Multi-omics approach to study the dual effects of novel proteins on the intestinal health of juvenile largemouth bass (Micropterus salmoides) under an alternate feeding strategy.

Introduction: In an effort to minimize the usage of fishmeal in aquaculture, novel protein diets, including Tenebrio molitor, cottonseed protein concentrate, Clostridium autoethanogenum, and Chlorella vulgaris were evaluated for their potential to replace fishmeal. Nevertheless, comprehensive examinations on the gut health of aquatic animals under an alternate feeding strategy when fed novel protein diets are vacant. Methods: Five isonitrogenous and isolipidic diets containing various proteins were manufactured, with a diet consisting of whole fishmeal serving as the control and diets containing novel proteins serving as the experimental diets. Largemouth bass (Micropterus salmoides) with an initial body weight of 4.73 ± 0.04g employed as an experimental animal and given these five diets for the first 29 days followed by a fishmeal diet for the next 29 days. Results: The results of this study demonstrated that the growth performance of novel protein diets in the second stage was better than in the first stage, even though only the C. vulgaris diet increased antioxidant capacity and the cottonseed protein concentrate diet decreased it. Concerning the intestinal barriers, the C. autoethanogenum diet lowered intestinal permeability and plasma IL-1ß/TNF-α. In addition, the contents of intestinal immunological factors, namely LYS and sIgA-like, were greater in C. vulgaris than in fishmeal. From the data analysis of microbiome and metabolome, the levels of short chain fatty acids (SCFAs), anaerobic bacteria, Lactococcus, and Firmicutes were significantly higher in the C. autoethanogenum diet than in the whole fishmeal diet, while the abundance of Pseudomonas, aerobic bacteria, Streptococcus, and Proteobacteria was lowest. However, no extremely large differences in microbiota or short chain fatty acids were observed between the other novel protein diets and the whole fishmeal diet. In addition, the microbiota were strongly connected with intestinal SCFAs, lipase activity, and tight junctions, as shown by the Mantel test and Pearson's correlation. Discussion: Taken together, according to Z-score, the ranking of advantageous functions among these protein diets was C. autoethanogenum diet > C. vulgaris diet > whole fishmeal diet > cottonseed protein concentrate > T. molitor diet. This study provides comprehensive data illustrating a mixed blessing effect of novel protein diets on the gut health of juvenile largemouth bass under an alternate feeding strategy.

Construction and efficacy of Aeromonas veronii mutant Δhcp as a live attenuated vaccine for the largemouth bass (Micropterus salmoides).

Aeromonas veronii is a human and animal co-pathogenic bacterium that could have a significant negative impact on both human health and aquaculture. In this study, a mutant strain of A. veronii with deletion of the hemolysin co-regulated protein (hcp) gene was constructed (Δhcp-AV). Compared with the wild strain, Δhcp-AV showed significantly reduced growth capacity and biofilm formation ability. Motility tests showed that the hcp gene had no significant effect on the swimming and swarming ability. In addition, the pathogenicity was also reduced. To evaluate the efficacy of Δhcp-AV as a live attenuated vaccine for prevention of Aeromonas veronii infection, we compared the immune response of largemouth bass (Micropterus salmoides) after immunization with 500 µL of 1.47 × 105 CFU/mL of Δhcp-AV and 4 × 108 CFU/mL of inactivated A. veronii. Obvious increases of serum immune related enzyme activity were observed in immunization groups. Expression levels of immune-related genes in Δhcp-AV group were up-regulated, and higher than those in inactivated A. veronii group. After challenging with live A. veronii, the relative percent survival (RPS) was 100% in Δhcp- AV group, whereas the RPS was 76.67% in inactivated A. veronii group. Our data suggest that the live attenuated vaccine Δhcp- AV could elicit a stronger immune response and provide a higher RPS than inactivated A. veronii. These data suggest that hcp gene is an important virulence factor of A. veronii, and the live attenuated vaccine Δhcp-AV is safe and effective for prevention A. veronii infection in M. salmoides farming.

Functional Characterization of Largemouth Bass (Micropterus salmoides) Soluble FcγR Homolog in Response to Bacterial Infection.

