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1.
Sci Total Environ ; 653: 1120-1127, 2019 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-30759552

RESUMO

Formalin is commonly used as a component of antiparasitic baths in fisheries. In this study the impact of this bath on the immune profile and oxidative stress parameters was evaluated. A formalin bath was prepared in the concentration of 185.3 mg L-1 (0.17 mL L-1) at a temperature of 20 °C. A total of 96 common carp Cyprinus carpio (Linnaeus, 1758) individuals were immersed in this bath for 60 min. The effects were monitored immediately, and then after 24, 48 h and 10 days following the treatment. The study revealed the most effects 10 days after the treatment, when we observed the decrease of lysozyme in skin mucus, the decrease of anti-inflammatory cytokine transforming growth factor beta in gill tissue and increase of interleukin 10 in cranial kidney tissue. The pro-inflammatory cytokine interleukin 1b showed an increase in gill tissue immediately after the bath and the increase in glutathione peroxidase in gill tissue was also observed 24 h and 10 days after bath treatment. The other investigated parameters did not show any significant changes. In conclusion, even though the formalin bath elevated some parameters as mentioned above, formalin used in the bath is probably safe as an antiparasitic treatment of fish.


Assuntos
Antiparasitários/uso terapêutico , Carpas/fisiologia , Formaldeído/uso terapêutico , Estresse Oxidativo , Animais , Antiparasitários/efeitos adversos , Carpas/imunologia , Carpas/parasitologia , Formaldeído/efeitos adversos , Brânquias/imunologia , Brânquias/metabolismo , Brânquias/fisiologia , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo
2.
Fish Shellfish Immunol ; 87: 307-314, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30677514

RESUMO

The gills of fish are large mucosal surfaces that are very important portals for pathogen entry. Investigations have shown that microRNAs (miRNAs) are key regulators of immune response to bacterial infections in the gills of fish; however, how miRNA expression changes in response to infection by Gram-positive bacteria remains largely unknown. To further investigate the immunological role of miRNAs in fish gills under pathogen stress induced by Gram-positive bacterial infection, this study investigated Staphylococcus aureus (SA)-induced changes in the miRNAs levels in gills of adult zebrafish (Danio rerio). miRNA microarrays were used to analyze expression profiles of known miRNA in the gills of zebrafish in response to SA infection and compared these to uninfected control fish. A total of 30 differentially expressed miRNAs (DEMs) were identified. Target genes likely regulated by DEMs were predicted, and functional enrichment analyses were performed. The results indicated that DEM targets were primarily involved in innate immune processes, apoptosis, defense responses, and antibacterial responses. Pathways involving bacterial infection, innate immunity, metabolic process, disease, and apoptosis were mediated by DEMs. Furthermore, real-time quantitative PCR experiments for nine key SA-responsive DEMs that regulated the "SA infection" pathway validated the accuracy of microarray results. Dynamic variations in gene expression were surveyed in detail for these key SA-responsive DEMs for PBS control and at 6, 12, 24, and 48 h after SA challenge in detail. This study provides novel insight into the mechanisms underlying the miRNA regulation during the SA-induced immune response in zebrafish gills, and provides basic knowledge on the innate immune response against Gram-positive bacterial infection in bony fish.


Assuntos
Imunidade Inata/genética , MicroRNAs/genética , MicroRNAs/imunologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Animais , Doenças dos Peixes/imunologia , Brânquias/imunologia , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/fisiologia
3.
Dev Comp Immunol ; 93: 93-102, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30630003

RESUMO

The parasitic ciliate Ichthyophthirius multifiliis (Ich), which infects almost all freshwater fish species, provides an optimal model for the study of immunity against extracellular protozoa. Ich invades the epithelia of mucosal tissues, forms white spots covering the whole body, and induces high mortality, while survivor fish develop both innate and adaptive immunity against Ich attack in systemic and mucosal tissues. Besides the protective roles of the Toll-like receptor (TLR)-mediated innate immune response, the critical immune functions of novel IgT in the skin, gut, gill, and olfactory organ of teleosts have been demonstrated in recent years, and all this information contributes to the ontogeny of the mucosal immune response in vertebrates. Especially in rainbow trout, Ich-infected fish exhibited higher IgT concentrations and titers in the mucosa and increased IgT+ B-lymphocyte proliferation in mucosal tissues. IgM mainly functions in the adaptive immune response in the systemic tissues of rainbow trout, accompanied with increased IgM+ B-lymphocyte proliferation in the head kidney of Ich-infected trout. However, little is known about the interaction between these mucosal tissues and systemic immune organs and the interaction between the inductive immune organs and functional immune organs. Immobilization antigens (Iags), located on the parasite cell and ciliary membranes, have been characterized to be targeted by specific antibodies produced in the host. The crosslinking of antigens mediated by antibodies triggers either an escape response or the immobilization of Ich. With more knowledge about the Iags of Ich and the immunity of teleosts, a more targeted vaccine, even a DNA vaccine, can be developed for the immune control strategy of Ich. Due to the high frequency of clinical fish ichthyophthiriasis, the study of fish immune responses to Ich provides an optimal experimental model for understanding immunity against extracellular protozoa.


