Rhodiola rosea L. improved intestinal digestive enzyme activities, inflammatory response, barrier and microbiota dysbiosis in Lateolabrax maculatus juveniles fed with high-carbohydrate diets.

A 56-d feeding trial was conducted to evaluate the influences of Rhodiola rosea L. on digestive enzyme activities, intestinal barrier, inflammatory response, and microbiota dysbiosis in Lateolabrax maculatus juveniles (9.37 ± 0.03 g) fed with high-carbohydrate diets. Six diets were designed: a control diet (20% corn starch, Control), high-carbohydrate diet (30% corn starch, HC1), and four high-carbohydrate diets supplemented with Rhodiola rosea L. at 30, 60, 90 and 120 mg/kg (HC2, HC3, HC4 and HC5, respectively). Compared with the control group, the HC1 diet remarkably increased α-amylase, lipase, and chymotrypsin activities in the intestine (p < 0.05), as well as the mRNA levels of Claudin-15, NF-κB, TNF-α, IL-1ß, and IL-8 (p < 0.05) and the relative abundance of Proteobacteria and Photobacterium in the intestine, which belong to the phylum and genus level, respectively. But the opposite trend was found in muscular thickness and villus lengths (p < 0.05), the mRNA levels of Occludin, ZO-1, and TGF-ß (p < 0.05), at the level of phylum and genus level in the HC1 group, and the relative abundance of Firmicutes, Bacteroidetes, and Bacillus in the intestine compared with the control group. Intestinal chymotrypsin activity was significantly higher in the HC3 group and intestinal muscular thickness and villus lengths were also significantly higher in the HC2, HC3, HC4, and HC5 groups compared to the HC1 group (p < 0.05). In addition, Occludin mRNA expression in the intestine was significantly increased in the HC2, HC4, and HC5 groups compared to the HC1 group. ZO-1 and TGF-ß mRNA expression in the intestine were significantly increased in the HC2, HC3, HC4, and HC5 groups compared to the HC1 group (p < 0.05). At the phylum level, the relative abundance of Firmicutes and Bacteroidetes was higher in the intestine in the HC2, HC3, HC4, and HC5 groups than that in the HC1 group. On the contrary, intestinal lipase and chymotrypsin activities were significantly decreased in the HC2 group compared to the HC1 group, respectively (p < 0.05). The Claudin-15, NF-κB, TNF-α, IL-1ß, and IL-8 mRNA expression in the intestine were significantly decreased in the HC2, HC3, HC4, and HC5 groups compared to the HC1 group (p < 0.05). Besides, at the genus level, compared to the HC1 group, the relative abundance of Photobacterium in the intestine and the diversity of the intestinal microbiota in the HC2, HC3, HC4, and HC5 groups were all decreased. In conclusion, these results demonstrated that the addition of Rhodiola rosea L. in high-carbohydrate diets can improve intestinal digestive enzyme activities, inflammatory response and intestinal barrier-related gene expression, and microbiota dysbiosis in L. maculatus. The suitable supplemental level of Rhodiola rosea L. in high-carbohydrate diets of L. maculatus is 60 mg/kg.

Black soldier fly pulp in the diet of golden pompano: Effect on growth performance, liver antioxidant and intestinal health.

Black soldier fly (Hermetia illucens) has been widely researched as a protein source for fish meal replacement in aquaculture, but few studies have focused on its potential as a feed additive for growth and immune enhancement. We conducted a 56-day culture experiment to determine the impact of feed addition of black soldier fly pulp (BSFP, with 86.2% small peptides in dry basis) on growth performance, plasma biochemistry, liver antioxidant levels, intestinal immunity, digestion and microbiota of juvenile golden pompano (Trachinotus ovatus, 5.63 ± 0.02 g). BSFP was added to the basal diet at 0%, 1%, 3%, 5%, 7% and 9% (named Control, BSFP-1, BSFP-3, BSFP-5, BSFP-7, BSFP-9), respectively. BSFP increased the weight gain rate, specific growth rate, protein efficiency ratio and reduced the feed conversion rate of juvenile T. ovatus, the optimal growth performance was reached at BSFP-1, after which a negative feedback phenomenon was observed. Low levels of BSFP upregulated the expression of hepatic antioxidant, intestinal tight junctions, anti-inflammatory related genes and enhanced antioxidant, immune and intestinal digestive enzyme activities, which simultaneously reduced hepatic malondialdehyde and plasma aspartate transaminase and alanine aminotransferase concentrations. However, at BSFP-7, catalase activity was significantly reduced, while NF-κB p65 and pro-inflammatory cytokines transcription was significantly enhanced (P < 0.05). The results suggest that high doses of BSFP addition may damage fish health by inhibiting small peptide uptake, decreasing the activity of antioxidant enzyme and activating the canonical NF-κB pathway. Conversely, low doses of BSFP enhanced intestinal tight junction protein transcription, digestive enzyme activity and immune performance, inhibited pathogenic microbiota, while enhancing liver antioxidant capacity, which was associated with activated Nrf2-Keap1 pathway and suppressed NF-κB pathway, showing its potential as a feed additive to aquafeeds.

