Complete mitochondrial genome of the giant triton snail Charonia tritonis (Tonnoidea: Charoniidae).

The giant triton snail, Charonia tritonis (Linnaeus, 1758), crucial for coral reef ecosystems as a primary predator of the crown-of-thorns sea star, is experiencing a significant decline due to overfishing for its ornamental shell, underscoring the urgent need for conservation and deeper understanding of its role within marine biodiversity. This study presents the first complete mitogenome sequence of C. tritonis. Spanning 15,346 bp, the C. tritonis mitogenome comprises 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes. Phylogenetic analysis of 88 Littorinimorpha mitogenomes confirms C. tritonis and C. lampas are grouped together within the family Charoniidae as a sister group to the remaining Tonnoidea families. This research not only enhances the taxonomic classification and conservation efforts for marine gastropods but also serves as a vital reference for future evolutionary and genetic studies within the Caenogastropoda.

Intestinal microbiota and gene expression alterations in leopard coral grouper (Plectropomus leopardus) under enteritis.

Enteritis poses a significant threat to fish farming, characterized by symptoms of intestinal and hepatic inflammation, physiological dysfunction, and dysbiosis. Focused on the leopard coral grouper (Plectropomus leopardus) with an enteritis outbreak on a South China Sea farm, our prior scrutiny did not find any abnormalities in feeding or conventional water quality factors, nor were any specific pathogen infections related to enteritis identified. This study further elucidates their intestinal flora alterations, host responses, and their interactions to uncover the underlying pathogenetic mechanisms and facilitate effective prevention and management strategies. Enteritis-affected fish exhibited substantial differences in intestinal flora compared to control fish (P = 0.001). Notably, norank_f_Alcaligenaceae, which has a negative impact on fish health, predominated in enteritis-affected fish (91.76 %), while the probiotic genus Lactococcus dominated in controls (93.90 %). Additionally, certain genera with pathogenesis potentials like Achromobacter, Sphingomonas, and Streptococcus were more abundant in diseased fish, whereas Enterococcus and Clostridium_sensu_stricto with probiotic potentials were enriched in control fish. At the transcriptomic level, strong inflammatory responses, accompanied by impaired metabolic functions, tissue damage, and iron death signaling activation were observed in the intestines and liver during enteritis. Furthermore, correlation analysis highlighted that potential pathogen groups were positively associated with inflammation and tissue damage genes while presenting negatively correlated with metabolic function-related genes. In conclusion, dysbiosis in the intestinal microbiome, particularly an aberrantly high abundance of Alcaligenaceae with pathogenic potential may be the main trigger for this enteritis outbreak. Alcaligenaceae alongside Achromobacter, Sphingomonas, and Streptococcus emerged as biomarkers for enteritis, whereas some species of Lactococcus, Clostridium_sensu_stricto, and Enterococcus showed promise as probiotics to alleviate enteritis symptoms. These findings enhance our understanding of enteritis pathogenesis, highlight intestinal microbiota shifts in leopard coral grouper, and propose biomarkers for monitoring, probiotic selection, and enteritis management.

Strain-Specific Benefits of Bacillus Probiotics in Hybrid Grouper: Growth Enhancement, Metabolic Health, Immune Modulation, and Vibrio harveyi Resistance.

In the realm of modern aquaculture, the utilization of probiotics has gained prominence, primarily due to their ability to enhance growth, boost immunity, and prevent diseases in aquatic species. This study primarily investigates the efficacy of Bacillus subtilis strains, both host-derived and from other sources, in influencing fish growth, immunity, lipid metabolism, and disease resistance. Employing a 42-day feeding trial, we divided hybrid grouper into four distinct groups: a control group on a basal diet and three experimental groups supplemented with 1 × 108 CFU/g of different Bacillus subtilis strains-BS, 6-3-1, and HAINUP40. Remarkably, the study demonstrated that the 6-3-1 and HAINUP40 groups exhibited significant enhancements across key growth parameters: final body weight (FBW), weight gain rate (WGR), feed intake (FI), feed efficiency ratio (FER), and feed conversion ratio (FCR). The investigation into lipid metabolism revealed that the 6-3-1 strain upregulated seven metabolism-related genes, HAINUP40 affected four metabolism-related genes, and the BS strain influenced two metabolism-related genes, indicating diverse metabolic impacts by different strains. Further, a notable reduction in liver enzymes AST and ALT was observed across all supplemented groups, implying improved liver health. Noteworthy was the BS strain's superior antioxidative capabilities, positively affecting all four measured parameters (CAT, GSH-Px, MDA). In the sphere of immune-related gene expression, the BS strain significantly decreased the expression of both inflammation and apoptosis-related genes, whereas the HAINUP40 strain demonstrated an upregulation in these genes. The challenge test results were particularly telling, showcasing improved survival rates against Vibrio harveyi infection in the BS and 6-3-1 groups, unlike the HAINUP40 group. These outcomes highlight the strain-specific nature of probiotics and their varying mechanisms of action within the host. In conclusion, this study reveals that probiotic strains, varying by source, demonstrate unique, strain-specific effects in promoting growth and modulating immunity in hybrid grouper. This research highlights the promise of tailored probiotic applications in improving aquaculture practices. Such advancements contribute to more sustainable and efficient fish farming methods.