Assuntos
Infecções por Cilióforos/veterinária , Doenças dos Peixes/imunologia , Hymenostomatida/imunologia , Membrana Mucosa/imunologia , Oncorhynchus mykiss/imunologia , Oncorhynchus mykiss/parasitologia , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Linfócitos B/imunologia , Proliferação de Células , Infecções por Cilióforos/imunologia , Infecções por Cilióforos/parasitologia , Doenças dos Peixes/parasitologia , Trato Gastrointestinal/imunologia , Brânquias/imunologia , Rim Cefálico/imunologia , Imunoglobulina M/imunologia , Imunoglobulinas/imunologia , Membrana Mucosa/parasitologia , Vacinas Protozoárias/imunologia , Pele/imunologia , Vacinas de DNA/imunologia
4.
Fish Shellfish Immunol ; 86: 635-640, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30528659

RESUMO

Vaccination is the most effective approach for prevention of infectious diseases in aquaculture. Although immersion vaccination is more applicable compared to in-feed/oral administration and injection, this method suffers from low potency as the efficiency of uptake of antigens through mucosal membranes is limited. In this study, we have successfully developed a mucoadhesive vaccine delivery system to enhance the efficacy of direct immersion vaccination against Flavobacterium columnare, the causative agent of columnaris disease in red tilapia. A formalin-killed negatively charged, bacterial cell suspension was used to prepare a mucoadhesive vaccine by electrostatic coating with positively charged chitosan. Our results demonstrate that the chitosan-complexed vaccine greatly increases its mucoadhesiveness, thus increasing the chances of vaccine uptake by the gill mucosa and improving the protection obtained against columnaris infection. The surface charge of the chitosan-complexed vaccine was altered from anionic to cationic after chitosan modification. Tilapia were vaccinated with the prepared chitosan-complexed vaccine by immersion. The challenge test was then carried out 30 and 60 days post vaccination, which resulted in a high level of mortalities in the non-vaccinated and uncomplexed vaccine groups. A high relative percentage survival (RPS) of vaccinated fish was noted with the mucoadhesive vaccine. Our results indicated that the naked vaccine failed to protect the fish from columnaris infection, which is consistent with the mucoadhesive assays performed during the study showing that the naked vaccine was unable to bind to mucosal surfaces. This system is therefore an effective method for immersion vaccination in order to deliver the antigen preparation to the mucosal surface membrane of the fish.


Assuntos
Vacinas Bacterianas/uso terapêutico , Doenças dos Peixes/prevenção & controle , Infecções por Flavobacteriaceae/veterinária , Polímeros/química , Tilápia/imunologia , Vacinação/métodos , Adesivos/química , Animais , Aquicultura , Vacinas Bacterianas/química , Quitosana/química , Infecções por Flavobacteriaceae/prevenção & controle , Flavobacterium , Brânquias/imunologia , Imersão , Membrana Mucosa/metabolismo , Eletricidade Estática , Propriedades de Superfície , Tilápia/microbiologia , Vacinas de Produtos Inativados/química , Vacinas de Produtos Inativados/uso terapêutico
5.
Fish Shellfish Immunol ; 86: 571-580, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30529463

RESUMO

Peptidoglycan (PGN) is an important target of recognition in invertebrate innate immunity. PGN recognition proteins (PGRPs) are responsible for PGN recognition. In this study, we cloned and functionally analyzed a short PGRP (HcPGRP2) from the triangle-shell pearl mussel Hyriopsis cumingii. The full-length cDNA sequence of HcPGRP2 gene was 1185 bp containing an open reading frame of 882 bp encoding a 293 amino acid protein. HcPGRP2 was predicted to have two SH3b domains and a conserved C-terminal PGRP domain. Quantitative real-time RT-PCR showed that HcPGRP2 was expressed in all examined tissues and its expression was induced most significantly by Staphylococcus aureus and Vibrio parahaemolyticus in the hepatopancreas and gills. RNA interference by siRNA results revealed that HcPGRP2 was involved in the regulation of whey acidic protein, theromacin, and defensin expression. As a pattern-recognition receptor, recombinant HcPGRP2 (rHcPGRP2) protein can bind and agglutinate (Ca2+ dependent) all tested bacteria. rHcPGRP2 exhibited specific binding to PGN but not to lipopolysaccharide. Moreover, rHcPGRP2 inhibited the growth activities of S. aureus and V. parahaemolyticus in vitro and accelerated the clearance of V. parahaemolyticus in vivo. Overall, our results indicated that HcPGRP2 may play an important role in the antibacterial immune mechanisms of H. cumingii.