Rhizoma curcumae Longae ameliorates high dietary carbohydrate-induced hepatic oxidative stress, inflammation in golden pompano Trachinotus ovatus.

In general, starch, as a complex carbohydrate, is the most economical energy source in aquaculture for its relatively low cost. However, excessive dietary levels of carbohydrate result in pathological conditions. An 8-week feeding trial with CT (control diet, containing 21% carbohydrate), HC (a high-carbohydrate diet, containing 50% carbohydrate) and HCR (a HC diet supplemented with 0.015% Rhizoma curcumae Longae) was performed to investigate the protective effect of curcumin on high-carbohydrate-induced hepatic oxidative stress and intestine lesion in juvenile Trachinotus ovatus. In the current study, HC group significantly decreased WGR, SGR, plasma CAT activity, intestinal C4 levels, hepatic Nrf2, Keap1, Bach1, HO1, CAT, and GPX mRNA expression as well as ZO-1, Occludin, and Claudin-3, TGF-ß mRNA transcription levels, while the opposite was true for plasma AST activity, hepatic MDA contents, intestinal Claudin-15, NF-κB, IL-1ß, IL-6, and TNF-α mRNA expression. In contrast with the HC group, the HCR group significantly increased the activities of hepatic CAT, SOD, intestinal C3, C4, IgG and LZM levels, hepatic Nrf2, Bach1, CAT, and GPX mRNA expression as well as intestinal ZO-1, Occludin, Claudin-3, TGF-ß and IL-10 mRNA expression levels, but the opposite trend was found in plasma triglyceride content, hepatic lipid deposition, hepatic Keap1 mRNA level as well as intestinal NF-κB, IL-6. In conclusion, high-carbohydrate diet can cause detrimental effect on physiological health status in Trachinotus ovatus, while adding Rhizoma curcumae Longae can improve hepatic and intestinal health status via attenuating the oxidative stress, inflammation, and reducing lipid deposition.

Characterization and functional study of nuclear factor erythroid 2-related factor 2 (Nrf2) in black tiger shrimp (Penaeus monodon).

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a member of the Cap'n'collar basic region leucine zipper (CNC-bZIP) transcription factor family, and is activated by diverse oxidants, pro-oxidants, antioxidants and chemopreventive agents. The full-length cDNA of Nrf2 from Penaeus monodon (PmNrf2; 2024 bp long with 729 bp coding region, GenBank accession no. MW390830) was cloned. The 242-amino-acid polypeptide encoded by this gene had a predicted molecular mass of 27.80 kDa. Sequence homology and phylogenetic analysis showed that PmNrf2 was similar to the insect Cap'n'Collar (CNC) transcription factor and mammalian Nrf2. Tissue expression profile analyzed by quantitative real-time RT-PCR (qRT-PCR) demonstrated that PmNrf2 was constitutively expressed in all examined tissues, with the highest expression observed in the intestines and the weakest expression observed in the hemocyte. PmNrf2 expression profiles were detected in the hepatopancreas of shrimp after bacterial challenge. The results suggested that PmNrf2 was involved in the responses to bacterial challenge, but the temporal expression pattern trend of PmNrf2 differed between the gram-negative and gram-positive bacterial challenges in the shrimp hepatopancreas. The recombinant PmNrf2 protein was expressed and purified through affinity chromatography. Furthermore, an anti-PmNrf2 polyclonal antibody was obtained, which was able to clearly detect PmNrf2 protein expression in the hepatopancreas of shrimp. Knockdown of PmNrf2 by RNA interference (RNAi) resulted in a reduction in the expression of PmGPx gene. Taken together, the results of our study indicated that PmNrf2 played a role in regulation the transcription of PmGPx antioxidant enzyme genes.

Effects of dietary leucine levels on intestinal antioxidant status and immune response for juvenile golden pompano (Trachinotus ovatus) involved in Nrf2 and NF-κB signaling pathway.