Variable Time-Step Physics Engine with Continuous Compliance Contact Model for Optimal Robotic Grinding Trajectory Planning.

In the transition from virtual environments to real-world applications, the role of physics engines is crucial for accurately emulating and representing systems. To address the prevalent issue of inaccurate simulations, this paper introduces a novel physics engine uniquely designed with a compliant contact model designed for robotic grinding. It features continuous and variable time-step simulations, emphasizing accurate contact force calculations during object collision. Firstly, the engine derives dynamic equations considering spring stiffness, damping coefficients, coefficients of restitution, and external forces. This facilitates the effective determination of dynamic parameters such as contact force, acceleration, velocity, and position throughout penetration processes continuously. Secondly, the approach utilizes effective inertia in developing the contact model, which is designed for multi-jointed robots through pose transformation. The proposed physics engine effectively captures energy conversion in scenarios with convex contact surface shapes through the application of spring dampers during collisions. Finally, the reliability of the contact solver in the simulation was verified through bouncing ball experiments and robotic grinding experiments under different coefficients of restitution. These experiments effectively recorded the continuous variations in parameters, such as contact force, verifying the integral stability of the system. In summary, this article advances physics engine technology beyond current geometrically constrained contact solutions, enhancing the accuracy of simulations and modeling in virtual environments. This is particularly significant in scenarios wherein there are constant changes in the outside world, such as robotic grinding tasks.

Construction and analysis of the immune effect of two different vaccine types based on Vibrio harveyi VgrG.

Vibrio harveyi poses a significant threat to fish and invertebrates in mariculture, resulting in substantial financial repercussions for the aquaculture sector. Valine-glycine repeat protein G (VgrG) is essential for the type VI secretion system's (T6SS) assembly and secretion. VgrG from V. harveyi QT520 was cloned and analyzed in this study. The localization of VgrG was determined by Western blot, which revealed that it was located in the cytoplasm, secreted extracellularly, and attached to the membrane. The effectiveness of two vaccinations against V. harveyi infection-a subunit vaccine (rVgrG) and a DNA vaccine (pCNVgrG) prepared with VgrG was evaluated. The findings indicated that both vaccines provided a degree of protection against V. harveyi challenge. At 4 weeks post-vaccination (p.v.), the rVgrG and pCNVgrG exhibited relative percent survival rates (RPS) of 71.43% and 76.19%, respectively. At 8 weeks p.v., the RPS for rVgrG and pCNVgrG were 68.21% and 72.71%, respectively. While both rVgrG and pCNVgrG elicited serum antibody production, the subunit vaccinated fish demonstrated significantly higher levels of serum anti-VgrG specific antibodies than the DNA vaccine group. The result of qRT-PCR demonstrated that the expression of major histocompatibility complex (MHC) class Iα, tumor necrosis factor-alpha (TNF-α), interferon γ (IFNγ), and cluster of differentiation 4 (CD4) were up-regulated by both rVgrG and pCNVgrG. Fish vaccinated with rVgrG and pCNVgrG exhibited increased activity of acid phosphatase, alkaline phosphatase, superoxide dismutase, and lysozyme. These findings suggest that VgrG from V. harveyi holds potential for application in vaccination.

Rationally Designed Self-Assembling Nanovaccines Elicit Robust Mucosal and Systemic Immunity against Rhabdovirus.