Fc receptors (FcRs) are key players in antibody-dependent cellular phagocytosis (ADCP) with their specific recognition of the Fc portion of an immunoglobulin. Despite reports of FcγR-mediated phagocytosis in mammals, little is known about the effects of soluble FcγRs on the immune response. In this study, FcγRIα was cloned from the largemouth bass (Micropterus salmoides) (MsFcγRIα). Without a transmembrane segment or a cytoplasmic tail, MsFcγRIα was identified as a soluble form protein and widely distributed in the spleen, head kidney, and intestine. The native MsFcγRIα was detected in the serum of Nocardia seriolae-infected largemouth bass and the supernatants of transfected HEK293 cells. Additionally, it was verified that the transfected cells' surface secreted MsFcRIα could bind to largemouth bass IgM. Moreover, the expression changes of MsFcγRIα, Syk, and Lyn indicated that MsFcγRIα was engaged in the acute phase response to bacteria, and the FcγR-mediated phagocytosis pathway was activated by Nocardia seriolae stimulation. Furthermore, recombinant MsFcγRIα could enhance both reactive oxygen species (ROS) and phagocytosis to Nocardia seriolae of leukocytes, presumably through the interaction of MsFcγRIα with a complement receptor. In conclusion, these findings provided a better understanding of the function of soluble FcγRs in the immune response and further shed light on the mechanism of phagocytosis in teleosts.

Nanoplastics Increase Fish Susceptibility to Nodavirus Infection and Reduce Antiviral Immune Responses.

Nanoplastics (NPs) might cause different negative effects on aquatic organisms at different biological levels, ranging from single cells to whole organisms, including cytotoxicity, reproduction, behavior or oxidative stress. However, the impact of NPs on disease resistance is almost unknown. The objective of this study was to assess whether exposure to 50 nm functionalized polystyrene NPs impacts fish susceptibility to viral diseases both in vitro and in vivo. In particular, we focused on the nervous necrosis virus (NNV), which affects many fish species, producing viral encephalopathy and retinopathy (VER), and causes great economic losses in marine aquaculture. In vitro and in vivo approaches were used. A brain cell line (SaB-1) was exposed to 1 µg mL-1 of functionalized polystyrene NPs (PS-NH2, PS-COOH) and then infected with NNV. Viral titers were increased in NP-exposed cells whilst the transcription of inflammatory and antiviral markers was lowered when compared to those cells only infected with NNV. In addition, European sea bass (Dicentrarchus labrax) juveniles were intraperitoneally injected with the same NPs and then challenged with NNV. Our results indicated that NPs increased the viral replication and clinical signs under which the fish died although the cumulate mortality was unaltered. Again, exposure to NPs produced a lowered inflammatory and antiviral response. Our results highlight that the presence of NPs might impact the infection process of NNV and fish resistance to the disease, posing an additional risk to marine organisms.

Two paralogs of CXCR4 in the Japanese sea bass (Lateolabrax japonica) are involved in the immune response of B lymphocytes.

CXC chemokine receptor 4 (CXCR4), a member of the G-protein-coupled receptor family, plays an important role in host immune responses. Within the teleost lineage, there are two paralogs of CXCR4; however, the role of CXCR4 in teleost B cells is poorly understood. In this study, we determined the cDNA sequences of the two CXCR4 paralogs from the Japanese sea bass (Lateolabrax japonica; LjCXCR4a and LjCXCR4b). Sequence and phylogenetic tree analyses revealed that LjCXCR4a and LjCXCR4b are most closely related to CXCR4a and CXCR4b, respectively, in the large yellow croaker (Larimichthys crocea). CXCR4 transcripts were mainly expressed in the gills, and their expression in different tissues was altered upon infection with Vibrio harveyi. LjCXCR4a and LjCXCR4b protein levels were upregulated in infected B cells. Knockdown of LjCXCR4a and LjCXCR4b in B cells by RNA interference, the phagocytic activity of B cells was not affected. Furthermore, knockdown of LjCXCR4a, not of LjCXCR4b, was observed to inhibit LjIgM expression in lipopolysaccharide-stimulated B cells. In addition, knockdown of LjCXCR4a, not of LjCXCR4b, was found to reduce reactive oxygen species levels in B cells. Our results indicate that LjCXCR4a and LjCXCR4b modulate the immune response of Japanese sea bass B cells against bacterial infection, albeit via different pathways.

Grouper TIA-1 functions as a crucial antiviral molecule against nervous necrosis virus infection.

