Specific biomarkers and neurons distribution of different brain regions in largemouth bass (Micropterus salmoides).

The brain regulates multiple physiological processes in fish. Despite this, knowledge about the basic structure and function of distinct brain regions in non-model fish species remains limited due to their diversity and the scarcity of common biomarkers. In the present study, four major brain parts, the telencephalon, diencephalon, mesencephalon and rhombencephalon, were isolated in largemouth bass, Micropterus salmoides. Within these parts, nine brain regions and 74 nuclei were further identified through morphological and cytoarchitectonic analysis. Transcriptome analysis revealed a total of 7153 region-highly expressed genes and 176 region-specifically expressed genes. Genes related to growth, reproduction, emotion, learning, and memory were significantly overexpressed in the olfactory bulb and telencephalon (OBT). Feeding and stress-related genes were in the hypothalamus (Hy). Visual system-related genes were predominantly enriched in the optic tectum (OT), while vision and hearing-related genes were widely expressed in the cerebellum (Ce) region. Sensory input and motor output-related genes were in the medulla oblongata (Mo). Osmoregulation, stress response, sleep/wake cycles, and reproduction-related genes were highly expressed in the remaining brain (RB). Three candidate marker genes were further identified for each brain regions, such as neuropeptide FF (npff) for OBT, pro-melanin-concentrating hormone (pmch) for Hy, vesicular inhibitory amino acid transporter (viaat) for OT, excitatory amino acid transporter 1 (eaat1) for Ce, peripherin (prph) for Mo, and isotocin neurophysin (itnp) for RB. Additionally, the distribution of seven neurotransmitter-type neurons and five types of non-neuronal cells across different brain regions were analyzed by examining the expression of their marker genes. Notably, marker genes for glutamatergic and GABAergic neurons showed the highest expression levels across all brain regions. Similarly, the marker gene for radial astrocytes exhibited high expression compared to other markers, while those for microglia were the least expressed. Overall, our results provide a comprehensive overview of the structural and functional characteristics of distinct brain regions in the largemouth bass, which offers a valuable resource for understanding the role of central nervous system in regulating physiological processes in teleost.

DC-SIGN of Largemouth Bass (Micropterus salmoides) Mediates Immune Functions against Aeromonas hydrophila through Collaboration with the TLR Signaling Pathway.

C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of Micropterus salmoides was identified. MsDC-SIGN is classified as a type II transmembrane protein. The extracellular segment of MsDC-SIGN possesses a coiled-coil region and a carbohydrate recognition domain (CRD). The key amino acid motifs of the extracellular CRD of MsDC-SIGN in Ca2+-binding site 2 were EPN (Glu-Pro-Asn) and WYD (Trp-Tyr-Asp). MsDC-SIGN-CRD can bind to four pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), glucan, peptidoglycan (PGN), and mannan. Moreover, it can also bind to Gram-positive, Gram-negative bacteria, and fungi. Its CRD can agglutinate microbes and displays D-mannose and D-galactose binding specificity. MsDC-SIGN was distributed in seven tissues of the largemouth bass, among which the highest expression was observed in the liver, followed by the spleen and intestine. Additionally, MsDC-SIGN was present on the membrane of M. salmoides leukocytes, thereby augmenting the phagocytic activity against bacteria. In a subsequent investigation, the expression patterns of the MsDC-SIGN gene and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) exhibited an up-regulated expression response to the stimulation of Aeromonas hydrophila. Furthermore, through RNA interference of MsDC-SIGN, the expression level of the DC-SIGN signaling pathway-related gene (RAF1) and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) was decreased. Therefore, MsDC-SIGN plays a pivotal role in the immune defense against A. hydrophila by modulating the TLR signaling pathway.

Fermented bile acids improved growth performance and intestinal health by altering metabolic profiles and intestinal microbiome in Micropterus salmoides.