Viral diseases have constantly caused great threats to global public health, resulting in an urgent need for effective vaccines. However, the current viral vaccines often show low immunogenicity. To counter this, we report a smart strategy of a well-designed modular nanoparticle (LSG-TDH) that recapitulates the dominant antigen SG, low-molecular-weight protamine, and tetralysine-modified H-chain apoferritin (TDH). The constructed LSG-TDH nanovaccine could self-assemble into a nanocage structure, which confers excellent mucus-penetrating, cellular affinity, and uptake ability. Studies demonstrate that the LSG-TDH nanovaccine could strongly activate both mucosal and systemic immune responses. Importantly, by immunizing wild-type and TLR2 knockout (TLR2-KO) zebrafish, we found that TLR2 could mediate LSG-TDH-induced adaptive mucosal and systemic immune responses by activating antigen-presenting cells. Collectively, our findings offer new insights into rational viral vaccine design and provide additional evidence of the vital role of TLR2 in regulating adaptive immunity.

Long-Term Phellodendri Cortex Supplementation in the Tiger Grouper (Epinephelus fuscoguttatus): Dual Effects on Intestinal Health Revealed by Transcriptome Analysis.

The tiger grouper (Epinephelus fuscoguttatus), an important mariculture fish in Southeast Asia, faces increasing health issues in recent years. Phellodendri Cortex (PC) is a traditional Chinese herbal medicine that exhibits a variety of beneficial effects on tiger groupers. The effects of PC, however, varies with the period of dietary intervention. This study aims to investigate the long-term effects of 1% PC supplementation on tiger groupers, focusing on growth, immunity, disease resistance, and intestinal gene expression. The tiger groupers (with an initial mean weight of 27.5 ± 0.5 g) were fed with a diet of Phellodendri Cortex supplementation and a control diet for 8 weeks. Our results indicate that the long-term PC supplementation did not affect growth or Vibrio disease resistance in tiger groupers. However, the transcriptome analysis revealed potential damage to the structural and functional integrity of the groupers' intestines. On the other hand, anti-inflammatory and cathepsin inhibition effects were also observed, offering potential benefits to fish enteritis prevention and therapy. Therefore, long-term PC supplementation in grouper culture should be applied with caution.

Honokiol induces apoptosis-like death in Cryptocaryon irritans Tomont.

BACKGROUND: Cryptocaryon irritans, a common parasite in tropical and subtropical marine teleost fish, has caused serious harm to the marine aquaculture industry. Honokiol was proven to induce C. irritans tomont cytoplasm shrinkage and death in our previous study, but the mechanism by which it works remains unknown. METHODS: In this study, the changes of apoptotic morphology and apoptotic ratio were detected by microscopic observation and AnnexinV-FITC/PI staining. The effects of honokiol on intracellular calcium ([Ca2+]i) concentration, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), quantity of DNA fragmentations (QDF) and caspase activities were detected by Fluo-3 staining, JC-1 staining, DCFH-DA staining, Tunel method and caspase activity assay kit. The effects of honokiol on mRNA expression levels of 61 apoptosis-related genes in tomonts of C. irritans were detected by real-time PCR. RESULTS: The results of the study on the effects of honokiol concentration on C. irritans tomont apoptosis-like death showed that the highest levels of prophase apoptosis-like death rate (PADR), [Ca2+]i concentration, ROS, the activities of caspase-3/9 and the lowest necrosis ratio (NER) were obtained at a concentration of 1 µg/ml, which was considered the most suitable for inducing C. irritans tomont apoptosis-like death. When C. irritans tomonts were treated with 1 µg/ml honokiol, the [Ca2+]i concentration began to increase significantly at 1 h. Following this, the ROS, QDF and activities of caspase-3/9 began to increase significantly, and the ΔΨm began to decrease significantly at 2 h; the highest PADR was obtained at 4 h. The mRNA expression of 14 genes was significantly upregulated during honokiol treatment. Of these genes, itpr2, capn1, mc, actg1, actb, parp2, traf2 and fos were enriched in the pathway related to apoptosis induced by endoplasmic reticulum (ER) stress. CONCLUSIONS: This article shows that honokiol can induce C. irritans tomont apoptosis-like death. These results suggest that honokiol may disrupt [Ca2+]i homeostasis in ER and then induce C. irritans tomont apoptosis-like death by caspase cascade or mitochondrial pathway, which might represent a novel therapeutic intervention for C. irritans infection.

Antibacterial Activity and Mechanisms of TroHepc2-22, a Derived Peptide of Hepcidin2 from Golden Pompano (Trachinotus ovatus).

