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1.
J Fish Dis ; 42(12): 1731-1743, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31631353

RESUMO

Atlantic lumpfish were vaccinated by intramuscular (im) or intraperitoneal (ip) injection with a multivalent oil-based vaccine, while control fish were injected with phosphate-buffered saline. Four lumpfish per group were sampled for skin/muscle and head kidney tissue at 0, 2, 7, 21 and 42 days post-immunization (dpi) for histopathology and immunohistochemistry (IHC). Gene expressions of secretory IgM, membrane-bound IgM, IgD, TCRα, CD3ε and MHC class IIß were studied in tissues by using qPCR. Im. vaccinated fish showed vaccine-induced inflammation with formation of granulomas and increasing number of eosinophilic granulocyte-like cells over time. On IHC sections, we observed diffuse intercellular staining of secretory IgM at the injection site at 2 dpi, while IgM + cells appeared in small numbers at 21 and 42 dpi. Skin/muscle samples from im. vaccinated fish demonstrated an increase in gene expression of IgM mRNA (secretory and membrane-bound) at 21 and 42 dpi and small changes for other genes. Our results indicated that im. vaccination of lumpfish induced local IgM production at the vaccine injection site, with no apparent proliferation of IgM + cells. Eosinophilic granulocyte-like cells appeared shortly after im. injection and increased in numbers as the inflammation progressed.


Assuntos
Formação de Anticorpos , Imunoglobulina M/imunologia , Inflamação/imunologia , Perciformes/imunologia , Vacinação/veterinária , Animais , Injeções Intramusculares , Vacinação/métodos
2.
Fish Shellfish Immunol ; 94: 819-832, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31597086

RESUMO

The present study is the first report of some representative innate immune genes in meagre (Argyrosomus regius) larvae. This study has specifically focused on the growth period from hatching to the juvenile stage, a critical time in marine fish development when reliance on innate immune mechanisms are required for survival. We report molecular cloning of partial open reading frames and expression patterns for some innate immune genes (c3, cox2, met, lyzc, mxp, myd88, nod2, nod3). In addition, phylogenetic analyses of some of the sequences obtained was performed where confusion among closely allied isoforms may have existed. These results show the met isoform from meagre is met II, an isoform more similar to a homolog described in Larimichthys crocea; lysozyme (lyzc) corresponds to the c-type and NOD isoforms (nod2, nod3) separate into different clades confirming their distinctness within a common evolutionary history. Gene expression profiles of innate genes were investigated, for nine developmental stages, from 8 days post-hatching (dph) to 120 dph. Present results demonstrated that c3, cox2, met II, lyzc, mxp, myd88, nod2, and nod3 were expressed in all stages of larval development and displayed distinct expression profiles in separate tissues (kidney, spleen gut and gill). Moreover, expression patterns suggested theses innate immune genes may be influenced by feeding practices, i.e. switching from live prey (rotifer and Artemia) and weaning onto an inert commercial diet. In addition to evaluating changes in gene expression during early development, this study evaluated the modulation of gene expression by means of in vivo trials in juveniles that were stimulated with PAMPs (LPS, poly I:C, ß-glucan). These results revealed significant changes in mRNA levels of target genes in the kidney, spleen, gut and gills. However, expression profiles differed in magnitude depending on the stimulant and/or tissue. These results are discussed in terms of their relevance and potential application in aquaculture practices.


Assuntos
Proteínas de Peixes/genética , Imunidade Inata/genética , Perciformes/genética , Transcriptoma/imunologia , Animais , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Padrões Moleculares Associados a Patógenos/metabolismo , Perciformes/imunologia , Filogenia
3.
Fish Shellfish Immunol ; 94: 389-397, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31520749

RESUMO

The aim of this study was to analyze the probiotic potential, fatty acid composition and immunostimulant activities of Kluyveromyces lactis M3 isolated from a hypersaline sediment. For this purpose, K. lactis M3 resistance to different pH, salinities and bile, as well as its antioxidant capability were assayed. Furthermore, total fatty acid composition of the yeast was determined where the dominant fatty acids were palmitic, palmitoleic, oleic and linoleic acids. K. lactis M3 showed no cytotoxic effects on peripheral blood leukocytes. During an in vivo experiment in gilthead seabream (Sparus aurata), dietary K. lactis M3 supplemented at 0.55 or 1.1% of the basal diet enhanced bactericidal activity against Vibrio parahaemolyticus N16, V. harveyi Lg 16/00, and V. anguillarum CECT 43442 compared to fish fed commercial diet (control group). Finally, nitric oxide production, peroxidase activity and skin mucus lectin union levels strongly increased in fish fed K. lactis M3 with respect to the control group. The results suggested that the yeast K. lactis M3 had exhibited high antioxidant capability, and its dietary administration at 0.55 or 1% basal diet had immunostimulant activity for gilthead seabream. For all these reasons, it should be considered an appropriate probiotic candidate for the aquaculture fish industry.


