Efficient RNA Virus Targeting via CRISPR/CasRx in Fish.

The emergence of the CRISPR/Cas system as a technology has transformed our ability to modify nucleic acids, and the CRISPR/Cas13 system has been used to target RNA. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that may have an interference effect on RNA viruses. However, the RNA virus-targeting activity of CasRx still needs to be verified in vivo in vertebrates. In this study, we successfully engineered a highly effective CasRx system for fish virus interference. We designed synthetic mRNA coding for CasRx and used CRISPR RNAs to guide it to target the red-spotted grouper nervous necrosis virus (RGNNV). This technique resulted in significant interference with virus infections both in vitro and in vivo. These results indicate that CRISPR/CasRx can be used to engineer interference against RNA viruses in fish, which provides a potential novel mechanism for RNA-guided immunity against other RNA viruses in vertebrates. IMPORTANCE RNA viruses are important viral pathogens infecting vertebrates and mammals. RNA virus populations are highly dynamic due to short generation times, large population sizes, and high mutation frequencies. Therefore, it is difficult to find widely effective ways to inhibit RNA viruses, and we urgently need to develop effective antiviral methods. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that can have an interference effect on RNA viruses. Nervous necrosis virus (NNV), a nonenveloped positive-strand RNA virus, is one of the most serious viral pathogens, infecting more than 40 cultured fish species and resulting in huge economic losses worldwide. Here, we establish a novel effective CasRx system for RNA virus interference using NNV and grouper (Epinephelus coioides) as a model. Our data showed that CasRx was most robust for RNA virus interference applications in fish, and we demonstrate its suitability for studying key questions related to virus biology.

Molecular characterization and functional study of the NLRP3 inflammasome genes in Tetraodon nigroviridis.

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is an important inflammasome in mammals, which is of great significance to eliminate pathogens. However, the research of the NLRP3 inflammasome in teleost is limited. Tetraodon nigroviridis has the characteristics of small genome and easy feeding, which can be used as a model for the study of fish immune function. In present study, three NLRP3 inflammasome component genes (NLRP3, ASC and caspase-1) in T. nigroviridis has been cloned. Real-time fluorescence quantitative PCR showed that TnNLRP3 (T. nigroviridis NLRP3), TnASC (T. nigroviridis ASC) and Tncaspase-1 (T. nigroviridis caspase-1) mRNA in various tissues from health T. nigroviridis were highly expressed in immune-related tissues, such as spleen, gill, head kidney and intestine. After Vibrio parahemolyticus infection, the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA in spleen, gill, head kidney reached a peak at 24 h, and the expression levels of these genes in intestine were the highest at 48 h. After the transfection of TnASC-pAcGFP-N1 monomer GFP plasmid into cos-7 cells, ASC specks, the activation marker of NLRP3 inflammasome, were observed. Bimolecular fluorescence complementarity and fluorescence colocation experiment showed that TnASC and Tncaspase-1 of TnNLRP3 inflammasome were co-located near the cell nucleus, and potentially interacted with each other. NLRP3 inflammasome inducer nigericin and agonist ATP could significantly induce the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA, and activation of NLRP3 inflammasome could promote the generation of mature TnIL-1ß (T. nigroviridis IL-1ß). These results uncover that T. nigroviridis NLRP3 inflammasome could participate in the antibacterial immune response and the generation of mature TnIL-1ß after activation.

The flagellin of Vibrio parahaemolyticus induces the inflammatory response of Tetraodon nigroviridis through TLR5M.