The aim of the study was to evaluate the effects of dietary leucine level on growth performance, intestinal antioxidant status and immune response involved in Nrf2 and NF-κB signaling pathway in juvenile golden pompano (Trachinotus ovatus). A total of 450 juvenile golden pompano (9.15 ± 0.04 g) were fed three isonitrogenous diets with graded leucine levels [1.25% (control), 2.77% and 5.84%] for 8 weeks. The results showed that, compared with the control group, the WG was significantly improved in fish fed with 2.77% of dietary leucine (P < 0.05), and the 5.84% dietary leucine group had a tendency to increase. Compared to control group, 5.84% dietary leucine group significantly decreased the moisture and ash contents of whole body (P < 0.05), meanwhile, 2.77% dietary leucine group significantly decreased moisture content of whole body, but significantly improved the whole body crude lipid content (P < 0.05). Compared with the control group, the ALP level was significantly improved in fish fed with 2.77% of dietary leucine (P < 0.05). Inversely, the AST and ALT activities were significantly decreased in fish fed with 2.77% dietary leucine level (P < 0.05). Compared with the control group, GPx, T-AOC, SOD activities in group of 2.77% dietary arginine level were significantly increased (P < 0.05). However, MDA level showed a reverse trend, which was significantly decreased in fish fed with 2.77% dietary leucine level (P < 0.05). 2.77% dietary leucine levels significantly increased the relative expressions of Nrf2, HO-1, Cu/Zn-SOD, Mn-SOD and CAT (P < 0.05). In contrast, the relative expression of Keap1 showed a converse trend. Compared with the control group, the relative expressions of NF-κB, TNF-α and IL1-ß were significantly lowered in fish fed with 2.77% of dietary leucine (P < 0.05). Additionally, 2.77% dietary leucine level significantly improved the relative expressions of TGF-ß and IL-10 (P < 0.05). The 2.77% dietary leucine level significantly increased the muscular thickness compared with 5.84% dietary leucine level (P < 0.05). Furthermore, compared with the control group, the villus height and goblet cell counts were significantly improved in fish fed with 2.77% of dietary leucine (P < 0.05). These results indicated that the optimum dietary leucine plays an important role in promoting growth, enhancing antioxidant and immunity to maintain the intestinal health status of juvenile golden pompano.

The effects of dietary Lycium barbarum extract on growth performance, liver health and immune related genes expression in hybrid grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀) fed high lipid diets.

Growth performance, hepatic morphology and antioxidant ability, and expressions of antioxidant, inflammatory and apoptosis related genes were investigated in hybrid grouper fed high lipid diets containing 0, 0.5, 1, 2 and 10 g kg-1Lycium barbarum extract (LBE) for 8-week feeding. The study showed that dietary LBE significantly increased weight gain rate (WGR) and specific growth rate (SGR) of fish (P < 0.05), the highest WGR and SGR were observed in fish fed 10.00 g kg-1 LBE diet. Dietary LBE improved liver morphology by decreasing hepatocyte necrosis and inflammatory cell infiltration induced by high lipid diets. Meanwhile, high lipid diets supplemented with 0.5-2 g kg-1 LBE improved hepatic antioxidant ability by increasing the expression of antioxidant genes (GPx and CAT) and decreasing Keap1 mRNA levels. Moreover, dietary supplementation with 0.50-2.00 g kg -1 LBE significantly decreased IL-8, caspase-3, caspase-8 and caspase-9 mRNA levels and significantly increased IL-10 and TGF-ß1 mRNA levels in the liver of fish fed high lipid diets. In conclusion, high lipid diets supplemented with LBE improved growth performance, feed utilization and liver health in hybrid groupers by increasing hepatic antioxidant enzymes activity and its genes expression, as well as inhibition of hepatic inflammatory response and apoptosis.

Molecular characterization and functional analysis of IL-12p40 from Chinese sea bass (Lateolabrax maculatus) under biotic and abiotic stresses.

Interleukins are critical cytokines that are ubiquitously present in both vertebrates and invertebrates and constitute the front line of host innate immunity. Here, we identified and analyzed IL-12p40 from the Chinese sea bass Lateolabrax maculatus (LmIL-12p40). The LmIL-12p40 gene is expressed as a 1386-base pair transcript that encodes a polypeptide of 321 amino acids. Transcriptional expression analysis indicated that LmIL-12p40 mRNA was ubiquitously expressed in all tested tissues and had a comparatively high expression level in immune-associated tissues (head-kidney and intestines). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) experiments showed that, after Vibro harveyi and Streptococus agalactiae infection, LmIL-12p40 mRNA expression was significantly up-regulated in the spleen, liver and head-kidney. To further clarify the immune function of LmIL-12p40 after bacterial challenge, the recombinant LmIL-12p40 protein was acquired using a prokaryotic expression method. Furthermore, the LmIL-12p40 dimer (LmIL-12p80) could be produced via protein-protein interactions by incubating p40 monomer expressed from the pET28a vector (pET28a-LmIL-12p40) with p40 monomer expressed from the pGEX4T-1 vector (pGEX4T-1-LmIL-12p40). The antimicrobial activity of the purified LmIL-12p40 and LmIL-12p80 proteins were further studied in vitro using a bacterial growth inhibition test (for both liquid and solid cultures) and in vivo (using a bacterial growth inhibition test with the head-kidney tissues). Furthermore, BL21 (DE3) E. coli cells transformed with the recombinant pET28a-LmIL-12p40 vector were dramatically protected in response to metal toxicity and H2O2-related oxidative stress. In summary, this study will provide foundational information regarding the role of LmIL-12p40 in defending against various biotic and abiotic stresses in fishes, which should help to further clarify the functional mechanism of interleukins.