Assuntos
Bivalves/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Animais , Bivalves/genética , Clonagem Molecular , DNA Complementar , Regulação da Expressão Gênica , Brânquias/imunologia , Brânquias/microbiologia , Hepatopâncreas/imunologia , Hepatopâncreas/microbiologia , Imunidade Inata , Lipopolissacarídeos/metabolismo , Peptidoglicano/metabolismo , Filogenia , Ligação Proteica , RNA Mensageiro , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Alinhamento de Sequência , Staphylococcus aureus , Vibrio parahaemolyticus
6.
Dev Comp Immunol ; 93: 66-77, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30590065

RESUMO

As pattern recognition receptors (PRRs), C-type lectins (CTLs) play crucial roles in recognizing and eliminating pathogens in innate immunity. In this study, a novel CTL (HcCUB-Lec) was identified from the triangle sail mussel Hyriopsis cumingii. The full-length of HcCUB-Lec cDNA was 1558 bp with an open reading frame of 1281 bp that encodes a putative protein of 426 amino acid residues, including an N-terminal signal peptide, a complement Uegf Bmp1 (CUB) domain, a single carbohydrate recognition domain (CRD), and a transmembrane domain. Quantitative real-time PCR analysis revealed that HcCUB-Lec transcript was distributed in all examined tissues with the highest levels in hepatopancreas and was significantly upregulated in gills and hepatopancreas after immune challenge with Staphyloccocus aureus and Vibrio parahaemolyticus. When HcCUB-Lec was silenced by RNAi, the expression levels of three antimicrobial peptides, including whey acidic protein (HcWAP), defensin (HcDef), and lysozyme (HcLyso), were dramatically decreased in gills. The recombinant HcCUB-Lec and its individual CUB and CRD domains can bind with Gram-positive bacteria (S. aureus and Bacillus subtilis), Gram-negative bacteria (V. parahaemolyticus and Aeromonas hydrophila), and polysaccharides (lipopolysaccharide and peptidoglycan). Moreover, rHcCUB-Lec and its domains could also agglutinate S. aureus and V. parahaemolyticus in the presence of Ca2+ and can clear V. parahaemolyticus in H. cumingii. Results of this study suggest that HcCUB-Lec acts as an antimicrobial PRR that participates in the innate immune responses of H. cumingii.


Assuntos
Bivalves/imunologia , Lectinas Tipo C/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Staphylococcus aureus/imunologia , Vibrio parahaemolyticus/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Defensinas/biossíntese , Brânquias/imunologia , Hepatopâncreas/imunologia , Imunidade Inata/imunologia , Lectinas Tipo C/genética , Proteínas do Leite/biossíntese , Muramidase/biossíntese , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Reconhecimento de Padrão/genética
7.
Dev Comp Immunol ; 90: 152-156, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30248360

RESUMO

Transforming growth factor-ß activated kinase 1 (TAK1) is a crucial signal transducer in multiple signaling pathways. TAK1 binds TAB1, TAB2, and TAB3, which act as activators and adaptors that specifically regulate the activation of TAK1. To date, the role of TABs is largely unknown in fish. In the present study, a TAB1 cDNA sequence was identified in grouper (Epinephelus coioides), and designated EcTAB1. The full-length open reading frame of EcTAB1 is 1, 521 bp; it encodes 506 amino acids that contains an N-terminal PP2C domain. Many important functional sites in mammalian TAB1 were conserved in TAB1 from grouper and from other fish. Multiple sequence alignment showed that EcTAB1 protein shared high sequence identity with TAB1 of other fish, especially with Stegastes partitus (95% identity). TAB1 was clustered into the same subgroup with other fish TAB1 in the phylogenetic tree. Tissue expression analysis indicated that TAB1 was widely distributed in different tissues. After infection with Cryptocaryon irritans, EcTAB1 expression was up-regulated in the infection site (gills). Besides, EcTAB1 was expressed throughout the grouper spleen (GS) cells and significantly enhanced the activation of NF-κB.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Infecções por Cilióforos/imunologia , Cilióforos/fisiologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Brânquias/imunologia , Perciformes/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Clonagem Molecular , Proteínas de Peixes/metabolismo , Brânquias/parasitologia , Humanos , NF-kappa B/metabolismo , Membro 2 do Grupo C da Subfamília 2 de Receptores Nucleares/metabolismo , Filogenia , Alinhamento de Sequência , Transdução de Sinais , Regulação para Cima
8.
Fish Shellfish Immunol ; 86: 1009-1018, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30586633

RESUMO

Autophagy plays a vital role in innate and adaptive immunity against invading microorganisms, such as virus and bacteria. However, the mechanism underlying autophagy in shrimp is still limited. In our study, we challenged white shrimp L. vannamei with rapamycin to induce autophagy and employed Solexa/Illumina high-throughput RNA-seq method to examine the differences of transcriptome from gills of shrimps treated with or without rapamycin. More than 22.64 Gb raw data were produced, which were assembled into 62, 503 unigenes, with 14,126 unigenes over 1 kb in length. We then performed differential expression analysis and identified a total of 3050 differentially expressed genes (DEGs). Among them, 1456 were upregulated and 1594 were downregulated. We further annotated DEGs by matching against non-redundant protein sequence (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups of proteins (COG), euKaryotic Orthologous Groups (KOG), Gene ontology (GO), and Pfam databases. The assembled and annotated DEGs will facilitate our understanding of the molecular mechanism underlying autophagy and promote the studies on the role of autophagy in innate immunity of L. vannamei and other crustaceans.