Vibrio parahaemolyticus is an important marine pathogen that cause inflammation even death in teleost. It has brought huge economic losses to aquaculture and serious threats to the sustainable development of marine fisheries. Here, we isolated the DNA, RNA, and total flagellin from V. parahaemolyticus, and obtained the primary spleen and head kidney cells (including leukocytes) from Tetraodon nigroviridis. V. parahaemolyticus DNA, RNA, and total flagellin were used to treat the T. nigroviridis primary cells described above. The results show that the nitric oxide (NO) production and respiratory burst response were significantly induced after stimulation with V. parahaemolyticus total flagellin in T. nigroviridis head kidney and spleen cells. And total flagellin could promote the gene expression and protein production of IL-1ß in T. nigroviridis leukocytes. T. nigroviridis TLR5M (TnTLR5M) and TLR5S (TnTLR5S) ORF sequences were obtained as the main recognition receptor for flagellin. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the expression of pattern recognition receptor TnTLR5M and TnTLR5S, the important signal molecule of inflammatory system TnMyD88 and TnTRAF6, and inflammatory cytokines TnIL-1ß and TnIFN-γ2. The results show that there were a significant upregulation after challenge with V. parahaemolyticus total flagellin. We further demonstrated that the total flagellin of V. parahaemolyticus could activate the luciferase activity of the NF-κB reporter gene mediated by TnTLR5M. Overall, our results suggest that V. parahaemolyticus total flagellin activated the NO production, respiratory burst response, and inflammatory cytokines expressions, such as TnIL-1ß and TnIFN-γ2, through the TnTLR5M-NF-κB signaling pathway in T. nigroviridis.

Recognition of DAP and activation of NF-κB by cytosolic sensor NOD1 in Oreochromis niloticus.

As a lower vertebrate, the immune defense mechanism of fish mainly depends on the innate immune system. Nucleotide-binding oligomerization domain-like receptors (NLRs) are an important class of pattern recognition receptors in the innate immune system. In this study, NOD1 gene was cloned and characterized in Nile tilapia (Oreochromis niloticus). The ORF of Nile tilapia NOD1 gene was 2826 bp long and encoded 941 amino acid residues with a structure of CARD-NACHT-LRRs that was similar to the other counterparts in mammals and fishes. Phylogenetic and synteny analysis showed that NOD1 was conserved among different fishes and existed at least in the early stage of fish evolution. Expression pattern revealed that NOD1 mRNA was constitutively expressed in the tested tissues, while had high expression level in main immune organs and mucosal immune tissues (liver, head kidney, spleen, blood, gill, and intestine). Following Streptococcus agalactiae challenge, Nile tilapia NOD1 mRNA expression levels were altered in immune organs (liver, head kidney, spleen, blood), and the expression pattern was similar in liver, spleen and blood. Furthermore, the ligand recognition and signaling pathway of Nile tilapia NOD1 were also analyzed, it showed that NOD1 could recognize Tri-DAP intracellularly and activated NF-κB signaling pathway. In summary, our results indicated that the Nile tilapia NOD1 may play an important role in innate immune system and provided a basis for the functional study of NOD1 in teleost.

Molecular characterization and functional analysis of IKKα in orange-spotted grouper (Epinephelus coioides).

Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays crucial roles in regulating activation of nuclear factor kappa-B (NF-κB) in response to pathogens infections. Here, we cloned and identified IKKα gene of orange-spotted grouper (Epinephelus coioides), named as EcIKKα. The gene transcript contained a 2262 bp open reading frame, which encoded 753 amino acids. The typically conserved IKKα structure, including serine kinase domain (KD), leucine chain (LZ) structure, helix-loop-helix (HLH) motif and IKKß-NEMO-binding domain, was identified in EcIKKα. Phylogenetic analysis suggested that EcIKKα had the closest relationship with large yellow croaker (Larimichthy crocea) IKKα. Ecikkα was ubiquitously expressed in all tissues tested and the highest expression level was in ovary. After lipopolysaccharide (LPS), flagellin, polyinosinic-polycytidylic acid (poly I:C), polyadenylic-polyuridylic acid (poly A:U), and Vibrio parahaemolyticus stimulation, the expression of Ecikkα increased in grouper spleen (GS) cells. In the luciferase assay, NF-κB-luc activity was significantly up-regulated when human embryonic kidney 293T (HEK 293T) cells were transfected with EcIKKα plasmid. Moreover, overexpression of EcIKKα significantly increased LPS- and flagellin-induced proinflammatory cytokines (interleukin-6 (il-6) and tumor necrosis factor-α (tnf-α)) expression, but did not significantly affect poly I:C- and poly A:U-induced cytokines (il-6 and tnf-α) expression. Overall, these results suggested that EcIKKα functions like that of mammals to activate NF-κB, and it could be involved in host defense against invading pathogens.