Effects of dietary ginkgo biloba leaf extract on growth performance, plasma biochemical parameters, fish composition, immune responses, liver histology, and immune and apoptosis-related genes expression of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) fed high lipid diets.

For thousands of years, leaves from the Ginkgo biloba tree have been a common treatment in Chinese medicine. The present study was conducted to investigate the effects of dietary ginkgo biloba leaf extract (GBE) supplementation on growth performance, plasma biochemical parameters, fish composition, immune responses, liver histology, and immune and apoptosis-related genes expression of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) fed high lipid diets. A basal diet supplemented with GBE at 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g kg-1 was fed to hybrid grouper for 8 weeks. The study indicated that dietary GBE did not improve growth performance and feed utilization but it reduced intraperitoneal fat rate. There were no significant differences in condition factor, viscerosomatic index, hepatosomatic index, spleen index, relative gut length, food intake, protein deposit rate and survival among all groups (P > 0.05). Dietary supplementation with 0.50-4.00 g GBE kg-1 diets effectively increased plasma HDL content and decreased plasma GLU, LDL and TG content in fish. Furthermore, dietary GBE had a significant effect on moisture, crude protein and lipid in the liver, and protein in the whole body of fish (P < 0.05). Dietary supplementation with 0.50-1.00 g GBE kg-1 diets effectively decreased occurrence rates of the hepatocyte swelling, hepatocyte vacuolization, and nuclei shifting to the cellular periphery cytoplasmic vacuolization, meanwhile hepatic antioxidant enzymes (SOD, CAT and T-AOC) activities significantly increased whereas MDA content significantly decreased in fish fed diets supplemented with GBE (P < 0.05). Moreover, dietary GBE up-regulated the expression of antioxidant genes (CAT, GPx and GR), immune-related genes (MHC2 and TLR3) and anti-inflammatory cytokines (IL-10 and TGF-ß1), while dietary supplementation with 0.50-4.00 g GBE kg-1 diets down-regulated apoptosis-related genes (p53, caspase-9, caspase-8 and caspase-3) expression in the head kidney of hybrid grouper. These results indicated that hybrid grouper fed diets supplemented with GBE did not improve growth performance and feed utilization but it had hypolipidemic effects, improved hepatic antioxidant status, maintained normal liver histology and preserved liver function, increased immune-related genes expression and decreased apoptosis-related genes expression in the head kidney of hybrid grouper.

Effects of dietary Spirulina platensis on growth performance, hematological and serum biochemical parameters, hepatic antioxidant status, immune responses and disease resistance of Coral trout Plectropomus leopardus (Lacepede, 1802).

The present study investigated the effects of dietary Spirulina platensis supplementation on growth performance, hematological and serum biochemical parameters, hepatic antioxidant status, immune responses and resistance to the pathogen infection in Coral trout Plectropomus leopardus. The fish were fed for 8-week with diets containing different levels of S. platensis: 0% (C), 2% (SP2), 4% (SP4), 6% (SP6), 8% (SP8) and 10% (SP10) as treatment groups, followed by a Vibrio harveyi infection test for 14 d. The study indicated that dietary supplementation with Spirulina platensis could significantly improve growth performance, and the highest weight gain rate (WGR) and specific growth rate (SGR) were observed in group SP10 (P < .05). Red cell count (RBC), white cell count (WBC), hemoglobin (Hb) and total antioxidant capacity (T-AOC) in the S. platensis supplemented groups were significantly higher than those of group C (P < .05). However, the levels of cholesterol, triglyceride and malondialdehyde (MDA) contents, and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities decreased with the increasing of dietary S. platensis levels. Compared with group C, the lysozyme (LYZ) and respiratory burst activities (RBA), and immunoglobulin (Ig) and complement contents in group SP4, SP6, SP8 and SP10 increased significantly than those of group C respectively (P < .05). After challenge with V. harveyi, the survival rate in group SP4, SP6, SP8 and SP10 was significantly higher than that of group C, and the highest survival rate was in group SP10 (P < .05). These results indicated that P. leopardus fed a diet supplemented with S. platensis (especially at 10%) could significantly promote its growth performance, improve its hepatic antioxidant status, and enhance its immune ability and resistance to V. harveyi infection.

Effects of dietary dandelion extract on intestinal morphology, antioxidant status, immune function and physical barrier function of juvenile golden pompano Trachinotus ovatus.