A type of fermented bile acids (FBAs) has been produced through a biological method, and its effects on growth performance, metabolism, and intestinal microbiota in largemouth bass were investigated. The results demonstrated that incorporating 0.03 %-0.05 % FBAs diet could improve the final weight, weight gain and specific growth rate, and decrease the feed conversion ratio. Dietary FBAs did not significantly affect the levels of high-density lipoprotein, low-density lipoprotein, and triglycerides, but decreased the activities of α-amylase in most groups. Adding FBAs to the diet significantly increased the integrity of the microscopic structure of the intestine, thickened the muscular layer of the intestine, and notably enhanced its intestinal barrier function. The addition of FBAs to the diet increased the diversity of the gut microbiota in largemouth bass. At the phylum level, there was an increase in the abundance of Proteobacteria, Firmicutes, Tenericutes and Cyanobacteria and a significant decrease in Actinobacteria and Bacteroidetes. At the genus level, the relative abundance of beneficial bacteria Mycoplasma in the GN6 group and Coprococcus in the GN4 group significantly increased, while the pathogenic Enhydrobacter was inhibited. Meanwhile, the highest levels of AKP and ACP were observed in the groups treated with 0.03 % FBAs, while the highest levels of TNF-α and IL-10 were detected in the group treated with 0.04 % FBAs. Additionally, the highest levels of IL-1ß, IL-8T, GF-ß, IGF-1, and IFN-γ were noted in the group treated with 0.06 % FBAs. These results suggested that dietary FBAs improved growth performance and intestinal wall health by altering lipid metabolic profiles and intestinal microbiota in largemouth bass.

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.

M1-type polarized macrophage contributes to brain damage through CXCR3.2/CXCL11 pathways after RGNNV infection in grouper.

The vertebrate central nervous system (CNS) is the most complex system of the body. The CNS, especially the brain, is generally regarded as immune-privileged. However, the specialized immune strategies in the brain and how immune cells, specifically macrophages in the brain, respond to virus invasion remain poorly understood. Therefore, this study aimed to examine the potential immune response of macrophages in the brain of orange-spotted groupers (Epinephelus coioides) following red-spotted grouper nervous necrosis virus (RGNNV) infection. We observed that RGNNV induced macrophages to produce an inflammatory response in the brain of orange-spotted grouper, and the macrophages exhibited M1-type polarization after RGNNV infection. In addition, we found RGNNV-induced macrophage M1 polarization via the CXCR3.2- CXCL11 pathway. Furthermore, we observed that RGNNV triggered M1 polarization in macrophages, resulting in substantial proinflammatory cytokine production and subsequent damage to brain tissue. These findings reveal a unique mechanism for brain macrophage polarization, emphasizing their role in contributing to nervous tissue damage following viral infection in the CNS.

Immune Rejection Mediated by prf1 and gzmb Affects the Colonization of Fat Greenling (Hexagrammos otakii) Spermatogonia in Heterotransplantation.

Fish germ cell transplantation holds great potential for conserving endangered species, improving cultured fish breeds, and exploring reproductive techniques. However, low transplantation efficiency is a common issue in heterotransplantation. This study transplanted fat greenling (Hexagrammos otakii) spermatogonia into the testes of spotted sea bass (Lateolabrax maculatus) to investigate factors that might affect the colonization and fixation of heterologous transplanted germ cells. Results indicated that transplanted fat greenling spermatogonia cells were successfully detected in the early transplantation phase in spotted sea bass. Their numbers gradually decreased over time, and after 10 days post-transplantation, more than 90% of the transplanted cells underwent apoptosis. Transcriptome sequencing analysis of the testes of spotted sea bass and fat greenling spermatogonia on days 1 and 10 post-transplantation revealed that this apoptosis process involved many immune-related genes and their associated signaling pathways. Acute immune rejection marker genes prf1 and gzmb were detected in the spotted sea bass testes, while immune tolerance genes lck and zap-70 were expressed in the fat greenling spermatogonia. Additionally, differential expression of prf1 and gzmb genes was screened from spotted sea bass, with experimental evidence indicating that PRF1 and GZMB protein from spotted sea bass primarily induce apoptosis in transplanted fat greenling spermatogonia via the mitochondrial apoptosis pathway, at the protein level. This suggests that the difficulties in heterotransplantation are primarily related to acute immune rejection, with PRF1 and GZMB playing significant roles.