T-cell intracellular antigen (TIA)-1 is a prion-related RNA-binding protein involved in splicing and translational repression, and regulates translation in response to stress conditions by isolating target mRNAs in stress granules (SGs). However, little is known about the potential roles of fish TIA-1 and how it works in viral infection. In this study, the TIA-1 (EcTIA-1) homolog from orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The open reading frame (ORF) sequence of EcTIA-1 encoded a 388 amino acid protein with predicted molecular mass of 42.73 kDa. EcTIA-1 contains three conserved domains of RNA recognition motif (RRM) that may interact with RNA via its second and third RRMs. Overexpression of EcTIA-1 inhibited red-spotted grouper nervous necrosis virus (RGNNV) replication and positively regulated interferon immune response, which was increased by knockdown of EcTIA-1. RGNNV induced formation of SGs in cells with EcTIA-1 overexpression. These results provide a novel insight into understanding the roles of fish TIA-1 in response to RNA viruses.

The transcription factor RFX5 positively regulates expression of MHCIa in the red-spotted grouper (Epinephelus akaara).

Regulatory factor X 5 (RFX 5) is a member of the RFX family, and it forms the transcription factor complex RFX with RFXANK/B and RFXAP. The RFX complex can activate MHC expression by binding to the MHC promoter. However, the regulate mechanism of RFX in fish species is not been fully elucidated. In this study, we investigated the transcriptional regulation of Epinephelus akaara RFX5 (EaRFX5) on EaMHCI, and its effect on immune pathways. The genomic sequence of EaRFX5 was 35,774 bp and consisted of ten exons and nine introns. The length of EaRFX5 ORF sequence is 2,160 bp, encoding 719 amino acids. By qRT-PCR, EaRFX5 was detected constitutively expressed in twelve selected tissues, showing a wide range of expression. EaRFX5 expression parttern in response to poly (I:C), LPS, Zymosan A, SGIV, and NNV challenges showed that EaRFX5 plays a differentiated immunomodulatory role in response to various stimuli in different tissues, and EaRFX5 was most significantly upregulated in the kidney after challenge with SGIV. Subcellular localization assays showed that EaRFX5 is a typical nuclear protein. Based on the in vitro overexpression experiments, EaRFX5 appeared to promote the expression of EaMHCIa gene, interferon signalling pathway and inflammatory cytokine. Luciferase reporter assay showed that the -267 bp to +82 bp region of EaMHCIa promoter was the core region where EaRFX5 modulated. Additionally, point mutations and electrophoretic mobility shift assays indicating M3 is the EaRFX5 binding sites in the EaMHCIa promoter. These results contribute to elucidating the function of EaRFX5 in fish immune response, and provide the first evidence of positive regulation of MHCIa expression by RFX5 in fish.

Molecular characterization and expression profiling of tandem-repeat galectin-8 from red-spotted grouper (Epinephelus akaara): Potential antibacterial, antiviral, and wound healing activities.

Galectin-8 is a typical ß-galactoside binding lectin, which primarily functions as a pattern recognition receptor and/or danger receptor that is engaged in pathogen recognition by the host innate immune system. Although several fish galectins have been identified, the role of galectin-8 in teleost immunity is still not fully understood. In this study, molecular, transcriptional, and immune-related functions of galectin-8 (EaGal8) from red-spotted grouper (Epinephelus akaara) were analyzed. The open reading frame of EaGal8 comprised 960 bp encoding 319 amino acids of a ∼35 kDa protein, composed of the N- and C-terminal carbohydrate recognition domains joined by a short hinge peptide. Phylogenetic analysis revealed that EaGal8 was closely related to the Epinephelus lanceolatus galectin-8-like protein. Although EaGal8 showed ubiquitous tissue expression, the highest expression level was observed in the blood. Immunostimulants, including lipopolysaccharide, poly(I:C), and nervous necrosis virus, significantly upregulated the EaGal8 transcription level in a time-dependent manner (p < 0.05). Furthermore, recombinant EaGal8 (rEaGal8) showed a binding affinity toward seven different carbohydrates in a concentration-dependent manner. In addition, rEaGal8 caused strong agglutination of fish red blood cells and several gram-positive and gram-negative bacteria, including Streptococcus iniae, Streptococcus parauberis, Lactococcus garvieae, Escherichia coli, Edwardsiella tarda, Vibrio alginolyticus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa. For the first time in teleosts, we report the wound healing ability of galectin-8 in this study. At low concentrations, rEaGal8 showed potential wound healing responses in FHM cells, in vitro. Thus, this study reinforces the role of EaGal8 in innate immune responses against bacterial and viral infections and wound healing in red-spotted grouper.