Hepcidin, a cysteine-rich antimicrobial peptide, has a highly conserved gene structure in teleosts, and it plays an essential role in host immune response against various pathogenic bacteria. Nonetheless, few studies on the antibacterial mechanism of hepcidin in golden pompano (Trachinotus ovatus) have been reported. In this study, we synthesized a derived peptide, TroHepc2-22, from the mature peptide of T. ovatus hepcidin2. Our results showed that TroHepc2-22 has superior antibacterial abilities against both Gram-negative (Vibrio harveyi and Edwardsiella piscicida) and Gram-positive (Staphylococcus aureus and Streptococcus agalactiae) bacteria. Based on the results of a bacterial membrane depolarization assay and propidium iodide (PI) staining assay in vitro, TroHepc2-22 displayed antimicrobial activity by inducing the bacterial membrane depolarization and changing the bacterial membrane permeability. Scanning electron microscopy (SEM) visualization illustrated that TroHepc2-22 brought about membrane rupturing and the leakage of the cytoplasm for the bacteria. In addition, TroHepc2-22 was verified to have hydrolytic activity on bacterial genomic DNA in view of the results of the gel retardation assay. In terms of the in vivo assay, the bacterial loads of V. harveyi in the tested immune tissues (liver, spleen, and head kidney) were significantly reduced in T. ovatus, revealing that TroHepc2-22 significantly enhanced the resistance against V. harveyi infection. Furthermore, the expressions of immune-related genes, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin 1-ß (IL-1ß), IL-6, Toll-like receptor 1 (TLR1), and myeloid differentiation factor 88 (MyD88) were significantly increased, indicating that TroHepc2-22 might regulate inflammatory cytokines and activate immune-related signaling pathways. To summarize, TroHepc2-22 possesses appreciable antimicrobial activity and plays a vital role in resisting bacterial infection. The observation of our present study unveils the excellent application prospect of hepcidin as a substitute for antibiotics to resist pathogenic microorganisms in teleosts.

Risk Assessment Model System for Aquatic Animal Introduction Based on Analytic Hierarchy Process (AHP).

The spread of invasive species (IS) has the potential to upset ecosystem balances. In extreme cases, this can hinder economical utilization of both aquatic (fisheries) and terrestrial (agricultural) systems. As a result, many countries regard risk assessment of IS as an important process for solving the problem of biological invasion. Yet, some IS are purposefully introduced for what is seen as their potential economic benefits. Thus, conducting IS risk assessments and then formulating policies based on scientific information will allow protocols to be developed that can reduce problems associated with IS incursions, whether occurring purposefully or not. However, the risk assessment methods currently adopted by most countries use qualitative or semiquantitative methodologies. Currently, there is a mismatch between qualitative and quantitative assessments. Moreover, most assessment systems are for terrestrial animals. What is needed is an assessment system for aquatic animals; however, those currently available are relatively rudimentary. To fill this gap, we used the analytic hierarchy process (AHP) to build a risk assessment model system for aquatic IS. Our AHP has four primary indexes, twelve secondary indexes, and sixty tertiary indexes. We used this AHP to conduct quantitative risk assessments on five aquatic animals that are typically introduced in China, which have distinct biological characteristics, specific introduction purposes, and can represent different types of aquatic animals. The assessment results show that the risk grade for Pterygoplichthys pardalis is high; the risk grade for Macrobrachium rosenbergii, Crassostrea gigas, and Trachemys scripta elegans is medium; and the grade risk for Ambystoma mexicanum is low. Risk assessment of the introduction of aquatic animals using our AHP is effective, and it provides support for the introduction and healthy breeding of aquatic animals. Thus, the AHP model can provide a basis for decision-making risk management concerning the introduction of species.

CpG ODN 1668 as TLR9 agonist mediates humpback grouper (Cromileptes altivelis) antibacterial immune responses.