Phylogenomic Characterization of Ranavirus Isolated from Wild Smallmouth Bass (Micropterus dolomieu).

In September 2021, 14 smallmouth bass (SMB; Micropterus dolomieu) with skin lesions were collected from Green Bay waters of Lake Michigan and submitted for diagnostic evaluation. All the skin samples tested positive for largemouth bass virus (LMBV) by conventional PCR. The complete genome of the LMBV (99,328 bp) isolated from a homogenized skin sample was determined using an Illumina MiSeq sequencer. A maximum likelihood (ML) phylogenetic analysis based on the 21 core iridovirus genes supported the LMBV isolated from SMB (LMBV-WVL21117) as a member of the species Santee-Cooper ranavirus. Pairwise nucleotide comparison of the major capsid protein (MCP) gene showed that LMBV-WVL21117 is identical to other LMBV reported from the United States and nearly identical to doctor fish virus and guppy virus 6 (99.2%) from Southeast Asia, as well as LMBV isolates from China and Thailand (99.1%). In addition, ML phylogenetic analysis based on the MCP gene suggests three genotypes of LMBV separated by region: genotype one from the United States, genotype two from Southeast Asia, and genotype three from China and Thailand. Additional research is needed to understand the prevalence and genetic diversity of LMBV strains circulating in wild and managed fish populations from different regions.

Molecular characterization of vascular endothelial growth factor b from spotted sea bass (Lateolabrax maculatus) and its potential roles in decreasing lipid deposition.

Vascular endothelial growth factor B (VEGFB), a member of the VEGF family, exhibits limited angiogenic activity in mammals but plays an unexpected role in targeting lipids to peripheral tissues. However, its role in lipid metabolism in fish is unknown. In this study, the vegfb gene was cloned and characterized from spotted sea bass (Lateolabrax maculatus). It encodes 254 amino acids and possesses the typical characteristics of the Vegfb family, demonstrating high homology with those from other vertebrate species. The vegfb gene exhibits the highest expression levels in the liver, followed by the gills, intestine, and adipose tissues in spotted sea bass. In vivo, high-lipid diets decreased vegfb expression and increased lipid deposition in liver of fish. In vitro, palmitic acid + oleic acid treatment or vegfb knockdown significantly increased TG and TC contents, promoting lipid droplet deposition in hepatocytes. Vegfb overexpression has the opposite effects, inhibiting lipid deposition and downregulating fatty acid transport and adipogenesis genes. In contrast, the vegfb knockdown significantly upregulated the expression levels of c/ebpα, plin2, and dgat1 (P < 0.05). These results demonstrate that Vegfb may play an important role in reducing lipid deposition by regulating fatty acid transport and adipogenesis in the hepatocytes of spotted sea bass.

Fish ELOVL7a is involved in virus replication via lipid metabolic reprogramming.