Beta glucan induced immune priming protects against nervous necrosis virus infection in sevenband grouper.

In the present study, we studied the effect of ß-glucan on the activation of antiviral immune responses against nervous necrosis virus (NNV) taking into consideration the role of innate immune training. Sevenband grouper primary macrophages showed an attenuated proinflammatory response and elevated antiviral response to NNV infection. In vitro, priming of ß-glucan enhanced macrophage viability against NNV infection which is associated with the activation of sustained inflammatory cytokines gene expression. Observations were clear to understand that NLR Family CARD Domain Containing 3 (NLRC3) and caspase-1 activation and subsequent IL-1ß production were reduced in ß-glucan-primed macrophages. Subsequent markers for training including Lactate and abundance of HIF-1α were elevated in the cells following training. However, the lactate dehydrogenase (LDH) concentrations remained stable among the ß-glucan stimulated infected and uninfected groups suggesting similar macrophage health in both groups. In vivo, the NNV-infected fish primed with ß-glucan had a higher survival rate (60%) than the control NNV-infected group (40%). Our findings demonstrate that ß-glucan induced protective responses against NNV infection and studies are underway to harness its potential applicability for prime and boost vaccination strategies.

Functional analysis of a novel MHC-Iα genotype in orange-spotted grouper: Effects on Singapore grouper iridovirus (SGIV) replication and apoptosis.

The classical major histocompatibility complex class I (MHC-Ⅰ) molecule plays a key role in vertebrate immune response for its important functions in antigen presentation and immune regulation. MHC pathway is closely related to many diseases involving autoimmunity, antigen intrusion and inflammation. However, rare literatures about the effect of MHC-I on fish cells apoptosis were reported. In this study, a novel type of MHC-Ⅰα genotype from orange-spotted grouper (named EcMHC-ⅠA*01) were cloned and characterized. It shared a 77% identity to its Epinephelus coioides MHC-Iα homology that has been uploaded to NCBI (ACZ97571.1). Molecular characterization analysis showed that EcMHC-ⅠA*01 encodes a 357-amino-acid protein, containing a signal peptide，α1，α2，α3, Cytoplasmic (Cyt) and Transmembrane (TM) domains. Tissue expression pattern showed that EcMHC-ⅠA*01 was extensively distributed in twelve selected tissues, with higher expression in the gill, intestine and skin. The expression of EcMHC-ⅠA*01 in grouper liver and spleen tissues were significantly induced by different stimuli (Zymosan A, LPS, Ploy I:C, RGNNV and SGIV). Comparing with the EcMHC-ⅠA*01 expression levels induced by Zymosan A, Ploy I:C and RGNNV, the effects induced by SGIV and LPS were more significant. Subcellular localization analysis showed that EcMHC-ⅠA*01 localizes throughout the cytoplasm appeared both diffuse and focal intracellular expression pattern. Overexpression of EcMHC-ⅠA*01 inhibited the CPE progression, the mRNA expression of the SGIV related genes (MCP, LITAF, ICP-18 and VP19) and the protein expression of MCP. Meanwhile, qRT-PCR result showed that EcMHC-ⅠA*01 overexpression upregulated the expression of interferon signaling molecules (IFN-γ, ISG56, MDA5 and MXI) and inflammatory cytokines (IL-1ß, IL-6, TNF-α and TRAF6). In addition, our results showed that overexpression of EcMHC-ⅠA*01 promoted the apoptosis of normal fathead minnow (FHM) cells as well as the apoptosis of FHM cells induced by SGIV. However, there was no significant change in the activity of caspase 3 between control group and EcMHC-ⅠA*01 overexpression group, suggesting that EcMHC-ⅠA*01-induced apoptosis may not depend on the caspase 3 pathway. Taken together, these data in our study provide new insights into the role of MHC-I in antiviral immune response and apoptosis in fish.

Grouper USP12 exerts antiviral activity against nodavirus infection.