Cromileptes altivelis (humpback grouper) is the main farmed species in the southern coastal area of China owing to its important economic value. Toll-like receptor 9 (TLR9) belongs to the toll-like receptor (TLR) family and functions as a pattern recognition receptor, recognising unmethylated oligodeoxynucleotides containing the CpG motif (CpG ODNs) in bacterial and viral genomes, thereby activating host immune response. In this study, the C. altivelis TLR9 (CaTLR9) ligand CpG ODN 1668 was screened and found to significantly enhance the antibacterial immunity of humpback grouper in vivo and head kidney lymphocytes (HKLs) in vitro. In addition, CpG ODN 1668 also promoted the cell proliferation and immune gene expression of HKLs and strengthened the phagocytosis activity of head kidney macrophages. However, when the CaTLR9 expression was knocked down in the humpback group, the expression levels of TLR9, myeloid differentiation factor 88 (Myd88), tumour necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-1ß (IL-1ß), IL-6, and IL-8 were significantly reduced, and the antibacterial immune effects induced by CpG ODN 1668 were mostly abolished. Therefore, CpG ODN 1668 induced antibacterial immune responses in a CaTLR9-dependent pathway. These results enhance the knowledge of the antibacterial immunity of fish TLR signalling pathways and have important implications for exploring natural antibacterial molecules in fish.

CpG ODN 2102 promotes antibacterial immune responses and enhances vaccine-induced protection in golden pompano (Trachinotusovatus).

CpG oligodeoxynucleotides (ODNs) are oligodeoxynucleotides containing CpG motifs and can be recognized by toll-like receptor 9 (TLR9), activating the host's immune responses. In this study, ten different CpG ODNs were designed and synthesized to study the antibacterial immune responses of CpG ODNs in golden pompano (Trachinotus ovatus). Results showed that CpG ODN 2102 significantly improved the immunity of golden pompano against bacteria. Besides, CpG ODN 2102 promoted the proliferation of head kidney lymphocytes and activated the head kidney macrophages. When TLR9-specific small interfering RNA (siRNA) was used to interfere with TLR9 expression, the immune responses were decreased. Moreover, the expression levels of myeloid differentiation primary response 88 (Myd88), p65, tumor necrosis factor receptor-associated factor 6 (TRAF6), and tumor necrosis factor-alpha (TNF-α) in the TLR9-knockdown golden pompano kidney (GPK) cells were significantly reduced. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) promoter activity of the TLR9-knockdown GPK cells was also significantly reduced. In vivo, the antibacterial immune effects induced by CpG ODN 2102 in golden pompano were mostly abolished when TLR9 expression was knocked down. These results suggested that TLR9 was involved in the immune responses induced by CpG ODN 2102. CpG ODN 2102 also enhanced the protective effect of the Vibrio harveyi vaccine pCTssJ, where the survival rate of golden pompano was significantly improved by 20%. In addition, CpG ODN 2102 enhanced the messenger RNA (mRNA) expression levels of TLR9, Myxovirus resistance (Mx), interferon γ (IFN-γ), TNF-α, interleukin (IL)-1ß, IL-8, major histocompatibility complex class (MHC) Iα, MHC IIα, Immunoglobulin D (IgD), and IgM. Therefore, TLR9 was involved in the antibacterial immune responses induced by CpG ODN 2102 and CpG ODN 2102 possessed adjuvant immune effects. These results enlarged our knowledge of the antibacterial immunity of fish TLRs signaling pathway and had important implications for exploring natural antibacterial molecules in fish and developing new vaccine adjuvants.

Molecular characterization and expression analysis of B-cell lymphoma-2 in Trachinotus ovatus and its role in apoptotic process.

