A rapid increase in tropical species of grouper (Perciformes: Serranidae) in the temperate waters, the Goto Islands, Japan.

Global warming has resulted in rapid poleward shifts in the geographical distributions of many tropical fish species. This study conducted daily market surveys from 2008 to 2013 to investigate catch trends of seven commercially important grouper species in the temperate Goto Islands, Japan. Our results revealed that the catch numbers of tropical grouper species increased rapidly by an average of 5.9-fold (12.3-fold at maximum) within six years, whereas the temperate and subtropical species did not exhibit substantial changes. Based on the findings of several previous studies, the rapid increase in the number of tropical groupers in temperate waters was most likely caused by the successful settlement of larvae transported from tropical waters. Large-scale ocean currents may facilitate larval transport from tropical waters because the Goto Islands face the Tsushima Warm Current, which branches from the Kuroshio Current. Meanwhile, the transition processes of size distribution in tropical groupers suggest a possible hypothesis that adults migrating from tropical waters first settle in temperate waters and then enhance their populations by reproduction. Further studies are required to determine how tropical grouper species settle and how their populations increase in temperate waters.

Lactobacillus casei displaying MCP2α and FlaC delivered by PLA microspheres effectively enhances the immune protection of largemouth bass (Micropterus salmoides) against LMBV infection.

Largemouth bass ranavirus (LMBV) seriously affects the development of largemouth bass (Micropterus salmoides) industry and causes huge economic losses. Oral vaccine can be a promising method for viral disease precaution. In this study, MCP2α was identified as a valuable epitope region superior to MCP and MCP2 of LMBV by neutralizing antibody experiments. Then, recombinant Lactobacillus casei expressing the fusion protein MCP2αC (MCP2α as antigen, C represents flagellin C from Aeromonas hydrophila as adjuvant) on surface was constructed and verified. Further, PLA microsphere vaccine loading recombinant MCP2αC L. casei was prepared. The PLA microspheres vaccine were observed by scanning electron microscopy and showed a smooth, regular spherical surface with a particle size distribution between 100 and 200 µm. Furthermore, we evaluated the tolerance of PLA-MCP2αC vaccine in simulated gastric fluid and simulated intestinal fluid, and the results showed that PLA-MCP2αC can effectively resist the gastrointestinal environment. Moreover, the protective effect of PLA-MCP2αC against LMBV was evaluated after oral immunization and LMBV challenge. The results showed that PLA-MCP2αC effectively up-regulated the activity of serum biochemical enzymes (T-SOD, T-AOC, LZM, complement C3) and induced the mRNA expression of representative immune genes (IL-1ß, TNF-α, IFN-γ, MHC-IIα, Mx, IgM) in spleen and head kidney tissues. The survival rate of largemouth bass vaccinated with PLA-MCP2αC increased from 24 % to 68 %. Meanwhile, PLA-MCP2αC inhibited the LMBV burden in spleen, head kidney and liver tissues and attenuated tissue damage in spleen. These results suggested that PLA-MCP2αC can be used as a candidate oral vaccine against LMBV infection in aquaculture.

17α-Ethinylestradiol exposure disrupts anxiety-like behaviours but not social preference in sea bass larvae (Dicentrarchus labrax).

Endocrine-disrupting chemicals (EDCs) are widespread pollutants known to interfere with hormonal pathways and to disrupt behaviours. Standardised behavioural procedures have been developed in common fish model species to assess the impact of various pollutants on behaviours such as locomotor activity and anxiety-like as well as social behaviours. These procedures need now to be adapted to improve our knowledge on the behavioural effects of EDCs on less studied marine species. In this context, the European sea bass (Dicentrarchus labrax) is emerging as a valuable species representative of the European marine environment. Here, we designed and validated a two-step procedure allowing to sequentially assess anxiety-like behaviours (novel tank test) and social preference (visual social preference test) in sea bass. Thereafter, using this procedure, we evaluated whether social behavioural disruption occurs in 2-month-old larvae after an 8-day exposure to a xenoestrogen, the 17α-ethinylestradiol (EE2 at 0.5 and 50 nM). Our results confirmed previous studies showing that exposure to 50 nM of EE2 induces a significant increase in anxiety-like behaviours in sea bass larvae. On the contrary, social preference seemed unaffected whatever the EE2 concentration, suggesting that social behaviour has more complex mechanical regulations than anxiety.