Assuntos
Autofagia/imunologia , Penaeidae/genética , Penaeidae/imunologia , Transcriptoma , Animais , Autofagia/efeitos dos fármacos , Perfilação da Expressão Gênica , Brânquias/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Imunidade Inata/genética , Sirolimo/farmacologia
9.
Mar Pollut Bull ; 133: 428-435, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30041332

RESUMO

Gold nanoparticles (AuNP) effects on Sparus aurata were evaluated on skin, gills and intestine by assessing the expression of immune genes and in peripheral blood evaluating genetic damage. Fish were exposed to 0.5 and 50 µg/L AuNP for 96 h. Results showed that exposure to 50 µg/L AuNP induced an upregulation in the expression of innate immune genes in gills (c3, lys, il1ß, tnfα, il6, il10 and tgfß) and intestine (il1ß, tnfα and il6). Furthermore, mRNA levels of hsp70 and hsp90 were increased in gills after exposure to 0.5 µg/L AuNP, when compared to 50 µg/L. Present data demonstrated the sensitivity of gills and intestines to AuNP exposure supporting their use in the study of fish responses to other nanoparticles. Genotoxic potential of AuNP was demonstrated by increased DNA strand breaks in red blood cells of fish exposed to AuNP, suggesting that AuNP represent a potential hazard to fish.


Assuntos
Proteínas de Peixes/genética , Nanopartículas Metálicas/toxicidade , Membrana Mucosa/efeitos dos fármacos , Dourada/imunologia , Animais , Dano ao DNA , Eritrócitos/efeitos dos fármacos , Proteínas de Peixes/imunologia , Brânquias/efeitos dos fármacos , Brânquias/imunologia , Ouro/química , Ouro/toxicidade , Interleucina-6/genética , Intestinos/efeitos dos fármacos , Intestinos/imunologia , RNA Mensageiro/metabolismo , Dourada/genética , Pele/efeitos dos fármacos , Pele/imunologia , Fator de Necrose Tumoral alfa/genética , Poluentes Químicos da Água/toxicidade
10.
Fish Shellfish Immunol ; 80: 22-30, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29859305

RESUMO

Kunitz-type serine protease inhibitor (KSPI) interacts with serine protease (SP) to regulate cascade reactions in vivo and plays essential roles in innate immunity. Theoretical considerations support various functions of kspi, but further studies are required for full characterization of these functions. In this study, a KSPI molecule was identified from Japanese flounder (Paralichthys olivaceus), and was named Pokspi. The full-length cDNA sequence of Pokspi was 2810 nt, containing an open reading frame of 1527 nt, which encoded a polypeptide of 509 amino acid residues. PoKspi protein contained five conversed domains, namely, MANEC, PKD, LDLa and two Kunitz domains. Homology analysis revealed that Pokspi shared the highest similarity (83%) with its homolog in Cynoglossus semilaevis. Phylogenetic analysis indicated that Pokspi clustered with the homologs in other fishes. The mRNA transcripts of Pokspi were detected in all tested tissues, with the highest expression level in gill, followed by kidney and intestine. Its elevated expression in response to the application of Edwardsiella tarda (in vivo) and pathogen-associated molecular pattern (in vitro) suggested the involvement of Pokspi in the essential immune defense against various pathogens. Recombinant PoKspi (rPoKspi) purified from Escherichia coli exhibited not only serine protease inhibitor activities but also a broad spectrum of anti-microbial effect in a manner that was independent of any host factors. In addition, the recombinant PoKspi protein could cause the down-regulation of pro-inflammatory factors TNF-α and IL-1ß. In conclusion, Pokspi is a biologically active serine protease inhibitor endowed with anti-bacterial and anti-inflammatory property. This study provides strong evidences for understanding the innate immune defense in Japanese flounder.


Assuntos
Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Linguado/genética , Linguado/imunologia , Inibidores de Serino Proteinase/genética , Inibidores de Serino Proteinase/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , DNA Complementar/genética , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Feminino , Doenças dos Peixes/imunologia , Brânquias/imunologia , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Positivas/imunologia , Imunidade Inata , Interleucina-1beta/genética , Intestinos/imunologia , Rim/imunologia , Masculino , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/genética
11.
Fish Shellfish Immunol ; 80: 397-404, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29859316