The ubiquitin-specific proteases (USPs) have attracted particular attention due to their multiple functions in different biological processes. USP12, a member of the USP family, has been demonstrated to exert critical roles in diverse cellular processes, including cell death, cancer and antiviral immunity. Here, we cloned a USP12 homolog from orange spotted grouper (Epinephelus coioides, E. coioides), and its roles in fish RNA virus replication were investigated. EcUSP12 contained a 1119-bp open reading frame (ORF) encoding a 372-amino acid polypeptide, which shared 100.00% and 91.32% identity with USP12 homolog of Etheostoma cragini and Homo sapiens, respectively. Sequence analysis indicated that EcUSP12 contained a conserved peptidase-C19G domain (aa 40-369). qPCR analysis showed that EcUSP12 transcript was most abundant in head kidney and spleen of grouper E. coioides. The expression of EcUSP12 was significantly upregulated in grouper spleen (GS) cells in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization analysis showed that EcUSP12 was evenly distributed throughout the cytoplasm, and mainly co-localized with endoplasmic reticulum (ER). Interestingly, during RGNNV infection, the endogenous distribution of EcUSP12 was obviously altered, and mostly overlapped with viral coat protein (CP). Co-Immunoprecipitation (Co-IP) assay indicated that EcUSP12 interacted with viral CP. In addition, overexpression of EcUSP12 significantly inhibited the replication of RGNNV in vitro, as evidenced by the decrease in viral gene transcription and protein synthesis during infection. Consistently, knockdown of EcUSP12 by small interfering RNA (siRNA) promoted the replication of RGNNV. Furthermore, EcUSP12 overexpression also increased the transcription level of inflammatory factors and interferon-related genes, including tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, IL-6, IL-8, interferon regulatory factor 3 (IRF3), and IRF7. Taken together, our results demonstrated that EcUSP12, as a positive regulator of IFN signaling, interacted with viral CP to inhibit virus infection.

Effects of fishmeal replacement by black soldier fly on growth performance, digestive enzyme activity, intestine morphology, intestinal flora and immune response of pearl gentian grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂).

An 8-week feeding trial was conducted to investigate the influence of partial replacement of fishmeal (FM) by black soldier fly (BSF) (Hermetia illucens) on the growth, distal intestine morphology, intestinal flora, and intestinal immune response of pearl gentian grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂). Four diets were formulated, 0% (0 g kg-1), 10% (50 g kg-1), 20% (100 g kg-1) and 30% (150 g kg-1) fishmeal were replaced with BSF, named as FM, BSF10, BSF20, BSF30, severally. The study found that, with the increasing dietary BSF levels, growth and feed conversion ratio of fish decreased significantly (P < 0.05). Chitinase and trypsin activities were significantly increased with increasing dietary BSF levels (P < 0.05). With the increasing dietary BSF levels, distal intestinal muscularis thickness and mucosal fold length decreased significantly (P < 0.05), as well as total abundance of intestinal flora. The relative abundance of four phyla and six genera among the top 20 genera were significantly affected by dietary BSF levels (P < 0.05). With the increasing dietary BSF levels, the mRNA levels of nf-κbem1, r-cel and il-10 up-regulated significantly (P < 0.05). For fish fed BSF30 diet, the mRNA levels of myd88 and tlr22 were significantly higher than fish fed FM diet (P < 0.05). In conclusion, replacement fishmeal with BSF increased activity of digestive enzymes, but negatively affected growth performance and intestinal health of pearl gentian grouper.

Yeast culture improved the growth performance, liver function, intestinal barrier and microbiota of juvenile largemouth bass (Micropterus salmoides) fed high-starch diet.

