 24 h significantly ameliorated ß-conglycinin-induced inflammation and apoptosis by up-regulating anti-inflammatory genes (IL-2, IL-4, IL-10 and TGF-ß1) expressions and down-regulated pro-inflammatory genes (IL-1ß, IL-8 and TNF-α) and apoptosis genes (caspase 3, caspase 8 and caspase 9) expressions (P < 0.05). In summary, both ß-conglycinin and glycinin can lead to inflammation and apoptosis in spotted seabass IECs, and ß-conglycinin is more effective; commensal B. siamensis LF4 can efficiently ameliorate ß-conglycinin induced inflammation and apoptosis in IECs.

Commensal Bacillus pumilus SE5-Derived Peptidoglycan and Lipoteichoic Acid Showed Synergistic Effects in Improving Growth, Immunity, and Intestinal Health of Grouper (Epinephelus coioides).

Commensal-derived peptidoglycan (PG) or lipoteichoic acid (LTA) can improve the growth, immunity, and intestinal health of fish, but it is not clear whether the two components have synergistic effects. To clarify this, grouper (Epinephelus coioides) was fed basal diet (CG) or diets containing 1.0 × 108 CFU/g heat-inactivated SE5 (HIB), PG (21.30 mg/kg), LTA (6.70 mg/kg), mixture (PL1) of PG (10.65 mg/kg) and LTA (3.35 mg/kg), and mixture (PL2) of PG (21.30 mg/kg) and LTA (6.70 mg/kg). Improved growth performance and feed utilization were observed in groups PG, LTA, PL1, and PL2, and the optimum growth performance was recorded in group PL1. Furthermore, improved serum alkaline phosphatase (AKP) activity and immunoglobulin M (IgM) and complement C3 (C3) contents were observed in all treatments, and the AKP activity in group PL1 was significantly superior to that of groups PG and LTA. Although PG and LTA alone or in combination exert comparable effects on intestinal microbiota and physical structure, obviously enhanced intestinal protease activity was observed in group PL1. The combined efficacy of PL1 could further potentiate the immune response by modulating the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and upregulating the expression of antimicrobial peptides (epinecidin-1, hepcidin-1, and ß-defensin) as well as IgM. At the same time, group PL1 could further mitigate intestinal inflammation by downregulating pro-inflammatory cytokines and upregulating anti-inflammatory cytokines. In conclusion, probiotic B. pumilus SE5-derived PG and LTA mixture (10.65 mg/kg PG and 3.35 mg/kg LTA) exhibits better potential for improving the growth performance, intestinal health, and immune function compared to another mixture (21.30 mg/kg PG and 6.70 mg/kg LTA) and PG or LTA alone in grouper.

Grape seed proanthocyanidin extract regulates lipid metabolism of the American eel (Anguilla rostrata).

The current study was aimed to examine the effect of grape seed proanthocyanidin extract (GSPE) on regulating lipid metabolism of American eels. A total of six cement tanks of fish were randomly divided into a control group fed with a commercial diet and a GSPE group fed with a commercial diet supplemented 400 mg/kg GSPE. There were three replicates in each group. Results suggested that GSPE could decrease the levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol, and increase the high-density lipoprotein cholesterol level in serum. GSPE might regulate lipid metabolism through upregulating linoleic acid metabolism and arachidonic acid metabolism along with downregulating metabolisms of phenylalanine, tyrosine, and tryptophan biosynthesis and valine, leucine, and isoleucine biosynthesis.

Effects of Dietary Supplementation of Peanut Skin Proanthocyanidins on Growth Performance and Lipid Metabolism of the Juvenile American Eel (Anguilla rostrata).

As a functional feed additive, grape seed proanthocyanidin extract has received a lot of attention due to its biological activity in the health of aquatic animals, but its high cost limits the application of this feed additive in the diet of many fish species. It is thus urgent to develop a new resource of proanthocyanidin extract. We aimed to investigate the effects of dietary supplementation with peanut skin proanthocyanidins (PSPc) on growth parameters and lipid metabolism of juvenile American eel (Anguilla rostrata). Four hundred and fifty juvenile eels were randomly divided into five groups fed diets with five PSPc supplementation levels. The trial lasted for 8 weeks. Dietary PSPc supplementation significantly improved weight gain and feed utilization, and the best growth performance was found in the group fed with 900 mg/kg PSPc. PSPc supplementation significantly affected the crude protein level of whole fish and serum lipid parameters, and the best lipid-lowering effect was found in the fish fed with 900 mg/kg PSPc. Dietary PSPc supplementation increased lipolytic enzyme activities and decrease lipid synthase levels in the liver. The lipid metabolites affected by 900 mg/kg PSPc in the liver were mainly upregulated phosphatidylethanolamine in autophagy, downregulated ceramides in sphingolipid metabolism, upregulated phosphatidylcholine and phosphatidylethanolamine, downregulated 2-lysophosphatidylcholine in glycerophospholipid metabolism, and upregulated phosphatidylcholine in linoleic acid metabolism. In conclusion, an appropriate level of PSPc might effectively improve growth performance and regulate the lipid metabolism of the juvenile American eel, and 900 mg/kg PSPc is recommended in the diet of this fish species.