RESUMO

Gyrodactylus cichlidarum and Cichlidogyrus sclerosus, two monogenean ectoparasite species commonly found on the body surface and gills of Nile tilapia (Oreochromis niloticus) respectively, inflicted considerable economic losses in intensive tilapia farming. In order to explore the immune response of tilapia against these two species of monogeneans, expression patterns of five immune-related genes were studied after singular G. cichlidarum or C. sclerosus infection and their coinfection. The transcription levels of IL-1ß were up-regulated in the skin after G. cichlidarum infection, reaching a peak at day 5 PI, and in the gills after C. sclerosus infection (peaking at day 8 PI), with significant elevation only detected in the gills after high-dose C. sclerosus infection. A trend favoring increased gill TNF-α expression at day 8 PI of C. sclerosus infection was statistically significant only in the low-dose infection group. TNF-α expression in the skin did not change significantly after G. cichlidarum infection. TGF-ß had extremely up-regulated expressions in the gills at day 8 PI after both high- and low-dose C. sclerosus infections, but its significantly promoted expression in the skin was observed only after infection of high-dose G. cichlidarum. Significantly increased expressions of HSP70 and COX-2 in the skin were detected after high-dose G. cichlidarum infections. In comparison to singular infection with either G. cichlidarum or C. sclerosus, concurrent infection resulted in significantly advanced expression of TGF-ß in both skin and gills, and lower expressions at day 8 PI, and similar patterns were observed in the expression of IL-1ß and TNF-α in the gills. G. cichlidarum infection on the body surface significantly down-regulated the expressions of TNF-α, TGF-ß and COX-2 in the gills. In addition, the intensity of G. cichlidarum was significantly positively correlated with that of C. sclerosus (correlation index 0.922, p = 0.000) at day 2 PI under concurrent infection. These results contribute to the understanding of mucosal immunity of fish against monogenean infection, particularly when two monogenean species infect concurrently.


Assuntos
Ciclídeos/genética , Ciclídeos/imunologia , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Platelmintos , Infecções por Trematódeos/genética , Infecções por Trematódeos/imunologia , Animais , Ciclídeos/parasitologia , Coinfecção/genética , Coinfecção/imunologia , Ciclo-Oxigenase 2/genética , Citocinas/genética , Proteínas de Peixes/genética , Expressão Gênica/imunologia , Brânquias/imunologia , Proteínas de Choque Térmico HSP70/genética , Pele/imunologia , Infecções por Trematódeos/veterinária
12.
Dev Comp Immunol ; 87: 109-115, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29909090

RESUMO

Antimicrobial peptides (AMPs) play important roles in innate immunity against pathogens and lysozymes are a particularly type of AMP. Lysozymes are hydrolytic enzymes that are characterized by their ability to cleave the beta-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, which is the major bacterial cell wall polymer. In this work, a lysozyme was identified from Procambarus clarkii and designated PcLys-i3. Quantitative RT-PCR was used to analyze the tissue distribution and expression profiles of PcLys-i3. PcLys-i3 was present in all tested tissues and had high expression levels in gills, stomach and intestine. The expression levels of PcLys-i3 were up-regulated in gills and intestine after challenge with Vibrio parahaemolyticus, Staphylococcus aureus and Aeromonas hydrophila. PcLys-i3 and PcFer proteins can enhance the bacterial elimination in crayfish, whereas the bacterial elimination was weakened when the expression level of PcLys-i3 or PcFer RNAs was suppressed by RNAi. Recombinant PcLys-i3 and PcFer significantly reduced the mortality of crayfish with bacterial infections. Further study found that PcLys-i3 could interact with PcFer in vitro. Finally, the PcLys-i3 and PcFer proteins could bind to bacteria and inhibit bacterial replication. These results suggest that both PcLys-i3 and PcFer play important roles in the antibacterial immunity of red swamp crayfish.


Assuntos
Antibacterianos/imunologia , Proteínas de Artrópodes/imunologia , Astacoidea/imunologia , Muramidase/imunologia , Aeromonas hydrophila/imunologia , Aeromonas hydrophila/fisiologia , Animais , Antibacterianos/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Astacoidea/genética , Astacoidea/microbiologia , Perfilação da Expressão Gênica/métodos , Brânquias/imunologia , Brânquias/metabolismo , Brânquias/microbiologia , Interações Hospedeiro-Patógeno , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Intestinos/microbiologia , Muramidase/genética , Muramidase/metabolismo , Interferência de RNA , Staphylococcus aureus/imunologia , Staphylococcus aureus/fisiologia , Regulação para Cima , Vibrio/imunologia , Vibrio/fisiologia
13.
Fish Shellfish Immunol ; 80: 357-375, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29909274