Assuntos
Imunidade Inata/imunologia , Kluyveromyces/química , Muco/imunologia , Perciformes/imunologia , Probióticos/farmacologia , Pele/imunologia , Ração Animal/análise , Animais , Antibacterianos/farmacologia , Antioxidantes/metabolismo , Sobrevivência Celular , Dieta/veterinária , Ácidos Graxos/análise , Concentração de Íons de Hidrogênio , Kluyveromyces/fisiologia , Leucócitos/microbiologia , Leucócitos/fisiologia , Muco/efeitos dos fármacos , Muco/microbiologia , Distribuição Aleatória , Salinidade , Pele/efeitos dos fármacos , Pele/microbiologia
4.
Fish Shellfish Immunol ; 94: 634-642, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31533082

RESUMO

Long non-coding RNA refers to an RNA transcript of a non-coding protein with a sequence length greater than 200 bp. More and more reports indicated that lncRNA was involved in the regulation of gene expression as a signalling molecule, an inducing molecule, a leader molecule and a scaffold molecule. Previous studies have sequenced the draft genome and several transcriptome data sets for protein-coding genes of the large yellow croaker (Larimichthys crocea), but little is known about the expression and function of lncRNAs in this species. In order to obtain a catalogue of lncRNAs for this croaker, Vibrio parahaemolyticus infection challenge experiment was conducted and long non-coding RNA sequences were obtained. Using high-throughput sequencing of lncRNA, a total of 73,233 high-confidence transcripts were reconstructed in 32,726 loci, recovering most of the expressed reference transcripts, and 6473 novel expressed loci were identified. The tissue expression profile revealed that most lacunas were specifically enriched in distinct tissues. A set of 163 lncRNAs were identified as being specifically expressed in the spleen and may be involved in the immune response. It is the first time to identify specific lncRNAs in the L. crocea systematically in this croaker, aiming to benefit the future genomic study of this species.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , RNA Longo não Codificante/genética , Animais , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , RNA Longo não Codificante/imunologia , Distribuição Aleatória , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrioses/veterinária , Vibrio parahaemolyticus/fisiologia
5.
Fish Shellfish Immunol ; 94: 539-547, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31533084

RESUMO

Myeloid differentiation factor 88 (MyD88) is a key adaptor of Toll-like receptors (TLR), an important pattern recognition receptor of the innate immune system. To study the origin and evolution of the vertebrate TLR signaling pathway in innate immune systems, we analyzed the biological characteristics and functions of the MyD88 gene in Northeast Chinese lamprey (Lethenteron morii) using PCR amplification, real-time PCR analysis, dual luciferase reporter gene assay, immunofluorescence assay, and other methods. Bioinformatics analysis showed that LmMyD88 has a modular structure consisting of Toll/IL-1R domain (TIR) and death domain (DD), which is typical of the MyD88 family. A phylogenetic tree showed that the homology of LmMyD88 was consistent with the phylogenetic status of lampreys. Tissue expression analysis indicated that the mRNA expression was expressed in some normal tissues of larval and adult L. morii. Real-time PCR analysis showed that the expression of LmMyD88 in tissues, such as gill and kidney, of the adult increased significantly after infection by Pseudomonas aeruginosa. Subcellular localization results showed that LmMyD88 was expressed in the nucleus, cytoplasm, and other parts. A dual luciferase reporter assay indicated that LmMyD88 activated nuclear factor kappa B downstream of the TLR signaling pathway. This study suggested that LmMyD88 might play an important role in the innate immune signal transduction process of L. morii.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Lampreias , Fator 88 de Diferenciação Mieloide/química , Filogenia , Pseudomonas aeruginosa/fisiologia , Alinhamento de Sequência/veterinária
6.
Fish Shellfish Immunol ; 93: 949-957, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31433996