DNA Methylation and Subgenome Dominance Reveal the Role of Lipid Metabolism in Jinhu Grouper Heterosis.

Heterosis of growth traits in economic fish has benefited the production of aquaculture for many years, yet its genetic and molecular basis has remained obscure. Nowadays, a new germplasm of hybrid Jinhu grouper (Epinephelus fuscoguttatus ♀ × E. tukula ♂), abbreviated as EFT, exhibiting paternal-biased growth heterosis, has provided an excellent model for investigating the potential regulatory mechanisms of heterosis. We integrated transcriptome and methylome to unravel the changes of gene expression, epigenetic modification, and subgenome dominance in EFT compared with maternal E. fuscoguttatus. Integration analyses showed that the heterotic hybrids showed lower genomic DNA methylation levels than the purebred parent, and the up-regulated genes were mostly DNA hypomethylation. Furthermore, allele-specific expression (ASE) detected paternal subgenome dominance-regulated paternal-biased heterosis, and paternal bias differentially expressed genes (DEGs) were wholly up-regulated in the muscle. Multi-omics results highlighted the role of lipid metabolism, particularly "Fatty acid synthesis", "EPA biosynthesis", and "Signaling lipids", in EFT heterosis formation. Coherently, our studies have proved that the eicosapentaenoic acid (EPA) of EFT was greater than that of maternal E. fuscoguttatus (8.46% vs. 7.46%). Finally, we constructed a potential regulatory network for control of the heterosis formation in EFT. Among them, fasn, pparg, dgat1, igf1, pomca, fgf8a, and fgfr4 were identified as key genes. Our results provide new and valuable clues for understanding paternal-biased growth heterosis in EFT, taking a significant step towards the molecular basis of heterosis.

The Fate of a Polygenic Phenotype Within the Genomic Landscapes of Introgression in the European Seabass Hybrid Zone.

Unraveling the evolutionary mechanisms and consequences of hybridization is a major concern in biology. Many studies have documented the interplay between recombination and selection in modulating the genomic landscape of introgression, but few have considered how associations with phenotype may affect this landscape. Here, we use the European seabass (Dicentrarchus labrax), a key species in marine aquaculture that undergoes natural hybridization, to determine how selection on phenotype modulates the introgression landscape between Atlantic and Mediterranean lineages. We use a high-density single nucleotide polymorphism array to assess individual local ancestry along the genome and improve the mapping of muscle fat content, a polygenic trait that is divergent between lineages. Taking into account variation in recombination rates, we reveal a purging of Atlantic ancestry in the admixed Mediterranean populations. While Atlantic individuals had higher muscle fat content, we observed that genomic regions associated with this trait in Mediterranean populations displayed reduced introgression of Atlantic ancestry. These results emphasize how selection against maladapted alleles shapes the genomic landscape of introgression.

Microalgae and phytase dietary supplementation improved growth and gut microbiota in juvenile European seabass (Dicentrarchus labrax).