RESUMO

Anti-lipopolysaccharide factor (ALF) is an immune-related protein that is crucially involved in immune defense mechanisms against invading pathogens in crustaceans. In the current study, three different ALFs of giant river prawn (Mr-ALF3, Mr-ALF8 and Mr-ALF9) were discovered. Based on sequence analysis, Mr-ALF3 and Mr-ALF9 were identified as new members of ALFs in crustaceans (groups F and G, respectively). Structurally, each newly identified Mr-ALF contained three α-helices packed against a four-stranded ß-sheet bearing the LPS-binding motif, which usually binds to the cell wall components of bacteria. Tissue expression analysis using quantitative real-time RT-PCR (qRT-PCR) demonstrated that Mr-ALF3 was expressed in most tissues, and the highest expression was in the heart and hemocytes. The Mr-ALF8 gene was highly expressed in the heart, hemocytes, midgut, hepatopacreas and hindgut, respectively, while the Mr-ALF9 gene was modestly expressed in the heart and hemocytes, respectively. The transcriptional responses of the Mr-ALFs to Aeromonas hydrophila and hot/cold temperatures were investigated by qRT-PCR in the gills, hepatopancreas and hemocytes. We found that all Mr-ALFs were clearly suppressed in all tested tissues when the experimental prawns were exposed to extreme temperatures (25 and 35 °C). Moreover, the expression levels of these genes were significantly induced in all examined tissues by 2 different concentrations of A. hydrophila (1 × 106 and 1 × 109 CFU/ml), particularly 12 and 96 h after the injection. Finally, binding activity analysis of LPS-motif peptides of each Mr-ALF revealed that the LPS peptide of Mr-ALF3 exhibited the strongest adhesion to two pathogenic Gram-negative bacteria, A. hydrophila and Vibrio harveyi, and the non-pathogenic Gram-positive Bacillus megaterium. The results also showed that the Mr-ALF8 and Mr-ALF9 peptides had mild antimicrobial effects against similar tested bacteria. Based on information obtained in this study, novel ALF genes were clearly identified. Analyses of their responses under pathogenic and temperature stresses demonstrated the binding and antimicrobial activities of these ALFs and the consequent physiological effects, indicating their crucial functional roles in the prawn immune system.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Proteínas de Artrópodes , Palaemonidae , Aeromonas hydrophila , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Bactérias/crescimento & desenvolvimento , Clonagem Molecular , Resposta ao Choque Frio/genética , Resposta ao Choque Frio/imunologia , DNA Complementar/genética , Expressão Gênica , Brânquias/imunologia , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/imunologia , Hemócitos/imunologia , Hepatopâncreas/imunologia , Lipopolissacarídeos , Palaemonidae/genética , Palaemonidae/imunologia , Filogenia , Estrutura Terciária de Proteína
14.
Fish Shellfish Immunol ; 80: 133-140, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29860069

RESUMO

Water temperature fluctuations are considered to be a major factor affecting the immune functions and metabolic processes of scallops. To better understand the immune defense mechanisms of Yesso scallop, Patinopecten yessoensis following exposure to water temperature fluctuations, transcriptomic profiles in the gills from high-frequency fluctuations (HF_G), low-frequency fluctuations (LF_G), and no fluctuations (NF_G) groups were obtained using HiSeq™ 2500 (Illumina). For HF_G, scallops were transferred directly between 18 and 8 °C every 4 h and for 10 fluctuations, while scallops in LF_G were transferred between 18 and 13 °C every 12 h, for a total of 4 fluctuations. A total of 442,922,590 clean reads were generated in 9 libraries and then assembled into 210,780 unigenes with an average length of 705 bp and an N50 of 1253 bp. Based on sequence similarity, 54,529 unigenes (25.87%) were annotated in at least one database. Comparative analysis revealed that 696 unigenes differentially expressed in temperature stressed groups compared with the control, including 229 unigenes between HF_G and NF_G, and 548 unigenes between LF_G and NF_G, respectively. Additionally, among these differentially expressed genes (DEGs), there were 41 immune-related unigenes and 16 protein metabolism-related unigenes. These results provide fundamental information on the molecular defense mechanisms in the Yesso scallop gills after exposure to water temperature fluctuations.


Assuntos
Brânquias/imunologia , Brânquias/metabolismo , Pectinidae/genética , Estresse Fisiológico/genética , Temperatura Ambiente , Animais , Perfilação da Expressão Gênica , Pectinidae/imunologia , Pectinidae/metabolismo , Estresse Fisiológico/imunologia , Estresse Fisiológico/fisiologia , Água
15.
Fish Shellfish Immunol ; 80: 335-347, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29920382

RESUMO

Tumor necrosis factor receptor-associated factor 6 (TRAF6) acts as a central intracellular signal adapter molecule that mediates the tumor necrosis factor receptor superfamily and the interleukin-1 receptor/Toll-like receptor family in vertebrates and invertebrates. In the present study, HcTRAF6, a molluscan homologue of TRAF6 from Hyriopsis cumingii, has been cloned and identified. The entire open reading frame of HcTRAF6 was found to comprise a 1965-bp region that encodes a predicted protein of 654 amino acids, which contains conserved characteristic domains including a RING domain, two TRAF-type zinc finger domains, a typical coiled coil and the MATH domain. Phylogenetic analysis revealed that HcTRAF6 was aggregated closely with CsTRAF6 from Cyclina sinensis in the invertebrate cluster of mollusks. Further, qRT-PCR analysis showed that HcTRAF6 mRNA was extensively distributed in mussel tissues with a high expression in gills. After immune stimulation with Aeromonas hydrophila and lipopolysaccharides, the transcription of HcTRAF6 was obviously induced in the gills and hemocytes. In addition, significant fluctuation in HcTRAF6 expression was observed in the pearl sac, gills and hemocytes after mantle implantation. These findings confirmed its role in the alloimmune response. Dual-luciferase reporter assay showed that over-expression of HcTRAF6 could enhance the activity of the NF-κB reporter in a dose-dependent manner. Further, the RNA interference showed that the up-regulation of antimicrobial peptides in anti-bacterial infection was strongly suppressed in HcTRAF6-silenced mussels and that depletion of HcTRAF inhibited the elimination of A. hydrophila. All these findings together prove that HcTRAF6 functions as an efficient regulator in innate immune mechanisms against invading pathogens and the alloimmune mechanism after mantle implantation in H. cumingii.