Intestinal morphology, antioxidant status, immune function and tight junction proteins mRNA expression were examined in golden pompano (Trachinotus ovatus) that fed respectively six diets containing dandelion extracts (DE) at 0, 0.5, 1, 2, 4 and 10 g kg-1 after 8 weeks feeding. The study indicated that dietary DE significantly improved intestinal antioxidant abilities by increasing SOD, CAT, T-AOC activities and up-regulating intestinal cat, gpx mRNA levels, but by decreasing MDA content and down-regulating intestinal keap1 mRNA levels in golden pompano. Meanwhile, dietary DE improved intestinal morphology, suggesting that enhances intestinal digestion and absorption, by increasing muscle thickness, villus length, villus width and villus number in the foregut and hindgut; as well as villus number, villus width and muscle thickness in the midgut (P < .05). Dietary DE enhanced intestinal barrier function by increasing intestinal zo-1 and occludin mRNA levels, but by decreasing the mRNA levels of claudin-12 and claudin-15. Furthermore, dietary DE improved intestinal immunity via increasing goblet cells numbers and regulating expression of immune-related genes. In conclusion, dietary DE supplementation promoted intestine health by improving intestine morphology, immunity, antioxidant abilities and intestinal barrier in golden pompano.

Effects of dietary hawthorn extract on growth performance, immune responses, growth- and immune-related genes expression of juvenile golden pompano (Trachinotus ovatus) and its susceptibility to Vibrio harveyi infection.

The present study was conducted to investigate the effects of dietary hawthorn extract (HTE) supplementation on growth performance, immune responses, hepatic antioxidant abilities, growth- and immune-related and heat shock protein genes expression and resistance to the pathogen Vibrio harveyi in Trachinotus ovatus. A basal diet supplemented with HTE at 0 (Diet 1), 0.50 (Diet 2), 1.00 (Diet 3), 2.00 (Diet 4), 4.00 (Diet 5) and 10.00 (Diet 6) g kg-1 were fed to golden pompano for 8 weeks. The highest final body weight, weight gain rate, specific growth rate, feed efficiency ratio and protein efficiency rate were observed in fish fed Diet 2 (P < 0.05). Dietary HTE significantly increased plasma complement 3, complement 4 and immunoglobulin M content (P < 0.05). Hepatic antioxidant enzymes (SOD, T-AOC, CAT, GPx, GR) significantly increased (P < 0.05), whereas MDA content decreased first and then increased in fish fed HTE supplement. After challenge with Vibrio harveyi, significant higher post-challenge survival was observed in fish fed Diet 2 and Diet 3 than the control group (P < 0.05). Transcription levels of growth-related genes (IGF-I and IGF-II) were significantly up-regulated in fish fed HTE supplement (P < 0.05), whereas HSP70 and HSP90 mRNA levels were significantly down-regulated (P < 0.05). With respect to immune-related genes, such as tumour necrosis factor-alpha (TNF-α), interleukin-8 (IL-8) and inhibitor protein κBα (IκB-α), upregulation was observed in the liver of fish fed with the diet supplemented with HTE. In contrast, the expression of antioxidant enzyme genes (CAT, GPx, MnSOD and Keap1) and cytokines (IL-10, TGF-ß1 and TOR) was downregulated. These results indicated that golden pompano fed a diet supplemented with 0.50 g kg-1 HTE could significantly promote growth performance and growth-related genes expression, strengthen immunity, and improve hepatic antioxidative abilities and resistance to Vibrio harveyi infection.

Effects of dietary dandelion extracts on growth performance, body composition, plasma biochemical parameters, immune responses and disease resistance of juvenile golden pompano Trachinotus ovatus.

The present study was conducted to investigate the effects of dietary dandelion extracts (DE) supplementation on growth performance, feed utilization, body composition, plasma biochemical indices, immune responses, hepatic antioxidant abilities, and resistance to the pathogen Vibrio harveyi in Trachinotus ovatus. A basal diet supplemented with DE at 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g kg-1 were fed to golden pompano for 8 weeks. The study indicated that dietary supplementation with DE could significantly improve final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), feed efficiency ratio (FER), feed intake (FI), protein efficiency ratio (PER) and protein deposit rate (PDR) (P < 0.05). The highest FBW, WGR, SGR, FI and PDR were observed in fish fed 1.00 g kg-1 dietary DE (P < 0.05). The highest FER and PER were recorded at 0.50 g kg-1 dietary DE (P < 0.05). Condition factor, viscerosomatic index, hepatosomatic index and survival were not significantly different among all groups. Fish fed 1.00 g kg-1 dietary DE showed significant increase in plasma total protein, complement 4 content and alkaline phosphatase, lysozyme, glutathione reductase (GSR) activity, but significant decrease in triglyceride, low density lipoprotein cholesterol, malondialdehyde (MDA) content and aspartate aminotransferase activities compared to the control group (P < 0.05). Hepatic antioxidant enzymes (SOD, T-AOC, CAT, GSH-Px, GSR) significantly increased whereas MDA content significantly decreased in fish fed 1.00 g kg-1 DE supplement (P < 0.05). After challenge with Vibrio harveyi, significant higher post-challenge survival was observed in fish fed DE supplement (P < 0.05). These results indicated that golden pompano fed a diet supplemented with DE (especially at 1.00 g kg-1 of fed supplement) could significantly promote its growth performance, feed utilization, body protein deposit, immune ability, hepatic and plasma antioxidative enzyme activities and improve its resistance to infection by Vibrio harveyi.