RESUMO

Pseudomonas plecoglossicida is a Gram-negative aerobic bacterium that causes high mortality and serious economic losses in some commercial marine fish. Expression of secY was found to be significantly upregulated at 18 °C compared to 28 °C by RNA-seq and qRT-PCR. All five tested recombinant vectors (pCM130/tac + shRNA) significantly reduced secY mRNA levels in P. plecoglossicida. The recombinant vector encoding shRNA-1165 exhibited the best gene-silencing efficiency, 82.4% and was used to create an RNAi strain for further studies. Compared with the wildtype strain, infections of Larimichthys crocea with the RNAi strain resulted in a 2-day delay in onset time and a 35% reduction in mortality, as well as the alleviation of spleen symptoms. The spleens of L. crocea infected by the wild type or RNAi strain of P. plecoglossicida were subjected to dual RNA-seq at 2 dpi. Compared with the wildtype strain, infection of P. plecoglossicida with the RNAi strain resulted in significant changes in the transcriptomes of both host and pathogen. KEGG analysis showed that the complement and coagulation cascade and the Toll-like receptor signalling pathway were the most enriched host pathways. In the pathogen, genes of the "Sec secretion system" were significantly downregulated. This downregulation of "Sec secretion system" genes hindered the secretion of bacterial proteins and reduced the virulence of P. plecoglossicida. Thus, it was easier for L. crocea to clear the RNAi strain of P. plecoglossicida, and the immune response was similarly reduced. The results indicated that secY was a virulence gene of P. plecoglossicida and played roles in the host-pathogen interactions of L. crocea and P. plecoglossicida.


Assuntos
Proteínas de Bactérias/genética , Doenças dos Peixes/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , Perciformes/imunologia , Canais de Translocação SEC/genética , Animais , Proteínas de Bactérias/metabolismo , Pseudomonas/fisiologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/veterinária , Canais de Translocação SEC/metabolismo
7.
Fish Shellfish Immunol ; 93: 1007-1017, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31449978

RESUMO

Pathogenic disease is a major factor affecting the aquaculture of the rockfish Sebastiscus marmoratus, an important commercial species inhabiting the nearshore waters of the Western Pacific Ocean. Antimicrobial peptides (AMPs), as critical components of innate immunity, have been considered as promising antibiotic substitutes. The aims of this study were 1) to identify major AMPs in the rockfish, 2) to assess their antimicrobial activity and 3) to evaluate their potential therapeutic application. Six AMPs were identified, Hepcidin 1, liver-expressed antimicrobial peptide 2 (LEAP-2), Piscidin, Moronecidin, NK-lysin and ß-defensin through analysis of the liver transcriptome of S. marmoratus. The transcriptional expression profiles of these AMPs were investigated by real-time quantitative PCR (RT-qPCR). These AMPs showed tissue-specific distribution patterns, and S. marmoratus displays a time-, dose- and tissue-dependent expression of AMPs in response to lipopolysaccharide (LPS) challenge. While the synthetic peptides of LEAP-2 and Moronecidin exerted broad-spectrum antimicrobial activity against important aquatic pathogens in vitro by directly disrupting microbial membrane, and no cytotoxicity against murine hepatic cells was observed at the effective concentrations from 5 µM to 40 µM. The existence of multiple AMPs and their distinct tissue distribution patterns and inducible expression patterns suggests a sophisticated, highly redundant, and multilevel network of antimicrobial defensive mechanisms of S. marmoratus. Therefore, S. marmoratus-derived AMPs appear to be potential therapeutic applications against pathogen infections in aquaculture.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Perciformes/genética , Perciformes/imunologia , Animais , Anti-Infecciosos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Linhagem Celular , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica/veterinária , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Perciformes/metabolismo
8.
Mol Immunol ; 114: 378-388, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31450183

RESUMO

Antimicrobial peptides (AMPs) play an important role in the innate immune response of vertebrates by creating a hostile environment for any invading pathogens. Piscidins are potent teleost specific AMPs, which have a broad spectrum activity. We have identified a novel piscidin active peptide, in the greater amberjack, Seriola dumerili, that consists of 25 aa, which forms an amphipathic helix with distinct hydrophobic and positively charged regions. Following homology and phylogenetic analysis the greater amberjack piscidin was deemed to belong to the group 3 family of piscidins. Piscidin was expressed constitutively at immune sites, with transcript level highest in the spleen and gut, at an intermediate level in the gills and lowest in the head kidney. Following in vivo stimulation with PAMPs (poly I:C, LPS and flagellin) piscidin transcript level increased in gills in response to flagellin, in gut and spleen in response to poly I:C, and in head kidney in response to poly I:C, LPS and flagellin. Head kidney and spleen cells were then isolated from greater amberjack and incubated with each of the PAMPs for 4, 12 and 24 h. Piscidin expression was unchanged at 4 and 12 h post PAMP stimulation in head kidney cells but at 24 h transcript level was markedly upregulated compared to control (unstimulated) cells, especially with the bacterial PAMPs. In contrast, spleen cells upregulated piscidin expression by 4 h post stimulation with poly I:C and flagellin, and remained upregulated to 24 h with flagellin exposure, but had returned to baseline levels by 12 h using poly I:C. To determine if piscidin expression could be modulated by diet, greater amberjack were fed diets supplemented with MOS and cMOS for 30 days when transcript level was determined. It was found that MOS supplemented diets increased expression in the spleen, cMOS supplemented diets upregulated transcript levels in the gills and head kidney, whilst a diet containing both MOS and cMOS upregulated transcript in the gut, when compared to fish fed the control diet. Finally, a synthetic greater amberjack piscidin was produced and showed bacteriostatic activity against a number of bacterial strains, including both Gram positive and Gram negative fish pathogens.