Type F scavenger receptor expressed by endothelial cells (SREC)-II from Epinephelus coioides is a potential pathogen recognition receptor in the immune response to Vibrio parahaemolyticus infection.

Scavenger receptors play a central role in defending against infectious diseases in mammals. However, the function of SRECII remains unknown in teleost fish. In this study, type F scavenger receptor expressed by endothelial cells-II (SRECII) cDNA sequence was first identified from Epinephelus coioides, named EcSRECII, which contained an N-terminal signal peptide, eight EGF/EGF-like cysteine-rich motifs and a C-terminal low-complexity region. The gene location maps revealed that EcSRECII has the conservation of synteny among selected species. Subcellular localization showed that EcSRECII was mainly located in the cytoplasm in HEK293T cells and GS cells. In healthy E. coioides, EcSRECII mRNA was highly expressed in spleen, skin, gill, thymus and head kidney. The relative EcSRECII mRNA expression after Vibrio parahaemolyticus infection was significantly up-regulated at 12 h in spleen, head kidney and thymus, but downregulated at 1 d in skin and reduced at 3 d and 1 w in spleen. Furthermore, overexpression of EcSRECII activated NF-κB and IFN-ß signaling pathway in vitro. Taken together, these results indicated that EcSRECII could be as the potential pathogen recognition receptor for involving in bacterial infection by regulating innate immunity responses in E. coioides.

MicroRNA-29b modulates the innate immune response by suppressing IFNγs production in orange-spotted grouper (Epinephelus coioides).

Interferon-γ (IFNγ), a type II interferon, is essential to host resistance against various infections. Unlike other vertebrates, fish have two types of IFNγs, IFNγ1 (also named IFNγ-rel) and IFNγ2. MicroRNAs (miRNAs) regulate multiple biological processes by suppressing mRNA translation or inducing mRNA degradation. Among them, miR-29 can directly target IFNγ and affact innate and adaptive immune responses in mice. There are five members of the miR-29 family in orange-spotted grouper (Epinephelus coioides), which share the same miRNA seed region. However, whether miR-29 directly targets E. coioides IFNγs and regulate IFNγ production is still unknown. In the present study, the negative correlation between miR-29b and both IFNγs in immune tissues of healthy E. coioides and grouper spleen cells (GS cells) stimulated with LPS or poly I:C was demonstrated. Moreover, dual-luciferase reporter assays and western blotting were performed to demonstrate that miR-29b suppressed E. coioides IFNγ production. Studies of NO production in GS cells after miR-29b transfection revealed that miR-29b overexpression affected NO production through the downregulation of IFNγ expression. Taken together, our results suggest that miR-29b may directly target E. coioides IFNγs and modulate IFNγ-mediated innate immune responses by suppressing E. coioides IFNγs production.

Vibrio parahaemolyticus flagellin induces cytokines expression via toll-like receptor 5 pathway in orange-spotted grouper, Epinephelus coioides.

Vibrio parahaemolyticus is the major pathogen of vibriosis in aquatic animals and causes inflammation that may be related to tissue damage. Here, we have established a V. parahaemolyticus flagellin stimulation model using grouper spleen (GS) cell line. Purified V. parahaemolyticus flagellin was used to stimulate GS cells. Our results showed that the mRNA levels of orange-spotted grouper (Epinephelus coioides) toll-like receptor 5M (EcTLR5M), EcTLR5S and downstream cytokines, such as interferon-γ2 (IFN-γ2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), were all significantly increased after stimulation with V. parahaemolyticus flagellin in GS cells. Gene silencing of the EcTLR5M and EcTLR5S in GS cells by using small interfering RNA resulted in suppression of the V. parahaemolyticus flagellin-induced cytokines expression. We further demonstrated that activation of both mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) were required for cytokines expression. We observed that the phosphorylation of NF-κB inhibitor-α (IκBα), extracellular signal-regulated kinase (ERK) and p38 were induced following treatment with flagellin. Additionally, most of p65, a NF-κB subunit, was found to translocate to the nucleus after 60 min stimulation. Overall, our results suggest that V. parahaemolyticus flagellin influences cytokines expression, such as IFN-γ2, IL-6 and TNF-α, via EcTLR5s recognition and MAPKs/NF-κB signaling pathway activation in GS cells.