Cloning and expression analysis of c-type lysozyme gene in golden pompano, Trachinotus ovatus.

It is well known that lysozymes are key proteins to teleosts in the innate immune system and possess high bactericidal properties. In the present study, a c-type lysozyme gene (To-lysC) was cloned from golden pompano, Trachinotus ovatus. The To-lysC cDNA is composed of 743 bp with a 36 bp of 5'-UTR, 432 bp open reading frame (ORF) and 275 bp 3'-UTR, encoding a polypeptide of 144 amino acids (GenBank accession no: KT935522). Phylogenetic analysis revealed that To-lysC showed highest similarity to Perca flavescens lysC. Quantitative real-time PCR (qRT-PCR) analysis showed that To-lysC had relatively high expression level in the head kidney, gill and brain. After Vibrio harveyi infection, transcripts of To-lysC increased and reached its peak at 12 h p.i. These results indicated that To-lysC may play an important role in innate immune response to bacteria.

Effects of dietary soybean isoflavones on non-specific immune responses and hepatic antioxidant abilities and mRNA expression of two heat shock proteins (HSPs) in juvenile golden pompano Trachinotus ovatus under pH stress.

This study determined the effect of dietary soybean isoflavones on non-specific immunity and on mRNA expression of two HSPs in juvenile golden pompano Trachinotus ovatus under pH stress. Six diets were formulated to contain 0, 10, 20, 40, 60 and 80 mg/kg of soybean isoflavones. Each diet was fed to triplicate groups of fish in cylindrical tanks. After 56 days of feeding, 15 fish per tank were exposed to pH stress (pH ≈ 9.2) for 24 h. Serum total protein (TP), respiratory burst activity (RBA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), lysozyme (LYZ), complement 3 (C3), complement 4 (C4), cortisol, hepatic total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT) and the relative mRNA expression of heat shock protein 70 (HSP70) and 90 (HSP90) were investigated. The results showed that after pH stress, serum TP, RBA, LYZ, C4, hepatic T-AOC and CAT levels were significantly reduced (P < 0.05) while serum ALT, hepatic MDA and HSP70 and HSP90 mRNA expression levels were significantly increased (P < 0.05). On the other hand, supplementation with soybean isoflavones significantly reduced levels of serum ALT (20, 40, 60 mg/kg soybean isoflavones groups) and hepatic MDA (40, 60 and 80 mg/kg soybean isoflavones groups). Supplemented groups had increased serum TP content (40 mg/kg soybean isoflavones groups), RBA (20 and 40 mg/kg soybean isoflavones groups), LYZ (40 and 60 mg/kg soybean isoflavones groups), C3(20, 40, 60 and 80 mg/kg soybean isoflavones groups), hepatic SOD activity (40, 60 and 80 mg/kg soybean isoflavones groups) as well as increased relative mRNA expression of hepatic HSP70 (40, 60 and 80 mg/kg soybean isoflavones groups) and HSP90 (40 and 60 mg/kg soybean isoflavones groups) (P < 0.05). These results indicate that ingestion of a basal diet supplemented with 40-60 mg/kg soybean isoflavones could enhance resistance against pH stress in T. Ovatus to some degree.

Effects of dietary chlorogenic acid on growth performance, antioxidant capacity of white shrimp Litopenaeus vannamei under normal condition and combined stress of low-salinity and nitrite.

An eight-week feeding trial followed by an acute combined stress test of low-salinity and nitrite were performed to evaluate effects of chlorogenic acid (CGA) on growth performance and antioxidant capacity of white shrimp Litopenaeus vannamei. Shrimp were randomly allocated in 12 tanks (30 shrimp per tank) and triplicate tanks were fed with a control diet or diets containing different levels of CGA (100, 200 and 400 mg kg(-1) feed) as treatment groups. Growth performance including weight gain (WG), biomass gain (BG), feed conversion ratio (FCR), and feed intake were determined after feeding for 56 days. Antioxidant capacity were evaluated by determining the activity of total antioxidant status (TAS), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) as well as the gene expression of GSH-Px and CAT in the hepatopancreas of shrimp at the end of feeding trial and again at the end of the combined stress test. The results indicated that supplemention of CGA had no significant effects on the growth performance and the activities of TAS, SOD, GSH-Px and CAT in hepatopancreas of shrimp cultured under normal conditions for 56 days. However, compared with the control group, CGA (200, 400 mg kg(-1) feed) significantly improved the resistance of L. vannamei against the combined stress of low-salinity and nitrite, as indicated by the significant (P < 0.05) higher survival, higher activities of TAS, GSH-Px and CAT, as well as higher transcript levels of GPx and CAT gene in shrimp treated with CGA in the combined tress test. Our findings suggested that CGA possessed dual-modulatory effects on antioxidant capacity of L. vannamei and could be a potential feed additive that can enhance shrimp resistance against environmental stresses. The recommended application dosage is 200 mg kg(-1) and further studies are needed to clarify the action model of CGA efficiency.