Fishmeal and fish oil have been the main sources of protein and fatty acid for aquaculture fish. However, their increasing price and low sustainability have led the aquafeed industry to seek sustainable alternative feedstuffs to meet the nutritional requirements of fish and improve their health and performance. Plant proteins have been successfully used to replace fishery derivatives in aquafeeds, but the presence of anti-nutritional substances is a potential drawback of this approach. Thus, it has been reported that phytate breakdown can be caused by feed supplementation with exogenous phytase. The inclusion of microalgae has been proposed to improve gut functionality in fish fed diets with a high vegetable protein content. The aim of this study was to evaluate the effect on the growth and gut microbiota of European seabass (Dicentrarchus labrax) juveniles of a diet containing a blend of microalgae (Arthrospira platensis and Nannochloropsis gaditana) and different concentrations of phytase. An 83-day feeding trial was conducted, comprising four experimental diets with 2.5% microalgae and 500, 1,000, 2,000, or 10,000 phytase units (FTU)/kg feed and a microalgae- and phytase-free control diet. At the end of the trial, a significantly increased body weight was observed in fish fed the diet with the highest phytase concentration (10,000 FTU/kg) versus controls, although the gut bacterial composition did not differ from controls in alpha or beta diversity with either majority (Weighted UniFrac) or minority bacterial strains (Unweighted UniFrac). In comparison to the control group, the groups fed diets with 1,000 or 2,000 FTU/kg diets had a lower alpha diversity (Shannon's diversity index), while those fed diets with 500 FTU/kg or 1,000 FTU/kg showed distinct clusters in beta diversity (involving minority ASVs). According to these findings, the diet containing the 2.5% microalgae blend with 10,000 FTU/kg may be useful to increase the aquafeed quality and sustain the growth performance of juvenile European seabass.

A naturaly attenuated largemouth bass ranavirus strain provided protection for Micropterus salmoides by immersion immunization.

Largemouth bass ranavirus (LMBV) causes disease outbreaks and high mortality at all stages of largemouth bass farming. Therefore, live vaccine development is critical for largemouth bass prevention against LMBV by immersion immunization. Herein, an attenuated LMBV strain with good immunogenicity, designated as LMBV-2007136, was screened from the natural LMBV strains bank through challenge assay and immersion immunization experiment. After determing the safe concentration range of LMBV-2007136, the minimum immunizing dose of immersion immunization was verified. When largemouth bass were vaccinated by immersion at the lowest concentration of 102.0 TCID50/mL, all of fish were survival post virulent LMBV challenge, and the relative percent survival (RPS) was 100 %. And the immune gene expression levels of IL-10, IL-12, IFN-γ, and IgM in the spleen and kidney post-vaccination were significantly up-regulated compared to the control group, but TNF-α expression showed no significant changes. The safety and efficacy of LMBV-2007136 at passages P8, P13, and P18 were futher assessed, and no death of largemouth bass was observed within 21 days post-immunization and RPS of three vaccination groups was 100 %, suggesting that the safety and efficacy of the attenuated strain at different passages was stable. Furthermore, in the virulence reversion test, the attenuated strain was propagated through 5 times in largemouth bass by intraperitoneal injection and no abnormality and mortality were observed, further proving the attenuated vaccine candidate LMBV-2007136 was safe. These results proved that LMBV-2007136 could be a promising candidate for a live vaccine to protect largemouth bass from LMBV disease.

The toxic effect of lead exposure on the physiological homeostasis of grouper: Insight from gut-liver axis.

The heavy metal lead (Pb) pollution in marine environment has been widely concerned. The liver and intestine are important for the health of fish. In this study, the grouper were exposed to 1 µg/L Pb for 14 days, and the physiological homeostasis changes were explored via gut-liver axis. The results showed that Pb stress caused liver morphological changes, oxidative stress, and the accumulation and peroxidation of the lipids. The liver metabolism were disturbed, especially amino acid metabolism and the synthesis and degradation of ketone bodies. Pb stress also caused intestinal mucosal ablation, tight junction dysfunction and inflammatory response. Additionally, intestinal microbial diversity was decreased, and the community composition was altered especially several bacteria genera (Ruminococcus UCG-005, Ruminococcus UCG-014, Oscillibacter, and Streptococcus) were significantly correlated with the physiological indexes and metabolites of the liver. These results reveal that Pb stress negatively affect the physiological homeostasis of the grouper via gut-liver axis.

Genome-Wide Association Study of Resistance to Largemouth Bass Ranavirus (LMBV) in Micropterus salmoides.