The elongation of very long chain fatty acids (ELOVL) proteins are key rate-limiting enzymes that catalyze fatty acid synthesis to form long chain fatty acids. ELOVLs also play regulatory roles in the lipid metabolic reprogramming induced by mammalian viruses. However, little is known about the roles of fish ELOVLs during virus infection. Here, a homolog of ELOVL7 was cloned from Epinephelus coioides (EcELOVL7a), and its roles in red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infection were investigated. The transcription level of EcELOVL7a was significantly increased upon RGNNV and SGIV infection or other pathogen-associated molecular patterns stimulation in grouper spleen (GS) cells. Subcellular localization analysis showed that EcELOVL7a encoded an endoplasmic reticulum (ER) related protein. Overexpression of EcELOVL7a promoted the viral production and virus release during SGIV and RGNNV infection. Furthermore, the lipidome profiling showed that EcELOVL7a overexpression reprogrammed cellular lipid components in vitro, evidenced by the increase of glycerophospholipids, sphingolipids and glycerides components. In addition, VLCFAs including FFA (20:2), FFA (20:4), FFA (22:4), FFA (22:5) and FFA (24:0), were enriched in EcELOVL7a overexpressed cells. Consistently, EcELOVL7a overexpression upregulated the transcription level of the key lipid metabolic enzymes, including fatty acid synthase (FASN), phospholipase A 2α (PLA 2α), and cyclooxygenases -2 (COX-2), LPIN1, and diacylglycerol acyltransferase 1α (DGAT1α). Together, our results firstly provided the evidence that fish ELOVL7a played an essential role in SGIV and RGNNV replication by reprogramming lipid metabolism.

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.

A novel vaccination strategy against Vibrio harveyi infection in Asian seabass (Lates calcarifer) with the aid of oxygen nanobubbles and chitosan.

Immersion vaccination, albeit easier to administer than immunization by injection, sometimes has challenges with antigen uptake, resulting in sub-optimal protection. In this research, a new strategy to enhance antigen uptake of a heat-inactivated Vibrio harveyi vaccine in Asian seabass (Lates calcarifer) using oxygen nanobubble-enriched water (ONB) and positively charged chitosan (CS) was explored. Antigen uptake in fish gills was assessed, as was the antibody response and vaccine efficacy of four different combinations of vaccine with ONB and CS, and two control groups. Pre-mixing of ONB and CS before introducing the vaccine, referred to as (ONB + CS) + Vac, resulted in superior antigen uptake and anti-V. harveyi antibody (IgM) production in both serum and mucus compared to other formulas. The integration of an oral booster (4.22 × 108 CFU/g, at day 21-25) within a vaccine trial experiment set out to further evaluate how survival rates post exposure to V. harveyi might be improved. Antibody responses were measured over 42 days, and vaccine efficacy was assessed through an experimental challenge with V. harveyi. The expression of immune-related genes IL1ß, TNFα, CD4, CD8, IgT and antibody levels were assessed at 1, 3, and 7-day(s) post challenge (dpc). The results revealed that antibody levels in the group (ONB + CS) + Vac were consistently higher than the other groups post immersion immunization and oral booster, along with elevated expression of immune-related genes after challenge with V. harveyi. Ultimately, this group demonstrated a significantly higher relative percent survival (RPS) of 63 % ± 10.5 %, showcasing the potential of the ONB-CS-Vac complex as a promising immersion vaccination strategy for enhancing antigen uptake, stimulating immunological responses, and improving survival of Asian seabass against vibriosis.

Molecular characterization of Lernathropus kroyeri from intensive aquaculture and pathophysiology of infested sea bass.

The copepod Lernathropus kroyeri constitutes one of the major parasites for the Mediterranean aquaculture, infesting the sea bass Dicentrarchus labrax causing thus disruptions of growth performance and occasionally mortalities. Despite the large spread and the high frequency of this parasite in mariculture farms of Eastern Mediterranean, L. kroyeri genetic profile from aquaculture as well as the pathophysiological response of D. labrax have not been studied so far. Keeping this in mind, in the present study we investigated the L. kroyeri infestation on D. labrax from two farms in Greece, examining both healthy and heavy parasitized individuals. Assays included histopathology, phylogenetic reconstruction of the parasite and physiological response of the fish by the means of antioxidant, inflammatory metabolic and stress related gene expression analysis at both mRNA and protein levels. Genetic analysis indicated that L. kroyeri composes a monophyletic group, highly phylogenetically distant from other congeneric groups. Heavy infested D. labrax witnessed a significantly increased immune response that further led to oxidative stress and metabolic alterations. Overall, our results demonstrate the, seasonally independent, high infestation of this parasitic copepods, which continue to affect Mediterranean intensive aquaculture systems.