The effects of dietary soybean isoflavones on growth, innate immune responses, hepatic antioxidant abilities and disease resistance of juvenile golden pompano Trachinotus ovatus.

The present study was conducted to investigate the effects of dietary soybean isoflavones (SI) supplementation on growth performance, innate immune responses, hepatic antioxidant abilities, heat shock protein 70 (HSP70) gene expression and resistance to the pathogen Vibrio harveyi in Trachinotus ovatus. A basal diet was supplemented with SI at 0, 10, 20, 40, 60, 80 mg kg(-1) feed for 8 weeks. Significantly maximum weight gain (WG) and specific growth rate (SGR) were observed in treatment with 40 mg kg(-1) SI supplement (P < 0.05). Feed conversion ratio (FCR), feeding rate (FR) and survival rate were not significantly different among treatments. Fish fed a diet with 40 mg kg(-1) SI showed significant increase in plasma total protein content, complement 3 content, lysozyme activity as well as respiratory burst activity, but decrease in alanine aminotransferase and aspartate aminotransferase activities (P < 0.05). Increased plasma alkaline phosphatase activity, hepatic total antioxidative capacity, catalase activity and superoxide dismutase activity were also noticed in fish fed SI at 40 or 60 mg kg(-1) (P < 0.05). On the contrary, the lowest hepatic malondialdehyde (MDA) content was observed in fish fed SI at 40 mg kg(-1) (P < 0.05). Compared with the control, the relative level of HSP70 mRNA in fish fed SI at 40-80 mg kg(-1) were significantly increased, respectively (P < 0.05). After challenge with V. harveyi, significant higher post-challenge survival was observed in fish fed diets with 40-80 mg kg(-1) SI supplement than that in control group (P < 0.05). These results indicated that dietary intake containing SI could enhance the immune ability of fish and improve its resistance to infection by V. harveyi. Especially supplementation with 40 mg kg(-1) SI to the fish for 8 weeks showed remarkable improvement in the growth, non-specific immune responses, hepatic antioxidant abilities and HSP70 gene expression.

Effect of dietary carbohydrate on non-specific immune response, hepatic antioxidative abilities and disease resistance of juvenile golden pompano (Trachinotus ovatus).

The present study was conducted to investigate the effects of dietary carbohydrate (CHO) levels on non-specific immune responses, hepatic antioxidative status and disease resistance of juvenile golden pompano. Fish were fed six isonitrogenous and isoenergetic diets containing various CHO levels for 8 weeks. After the feeding trial, fish were challenged by Vibrio harveyi and survival rate was recorded for the next 12 days. Plasma total protein and albumin content, respiratory burst activity, alkaline phosphatase, slightly increased with dietary starch level from 0% to 16.8%, but significantly decreased at dietary starch levels of 16.8%-28%. Plasma lysozyme, complement 3 and complement 4 levels increased with increasing dietary carbohydrate up to 11.2% and then declined (P < 0.05). Contrary to glutamic-oxalacetic transaminase and triiodothyronine, plasma cortisol content increased with increasing dietary carbohydrate up to 22.4%, and then levelled off. The hepatic total antioxidative capacity, reduced glutathione and catalase levels reached the peak at the fish fed diet with 16.8% carbohydrate (P < 0.05). This also held true for hepatic superoxide dismutase activities, whereas the hepatic malondialdehyde content of fish fed dietary starch level of 16.8% was significantly lower than that of fish fed no CHO diet, but showed little difference (P > 0.05) with those of the other treatments. After challenge, fish fed 11.2% and 16.8% dietary CHO showed higher survival rate than that of fish in 0% CHO group (P < 0.05). However, survival rate showed little difference among 0%, 5.6%, 22.4% and 28% CHO groups (P > 0.05). The results of this study suggest that ingestion of 11.2-16.8% dietary CHO can enhance the non-specific immune responses, increase the hepatic antioxidant abilities, and improve resistance to V. harveyi infection of juvenile golden pompano.

Effect of guava leaves on growth and the non-specific immune response of Penaeus monodon.