MicroRNA-182-3p negatively regulates cytokines expression by targeting TLR5M in orange-spotted grouper, Epinephelus coioides.

Toll-like receptors (TLRs) as essential pattern recognition receptors in innate immunity, can recognize pathogens and trigger immune response to eliminate invading pathogens. MicroRNAs regulates multiple biological processes by suppressing mRNA translation or resulting in mRNA degradation. MiR-182 has previously been implicated in DNA repair, disease and cancer aspects. The potential role of miR-182-3p in TLR signaling pathway against pathogens is unclear. In this study, we found that the expression of miR-182-3p was up-regulated after Vibrio parahaemolyticus flagellin stimulation in grouper spleen (GS) cells, and negatively correlated with the expression of orange-spotted grouper (Epinephelus coioides) TLR5M (EcTLR5M). Then we found that miR-182-3p could directly target EcTLR5M by using bioinformatic analysis and dual-luciferase reporter assay. Dual-luciferase reporter assay also showed that miR-182-3p down-regulated the wild-type EcTLR5M 3'UTR in luciferase activity rather than the mutant group in HEK 293T cells. We further verified the effect of miR-182-3p on the activation of Nuclear factor-κB (NF-κB) signaling pathway, and found that miR-182-3p inhibitors significantly augmented flagellin-induced NF-κB phosphorylation. Additionally, we also demonstrated that the increased expression of miR-182-3p significantly suppressed the flagellin-induced EcTLR5M, pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) mRNA expression. And the endogenous miR-182-3p knockdown experiments reversely verified the regulatory effect of miR-182-3p. These results suggested that miR-182-3p post-transcriptionally controls EcTLR5M expression and thereby suppresses the expression of pro-inflammatory cytokines. This study is the first to demonstrate that miR-182-3p suppresses pro-inflammatory cytokines expression by regulating the TLR signaling pathway.

Physicochemical stability of extemporaneously prepared clonidine solutions for use in neonatal abstinence syndrome.

WHAT IS KNOWN AND OBJECTIVE: Extemporaneously prepared clonidine admixture is increasingly used for the management of neonatal abstinence syndrome. However, its stability beyond 15 minutes at room temperature is currently unknown. Therefore, healthcare professionals must prepare clonidine admixtures multiple times a day while the treatment is indicated, resulting in subsequent limitations and problems. The aim of this study was to investigate the physicochemical stability of clonidine in commonly used pharmaceutical diluents at clinically relevant concentrations and temperatures. METHODS: Glass bottles (n = 18) and plastic syringes (n = 18) containing 0.5 and 5 µg/mL of clonidine in either 5% glucose, 10% glucose or 0.9% normal saline were prepared and stored at 4°C for 7 days and at 35°C for 24 hours, respectively. Aliquots were withdrawn at predefined time points and analysed for the concentration of clonidine, changes in pH and colour, and particle content. RESULTS AND DISCUSSION: No evidence of particle formation, or colour or pH change was observed throughout the study period. Clonidine retained more than 98% of its initial concentration when stored in the tested diluents at 4°C for 7 days and at 35°C for 24 hours. WHAT IS NEW AND CONCLUSION: Our findings will allow healthcare professionals to prepare weekly dose of clonidine in glass bottles for storage in a refrigerator. The daily required dose of clonidine can be drawn aseptically from the glass bottle each day and stored in a plastic syringe at room temperature. Clonidine present in a plastic syringe can be administered via the nasogastric route 4-6 times a day. This practice would not only save nursing time and avoid delays in the timely administration of clonidine, but also reduce the risk of potential medication errors as well as preparation-associated costs.