Introduction: B-cell lymphoma-2 (Bcl-2) is the first identified member of the Bcl-2 family that performs an anti-apoptotic function in mammals. However, its role in teleosts is not fully understood. In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned, and its role in apoptosis was investigated. Methods: In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect its mRNA expression level in healthy condition and after LPS stimulation. Subcellular localization was performed by transfecting the pTroBcl2-N3 plasmid into golden pompano snout (GPS) cells and observed under an inverted fluorescence microscope DMi8 and further verified by immunoblotting. In vivo overexpression and RNAi knockdown method were performed to evaluate the role of TroBcl2 in apoptosis. The anti-apoptotic activity of TroBcl2 was detected by flow cytometry. The effect of TroBcl2 on the mitochondrial membrane potential (MMP) was measured by an enhanced mitochondrial membrane potential assay kit with JC-1. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was performed to evaluate the role of TroBcl2 in the DNA fragmentation. Immunoblotting was used to verify whether TroBcl2 inhibits the release of cytochrome c from mitochondria into the cytoplasm. The Caspase 3 and Caspase 9 Activity Assay Kits were used to investigate the effect of TroBcl2 on caspase 3 and caspase 9 activities. The effects of TroBcl2 on the expression of apoptosis-related and nuclear factor- κB (NF-κB) signaling pathway-related genes in vitro were evaluated by qRT-PCR and Enzyme linked immunosorbent assay (ELISA). Luciferase reporter assay was used to evaluate the activity in NF-κB signaling pathway. Results and discussion: The full-length coding sequence of TroBcl2 contains 687 bp and encodes a protein containing 228 amino acids. Four conserved Bcl-2 homology (BH) domains and one invariant "NWGR" motif located in BH1 were identified in TroBcl2. In healthy T. ovatus, TroBcl2 was widely distributed in the eleven tested tissues, and higher expression levels were found in immune-related tissues, such as spleen and head kidney tissues. After stimulation with lipopolysaccharide (LPS), the expression of TroBcl2 in the head kidney, spleen, and liver was significantly upregulated. In addition, subcellular localization analysis revealed that TroBcl2 was localized in both the cytoplasm and nucleus. Functional experiments showed that TroBcl2 inhibited apoptosis, possibly by reducing mitochondrial membrane potential loss, decreasing DNA fragmentation, preventing cytochrome c release into cytoplasm, and reducing the caspase 3 and caspase 9 activations. Moreover, upon LPS stimulation, overexpression of TroBcl2 suppressed the activation of several apoptosis-related genes, such as BOK, caspase-9, caspase-7, caspase-3, cytochrome c, and p53. Furthermore, knockdown of TroBcl2 significantly increased the expression of those apoptosis-related genes. In addition, TroBcl2 overexpression or knockdown induced or inhibited, respectively, the transcription of NF-κB and regulated the expression of genes (such as NF-κB1 and c-Rel) in the NF-κB signaling pathway as well as the expression of the downstream inflammatory cytokine IL-1ß. Overall, our study suggested that TroBcl2 performs its conserved anti-apoptotic function via the mitochondrial pathway and may serve as an anti-apoptotic regulator in T. ovatus.

Insulin-like growth factor binding protein 5b of Trachinotus ovatus and its heparin-binding motif play a critical role in host antibacterial immune responses via NF-κB pathway.

Introduction: Insulin-like growth factor binding protein 5 (IGFBP5) exerts an essential biological role in many processes, including apoptosis, cellular differentiation, growth, and immune responses. However, compared to mammalians, our knowledge of IGFBP5 in teleosts remains limited. Methods: In this study, TroIGFBP5b, an IGFBP5 homologue from golden pompano (Trachinotus ovatus) was identified. Quantitative real-time PCR (qRT-PCR) was used to check its mRNA expression level in healthy condition and after stimulation. In vivo overexpression and RNAi knockdown method were performed to evaluate the antibacterial profile. We constructed a mutant in which HBM was deleted to better understand the mechanism of its role in antibacterial immunity. Subcellular localization and nuclear translocation were verified by immunoblotting. Further, proliferation of head kidney lymphocytes (HKLs) and phagocytic activity of head kidney macrophages (HKMs) were detected through CCK-8 assay and flow cytometry. Immunofluorescence microscopy assay (IFA) and dual luciferase reporter (DLR) assay were used to evaluate the activity in nuclear factor-κB (NF-κß) pathway. Results: The TroIGFBP5b mRNA expression level was upregulated after bacterial stimulation. In vivo, TroIGFBP5b overexpression significantly improved the antibacterial immunity of fish. In contrast, TroIGFBP5b knockdown significantly decreased this ability. Subcellular localization results showed that TroIGFBP5b and TroIGFBP5b-δHBM were both present in the cytoplasm of GPS cells. After stimulation, TroIGFBP5b-δHBM lost the ability to transfer from the cytoplasm to the nucleus. In addition, rTroIGFBP5b promoted the proliferation of HKLs and phagocytosis of HKMs, whereas rTroIGFBP5b-δHBM, suppressed these facilitation effects. Moreover, the in vivo antibacterial ability of TroIGFBP5b was suppressed and the effects of promoting expression of proinflammatory cytokines in immune tissues were nearly lost after HBM deletion. Furthermore, TroIGFBP5b induced NF-κß promoter activity and promoted nuclear translocation of p65, while these effects were inhibited when the HBM was deleted. Discussion: Taken together, our results suggest that TroIGFBP5b plays an important role in golden pompano antibacterial immunity and activation of the NF-κß signalling pathway, providing the first evidence that the HBM of TroIGFBP5b plays a critical role in these processes in teleosts.