Assuntos
Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Sequência de Bases , Brânquias/imunologia , Rim Cefálico/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Padrões Moleculares Associados a Patógenos/imunologia , Filogenia , Alinhamento de Sequência
9.
Fish Shellfish Immunol ; 94: 157-165, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31465874

RESUMO

A feeding experiment was conducted to determine the effects of fish oil replaced by olive oil (OO) on growth performance, serum biochemical, antioxidant capacity and inflammatory response in large yellow croaker (Larimichthys crocea). Four iso-nitrogenous and iso-lipidic diets were formulated by replacing fish oil (FO) with 0% (the control group), 33.3%, 66.7% and 100% OO. Fish fed the diet with 100% OO had the lowest growth performance among dietary treatments. However, there were no significant differences in SGR and FI among fish fed diets with 0% (the control group), 33.3% and 66.7% OO (P > 0.05). As to morphological parameters, HSI was significantly increased in fish fed the diet with 100% OO than the control group (P < 0.05). Furthermore, the lipid content of the liver in fish fed the diet with 100% OO was significantly higher than the control group (P < 0.05). Fish fed the diet with 100% OO had the highest content of C18:1n-9 among dietary treatments. Serum total triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C) levels and activity of serum alanine transaminase (ALT) were significantly increased in fish fed the diet with 100% OO compared with the control group (P < 0.05). Meanwhile, dietary OO decreased the activity of superoxide dismutase (SOD) and the total antioxidant capacity (T-AOC) in fish fed diets with increasing dietary OO levels. However, the content of malondialdehyde (MDA) was significantly increased in fish fed the diet with 100% OO compared with the control group (P < 0.05). The expression of pro-inflammatory genes, COX-2, IL-1ß and TNFα, were significantly increased in the liver of fish fed the diet with 100% OO compared with the control group (P < 0.05), which was probably due to the activation of p38 mitogen-activated protein kinase (p38 MAPK) pathways and Jun N-terminal kinase (JNK) as the increased protein ratio of p-p38 MAPK to p38 MAPK and p-JNK to JNK. These results suggested that high level of dietary OO decreased the growth performance and antioxidant capacity but induced inflammation via the activation of p38 MAPK and JNK pathways in large yellow croaker.


Assuntos
Gorduras Insaturadas na Dieta/metabolismo , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Azeite de Oliva/metabolismo , Perciformes/imunologia , Ração Animal/análise , Animais , Dieta/veterinária , Fígado/efeitos dos fármacos , Perciformes/crescimento & desenvolvimento , Perciformes/metabolismo
10.
Fish Shellfish Immunol ; 93: 623-630, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31400512

RESUMO

Cathepsin S belong to the cathepsin L-like family of cysteine cathepsins. It is well known that Cathepsin S participate in various physiological processes and host immune defense in mammals. However, in teleost fish, the function of cathepsin S is less investigated. In the present study, a cathepsin S homologue (SsCTSS) from the teleost fish black rockfish (Sebastes schlegelii) were identified and examined at expression and functional levels. In silico analysis showed that three domains, including signal peptide, cathepsin propeptide inhibitor I29 domain, and functional domain Pept_C1, were existed in the cathepsin. SsCTSS possesses a peptidase domain with three catalytically essential residues (Cys25, His162, and Asn183). Phylogenetic profiling indicated that SsCTSS are evolutionally close to the cathepsin S of other teleost fish. The expression of SsCTSS in immune-related tissues was upregulated in a time-dependent manner upon bacterial pathogen infection. Purified recombinant SsCTSS (rSsCTSS) exhibited apparent peptidase activity, which was remarkably declined in the presence of the cathepsin inhibitor E-64. rSsCTSS showed strong binding ability to LPS and PGN, the major constituents of the outer membranes of Gram-negative and Gram-positive bacteria, respectively. rSsCTSS also exhibited the capability of agglutination to different bacteria. The knockdown of SsCTSS attenuated the ability of host to eliminate pathogenic bacteria. Taken together, our results suggested that SsCTSS functions as cysteine protease which might be involved in the antibacterial immunity of black rockfish.