Development of attenuated erythromycin-resistant Streptococcus agalactiae vaccine for tilapia (Oreochromis niloticus) culture.

Streptococcus agalactiae is an important pathogen in fish, causing great losses of intensive tilapia farming. To develop a potential live attenuated vaccine, a re-attenuated S. agalactiae (named TFJ-ery) was developed from a natural low-virulence S. agalactiae strain TFJ0901 through selection of resistance to erythromycin. The biological characteristics, virulence, stability and the immunization protective efficacy to tilapia of TFJ-ery were determined. The results indicated that TFJ-ery grew at a slower rate than TFJ0901. The capsule thickness of TFJ-ery was significantly less (p < 0.05) than TFJ0901. When Nile tilapia were intraperitoneally (IP) injected with TFJ-ery, the mortality of fish was decreased than that injected with TFJ0901. The RPS of fish immunized with TFJ-ery at a dose of 5.0 × 107 CFU was 95.00%, 93.02% and 100.00% at 4, 8 and 16 weeks post-vaccination, respectively. ELISA results showed that the vaccinated fish produced significantly higher (p < 0.05) antibody titres compared to those of control at 2 or 4 weeks post-vaccination. Taken together, our results suggest that erythromycin could be used to attenuate S. agalactiae, and TFJ-ery is a potent attenuated vaccine candidate to protect tilapia against S. agalactiae infections.

Intracellular TLR22 acts as an inflammation equalizer via suppression of NF-κB and selective activation of MAPK pathway in fish.

TLR22, a typical member of the fish-specific TLRs, is a crucial sensor in virally triggered innate immune signalling retained from natural selection. To elucidate the role of the TLR22-specific signalling cascade mechanism, we provide evidence that the double-stranded (ds) RNA-sensor TLR22 positively regulates the ERK pathway and negatively regulates the JNK, p38 MAP kinase and NF-κB pathway. Here, we show that TLR22 restrains NF-κB activation and IFN (interferon) ß and AP-1 (activator protein-1) promoter binding (impairing "primary response" genes (TNF and IL-1)), induces "secondary response" genes (IL-12 and IL-6) and mediates the irregular expression of inflammatory genes. Therefore, TLR22 promotes ERK phosphorylation but impairs the JNK and p38 MAP kinases and IκB phosphorylation. Additionally, TLR22 controls the excessive generation of reactive oxygen species (ROS) to avoid damaging the organism. The specific kinetics of TLR22 depends on its distinct cellular localization. We demonstrate that TLR22 is an intracellular receptor localized in the endosome, and the TLR22-TIR domain is the functional structure inducing the signalling cascade post-viral replication in the body. As mentioned above, our data reveal a novel mechanism whereby TLR22-induced positive adjustment and negative regulation evolved independently to avoid harmful and inappropriate inflammatory responses.

Vaccine-induced antibody level as the parameter of the influence of environmental salinity on vaccine efficacy in Nile tilapia.

To effectively increase production and improve economic returns, the co-culture of Nile tilapia (Oreochromis niloticus) and marine shrimp has been adopted in many countries, including China. Although O. niloticus is an euryhaline fish that can tolerate elevated salinities and even full-strength seawater, fluctuations in salinity levels can undoubtedly induce stress and affect the immune response of this fish. Therefore, this study assessed the impact of salinity on vaccine efficacy in Nile tilapia, which used serum antibody level as a surrogate marker to detect vaccine efficacy. Nile tilapia were acclimatized to 0, 10, 20, or 30 ppt salinity, and then immunized with a formalin-inactivated Streptococcus agalactiae vaccine. Significantly lower levels of antibody in vaccinated fish were found at 20 and 30 ppt salinity compared to 0 and 10 ppt salinity. White blood cell counts, absolute blood lymphocyte counts, and serum bactericidal activity levels were all significantly lower in vaccinated fish at 20 and 30 ppt salinity. Elevated cortisol levels were detected in all of the fish exposure to salinity. Concentrations of serum electrolytes (Na+ and Cl-) were significantly higher in fish at 30 ppt salinity, as compared to fish at lower salinities. Furthermore, the mRNA transcription levels of three of the immune-related genes analyzed (IgM, IL-1ß, and IFN-γ, but not Hsp70) were significantly inhibited in the vaccinated fish at 20 and 30 ppt salinity. A suppressed immune response and decreased vaccine efficacy were also indicated by the lower survival rate of vaccinated fish at 20 ppt salinity when challenged with S. agalactiae. Therefore, salinities ≥20 ppt negatively affected antibody production in Nile tilapia, ultimately affecting vaccine efficacy.