The disease caused by Largemouth bass ranavirus (LMBV) is one of the most severe viral diseases in largemouth bass (Micropterus salmoides). It is crucial to evaluate the genetic resistance of largemouth bass to LMBV and develop markers for disease-resistance breeding. In this study, 100 individuals (45 resistant and 55 susceptible) were sequenced and evaluated for resistance to LMBV and a total of 2,579,770 variant sites (SNPs-single-nucleotide polymorphisms (SNPs) and insertions-deletions (InDels)) were identified. A total of 2348 SNPs-InDels and 1018 putative candidate genes associated with LMBV resistance were identified by genome-wide association analyses (GWAS). Furthermore, GO and KEGG analyses revealed that the 10 candidate genes (MHC II, p38 MAPK, AMPK, SGK1, FOXO3, FOXO6, S1PR1, IL7R, RBL2, and GADD45) were related to intestinal immune network for IgA production pathway and FoxO signaling pathway. The acquisition of candidate genes related to resistance will help to explore the molecular mechanism of resistance to LMBV in largemouth bass. The potential polymorphic markers identified in this study are important molecular markers for disease resistance breeding in largemouth bass.

Temporal analysis of water chemistry and smallmouth bass (Micropterus dolomieu) health at two sites with divergent land use in the Susquehanna River watershed, Pennsylvania, USA.

Monitoring wild fish health and exposure effects in impacted rivers and streams with differing land use has become a valuable research tool. Smallmouth bass (Micropterus dolomieu) are a sensitive, indicator species that exhibit signs of immunosuppression and endocrine disruption in response to water quality changes and contaminant exposure. To determine the impact of agriculture and development on smallmouth bass health, two sites (a developed/agriculture site and a forested site) in the Susquehanna River watershed, Pennsylvania were selected where bass and water chemistry were sampled from 2015 to 2019. Smallmouth bass were sampled for histopathology to assess parasite and macrophage aggregate density in the liver and spleen, condition factor (Ktl), hepatic gene transcript abundance, hepatosomatic index (HSI), and a health assessment index (HAI). Land use at the developed/agriculture site included greater pesticide application rates and phytoestrogen crop cover and more detections and higher concentrations of pesticides, wastewater-associated contaminants, hormones, phytoestrogens, and mycotoxins than at the forested site. Additionally, at the developed/agriculture site, indicators of molecular changes, including oxidative stress, immune/inflammation, and lipid metabolism-related hepatic gene transcripts, were associated with more contaminants and land use variables. At both sites, there were multiple associations of contaminants with liver and/or spleen macrophage aggregate density, indicating that changes at the molecular level seemed to be a better indicator of exposures unique to each site. The findings illustrate the importance of timing for land management practices, the complex mixtures aquatic animals are exposed to, and the temporal changes in contaminant concentration. Agricultural practices that affect hepatic gene transcripts associated with immune function and disease resistance were demonstrated which could negatively affect smallmouth bass populations.

Investigating dynamics, etiology, pathology, and therapeutic interventions of Caligus clemensi and Vibrio alginolyticus co-infection in farmed marine fish.

This study investigated a disease outbreak characterized by caligid copepod infestations and subsequent secondary bacterial infections in European seabass (Dicentrarchus labrax) and flathead grey mullet (Mugil cephalus) cultivated at a private facility in the Deeba Triangle region of Egypt. Moribund fish displayed brown spots on the skin, tongue, and gills, along with lethargy and excess mucus. The fish suffered severe infections, exhibiting external hemorrhages, ulcers, and ascites. The fish had pale, enlarged livers with hemorrhaging. Comprehensive parasitological, bacteriological, molecular, immunity and histopathological analyses were conducted to identify the etiological agents and pathological changes. Caligid copepod infestation was observed in wet mounts from the buccal and branchial cavities of all examined fish, and the caligids were identified as Caligus clemensi through COI gene sequencing and phylogenetic analysis. Vibrio alginolyticus was confirmed as a secondary bacterial infection through biochemical tests, recA gene sequencing, and phylogenetic analyses. Antibiotic susceptibility testing revealed resistance to ß-lactams, aminoglycosides, and trimethoprim-sulfamethoxazole in V. alginolyticus isolates. Upregulation of the inflammatory marker IL-1ß in gill and skin tissues indicated a robust cell-mediated immune response against the pathogens. Histopathological examination revealed severe tissue damage, hyperplasia, hemorrhage, and congestion in the gills, along with hepatocellular degeneration and steatosis in the liver, providing initial insights into this outbreak. A comprehensive therapeutic regimen was implemented, comprising prolonged hydrogen peroxide immersion baths, followed by the application of the nature-identical plant-based compound Lice-less and probiotic Sanolife Pro-W supplementation. This integrated approach effectively eliminated C. clemensi infestations, controlled secondary bacterial infections, and restored fish health, reducing morbidity and mortality rates to minimal levels.