Effects of Long-Term Cryopreservation on the Transcriptomes of Giant Grouper Sperm.

The giant grouper fish (Epinephelus lanceolatus), one of the largest and rarest groupers, is a fast-growing economic fish. Grouper sperm is often used for cross-breeding with other fish and therefore sperm cryopreservation is important. However, freezing damage cannot be avoided. Herein, we performed a transcriptome analysis to compare fresh and frozen sperm of the giant grouper with frozen storage times of 0, 23, 49, and 61 months. In total, 1911 differentially expressed genes (DEGs), including 91 in El-0-vs-El-23 (40 upregulated and 51 downregulated), 251 in El-0-vs-El-49 (152 upregulated and 69 downregulated), and 1569 in El-0-vs-El-61 (984 upregulated and 585 downregulated), were obtained in the giant grouper sperm. DEGs were significantly increased at 61 months of cryopreservation (p < 0.05). GO and KEGG enrichment analyses of the DEGs revealed significant enrichment in the pilus assembly, metabolic process, MAPK signaling pathway, apoptosis, and P53 signaling pathway. Time-series expression profiling of the DEGs showed that consistently upregulated modules were also significantly enriched in signaling pathways associated with apoptosis. Four genes, scarb1, odf3, exoc8, and atp5f1d, were associated with mitochondria and flagella in a weighted correlation network analysis. These genes may play an important role in the response to sperm freezing. The experimental results show that long-term cryopreservation results in freezing damage to the giant grouper sperm. This study provides rich data for studies of the mechanism underlying frozen fish sperm damage as well as a technical reference and evaluation index for the long-term cryopreservation of fish sperm.

Complete genome sequence and genome-wide transposon mutagenesis enable the determination of genes required for sodium hypochlorite tolerance and drug resistance in pathogen Aeromonas veronii GD2019.

Aeromonas veronii, a significant pathogen in aquatic environments, poses a substantial threat to both human and animal health, particularly in aquaculture. In this study, we isolated A. veronii strain GD2019 from diseased largemouth bass (Micropterus salmoides) during a severe outbreak of aeromonad septicemia in Guangdong Province, China. The complete genome sequence of A. veronii GD2019 revealed that GD2019 contains a single chromosome of 4703,168 bp with an average G+C content of 58.3%. Phylogenetic analyses indicated that GD2019 forms a separate sub-branch in A. veronii and comparative genomic analyses identified the existence of an intact Type III secretion system. Moreover, to investigate the genes that are required for the conditional fitness of A. veronii under various stresses, a high-density transposon insertion library in GD2019 was generated by a Tn5-based transposon and covers 6311 genomic loci including 4155 genes and 2156 intergenic regions. Leveraging this library, 630 genes were classified as essential genes for growth in rich-nutrient LB medium. Furthermore, the genes GE001863/NtrC and GE002550 were found to confer tolerance to sodium hypochlorite in A. veronii. GE002562 and GE002614 were associated with the resistance to carbenicillin. Collectively, our results provide abundant genetic information on A. veronii, shedding light on the pathogenetic mechanisms of Aeromonas.

Estradiol-17ß and bisphenol A affect growth and mineralization in early life stages of seabass.