Identification and functional characterization of Toll-like receptor 13 from orange-spotted grouper (Epinephelus coioides).

Toll-like receptors (TLRs) are one of the most important innate immune receptors, which recognize various pathogen-associated molecular patterns and activate the downstream immune response. Mouse TLR13 has been found to recognize a highly conserved sequence from bacterial or viral RNA and activate the myeloid differentiation primary response gene 88-dependent signaling response. The function of teleost tlr13 is still not fully understood, especially its relationship with bacterial RNA. In our study, we identified and characterized a tlr13 from Epinephelus coioides (orange-spotted grouper). The full-length cDNA of Eco. tlr13 contained a 2844 bp open reading frame, encoding 947 amino acids. The polypeptide was constitutive of a signal peptide, 13 leucine-rich repeats domains, a C-terminal leucine-rich repeats, a transmembrane domain and a conserved Toll/interleukin (IL)-1 receptor domain, indicating that Eco. Tlr13 exhibited a typical TLR structure. Multiple alignments showed that the Toll/IL-1 receptor domain of Eco. Tlr13 was identical with other homologues, and the phylogenetic tree suggested that Eco. Tlr13 was clustered with other TLR13s and had the closest relationship with predicted Lates calcarifer (sea bass) Tlr13. Subcellular localization analysis revealed that Eco. Tlr13 colocalized with the endoplasmic reticulum and early endosome. Moreover, Eco. tlr13 was broadly observed in all tested tissues with the relatively high expressions in the brain and immune-related tissues. After challenged with 19-mer Staphylococcus aureus 23S ribosomal RNA-derived oligoribonucleotide (ORN Sa19), the expression of Eco. tlr13 was significantly up-regulated in grouper spleen cells. Also, the luciferase assay further revealed that with the overexpression of Eco. Tlr13 in human embryonic kidney 293T cells, ORN Sa19 activated the promoter activity of interferon-ß in a dose-dependent pattern. These results indicate that Eco. tlr13 may involve in the recognition of bacterial RNA.

The effect of intermittent hypoxia under different temperature on the immunomodulation in Streptococcus agalactiae vaccinated Nile tilapia (Oreochromis niloticus).

Dissolved oxygen (DO) and temperature are the potential immunomodulators in fish and play the important roles in regulating immunity. We studied the effect of intermittent hypoxia under different temperature on the immunomodulation in vaccinated Nile tilapia (Oreochromis niloticus). The expression of immune-related genes, enzymatic activities, histology, cumulative mortality, and S. agalactiae clearance were assessed. Study conditions were intermittently hypoxic (4.0 ±â€¯1.0 mg/L DO) at 30 ±â€¯0.5 °C or 35 ±â€¯0.5 °C. Interleukin-1beta (IL-1ß), tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) mRNA expression in spleen and head kidney were significantly lower in vaccinated hypoxic fish compared to the vaccinated normoxic fish. Levels of heat shock protein 70 (HSP70) in tissues showed an opposite tendency. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities were significantly lower in vaccinated hypoxic fish. Malondialdehyde levels were significantly greater under hypoxic conditions. In vitro studies evaluated the effects of intermittent hypoxia at different temperatures on cells of vaccinated O. niloticus. Phagocytic activity of peripheral blood leucocytes (PBLs) and intracellular reactive oxygen species (ROS) production in head kidney cells were significantly decreased by intermittent hypoxia at either 30 °C or 35 °C, while nitric oxide levels in tissues cells increased significantly under hypoxic conditions. These changes were well reflected by the further suppression modulation on S. agalactiae clearance in vaccinated O. niloticus and higher cumulative mortality by intermittent hypoxia. Taken together, intermittent hypoxia at either 30 °C or 35 °C could suppress immunomodulation in vaccinated Nile tilapia.