Antiviral immunity of grouper MAP kinase phosphatase 1 to Singapore grouper iridovirus infection.

Singapore grouper iridovirus (SGIV), with various mechanisms for evading and modulating host, has inflicted heavy economic losses in the grouper aquaculture. MAP kinase phosphatase 1 (MKP-1) regulates mitogen-activated protein kinases (MAPKs) to mediate the innate immune response. Here, we cloned EcMKP-1, an MKP-1 homolog from the orange-spotted grouper Epinephelus coioides, and investigated its role in the infection of SGIV. In juvenile grouper, EcMKP-1 was highly upregulated and peaked at different times after injection with lipopolysaccharide, polyriboinosinic polyribocytidylic acid and SGIV. EcMKP-1 expression in heterologous fathead minnow cells was able to suppress SGIV infection and replication. Furthermore, EcMKP-1 was a negative regulator of c-Jun N-terminal kinase (JNK) phosphorylation early in SGIV infection. EcMKP-1 decreased the apoptotic percentage and caspase-3 activity during the late stage of SGIV replication. Our results demonstrate critical functions of EcMKP-1 in antiviral immunity, JNK dephosphorylation and anti-apoptosis during SGIV infection.

Effects of Five Prebiotics on Growth, Antioxidant Capacity, Non-Specific Immunity, Stress Resistance, and Disease Resistance of Juvenile Hybrid Grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂).

To explore the short-term health benefits of five prebiotics on hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂), six experimental groups fed with different diets (basal diet, diet control (CON); basal diet + 0.2% fructooligosaccharide (FOS), diet FOS; basal diet + 0.5% chitosan, diet chitosan (CTS); basal diet + 0.2% mannan-oligosaccharide (MOS), diet MOS; basal diet + 0.1% ß-glucan (GLU), Diet GLU; basal diet + 0.05% xylooligosaccharide (XOS), diet XOS) were set up, and a 4-week feeding trial was conducted. MOS and XOS significantly improved the growth of hybrid grouper compared to the CON group (p < 0.05). Antioxidant enzyme assay showed that the activity of glutathione peroxidase (GPx) was significantly enhanced in the MOS group, and the content of malondialdehyde (MDA) in the XOS group was significantly lower than in the CON group (p < 0.05). The catalase (CAT) activities were significantly enhanced in all prebiotic-supplemented groups compared with the CON group (p < 0.05). Non-specific immunity assay showed that the activities of alkaline phosphatase (AKP) and lysozyme (LZM) were significantly increased in all prebiotic-supplemented groups compared with the CON group (p < 0.05). The total protein content in the XOS group was significantly increased (p < 0.05), and the albumin (ALB) activity in the MOS group was more significantly increased than that in the CON group. Histological examination of the intestine revealed that muscle thickness was significantly increased in all prebiotic-supplemented groups compared to the CON group (p < 0.05). Villi length, villi width, muscle thickness all increased significantly in the MOS group (p < 0.05). In addition, the crowding stress and ammonia nitrogen stress experiments revealed that the survival rates of the MOS and XOS groups after stresses were significantly higher than those of the CON group (p < 0.05). Though MOS and XOS exhibited similar anti-stress effects, the antioxidant and non-specific immunity parameters they regulated were not the same, indicating that the specific mechanisms of MOS and XOS's anti-stress effects were probably different. After being challenged with Vibrio harvey, MOS and GLU groups showed significantly higher post-challenge survival rates than the CON group (p < 0.05). These findings indicated that among the five prebiotics, MOS and XOS showed the best overall short-term beneficial effects and could be considered promising short-term feed additives to improve the stress resistance of juvenile hybrid grouper.

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing.

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

IFN regulatory factor 3 of golden pompano and its NLS domain are involved in antibacterial innate immunity and regulate the expression of type I interferon (IFNa3).