Assuntos
Catepsinas/genética , Catepsinas/imunologia , Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Catepsinas/química , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Lipopolissacarídeos/farmacologia , Peptidoglicano/farmacologia , Perciformes/genética , Perciformes/imunologia , Filogenia , Alinhamento de Sequência/veterinária
11.
Fish Shellfish Immunol ; 93: 823-831, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31422181

RESUMO

Calreticulin (CRT) is a highly conserved and multi-functional protein with diverse localizations. CRT has lectin-like properties and possesses important immunological activities in mammalian. In teleost, very limited studies on CRT immunologic function have been documented. In the present study, a CRT homologue (SsCRT) was cloned, identified and characterized from black rockfish, Sebastes schlegeli, an important aquaculture species in East Asia. The full length of SsCRT cDNA is 2180 bp and encoded a polypeptide of 425 amino acids. SsCRT contains a signal peptide, three distinct structural and functional domains (N-, P- and C-domains), and an endoplasmic reticulum (ER) retrieval signal sequence (KDEL). The deduced amino acid sequence of SsCRT shares 89-92% overall sequence identities with the CRT proteins of several fish species. SsCRT was distributed ubiquitously in all the detected tissues and was highly expressed in the spleen, muscle and liver. After the infection of fish extracellular bacterial pathogen Vibrio anguillarum and intracellular bacterial pathogen Edwardsiella tarda, the mRNA transcripts of SsCRT in spleen, liver, and head kidney were significantly up-regulated. The expression patterns were time-dependent and tissue-dependent. Recombinant SsCRT (rSsCRT) exhibited apparent binding activities against different bacteria and PAMPs. In vivo studies showed that the expressions of multiple immune-related genes such as TNF13B, IL-1ß, IL-8, SAA, Hsp70, and ISG15 in head kidney were significantly enhanced when black rockfish were treated with rSsCRT. Furthermore, rSsCRT reduced pathogen dissemination and replication in fish kidney and spleen. These results indicated that SsCRT served as an immune receptor to recognize and eliminate the invading pathogens, which played a vital role in the immune response of Sebastes schlegeli. These findings provide new insights into understanding the roles of CRT proteins in immune response and pathogen infection in teleost.


Assuntos
Calreticulina/genética , Calreticulina/imunologia , Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Calreticulina/química , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Padrões Moleculares Associados a Patógenos/farmacologia , Perciformes/genética , Perciformes/imunologia , Filogenia , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
12.
Fish Shellfish Immunol ; 93: 344-353, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31352116

RESUMO

Large yellow croaker (Larimichthys crocea) is an economical important farmed fish in China. "Visceral White Spot Disease" caused by Pseudomonas plecoglossicida is a disease with a high mortality rate in cage-cultured L. crocea in recent years and resulted in heavy economy lossess. The dual RNA-seq results of previous study showed that the expression of clpV gene in P. plecoglossicida was significantly up-regulated during infection. RNAi significantly reduced the expression of clpV in P. plecoglossicida with maximum silencing efficiency of 96.1%. Compared with the wild type strain, infection of clpV-RNAi strain resulted in a delayed onset time and a 25% reduction in mortality of L. crocea, as well as lessening the symptoms of the spleen. The results of dual RNA-seq of L. crocea infected by clpV-RNAi strain of P. plecoglossicida changed considerably, compared with the counterpart infected with the wild strain. The KEGG enrichment analysis showed that Cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway and MAPK signaling pathway of L. crocea were most affected by the silence of clpV in P. plecoglossicida. RNAi of clpV resulted in the downregulation of genes in flagella assembly pathway and a weaker immune response of host.


Assuntos
Proteínas de Bactérias/genética , Doenças dos Peixes/imunologia , Interações Hospedeiro-Patógeno/imunologia , Perciformes/imunologia , Transcriptoma/imunologia , Animais , Proteínas de Bactérias/metabolismo , Pseudomonas/fisiologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/veterinária , Interferência de RNA , Baço/metabolismo , Baço/microbiologia
13.
Fish Shellfish Immunol ; 93: 449-462, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31352119