Molecular characterization of the SP3a gene, a negative regulator of viral infection in the orange-spotted grouper, Epinephelus coioides.

SP3 (specificity protein 3) is a transcription factor characterized by three conserved Cys2His2 zinc finger motifs that exert a transregulatory effect by binding to GC boxes, either upregulating or downregulating multiple genes or by co-regulating gene expression in coordination with other proteins. SP3 potentially regulates a series of processes, such as the cell cycle, growth, metabolic pathways, and apoptosis, and plays an important role in antiviral effect. The function of sp3 in fish is poorly understood. In this study, the Sp3a open reading frame was cloned from the orange-spotted grouper, Epinephelus coioides. The full-length open reading frame of Sp3a was 2034 bp, encoding 677 amino acids, with a predicted molecular weight of 72.34 kDa and an isoelectric point of 5.05. Phylogenetically, Sp3a in Epinephelus coioides was the most closely related to Sp3a in the Malabar grouper, Epinephelus malabaricus. RT-qPCR revealed ubiquitous expression of Sp3a in all examined grouper tissues, with no significant differences in expression levels among tissues. A eukaryotic expression vector, pEGFP-Sp3a, was constructed and transfected into grouper spleen (GS) cells. Subcellular localization of Sp3a was observed using an inverted fluorescence microscope. When Spa3 was overexpressed in GS cells, the expression of orange-spotted grouper nerve necrosis virus (RGNNV) genes (CP and RdRp) decreased significantly, indicating that Sp3a significantly inhibited RGNNV replication. siRNA inhibition of Sp3a accelerated the intracellular replication of RGNNV, implying the antiviral effect of Sp3a. Conclusively, our findings contribute to further research on the antiviral capabilities of Sp3a in grouper and other fish. Therefore, our research has potential implications on the development of the aquaculture industry.

Development and characterization of primary cell culture from the spinal cord of Asian seabass, Lates calcarifer.

Asian seabass, Lates calcarifer, is one of the most important fish species in aquaculture. An attempt was made to develop a primary cell culture from the spinal cord of Lates calcarifer by the enzymatic and mechanical dissociation method. The primary cell culture was sub-cultured for 20 times in Leibovitz's L-15 medium with 20% fetal bovine serum (FBS) and 0.5 nM of human neurotrophin-3 at 28°C. The primary cell culture was cryopreserved at different passage levels and recovery of cells after long-term storage was estimated about 75-85%. The authenticity of origin of primary cell culture from L. calcarifer was confirmed by polymerase chain reaction assay using species-specific mitochondrial 12S rRNA primer. The primary cell culture was designated as seabass spinal cord cells (SBSC). The cells morphologically resembled the neurons due to their neural-like prolongations and star-like structure. Immunophenotypic analysis of the SBSC revealed that they are of neuronal origin. The SBSC were found to be highly susceptible to striped jack nervous necrosis virus (SJNNV) and infection in the cells was confirmed by RT-PCR. In conclusion, this is the first innovative euryhaline fish neuronal primary cell culture of L. calcarifer now available for neurophysiological and neurotoxicological studies.

Molecular characterization and gene expression of pattern recognition receptors in brown-marbled grouper (Epinephelus fuscoguttatus) fingerlings responding to vibriosis infection.