Natural and synthetic estrogens are contaminants present in aquatic ecosystems. They can have significant consequences on the estrogen-sensitive functions of organisms, including skeletal development and growth of vertebrate larvae. Synthetic polyphenols represent a group of environmental xenoestrogens capable of binding the receptors for the natural hormone estradiol-17ß (E2). To better understand how (xeno-)estrogens can affect the skeleton in fish species with high ecological and commercial interest, 16 days post-hatch larvae of the seabass were experimentally exposed for 7 days to E2 and Bisphenol A (BPA), both used at the regulatory concentration of surface water quality (E2: 0.4 ng.L-1, BPA: 1.6 µg.L-1) or at a concentration 100 times higher. Skeletal mineralization levels were evaluated using Alizarin red staining, and expression of several genes playing key roles in growth, skeletogenesis and estrogen signaling pathways was assessed by qPCR. Our results show that E2 exerts an overall negative effect on skeletal mineralization at the environmental concentration of 0.4 ng.L-1, correlated with an increase in the expression of genes associated only with osteoblast bone cells. Both BPA exposures inhibited mineralization with less severe effects and modified bone homeostasis by regulating the expression of gene encoding osteoblasts and osteoclasts markers. Our results demonstrate that environmental E2 exposure inhibits larval growth and has an additional inhibitory effect on skeleton mineralization while both BPA exposures have marginal inhibitory effect on skeletal mineralization. All exposures have significant effects on transcriptional levels of genes involved in the skeletal development of seabass larvae.

Response signatures of intestinal microbiota and gene transcription of the pearl gentian grouper to Vibrio harveyi infection.

Vibrio harveyi causes high mortality and severely limits grouper culture. The gut microbiota is an important biological barrier against pathogen invasion. In this study, we investigated dynamic changes in the intestinal microbial community, gene transcription and immune responses signatures of pearl gentian grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀) at 0, 3 and 7 days (referred to as d0, d3 and d7 groups, respectively) after infection with V. harveyi. The results demonstrated that the d7 treatment reduced the gut microbial diversity and increased the proportion of Proteobacteria and Cyanobacteria. Notably, several putative pathogenic genera (Sphingomonas and Bacteroides) proliferated, while putative probiotic genera (Rhodococcus and Lactobacillus) reduced, and these changes in intestinal bacteria might be correlated to the alterations of host immune-related molecules. The d3 and d7 treatments also altered the histomorphology and gene transcription profiles mainly associated with immune function in intestine, such as 'MAPK signaling pathway', 'Apoptosis' and 'Toll-like receptor (TLR) signaling pathway'. Furthermore, d3 group induced a homeostatic dysregulation of the antioxidant system, cytokines and TLR signaling, with a tendency to gradually return to a normal state in d7 group, along with the apoptosis process. The pathogenic infection suppressed the expression of JNK pathway and enhanced the ERK pathway. In conclusion, the dysbiosis of the intestinal bacterial communities caused by the immune changes that occurred during V. harveyi infection disrupted the intestine health in the pearl gentian grouper. These results provided a comprehensive understandings of the immune defense mechanisms in fish and valuable references to develop disease control strategies in grouper aquaculture.

Occurrence of microplastics and metals in European seabass produced in different aquaculture systems: Implications for human exposure, risk, and food safety.

Microplastics (MPs) are emerging contaminants of increasing concern as they may cause adverse effects and carry other contaminants, which may potentially compromise human health. Despite occurring in aquatic ecosystems worldwide, the knowledge about MP presence in different aquaculture systems and their potential impact on seafood products is still limited. This study aimed to determine the levels of MPs in water, feed, and European seabass (Dicentrarchus labrax) from three relevant aquaculture systems and estimate human exposure to MPs and metals through seabass consumption. The recirculating aquaculture system (RAS) had the highest MP occurrence in water and feed. MP levels in seabass followed the aquaculture system's levels in water and feed, with RAS-farmed fish presenting the highest MP load, both in the fish gastrointestinal tract (GIT) and muscle, followed by pond-, and cage-farmed fish. MPs' characteristics across aquaculture systems and fish samples remained consistent, with the predominant recovered particles falling within the MP size range. The particles were visually characterized and chemically identified by micro-Fourier Transform Infrared Spectroscopy (µFTIR). Most of these particles were fibres composed of man-made cellulose and PET. MP levels in GIT were significantly higher than in muscle for pond- and RAS-farmed fish, MPs' bioconcentration factors >1 indicated bioconcentration in farmed seabass. Metal concentrations in fish muscle were below permissible limits, posing low intake risks for consumers according to the available health-based guidance values and estimated dietary scenarios.