The present study was conducted to investigate the effects of yeast culture on the growth, health and microflora of the juvenile largemouth bass fed high-starch diet. The experiment set three isonitrogenous and isolipidic diets, control (high-starch diet), HSY1 (high-starch diet with 1% yeast culture) and HSY3 (high-starch diet with 3% yeast culture). A feeding trial was conducted in largemouth bass juveniles for 8 weeks. The results indicated fish fed with 3% yeast culture not only could improve specific growth rate (SGR), but also significantly decreased hepatic lipid content, hepatic glycogen content, and hepatopancreas somatic index (HSI) compared with the control group (p<0.05). The total superoxide dismutase (T-SOD) and catalase (CAT) activities of HSY3 group significantly increased while malondialdehyde (MDA) content significantly reduced in liver compared with the control group (p<0.05). Meanwhile, the mRNA expression levels of hepatic Sod and Cat were up-regulated (p<0.05), and liver metabolism showed 111 metabolites were significantly changed in HSY3 group, liver lipid metabolism pathway remarkably changed. Besides, the intestinal anti-inflammatory cytokines were significantly up-regulated, and the pro-inflammatory cytokines were significantly down-regulated as the inclusion of yeast culture (p<0.05). Notably, HSY3 group diet up-regulated the expression of Zo-1, Claudin and Occludin in intestine compared with the other groups (p<0.05). Serum d-lactate (D-lac), diamine oxidase (DAO) and lipopolysaccharide (LPS) decreased significantly with the inclusion of yeast culture (p<0.05). Furthermore, the abundance of probiotics (such as Lactobacillus, Bacillus and Bifidobacterium) increased significantly, and the abundance of intestinal potential pathogenic bacteria (Plesiomonas) decreased in HSY3 group (p<0.05). The phenotypic analysis showed that gram-negative bacteria significantly decreased while gram-positive bacteria increased in HSY3 group (p<0.05). All in all, this study revealed that supplementation of 3% yeast culture can improve the growth performance and the health of juvenile largemouth bass, and has the potential to be used as an effective synbiotics for M. salmoides.

Immune responses and inorganic ion transport regulations of Epinephelus coioides in response to L321_RS13075 gene of Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida is a well-known pathogen of viscera granulomas disease in fish, which has led to severe economic losses. In our previous study, L321_RS13075 was predicted to be a key virulence gene of P. plecoglossicida during the host-pathogen interaction with Epinephelus coioides. To investigate the role of L321_RS13075 in the regulation of virulence in P. plecoglossicida, a L321_RS13075 knock-down strain was constructed. And a significant reduction in the ability of colonization, intracellular survival, motility, biofilm formation, and adhesion was detected in the L321_RS13075 knock-down strain. Compared with the wild-type strain, the silence of L321_RS13075 in P. plecoglossicida resulted in a significant change in the transcriptome of infected Epinephelus coioides (E. coioides). Results of COG and GO analysis on E. coioides showed that genes related to immune responses and inorganic ion transport were significantly affected by L321_RS13075 of P. plecoglossicida. Meanwhile, the interactions of the genes related to immune responses and inorganic ion transport were predicted, and the important hub genes were identified. Taken together, the results indicated that L321_RS13075 was a virulent gene of P. plecoglossicida, which significantly affected the immune responses and inorganic ion transport in E. coioides.

Effects of dietary dandelion extract on the growth performance, serum biochemical parameters, liver histology, and immune and apoptosis-related genes expression of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) at different feeding period.

The study explored on the effects of dietary 0.4% dandelion extract on the growth performance, serum biochemical parameters, liver histology and the expression levels of immune and apoptosis-related genes in the head kidney and spleen of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) at different feeding period. The results showed that the weight gain rate (WGR) of the hybrid grouper were significantly increased at the second and fourth weeks (P < 0.05), but there was no significant difference in WGR at the eighth week (P > 0.05). Compared with the control group, dietary dandelion extracts supplementation improve lipid metabolism, reduce lipid accumulation in liver and maintain normal liver histology at the second and fourth weeks. At the end of the second week, the relative expression levels of antioxidant related genes (MnSOD, GPX and GR) in the head kidney of hybrid grouper fed with dandelion extract increased significantly; at the end of week 4 and week 8, the relative expression levels of antioxidant related genes other than MnSOD did not change significantly. However, in the spleen of hybrid grouper, the expression of these antioxidant genes showed the opposite trend. At the end of the eighth week, dietary dandelion extract supplementation significantly increased the expression of inflammatory response related genes in head kidney of hybrid grouper, but showed the opposite trend in spleen. In conclusion, the short-term (2 or 4 weeks) application of 0.4% dandelion extract in feed had the effects of growth improvement, liver protection and immune stimulation on hybrid grouper due to its antioxidant and anti-inflammatory activities. The beneficial effect of dandelion extract on hybrid grouper was time-dependent, and its action time on different immune organs of hybrid grouper was not synchronous.

A study of the potential effect of yellow mealworm (Tenebrio molitor) substitution for fish meal on growth, immune and antioxidant capacity in juvenile largemouth bass (Micropterus salmoides).