The pathogen recognition system involves receptors and genes that play a crucial role in activating innate immune response in brown-marbled grouper (Epinephelus fuscoguttatus) as a control agent against various infections including vibriosis. Here, we report the molecular cloning of partial open reading frames, sequences characterization, and expression profiles of Pattern Recognition Receptors (PRRs) in brown-marbled grouper. The PRRs, namely pglyrp5, tlr5, ctlD, and ctlE in brown-marbled grouper, possess conserved domains and showed shared evolutionary relationships with other fishes, humans, mammals, birds, reptilians, amphibians, and insects. In infection experiments, up to 50% mortality was found in brown-marbled grouper fingerlings infected with Vibrio alginolyticus compared to 27% mortality infected Vibrio parahaemolyticus and 100% survival of control groups. It is also demonstrated that all four PRRs had higher expression in samples infected with V. alginolyticus compared to V. parahaemolyticus. This PRRs gene expression analysis revealed that all four PRRs expressed rapidly at 4-h post-inoculation even though the Vibrio count was only detected earliest at 12-h post-inoculation in samples. The highest expression recorded was from V. alginolyticus inoculated fish spleen with up to 73-fold change for pglyrp5 gene, followed by 14 to 38-fold expression for the same treatment in spleen, head kidney, and blood samples for other PRRs, namely tlr5, ctlD, and ctlE genes. Meanwhile less than a 10% increase in expression of all four genes was detected in spleen, head kidney, and blood samples inoculated with V. parahaemolyticus. These findings indicated that pglyrp5, tlr5, ctlD, and ctlE play important roles in the early immune response to vibriosis infected, brown-marbled grouper fingerlings.

Characterization and immune role of class B scavenger receptor member 1 in spotted sea bass (Lateolabrax maculatus).

Scavenger receptors (SRs) are integral to the innate immune system and function as pattern-recognition receptors that facilitate pathogen clearance and mediate anti-inflammatory responses. However, the role of SRs in the immune response of Lateolabrax maculatus against Aeromonas veronii is unclear. Here, we cloned scavenger receptor B1 from L. maculatus (LmSRB1) and performed bioinformatics analysis to study its potential functions. The open reading frame spans 1530 base pairs and encodes a 509-amino acid protein with a molecular mass of 57.44 kDa. Comparative analysis revealed high sequence conservation among fish species. Expression profiling revealed strong LmSRB1 transcription in various tissues, especially in head kidney and spleen. Following A. veronii exposure, LmSRB1 expression initially increased, peaking after 4-8 h, with a notable secondary peak at 72 h. Fluorescence in situ hybridization indicated that LmSRB1 mainly localized to the cytoplasm, and subcellular-localization studies confirmed LmSRB1 protein expression in the cytoplasm and cell membrane. Enzyme-linked immunosorbent assay data showed dose-dependent binding of LmSRB1 to A. veronii. Modulating LmSRB1 expression significantly altered the levels of IL-8, IL-1ß, TRAF6, and NIK. These results highlight the crucial role of LmSRB1 in L. maculatus's innate immune response to A. veronii and offer insights into improving the management of bacterial infections in aquaculture.

Grouper TIM-1 promotes nodavirus infection by inhibiting immune and inflammation response.

T-cell/transmembrane immunoglobulin and mucin domain-containing (TIM) protein family has attracted particular attention because of their broad immune functions and the response to viral infections. TIM-1, a member of the TIM family, has been demonstrated to play an important role in viral infections. However, its roles during fish nodavirus infection still remained largely unknown. In this study, a homolog of TIM-1 from orange-spotted grouper (Epinephelus coioides) (EcTIM-1) was identified, and characterized. EcTIM-1 encoded a 217-amino acids protein, containing one Immunoglobulin domain. Homology analysis showed that EcTIM-1 shared 98.62 % and 42.99 % identity to giant grouper (E. lanceolatus) and human (Homo sapiens). Quantitative Real-time PCR analyses indicated that EcTIM-1 was expressed in all examined tissues, with higher expression in liver, spleen, skin, and heart, and was significantly up-regulated in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. EcTIM-1 was distributed in the cytoplasm, and partly co-localized with Golgi apparatus and lysosomes in vitro. The ectopic expression of EcTIM-1 promoted RGNNV replication by increasing the level of viral genes transcription and protein synthesis. Besides, overexpression of EcTIM-1 decreased the luciferase activity of type I interferon (IFN1), interferon stimulated response elements (ISRE) and nuclear factor kappa-B (NF-κB) promoters, as well as the transcription of pro-inflammatory factors and interferon related genes. EcTIM-1 significantly suppressed the luciferase activity of IFN1, ISRE and NF-κB promoters evoked by Epinephelus coioides melanoma differentiation-associated gene 5 (EcMDA5), mitochondrial antiviral signaling protein (EcMAVS), stimulator of IFN genes (EcSTING) or TANK-binding kinase 1 (EcTBK1). Collectively, EcTIM-1 negatively regulated interferon and inflammatory response to promote RGNNV infection. These results provide a basis for a better understanding of the innate immune response of TIM-1 in fish.