Tetraodon nigroviridis: A model of Vibrio parahaemolyticus infection.

Vibriosis is the most common bacterial diseases and brings great economic loss on aquaculture. Vibrio parahaemolyticus (V. parahaemolyticus), a gram-negative bacterium, has been identified as one main pathogens of Vibriosis. The pathogenic mechanism of V. parahaemolyticus is not entirely clear now. In our study, a model of V. parahaemolyticus infection of green-spotted puffer fish (Tetraodon nigroviridis) was established. T. nigroviridis were injected intraperitoneally (i.p.) with 200 µL of V. parahaemolyticus (8 × 10(10) CFU/mL). V. parahaemolyticus infection caused 64% mortality and infected some organs of T. nigroviridis. Histopathology studies revealed V. parahaemolyticus infection induced tissue structural changes, including adipose hollow space in the liver. Immunohistochemistry showed V. parahaemolyticus were present in infected tissue such as liver, head kidney and spleen. In livers of T. nigroviridis infected by V. parahaemolyticus, the alkaline phosphatases (ALP) activity first gradually increased and then backed to normal level, a trend that was on the contrary to the expression profile of the miR-29b. Quantitative real-time PCR analysis showed that the expression level of TLR1, TLR2, TLR5, TLR9, TLR21, NOD1, NOD2 and IL-6 in response to V. parahaemolyticus infection decreased compared to that of non-infected fish. The establishment of the T. nigroviridis model of V. parahaemolyticus infection further confirmed V. parahaemolyticus spreads through the blood circulation system primary as an extracellular pathogen. Meanwhile, liver is an important target organ when infected by V. parahaemolyticus. miR-29b in liver was involved in the progress of liver steatosis during V. parahaemolyticus infection. Moreover, V. parahaemolyticus infection in vivo may have an effect of immunosuppression on host.

UNC93B1 facilitates the localization and signaling of TLR5M in Epinephelus coioides.

Toll-like receptor 5 (TLR5), serving as a sensor of bacterial flagellin, mediates the innate immune response to actively engage in the host's immune processes against pathogen invasion. However, the mechanism underlying TLR5-mediated immune response in fish remains unclear. Despite the presumed cell surface expression of TLR5 member form (TLR5M), its trafficking dynamics remain elusive. Here, we have identified Epinephelus coioides TLR5M as a crucial mediator of Vibrio flagellin-induced cytokine expression in grouper cells. EcTLR5M facilitated the activation of NF-κB signaling pathway in response to flagellin stimulation and exerted a modest influence on the mitogen-activated protein kinase (MAPK)-extracellular regulated kinase (ERK) signaling. The trafficking chaperone Unc-93 homolog B1 (EcUNC93B1) participated in EcTLR5M-mediated NF-κB signaling activation and downstream cytokine expression. In addition, EcUNC93B1 combined with EcTLR5M to mediate its exit from the endoplasmic reticulum, and also affected its post-translational maturation. Collectively, these findings first discovered that EcTLR5M mediated the flagellin-induced cytokine expression primarily by regulating the NF-κB signaling pathway, and EcUNC93B1 mediated EcTLR5M function through regulating its trafficking and post-translational maturation. This research expanded the understanding of fish innate immunity and provided a novel concept for the advancement of anti-vibrio immunity technology.

Sexual dimorphism of steroidogenesis regulated by GnIH in the goldfish, Carassius auratus.