Assuntos
Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/imunologia , Unionidae/genética , Unionidae/imunologia , Aeromonas hydrophila , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Brânquias/imunologia , Células HEK293 , Hemócitos/imunologia , Humanos , Imunidade Inata , Lipopolissacarídeos , Filogenia , RNA Interferente Pequeno/genética
16.
Dev Comp Immunol ; 86: 9-16, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29723812

RESUMO

Among its other physiological roles, C-type lectins functioned as pattern recognition receptors (PRR) in innate immunity received much attention. In the present study, a novel C-type lectin was identified and characterized from the invertebrate razor clam Sinonovacula constrict and designated as ScCTL. The complete cDNA sequence of ScCTL was 828 bp in length and coded a secreted polypeptide of 158 amino acids with a typical CRD domain. Multiple sequence alignments combined with phylogenetic analysis both collectively confirmed that ScCTL was a novel member belong to lectin family. Spatial expression distribution analysis revealed that ScCTL was extensively expressed in all of the examined tissues, and the highest expression was detected in the hepatopancreas. After 1 × 107 CFU/mL Vibrio parahaemolyticus challenge by immersion infection, the ScCTL transcript in hepatopancreas and gill were markedly upregulated and arrived the maximum levels at 24 or 12 h after challenge, respectively. Recombinant ScCTL could agglutinate not only all tested bacteria but sheep and mouse erythrocyte in the presence of Ca2+. All of our studies suggested that ScCTL performed important roles in protecting cells from pathogenic infection in S. constrict.


Assuntos
Aglutinação/imunologia , Bactérias/imunologia , Bivalves/metabolismo , Cálcio/metabolismo , Eritrócitos/imunologia , Lectinas Tipo C/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/metabolismo , Brânquias/imunologia , Hepatopâncreas/imunologia , Imunidade Inata/imunologia , Imunidade Inata/fisiologia , Camundongos , Filogenia , Receptores de Reconhecimento de Padrão/imunologia , Alinhamento de Sequência , Ovinos/imunologia , Vibrio parahaemolyticus/imunologia
17.
Fish Shellfish Immunol ; 78: 109-113, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29684599

RESUMO

Shrimp, as invertebrates, have an open vasculature that allows circulating hemocytes to infiltrate the tissues, where they are referred to as sessile hemocytes. Sessile hemocytes are known to express immune-related genes, but it is not known whether their functions differ from those of circulating hemocytes. To answer this question, we enriched them from suspensions of different tissues using discontinuous density gradient centrifugation and analyzed their transcripts by RNA-seq. The results suggest that circulating hemocytes and sessile hemocytes of the gills are in a state that could react quickly to pathogens, immune-related genes expression of sessile hemocytes differ from circulating hemocytes, and the gills, heart and lymphoid organs have cells that express immune-related genes that are different from hemocytes.


Assuntos
Centrifugação com Gradiente de Concentração/métodos , Hemócitos/metabolismo , Penaeidae/imunologia , Animais , Proteínas de Artrópodes/metabolismo , Brânquias/imunologia , Brânquias/metabolismo , Hemócitos/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Miocárdio/imunologia , Miocárdio/metabolismo , Penaeidae/metabolismo , Análise de Sequência de RNA
18.
Dev Comp Immunol ; 85: 134-141, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29680689

RESUMO

Lysozymes possess antibacterial activities, making them crucial defense proteins in innate immunity. In this study, a chicken-type (c-type) lysozyme (designated PcLyzc) was cloned and characterized from red swamp crayfish Procambarus clarkii. The full-length cDNA had an open reading frame of 435 base pairs encoding a polypeptide of 144 amino acid residues. Multiple alignments and phylogenetic analysis revealed that PcLyzc shared high similarity to the other known invertebrate c-type lysozymes. PcLyzc transcripts were steadily expressed in a wide range of tissues in healthy crayfish, and were prominently up-regulated in the hepatopancreas and gills after Vibrio anguillarum or Aeromonas hydrophila challenge. Recombinant PcLyzc showed inhibitory activity in vitro against both Gram-positive bacteria, including Staphylococcus aureus, Micrococcus luteus and Bacillus thuringiensis, and Gram-negative bacteria, including A. hydrophila, V. anguillarum and Escherichia coli. By overexpressing PcLyzc through introducing exogenous recombinant protein, or silencing PcLyzc expression through injecting double strand RNA, it was found that PcLyzc could help eliminate the invading bacteria in crayfish hemolymph and could protect crayfish from death, possibly by promoting the hemocytic phagocytosis. These results indicated that PcLyzc played a role in the antibacterial immunity of crustaceans, and laid a foundation of developing new therapeutic agents in aquaculture.