Molecular structure and functional responses of IgM, IgT and IgD to Flavobacterium covae and Streptococcus iniae infection in Asian seabass (Lates calcarifer Bloch, 1790).

The Asian seabass (Lates calcarifer) faces significant disease threats, which are exacerbated by intensive farming practices and environmental changes. Therefore, understanding its immune system is crucial. The current study presents a comprehensive analysis of immune-related genes in Asian seabass peripheral blood leukocytes (PBLs) using Iso-seq technology, identifying 16 key pathways associated with 7857 immune-related genes, comprising 634 unique immune-related genes. The research marks the first comprehensive report on the entire immunoglobulin repertoire in Asian seabass, revealing specific characteristics of immunoglobulin heavy chain constant region transcripts, including IgM (Cµ, ighm), IgT (Cτ, ight), and IgD (Cδ, ighd). The study confirms the presence of membrane-bound form, ighmmb, ightmb, ighdmb of IgM, IgT and IgD and secreted form, ighmsc and ightsc of IgM and IgT, respectively, with similar structural patterns and conserved features in amino acids across immunoglobulin molecules, including cysteine residues crucial for structural integrity observed in other teleost species. In response to bacterial infections by Flavobacterium covae (formerly F. columnare genomovar II) and Streptococcus iniae, both secreted and membrane-bound forms of IgM (ighmmb and ighmsc) and IgT (ightmb and ightsc) show significant expression, indicating their roles in systemic and mucosal immunity. The expression of membrane-bound form IgD gene, ighdmb, predominantly exhibits targeted upregulation in PBLs, suggesting a regulatory role in B cell-mediated immunity. The findings underscore the dynamic and tissue-specific expression of immunoglobulin repertoires, ighmmb, ighmsc, ightmb, ightsc and ighdmb in Asian seabass, indicating a sophisticated immune response to bacterial pathogens. These findings have practical implications for fish aquaculture, and disease control strategies, serving as a valuable resource for advancing research in Asian seabass immunology.

Molecular features, antiviral activity, and immunological expression assessment of interferon-related developmental regulator 1 (IFRD1) in red-spotted grouper (Epinephelus akaara).

Interferon-related developmental regulator 1 (IFRD1) is a viral responsive gene associated with interferon-gamma. Herein, we identified the IFRD1 gene (EaIFRD1) from red-spotted grouper (Epinephelus akaara), evaluated its transcriptional responses, and investigated its functional features using various biological assays. EaIFRD1 encodes a protein comprising 428 amino acids with a molecular mass of 48.22 kDa. It features a substantial domain belonging to the interferon-related developmental regulator superfamily. Spatial mRNA expression of EaIFRD1 demonstrated the highest expression levels in the brain and the lowest in the skin. Furthermore, EaIFRD1 mRNA expression in grouper tissues exhibited significant modulation in response to immune stimulants, including poly (I:C), LPS, and nervous necrosis virus (NNV) infection. We analyzed downstream gene regulation by examining type Ⅰ interferon pathway genes following EaIFRD1 overexpression. The results demonstrated a significant upregulation in cells overexpressing EaIFRD1 compared to the control after infection with viral hemorrhagic septicemia virus (VHSV). A subcellular localization assay confirmed the nuclear location of the EaIFRD1 protein, consistent with its role as a transcriptional coactivator. Cells overexpressing EaIFRD1 exhibited increased migratory activity, enhancing wound-healing capabilities compared to the control. Additionally, under H2O2 exposure, EaIFRD1 overexpression protected cells against oxidative stress. Overexpression of EaIFRD1 also reduced poly (I:C)-mediated NO production in RAW267.4 macrophage cells. In FHM cells, EaIFRD1 overexpression significantly reduced VHSV virion replication. Collectively, these findings suggest that EaIFRD1 plays a crucial role in the antiviral immune response and immunological regulation in E. akaara.