A nanobody-mediated drug system against largemouth bass virus delivered by bacterial nanocellulose in Micropterus salmoides.

Diseases caused by pathogens severely hampered the development of aquaculture, especially largemouth bass virus (LMBV) has caused massive mortality and severe economic losses to the culture of largemouth bass (Micropterus salmoides). Considering the environmental hazards and human health, effective and environmentally friendly therapy strategy against LMBV is of vital importance and in pressing need. In the present study, a novel nanobody (NbE4) specific for LMBV was selected from a phage display nanobody library. Immunofluorescence and indirect ELISA showed that NbE4 could recognize LMBV virions and had strong binding capacity, but RT-qPCR evidenced that NBE4 did not render the virus uninfectious. Besides, antiviral drug ribavirin was used to construct a targeted drug system delivered by bacterial nanocellulose (BNC). RT-qPCR revealed that NbE4 could significantly enhance the antiviral activity of ribavirin in vitro and in vivo. The targeted drug delivery system (BNC-Ribavirin-NbE4, BRN) reduced the inflammatory response caused by LMBV infection and improved survival rate (BRN-L, 33.3 %; BRN-M, 46.7 %; BRN-H, 56.7 %)compared with control group (13.3 %), ribavirin group (RBV, 26.7 %) and BNC-ribavirin (BNC-R, 40.0 %), respectively. This research provided an effective antiviral strategy that improved the drug therapeutic effect and thus reduced the dosage.

Study of the antivirus function mediated by STING in Micropterus salmoides.

Stimulator of interferon genes (STING) has been demonstrated as a critical mediator in the innate immune response to cytosolic DNA and RNA derived from different pathogens. While the role of Micropterus salmoides STING (MsSTING) in largemouth bass virus is still unknown. In this study, RT-qPCR assay and Western-blot assay showed that the expression levels of MsSTING and its downstream genes were up-regulated after LMBV infection. Pull down experiment proved that a small peptide called Fusion peptide (FP) that previously reported to target to marine and human STING as a selective inhibitor also interacted with MsSTING in vitro. Comparing with the RNA-seq of Largemouth bass infected with LMBV singly, 326 genes were significantly up-regulated and 379 genes were significantly down-regulated in the FP plus LMBV group in which Largemouth bass was treatment with FP before LMBV-challenged. KEGG analysis indicated that the differentially expressed genes (DEGs) were mainly related to signaling transduction, infectious disease viral, immune system and endocrine system. Besides, the survival rate of LMBV-infected largemouth bass was highly decreased following FP treatment. Taken together, our study showed that MsSTING played an important role in immune response against LMBV infection.

Identification and functional analysis of perforin 1 from largemouth bass (Micropterus salmoides).

In this study, we present the first cloning and identification of perforin (MsPRF1) in largemouth bass (Micropterus salmoides). The full-length cDNA of MsPRF1 spans 1572 base pairs, encoding a 58.88 kDa protein consisting of 523 amino acids. Notably, the protein contains MACPF and C2 structural domains. To evaluate the expression levels of MsPRF1 in various healthy largemouth bass tissues, real-time quantitative PCR was employed, revealing the highest expression in the liver and gut. After the largemouth bass were infected by Nocardia seriolae, the mRNA levels of MsPRF1 generally increased within 48 h. Remarkably, the recombinant protein MsPRF1 exhibits inhibitory effects against both Gram-negative and Gram-positive bacteria. Additionally, the largemouth bass showed a higher survival rate in the N. seriolae challenge following the intraperitoneal injection of rMsPRF1, with observed reductions in the tissue bacterial loads. Moreover, rMsPRF1 demonstrated a significant impact on the phagocytic and bactericidal activities of largemouth bass MO/MΦ cells, concurrently upregulating the expression of pro-inflammatory factors. These results demonstrate that MsPRF1 has a potential role in the immune response of largemouth bass against N. seriolae infection.