Assuntos
Antibacterianos/imunologia , Astacoidea/imunologia , Galinhas/metabolismo , Imunidade Inata/imunologia , Muramidase/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Brânquias/imunologia , Brânquias/microbiologia , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Positivas/imunologia , Hemócitos/imunologia , Hemócitos/microbiologia , Hepatopâncreas/imunologia , Hepatopâncreas/microbiologia , Peptídeos/imunologia , Filogenia , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Vibrio/imunologia
19.
Dev Comp Immunol ; 85: 61-70, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29649551

RESUMO

The receptor for activated C kinase 1 (RACK1) is a intracellular receptor for the protein kinase C family which mediates various biological processes. Here, a novel RACK1 gene termed Mc-RACK1 was identified from thick shell mussel, Mytilus coruscus. Mc-RACK1 shared typical RACK1 domains containing WD repeats, PKC phosphorylation sites, N-myristoylation sites, PKC activation sites, TK phosphorylation site and WD motifs. Mc-RACK1 was constitutively expressed in all examined tissues, and its expression in gills, haemocytes and digestive glands were significantly up-regulated upon LPS challenge. Mc-RACK1 showed a significantly down-regulated expression in gills and haemocytes at the early phase upon copper exposure. Mc-RACK1 in haemocytes was silenced after receiving its dsRNA, meanwhile, the increases of SOD and CAT activity were investigated. Further, Mc-RACK1 could activate the NF-κB and ISRE reporter in HEK-293T cells. These suggested that Mc-RACK1 had a deeper involvement in mollusc immunity, and played an important role in antioxidant system.


Assuntos
Mytilus/imunologia , Mytilus/metabolismo , Receptores de Quinase C Ativada/imunologia , Animais , Antioxidantes/metabolismo , Sistema Digestório/imunologia , Sistema Digestório/metabolismo , Regulação para Baixo/imunologia , Brânquias/imunologia , Brânquias/metabolismo , Células HEK293 , Humanos , NF-kappa B/metabolismo , Superóxido Dismutase/metabolismo , Regulação para Cima/imunologia
20.
Fish Shellfish Immunol ; 74: 637-648, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29360541

RESUMO

This study was conducted to investigate the effects of methionine hydroxy analogue (MHA) on the physical barrier and immune defence in the gill of young grass carp (Ctenopharyngodon idella). A total 630 young grass carp with an average initial weight of 259.70 ±â€¯0.47 g were fed graded levels of MHA (0, 2.4, 4.4, 6.4, 8.5 and 10.5 g/kg diet) and one DL-methionine (DLM) group (6.4 g/kg diet) for 8 weeks. After feeding trial, 15 fish from each treatment were challenged with Flavobacterium columnare. Compared to the basal diet, optimal MHA improved cellular structure integrity of gill via repressing death receptor and mitochondria pathways induced apoptosis, which might be related to the down-regulation of c-Jun-N-terminal kinase mRNA levels (P < .05). Simultaneously, optimal MHA supplementation improved cellular structure integrity of gill via elevating glutathione contents, antioxidant enzymes activities and corresponding isoforms mRNA levels to attenuate oxidative damage, which might be to the up-regulation of NF-E2-related factor 2 mRNA levels and down-regulation of Kelch-like ECH-associating protein 1a mRNA levels (P < .05). Besides, optimal MHA improved intercellular structure integrity of immune organs via up-regulating the mRNA levels of intercellular tight junctions-related genes, which might be owing to the down-regulation of myosin light chain kinase (MLCK) mRNA levels (P < .05). Summarily, MHA could improve the physical barrier of fish gill. In addition, optimal MHA supplementation increased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M contents and up-regulated mRNA levels of liver-expressed antimicrobial peptide 2, hepcidin and ß-defensin, suggesting that MHA could enhance antimicrobial ability of fish gill. Meanwhile, optimal MHA supplementation enhanced the immune defence of gill via down-regulating pro-inflammatory cytokines mRNA levels and up-regulated anti-inflammatory cytokines mRNA levels, which might be attributed to the down-regulation of nuclear factor κB p65, c-Rel, IκB kinase ß, p38 mitogen activated protein kinase, eIF4E-binding protein1 (4E-BP1) and 4E-BP2 mRNA levels and up-regulation of inhibitor of κBα, ribosomal protein S6 kinase 1 and target of rapamycin mRNA levels (P < .05). In conclusion, the positive effect of MHA on gill health is associated with the improvement of the defence against apoptosis, antioxidant status, tight junctions and immune defence of fish gill. Meanwhile, MHA was superior to DLM on improving the physical barrier of fish gill. For the direction to healthy breeding of young grass carp, the optimal MHA supplementation levels on the premise of 4.01 g/kg methionine basal were estimated by quadratic regression curve, such as 5.49, 6.17 and 6.02 g/kg diet bases on the defence against gill-rot, malondialdehyde content and LZ activity in the gill, respectively.


Assuntos
Carpas/imunologia , Carpas/metabolismo , Doenças dos Peixes/imunologia , Imunidade Inata/efeitos dos fármacos , Metionina/análogos & derivados , Ração Animal/análise , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Dieta/veterinária , Suplementos Nutricionais/análise , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Infecções por Flavobacteriaceae/imunologia , Flavobacterium/fisiologia , Brânquias/enzimologia , Brânquias/imunologia , Metionina/administração & dosagem , Metionina/metabolismo , Distribuição Aleatória , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo
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