RESUMO

Galectins are ß-galactoside-binding lectins, which are involved in pattern recognition, cell adhesion, and stimulation of the host innate immune responses against microbial pathogens. In spite of several functional studies on different galectins isolated from vertebrates and invertebrates, this is the first report to present functional studies for galectin-8 from the marine teleost tissues. In the present study, we characterized galectin-8 homolog from black rockfish (Sebastes schlegelii), in molecular and functional aspects. Rockfish galectin-8 (SsGal8) was found to consist of a 969 bp long open reading frame (ORF), encoding a protein of 322 amino acids and the predicted molecular weight was 35.82 kDa. In silico analysis of SsGal8 revealed the presence of two carbohydrate binding domains (CRDs), at both N and C-termini and a linker peptide of 40 amino acids, in between the two domains. As expected, the phylogenetic tree categorized SsGal8 as a tandem-repeat galectin, and ultimately positioned it in the sub-clade of fish galectin-8. rSsGal8 was able to strongly agglutinate fish erythrocytes and the inhibition of agglutination was successfully exhibited by lactose and d-galactose. Bacterial agglutination assay resulted in agglutination of both Gram (+) and Gram (-) bacteria, including Escherichia coli, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus parauberis, Lactococcus garvieae, Streptococcus iniae and Vibrio tapetis. The tissue distribution analysis based on qPCR assays, revealed a ubiquitous tissue expression of SsGal8 for the examined rockfish tissues, with the most pronounced expression in blood, followed by brain, intestine, head kidney and kidney. Furthermore, the mRNA transcription level of SsGal8 was significantly up-regulated in spleen, liver and head kidney, upon immune challenges with Streptococcus iniae, LPS and poly I:C, in a time dependent manner. Taken together, these findings strongly suggest the contribution of SsGal8 in regulating innate immune responses to protect the rockfish from bacterial infections.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Galectinas/genética , Galectinas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Galectinas/química , Perfilação da Expressão Gênica/veterinária , Bactérias Gram-Negativas/fisiologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Bactérias Gram-Positivas/fisiologia , Infecções por Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/veterinária , Perciformes/genética , Perciformes/imunologia , Filogenia , Alinhamento de Sequência/veterinária
14.
Fish Shellfish Immunol ; 92: 655-666, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31252045

RESUMO

Viperin, also known as RSAD2 (Radical S-adenosyl methionine domain containing 2), is an interferon-induced endoplasmic reticulum-associated antiviral protein. Previous studies have shown that viperin levels are elevated in the presence of viral RNA, but it has rarely been characterized in marine organisms. This study was designed to functionally characterize rockfish viperin (SsVip), to examine the effects of different immune stimulants on its expression, and to determine its subcellular localization. SsVip is a 349 amino acid protein with a predicted molecular mass of 40.24 kDa. It contains an S-adenosyl l-methionine binding conserved domain with a CNYKCGFC sequence. Unchallenged tissue expression analysis using quantitative real time PCR (qPCR) revealed SsVip expression to be the highest in the blood, followed by the spleen. When challenged with poly I:C, SsVip was upregulated by approximately 60-fold in the blood after 24 h, and approximately 50-fold in the spleen after 12 h. Notable upregulation was detected throughout the poly I:C challenge experiment in both tissues. Significant expression of SsVip was detected in the blood following Streptococcus iniae and lipopolysaccharide challenge, and viral hemorrhagic septicemia virus (VHSV) gene transcription was significantly downregulated during SsVip overexpression. Furthermore, cell viability assay and virus titer quantification with the presence of SsVip revealed a significant reduction in virus replication. As with previously identified viperin counterparts, SsVip was localized in the endoplasmic reticulum. Our findings show that SsVip is an antiviral protein crucial to innate immune defense.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Peixes , Perfilação da Expressão Gênica/veterinária , Septicemia Hemorrágica Viral/imunologia , Proteínas com Ferro-Enxofre/química , Proteínas com Ferro-Enxofre/genética , Proteínas com Ferro-Enxofre/metabolismo , Lipopolissacarídeos/farmacologia , Novirhabdovirus/fisiologia , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência/veterinária , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus iniae/fisiologia
15.
Fish Shellfish Immunol ; 92: 216-223, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31154113

RESUMO

Galectin-9 is a -galactoside-binding lectin which could modulate a variety of biological functions including recognition, aggregation and clearance of pathogen. In this study, a galectin-9 homologue (OnGal-9) was identified from Nile tilapia (Oreochromis niloticus) and its expression model and biological effects on bacterial infection were analyzed. The open reading frame of OnGal-9 sequence was 975 bp encoding 324 amino acids. It shares 45%-92% identities with other galectin-9 proteins. The deduced mature peptide of OnGal-9 possesses two conserved carbohydrate recognition domain (CRD) that connected with a linker peptide. Expression analysis indicated that OnGal-9 was distributed in all the tested tissues of healthy tilapia. The OnGal-9 expression was significantly up-regulated in spleen, head kidney, and intestine after challenged by Streptococcus agalactiae. Meanwhile, the recombinant OnGal-9 (rOnGal-9) protein displayed strong binding and agglutination activity toward both Streptococcus agalactiae and Aeromonas hydrophila. Moreover, rOnGal-9 could promote phagocytosis of macrophages. Taken together, the results here indicate that OnGal-9 might be involved in the immune response of Nile tilapia against bacterial infection.