The transcriptional landscape underlying larval development and metamorphosis in the Malabar grouper (Epinephelus malabaricus).

Most teleost fishes exhibit a biphasic life history with a larval oceanic phase that is transformed into morphologically and physiologically different demersal, benthic, or pelagic juveniles. This process of transformation is characterized by a myriad of hormone-induced changes, during the often abrupt transition between larval and juvenile phases called metamorphosis. Thyroid hormones (TH) are known to be instrumental in triggering and coordinating this transformation but other hormonal systems such as corticoids, might be also involved as it is the case in amphibians. In order to investigate the potential involvement of these two hormonal pathways in marine fish post-embryonic development, we used the Malabar grouper (Epinephelus malabaricus) as a model system. We assembled a chromosome-scale genome sequence and conducted a transcriptomic analysis of nine larval developmental stages. We studied the expression patterns of genes involved in TH and corticoid pathways, as well as four biological processes known to be regulated by TH in other teleost species: ossification, pigmentation, visual perception, and metabolism. Surprisingly, we observed an activation of many of the same pathways involved in metamorphosis also at an early stage of the larval development, suggesting an additional implication of these pathways in the formation of early larval features. Overall, our data brings new evidence to the controversial interplay between corticoids and thyroid hormones during metamorphosis as well as, surprisingly, during the early larval development. Further experiments will be needed to investigate the precise role of both pathways during these two distinct periods and whether an early activation of both corticoid and TH pathways occurs in other teleost species.

Multiple effects of dietary supplementation with Lactobacillus reuteri and Bacillus subtilis on the growth, immunity, and metabolism of largemouth bass (Micropterus salmoides).

Probiotics play an essential role in the largemouth bass (Micropterus salmoides) aquaculture sector. They aid the fish in sickness prevention, intestinal structure improvement, food absorption, and immune system strengthening. In this experiment, Bacillus subtilis (BS, 107 CFU/g) and Lactobacillus reuteri (LR, 107 CFU/g) were added to the feed and then fed to M. salmoides for 35 days. The effects of two probiotics on the growth, immunity, and metabolism of M. salmoides organisms were studied. The results revealed that the BS group significantly increased the growth rate and specific growth rate of M. salmoides, while both the BS and LR groups significantly increase the length of villi M. salmoides intestines. The BS group significantly increased the levels of AKP, T-AOC, and CAT in the blood of M. salmoides, as well as AKP levels in the intestine. Furthermore, the BS group significantly increased the expression of intestinal genes Nrf2, SOD1, GPX, and CAT, while significantly decreasing the expression of the keap1 gene. M. salmoides gut microbial analysis showed that the abundance of Planctomycetota was significantly different in both control and experimental groups. Analyzed at the genus level, the abundance of Citrobacter, Paracoccus, Luedemannella， Sphingomonas, Streptomyces and Xanthomonas in the both control and experimental groups were significantly different. The BS group's differentially expressed genes were predominantly enriched in oxidative phosphorylation pathways in the intestine, indicating that they had a good influence on intestinal metabolism and inflammation suppression. In contrast, differentially expressed genes in the LR group were primarily enriched in the insulin signaling and linoleic acid metabolism pathways, indicating improved intestine metabolic performance. In conclusion, B. subtilis and L. reuteri improve the growth and health of M. salmoides, indicating tremendous potential for enhancing intestinal metabolism and providing significant application value.