Gonadotropin-inhibitory hormone (GnIH) has been shown to inhibit reproduction in several species. GnIH suppresses gonadotropin synthesis/release at the hypothalamic and pituitary levels; however, increasing evidence suggests that GnIH has a putative function in the gonad. In this study, we demonstrated that GnIH receptors localize to the ovary and testis in goldfish. In situ hybridization illustrated that goldfish GnIHRs were localized exclusively to the oocytes before the cortical alveolus stage and to the interstitial tissue to the testis. Implantation of goldfish GnIH peptides did not affect the serum estradiol levels in female goldfish, but it did enhance the serum testosterone levels in males. Conversely, injecting goldfish GnIH peptides increased the expression of StAR and 3bHSD mRNA and decreased the expression of CYP19 mRNA significantly in the testis, but these genes remained unchanged in the ovary. In addition, goldfish GnIH peptides not only increased the expression of StAR and 3bHSD and decreased CYP19 mRNA, but they also increased the expression of FSHR and LHR mRNA in testicular cells. However, they did not affect the expression of these genes in ovarian cells in vitro. Thus, we suggest that GnIH may contribute to the sexual dimorphism of steroidogenesis in goldfish.

Molecular cloning, characterization and expression profiles of multiple leptin genes and a leptin receptor gene in orange-spotted grouper (Epinephelus coioides).

Leptin plays key roles in body weight regulation, energy metabolism, food intake, reproduction and immunity in mammals. However, its function in teleosts is still unclear. In the present study, two leptin genes (gLepA and gLepB) and one leptin receptor gene (gLepR) were cloned and characterized in orange-spotted grouper (Epinephelus coioides). The cDNAs of gLepA and gLepB were 671 bp and 684 bp in length, encoding for proteins of 161 amino acid (aa) and 158 aa, respectively. The three-dimensional (3D) structures modeling of gLepA and gLepB showed strong conservation of tertiary structure with that of other vertebrates. The total length of gLepR cDNA was 4242 bp, encoding a protein of 1169 aa which contained all functionally important domains conserved among vertebrate LEPR. Tissue distribution analysis showed that gLepA was highly expressed in cerebellum, liver and ovary, while gLepB mRNA abundantly in the brain regions, as well as in the ovary with some extend. The gLepR was mainly expressed in kidney, head kidney and most of brain regions. Analysis of expression profiles of gLep and gLepR genes during the embryonic stages showed that high expression of gLepR was observed in the brain vesicle stage, while neither gLepA nor gLepB mRNA was detected during different embryonic stages. Finally, fasting and refeeding experiments were carried out to investigate the possible function of leptin genes in food intake and energy metabolism, and the results showed that a significant increase of gLepA expression in the liver was induced by food deprivation in both short-term (7 days) and long-term (3 weeks) fasting and gLepA mRNA upregulation was eliminated after refeeding, while gLepB wasn't detected in the liver of grouper during fasting. No significant differences in hypothalamic leptin and leptin receptor expression were found during short-term fasting and refeeding. Hepatic expression of gLepA mRNA increased significantly 9h after a single meal. These results suggested gLepA, other than gLepB, functioned in the regulation of energy metabolism and food intake in this Perciform fish.

Identification and characterization of a motilin-like peptide and its receptor in teleost.

Although putative motilin receptor sequences have been reported in teleost, there is no proof for the existence of the motilin gene in teleost. In this study, we have identified a motilin-like gene in the genome of several fish species and cloned its cDNA sequence from zebrafish. The zebrafish motilin-like precursor shares very low amino acid (aa) identities with the previously reported motilin precursors. Processing of the zebrafish motilin-like precursor may generate a 17-aa C-terminal amidated mature peptide, the motilin-like peptide (motilin-LP). A putative zebrafish motilin receptor (MLNR) was also identified in zebrafish. In cultured eukaryotic cells transfected with the zebrafish MLNR, zebrafish motilin-LP could enhance both CRE-driven and SRE-driven promoter activities. Tissue distribution studies indicated that the zebrafish motilin-like gene is mainly expressed in the intestine and liver while the zebrafish MLNR gene is highly expressed in brain regions, suggesting that motilin-LP behaves like other gut hormones to regulate brain functions. These data suggest that the presence of a unique motilin/MNLR system in teleost.