Assuntos
Doenças dos Peixes/imunologia , Galectinas/genética , Galectinas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Ciclídeos , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Galectinas/química , Perfilação da Expressão Gênica/veterinária , Filogenia , Alinhamento de Sequência/veterinária , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae/fisiologia
16.
Fish Shellfish Immunol ; 92: 133-140, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31173860

RESUMO

Infectious spleen and kidney necrosis virus (ISKNV) cause a high mortality disease which lead to significant economic loss on mandarin fish in China. There is no effective drug or vaccine against this fatal disease at present. Meanwhile, many drugs and vaccines had no effect in many cases account of several impenetrable barriers (cell, skin and gastrointestinal tract). Here we reported an immersion subunit vaccine system (SWCNTs-MCP) encoding MCP gene of ISKNV based on single-walled carbon nanotubes (SWCNTs). To evaluate its efficacy against ISKNV, we found a stronger and longer duration immune response (serum antibody production, enzyme activities and immune-related genes expression) can be induced in fish vaccinated with SWCNTs-MCP in comparison with those vaccinated with MCP alone. Importantly, SWCNTs can increase the immune protective effect of naked subunit vaccine by ca. 23.8%. Thereby, this study demonstrates that SWCNTs as a promising carrier for subunit vaccine might be used to vaccinate large-scale juvenile mandarin fish by bath administration approach.


Assuntos
Doenças dos Peixes/imunologia , Imunidade Inata , Iridoviridae/imunologia , Nanotubos de Carbono , Perciformes/imunologia , Vacinação/veterinária , Vacinas Virais/imunologia , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Vacinas de Subunidades/imunologia
17.
Fish Shellfish Immunol ; 92: 570-582, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31202963

RESUMO

Peroxiredoxin (Prx), also named thioredoxin peroxidase (TPx), is a selenium independent antioxidant enzyme that can protect organisms from oxidative damage caused by reactive oxygen species (ROS) and is important for immune responses. In this study, the molecular cloning and characterization of a Prx2 homologue (CiPrx2) were described from grass carp (Ctenopharyngodon idella). The full-length cDNA of CiPrx2 was 1163 bp containing 5'-untranslated region (UTR) of 52 bp, a 3'-UTR of 517 bp with the putative polyadenylation consensus signal (AATAAA), an open reading frame (ORF) of 594 bp encoding polypeptides of 197 amino acids with a predicted molecular mass of 21.84 kDa and theoretical isoelectric point of 5.93. The analysis results of multiple sequence alignment and phylogenetic tree confirmed that CiPrx2 belong to the typical 2-Cys Prx subfamily. The CiPrx2 mRNA was ubiquitously expressed in all tested tissues. The temporal expression of CiPrx2 were differentially induced infected with grass carp reovirus (GCRV), polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) in liver and spleen. Subcellular localization of CiPrx2-GFP fusion proteins were only distributed in the cytoplasm. The purified recombinant CiPrx2 possessed an apparent antioxidant activity and could protect DNA against oxidative damage. Finally, CiPrx2 proteins could obviously inhibit H2O2 and heavy metal toxicity. However, further researches are needed to better understand the regulation of CiPrx2 under oxidative stresses.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Peroxirredoxinas/genética , Peroxirredoxinas/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Carpas , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Padrões Moleculares Associados a Patógenos/administração & dosagem , Peroxirredoxinas/química , Filogenia , Poli I-C/farmacologia , Distribuição Aleatória , Reoviridae/fisiologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/veterinária , Alinhamento de Sequência/veterinária , Baço/metabolismo
18.
Fish Shellfish Immunol ; 92: 341-347, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31202964