Guava (Psidium guajava L.) leaf extracts have antiviral and antibacterial activity against shrimp pathogens such as yellow-head virus (YHV), white spot syndrome virus (WSSV), and Vibrio harveyi, which make it a potential water disinfectant for use in shrimp culture. In this study, the safety of guava leaf supplementation in shrimp was evaluated by studying its influence on growth and the non-specific immune response of Penaeus monodon. Six diets containing different levels of guava leaves (0% [basal diet], 0.025% [G1], 0.05% [G2], 0.1% [G3], 0.2% [G4], and 0.4% [G5]) were fed to groups of shrimp (1.576 ± 0.011 g body weight) in triplicate for 56 days. Growth performance (final body weight, WG, PWG, SGR) of shrimp fed guava leaf diets was significantly higher (P < 0.05) than that of shrimp fed on the basal diet. The G1 diet resulted in the highest body weight gain (308.44%), followed by the G2 (295.45%), G3 (283.05%), G5 (281.29%), G4 (276.11%), and finally the basal diet (214.58%). Survival of shrimp in the G1 diet group was higher than that of shrimp in the control and the other experimental groups; however, no statistical differences (P > 0.05) were found. Dietary supplementation with guava leaf improved the activities of prophenoloxidase (PO) and nitric oxide synthase (NOS) in serum, and of superoxide dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LSZ) both in serum and hepatopancreas of shrimp. In the experimental groups, the activities of these enzymes followed a similar pattern of change; they increased initially at low levels of dietary supplementation and then decreased with increasing concentrations of dietary guava leaf. Serum PO and SOD activities in shrimp fed the G1 diet reached 7.50 U ml(-1) and 178.33 U ml(-1), respectively, with PO activity being significantly higher than in controls. In shrimp fed the G1 diet, SOD, ACP, and AKP activities in hepatopancreas were significantly higher than in the controls, reaching 57.32 U g(-1), 23.28 U g(-1), and 19.35 U g(-1) protein, respectively. The highest activities of serum ACP, AKP, LSZ, and of hepatopancreas LSZ, were observed in the G3 diet group. Total nitric oxide synthase (TNOS) activity was highest (64.80 U ml(-1)) in the G4 diet group, which was significantly higher than that observed in the control group. These results suggest that dietary guava leaf supplementation could enhance the growth performance and non-specific immune response of P. monodon. Therefore, guava leaf is considered safe for use as a water disinfectant in shrimp culture.

Effects of Dietary Multi-Strain Probiotics on Growth Performance, Antioxidant Status, Immune Response, and Intestinal Microbiota of Hybrid Groupers (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂).

This study aims to examine the effects of the mixture of Bacillus cereus G1-11 and Exiguobacterium acetylicum G1-33, isolated from the gut of hybrid groupers (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), on the host. The hybrid groupers were divided into a control (C, without any probiotics), B. cereus (BC, 1010 cfu/g), E. acetylicum (EA, 108 cfu/g), compound (mix, a 1:1 mixture of B. cereus and E. acetylicum), and positive reference group (P, Lactobacillus acidophilus, 5 × 108 cfu/L). Each group had four replicates, with 30 fish per replicate (53.30 ± 0.50 g), and were fed for 60 days. The results showed that adding probiotics to the feed significantly improved the weight gain, weight growth rate, specific growth rate, and digestive enzyme activities of hybrid groupers compared to the C group. The compound group was the most significant. In addition, composite probiotics added to feed significantly upregulated the expression levels of several growth-related genes in the liver and muscles. The activities of alkaline phosphatase, catalase, glutathione peroxidase, glutathione transferase, lysozyme, and total antioxidant capacity in the serum and liver were significantly influenced through mixed probiotic feeding. Moreover, the expression levels of several immune-related genes in the liver, spleen, and head kidney were significantly enhanced by adding single and mixed probiotics to feed, with the synergy of mixed probiotics being the best. An analysis of the gut microbiota showed that adding composite bacteria enhanced the richness and diversity of the gut microbiota, significantly increasing the relative abundance of potential probiotics (Cetobacterium and Microbacterium) while decreasing the presence of potential pathogens (Mycoplasma). Overall, our findings highlighted the efficacy of mixed probiotics (B. cereus and E. acetylicum) in enhancing growth performance, nutritional value of hybrid grouper feed, antioxidant capacity, immune response, and intestinal health, in finding the best combination of functional feed additives.

De Novo Assembly, Characterization, and Comparative Transcriptome Analysis of Mature Male and Female Gonads of Rabbitfish (Siganus oramin) (Bloch & Schneider, 1801).

The rabbitfish, Siganus oramin, is a commercially important table fish in southeastern China. However, there have been few studies on its gonad development and reproduction regulation. Comparative transcriptome analysis was first performed on adult male and female gonads of S. oramin. In total, 47,070 unigenes were successfully assembled and 22,737 unigenes were successfully annotated. Through comparative transcriptome analysis of male and female gonads, a total of 6722 differentially expressed genes were successfully identified, with 3528 upregulated genes and 3154 downregulated genes in the testes. In addition, 39 differentially expressed reproduction-related genes were identified. Finally, quantitative real-time PCR was used to validate the expression levels of several differentially expressed genes. These results provide important data for further studying the function of reproduction-related genes and the molecular mechanism regulating gonad development and reproduction in S. oramin.