This study aimed to evaluate the effects of partial replacement of fish meal (FM) with yellow mealworm (Tenebrio molitor, TM) on the growth performance, food utilization and intestinal immune response of juvenile largemouth bass (Micropterus salmoides). Seven diets containing increasing levels of TM (FM substitution) were designed (approximately 0% (0%), 4% (11.1%), 8.1% (22.2%), 12.2% (33.3%), 16.3% (44.4%), 20.4% (55.5%), and 24.5% (66.6%), designated TM0, TM11, TM22, TM33, TM44, TM55, and TM66, respectively). 420 fish were randomly selected and placed in 21 cages (1 m*1 m*1 m, 7 treatments for triplicate, 20 fish per cage). Fish (initial weight 6.25 ± 0.03 g) were fed seven isonitrogenous (47%) and isocaloric (19 MJ kg-1) diets to satiety twice daily for 8 weeks. Compared to the control group (TM0), TM11 showed no significant difference in the weight gain rate (WGR), specific growth rate (SGR) or feed conversion ratio (FCR), while all other TM inclusion groups presented different degrees of decline. There was no significant difference in the whole-body composition among all groups (P > 0.05). Plasma total protein (TP), triglyceride (TG) and albumin (ALB) contents were significantly decreased in TM55 and TM66 (P < 0.05). The highest plasma aspartate transaminase (AST) activity was observed in TM66 (P < 0.05). TM33, TM44 and TM55 showed the lowest activities of plasma alanine amiotransferase (ALT) and alkaline phosphatase (ALP) (P < 0.05). Moreover, increased mRNA levels of superoxide dismutase (SOD) and catalase (CAT) were measured in the TM11 to TM55 groups, while intestinal SOD activity peaked in TM11 (P < 0.05). With the exception of TM11, the other TM inclusion groups showed significant inhibition of the relative expression of RelA, C3 and TNF-α (P < 0.05). All experimental groups exhibited lower expression of IL-10 than TM0 (P < 0.05). The TM11 group showed significantly upregulated expression of IL-1ß and TGF-ß (P < 0.05). In addition, TLR2 expression was increased in TM11 and TM22 (P < 0.05). Considering enzyme activities and immune-related gene expression, TM supplementation levels should not exceed 4% (TM11).

Seawater activates l-amino acid oxidase from the serum of the red-spotted grouper Epinephelusakaara.

l-amino acid oxidases (LAOs) catalyze the oxidative deamination of l-amino acid and generate α-keto acid, ammonia, and hydrogen peroxide as byproducts. LAOs showed the variety of bioactivity by the resulting hydrogen peroxide. The serum of the red-spotted grouper Epinephelus akaara contains an LAO (Ea-LAO) with the potential to kill bacterial pathogens Aeromonas salmonicida and Vibrio anguillarum via hydrogen peroxide. However, it is unknown how the grouper tolerates the harmful effects of the serum Ea-LAO byproducts. In this study, we analyzed the kinetics of fish LAOs to understand how they escape the toxicity of byproducts. The LAO activity of grouper serum was suppressed in low-salt solutions such as NaCl, CaCl2, MgCl2, and diluted seawater. The activity was non-linearly increased and fitted to the four-parameter log-logistic model. The EC50 of the seawater was calculated to have a 0.72-fold concentration. This result suggested that the Ea-LAO could be activated by mixing with seawater. The results of circular dichroism spectroscopy showed that the α helix content was estimated to be 12.1% and 5.3% in a salt-free buffer (inactive condition) and the original concentration of seawater (active condition), respectively, indicating that the secondary structure of the Ea-LAO in the active condition was randomized. In addition, the Ea-LAO showed reversible LAO activity regulation according to the salt concentration in the environment. Taken together, this indicates that the Ea-LAO is normally on standby as an inactive form, and it could activate as a host-defense molecule to avoid pathogen invasion via a wound when mixed with seawater.

Defensing role of novel piscidins from largemouth bass (Micropterus salmoides) with evidence of bactericidal activities and inducible expressional delineation.