RESUMO

The yellow drum (Nibea albiflora) is an economically important maricultured fish in China, but the aquaculture of this species has recently been limited by an increase in overwinter mortalities associated with cold and starvation stress due to global climate changes. To better understand the interaction between starvation and cold-stress-driven overwinter mortality, we investigated the effects of these stresses on the growth performance, liver lesions, and immune response of yellow drum fish. The fish were subjected to different cold treatments and under starvation stress. The experiment lasted 30 days and involved four experimental groups: a fed group and a fasted group maintained at 16 °C (control), and a fed group and a fasted group subjected to cold stress at 8 °C. We found that the growth of yellow drum was severely affected by cold temperatures and starvation. Throughout the experimental period, the body weights were significantly lower in the groups subjected to starvation and cold stress than in the control group. The liver cells showed irregular shapes and disorderly arrangements in the stress groups; indicating liver lesions. The gene expressions of antioxidant enzymes (copper, zinc superoxide dismutase, manganese superoxide dismutase, iron superoxide dismutase, and catalase) in the liver were lower in the groups subjected to starvation and cold stress than in the control groups. These results were basically consistent with the enzyme activities of superoxide dismutase and catalase tested in the livers. In addition, activities of immunomodulatory enzymes (alkaline phosphatase and acid phosphatase) were also inhibited in groups subjected to stress throughout the experiment period. These findings suggested that starvation and cold stress inhibited growth, depressed liver function, and suppressed the immune system of yellow drum, which likely would lead to physiological failure and increased susceptibility to infection. The present study offers insights into the physiological and immune response of yellow drum under cold and starvation stress. These insights not only provide baseline information from which effective strategies can be established and appropriate management decisions formulated, but can also be used to improve the overwinter survival of this important fish species in China.


Assuntos
Privação de Alimentos , Imunidade Inata , Longevidade , Perciformes/imunologia , Animais , Temperatura Baixa/efeitos adversos , Fígado/anatomia & histologia , Fígado/fisiologia , Perciformes/crescimento & desenvolvimento
19.
Fish Shellfish Immunol ; 92: 141-150, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31176007

RESUMO

Mandarin fish (Siniperca chuatsi) is a popular cultured freshwater fish species due to its high market value in China. With increasing density of breeding, mandarin fish is often cultured under low environmental oxygen concentrations (hypoxia). In this study, the relative expression levels of hypoxia response element (HRE)-luciferase reporter and the HIF signaling pathway downstream genes (scldha, scvegf, and scglut-1) were significantly increased by hypoxic stress, thereby indicating that mandarin fish has an HIF signaling pathway. The mandarin fish HIF-1α (scHIF-1α) was also characterized. Multiple sequence alignments showed that scHIF-1α presented similar architectures to other known vertebrates. Subcellular localization analysis showed that scHIF-1α was mainly located in the nucleus of the mandarin fish fry-1 (MFF-1) cells. The role of scHIF-1α in the regulation of the HIF signaling pathway was confirmed. Overexpression of scHIF-1α could induce the HIF signaling pathway, whereas knockdown of scHIF-1α inhibited the activity of the HIF-1 signaling pathway. Tissue distribution analysis showed that schif-1α was significantly highly expressed in the blood, heart, and liver, which indicated that the main function of scHIF-1α was closely related to the circulatory system. Furthermore, scHIF-1α expression was significantly induced by poly I:C, poly dG:dC or PMA, thereby indicating that scHIF-1α was involved in the immune response. HIF-1α plays an important role in pathogen infections in mammals, but its role in fish is rarely investigated. Overexpression of scHIF-1α could inhibit MRV and SCRV infections, whereas knockdown of scHIF-1α could promote such infections. Those results suggested that scHIF-1α played an important role in fish virus infection. Our study will help understand the hypoxia associated with the outbreaks of aquatic viral disease.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Filogenia , Poli I-C/farmacologia , Polidesoxirribonucleotídeos/farmacologia , Alinhamento de Sequência/veterinária , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia
20.
Fish Shellfish Immunol ; 91: 172-179, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31103555

RESUMO

The inflammatory response of fish to LPS is subdued, attributed to absence of TLR4, a key pro-inflammatory receptor for LPS in mammals. Nevertheless, LPS is processed in fish in a T-independent manner and is a protective antigen in fish vaccines, yet pathways for processing LPS in fish remain to be elucidated. Here, we report that caspases and NOD-like receptor inflammasomes typically responsible for LPS recognition and processing in mammals lack critical domains or are absent in barramundi (Lates calcarifer). On the other hand, leucocyte integrins MAC-1 and LFA-1 were detected on the surface of neutrophil- and lymphocyte-like cells respectively in the barramundi spleen by immunocytochemistry, and leucocytes displaying MAC-1 or LFA-1 bound to Factor X and ESM-1 respectively. Exposure to MAC-1 and LFA-1 induced significant IL-1ß expression post-stimulation with LPS compared to unstimulated and isotype controls, but the differences observed in TNF-α expression were inconclusive. Our findings implicate MAC-1 and LFA-1 involvement in immune processing of LPS in barramundi and in antigen processing in fish.


Assuntos
Imunidade Inata/genética , Inflamassomos/imunologia , Leucócitos/imunologia , Lipopolissacarídeos/farmacologia , Perciformes/imunologia , Animais , Caspases/imunologia , Integrinas/imunologia , Proteínas NLR/imunologia , Filogenia
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