Introduction: The transcription factor interferon regulatory factor 3 (IRF3) plays an important role in host defence against viral infections. However, its role during bacterial infection in teleosts remains unclear. In the present study, we evaluated the antibacterial effects of Trachinotus ovatus IRF3 (TroIRF3) and how it regulates type I interferon (IFN). Methods: Subcellular localisation experiments, overexpression, and quantitative real-time PCR (qRT-PCR) were performed to examine the nuclear localisation signal (NLS) of TroIRF3 and its role in the antibacterial regulatory function of TroIRF3. We assessed the binding activity of TroIRF3 to the IFNa3 promoter by luciferase reporter assay. Results and Discussion: The results showed that TroIRF3 was constitutively expressed at high levels in the gill and liver. TroIRF3 was significantly upregulated and transferred from the cytoplasm to the nucleus after Vibrio harveyi infection. By overexpressing TroIRF3, the fish were able to inhibit the replication of V. harveyi, whereas knocking it down increased bacterial replication. Moreover, the overexpression of TroIRF3 increased type I interferon (IFNa3) production and the IFN signalling molecules. The NLS, which is from the 64-127 amino acids of TroIRF3, contains the basic amino acids KR74/75 and RK82/84. The results proved that NLS is required for the efficient nuclear import of TroIRF3 and that the NLS domain of TroIRF3 consists of the key amino acids KR74/75 and RK82/84. The findings also showed that NLS plays a key role in the antibacterial immunity and upregulation of TroIFNa3 induced by TroIRF3. Moreover, TroIRF3 induces TroIFNa3 promoter activity, whereas these effects are inhibited when the NLS domain is deficient. Overall, our results suggested that TroIRF3 is involved in the antibacterial immunity and regulation of type I IFN in T. ovatus and that the NLS of TroIRF3 is vital for IRF3-mediated antibacterial responses, which will aid in understanding the immune role of fish IRF3.

TroTNFSF6, a tumor necrosis factor ligand superfamily member, promotes antibacterial immune response of golden pompano, Trachinotus ovatus.

Tumor necrosis factor ligand superfamily member 6 (TNFSF6), also known as FasL/CD95L, is essential for maintaining the body's immune homeostasis. However, the current reports on TNFSF6 in fish are relatively scarce. In the present study, we conducted functional analyses of a TNFSF6 (TroTNFSF6) from the teleost fish golden pompano (Trachinotus ovatus). TroTNFSF6 is composed of 228 amino acids and has a low similarity with other species (9.65%-58.79%). TroTNFSF6 was expressed in the 11 tissues tested and was significantly up-regulated after Edwardsiella tarda infection. In vivo, overexpression of TroTNFSF6 effectively stimulated the AKP and ACP activities, and reduced bacterial infection in fish tissues. Correspondingly, knockdown of TroTNFSF6 expression resulted in increasing bacterial dissemination and colonization in fish tissues. In vitro, recombinant TroTNFSF6 protein promoted the proliferation of T. ovatus head kidney lymphocytes (HKLs), and promoted the apoptosis of murine liver cancer cells (Hepa1-6). The results indicated that TroTNFSF6 plays an important role in the T. ovatus antibacterial immunity. These observations will facilitate the future in-depth study of teleost TNFSF6.

Molecular characterization and functional analysis of tartrate-resistant acid phosphatase (ACP5) gene in red drum (Sciaenops ocellatus).

Tartrate-resistant acid phosphatase (ACP5) plays an important biological function in immune defense and is highly expressed in activated macrophages, osteoclasts and dendritic cells. In teleost, the functionality of ACP5 remains to be revealed. In this study, we cloned and identified SoACP5 from red drum (Sciaenops ocellatus) and analyzed its function in vivo and in vitro. The open reading frame of SoACP5 is 1002 bp in length, encoding 333 amino acids. SoACP5 shares high sequence identities (96.70%-49.25%) with ACP5 of other species. The SoACP5 mRNA was widely distributed in collected tissues of healthy red drum, and with the maximum in gills. The expression of SoACP5 increased significantly in vivo following challenge with Edwardsiella tarda. Moreover, the recombinant SoACP5 protein (rSoACP5) was purified with his-tag band resin columns, and confirmed to have phosphatase activity which was optimal at pH 5 and 55 °C. Various metal ions (K+, Zn2+, Mn2+, Mg2+, Ca2+, Cu2+, Fe2+ and Fe3+) have different effects on phosphatase activity. rSoACP5 induced the cellular proliferation of peripheral blood leukocytes. The over-expression and knockdown of SoACP5 in vivo had a significant effect on bacterial proliferation. Furthermore, both of the antibacterial activity and phosphatase activity were decreased when the reducedSoACP5 was oxidized by H2O2. In summary, the present study indicated that SoACP5 is likely involved in host defense against bacterial infection in S. ocellatus.