Micropterus salmoides is an economical important species of freshwater-cultured fish, the in-depth knowledge of its immune system is in urgent development to cope with serious infectious diseases. Piscidin is an important antimicrobial peptide (AMP) family existing in almost all teleosts. However, no piscidin has been reported in largemouth bass. In this study, three novel piscidins (MSPiscidin-1, -2, and -3) were firstly identified and characterized from the largemouth bass. The predicted mature peptides of MSPiscidin-1, -2, and -3 (consists of 24, 27, 25 amino acid residues, respectively) all adopted an amphipathic α-helical conformation representative of cationic AMPs that are important for membrane permeabilization and antibacterial activity. MSPiscidin-2 and -3 indeed displayed strong, broad-spectrum, and highly efficient antimicrobial activities in vitro against aquatic pathogens, but MSPiscidin-1 didn't show direct antimicrobial activity. MSPiscidin-2 and -3 killed bacteria mainly by inducing membrane permeabilization, in addition, they also can interact with bacterial genomic DNA, which might influence the DNA replication and transcription. Besides, MSPiscidin-2 and -3 could effectively inhibit the formation of the bacterial biofilm and eliminate the preformed biofilms. In vivo, MSPiscidin-1-3 genes showed an inducible expression pattern in the tested tissues upon Vibrio harveyi infection, which further indicated the key roles of piscidins in innate immunity in largemouth bass. Overall, this study will supplement the understanding of M. salmoides innate immune system and provide candidates for the design of novel peptide antibacterial agents used in aquaculture.

Pharmacological characterization and biological function of the interleukin-8 receptor, CXCR2, in largemouth bass (Micropterus salmoides).

Interleukin-8 (IL-8 or C-X-C motif chemokine ligand 8, CXCL8) is a cytokine secreted by numerous cell types and is best known for its functional roles in inflammatory response by binding to specific receptors (the interleukin-8 receptors, IL-8Rs). From the transcriptomic data of largemouth bass (Micropterus salmoides), we identified an IL-8R that is highly homologous to the functionally validated teleost IL-8Rs. The M. salmoides IL-8 receptor (MsCXCR2) was further compared with the C-X-C motif chemokine receptor 2 subfamily by phylogenetic analysis. Briefly, the full-length CDS sequence of MsCXCR2 was cloned into the pEGFP-N1 plasmid, and the membrane localization of fusion expressing MsCXCR2-EGFP was revealed in HEK293 cells. To determine the functional interaction between IL-8 and MsCXCR2, secretory expressed Larimichthys crocea IL-8 (LcIL-8) was used to stimulate MsCXCR2 expressing cells. MsCXCR2 was demonstrated to be activated by LcIL-8, leading to receptor internalization, which was further revealed by the detection of extracellular regulated protein kinase (ERK) phosphorylation. Quantitative real-time PCR was used to evaluate the expressional distribution and variation of MsCXCR2 in healthy and Nocardia seriolae infected fish. Based on our findings, MsCXCR2 was constitutively expressed in all examined tissues, despite at different levels. Furthermore, gene expression was found to be significantly upregulated in the liver and head kidney of diseased fish. Collectively, our findings reveal the molecular activity of MsCXCR2 and indicate the functional involvement of this IL-8R in the immune response induced by N. seriolae in M. salmoides.

Epinephelus coioides Hsp27 negatively regulates innate immune response and apoptosis induced by Singapore grouper iridovirus (SGIV) infection.

Heat shock proteins (Hsps) are important for maintaining protein homeostasis and cell survival. In this study, Hsp27 of Epinephelus coioides, an economically important marine fish in China and Southeast Asian countries, was characterized. E. coioides Hsp27 contains the consered ACD_HspB1_like domain and three p38 MAPK phosphorylation sites, located at Thr-13, Thr-60 and Ser-167. E. coioides Hsp27 was distributed in both the cytoplasm and nucleus, its mRNA was detected in all 14 tissues examined, and its expression was up-regulated after challenge with Singapore grouper iridovirus (SGIV), an important E. coioides pathogen. Over-expression of E. coioides Hsp27 significantly upregulated the expressions of the key SGIV genes (VP19, LITAF, MCP, and ICP18), downgraded the expressions of the E. coioides immune factors (IRF3, IRF7, ISG15, and TRAF6) and proinflammatory factors (TNF-α, IL-8), downgraded the activation of nuclear factor kappa-B (NF-κB) and activator protein-1 (AP-1), and substantially inhibited the cell apoptosis induced by SGIV infection. These data illustrated that E. coioides Hsp27 might be involved in SGIV infection by negatively regulating the innate immune response.