RESUMO
Prohibitin 1 (PHB1) is ubiquitously expressed in multiple compartments within cells and is involved in the cell cycle, cell signaling, apoptosis, transcriptional regulation, and mitochondrial biogenesis at the cellular level and in the inflammation-associated and immunological functions of B and T lymphocytes. PHB1 is an important protein that performs antioxidant regulation and immune functions inside and outside cells but has not been sufficiently studied in teleost fish. Our study aimed to elucidate the functional properties and gain new insights into the biological processes and immune system of red seabream (Pagrus major), a commercially important fish cultured in South Korea and East Asia. PHB1 mRNA was most abundantly expressed in the head kidney of healthy red seabream, and significant changes in its expression were observed after artificial infection with bacteria and viruses. On analysis, reporter gene was also significantly upregulated by polyinosinic-polycytidylic acid, lipopolysaccharides, and hydrogen peroxide. Consequent to the functional characterization of PHB1 in cells via recombinant protein preparation, the activity of leukocytes was enhanced and the reactive oxygen species-induced stress in red blood cells was reduced. The results reveal the functional characteristics of PHB1 and provide new insights into the biological processes and immune system of P. major, with beneficial implications in the study of stress responses.
Assuntos
Doenças dos Peixes , Proteínas de Peixes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Imunidade Inata , Proibitinas , Proteínas Repressoras , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/imunologia , Doenças dos Peixes/imunologia , Imunidade Inata/genética , Regulação da Expressão Gênica/imunologia , Perfilação da Expressão Gênica/veterinária , Poli I-C/farmacologia , Filogenia , Dourada/imunologia , Dourada/genética , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Sequência de Aminoácidos , Alinhamento de Sequência/veterinária , Lipopolissacarídeos/farmacologia , Perciformes/imunologia , Perciformes/genética , Iridoviridae/fisiologia , Vibrio/fisiologiaRESUMO
Astaxanthin (AX) is a carotenoid known to have one of the highest documented antioxidant capacities and has attracted considerable scientific and commercial interest. The incorporation of AX into aquaculture practices has been associated with improved pigmentation, modulation of the immune and endocrine systems, stress reduction, reproductive efficiency and general fish health. This study describes the effects of dietary AX (0, control, 20, 100 and 500 mg kg-1 AX per kg of diet) for 15 and 30 days on growth performance, immune and antioxidant status, histology and gene expression in gilthead seabream (Sparus aurata). Fish fed diets enriched with 500 mg kg-1 of AX for 15 days decreased in skin mucus peroxidase activity while at 30 days of trial, fish fed a diet supplemented with 20 mg kg-1 AX increased the peroxidase activity in serum. In addition, bactericidal activity against Vibrio harveyi increased in the skin mucus of fish fed any of the AX supplemented diets. Regarding antioxidant activities in the liver, catalase and glutathione reductase were decreased and increased, respectively, in fish fed a diet supplemented with 500 mg kg-1 of AX. Finally, although the expression of up to 21 inflammatory and lipid metabolism-related genes was analysed in visceral adipose tissue, only the expression of the interleukin 6 (il6) gene was up-regulated in fish fed a diet supplemented with 20 mg kg-1 of AX. The present results provide a detailed insight into the potent antioxidant properties of AX and its possible modulatory effects on the immune status and lipid metabolism of seabream, which may be of interest to the aquaculture sector.
Assuntos
Ração Animal , Dieta , Suplementos Nutricionais , Metabolismo dos Lipídeos , Dourada , Xantofilas , Animais , Dourada/imunologia , Dourada/crescimento & desenvolvimento , Dourada/metabolismo , Xantofilas/administração & dosagem , Xantofilas/farmacologia , Ração Animal/análise , Dieta/veterinária , Suplementos Nutricionais/análise , Metabolismo dos Lipídeos/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Relação Dose-Resposta a Droga , Antioxidantes/metabolismo , Distribuição Aleatória , VibrioRESUMO
IRF9 can play an antibacterial role by regulating the type I interferon (IFN) pathway. Streptococcus iniae can cause many deaths of yellowfin seabream, Acanthopagrus latus in pond farming. Nevertheless, the regulatory mechanism of type I IFN signalling by A. latus IRF9 (AlIRF9) against S. iniae remains elucidated. In our study, AlIRF9 has a total cDNA length of 3200 bp and contains a 1311 bp ORF encoding a presumed 436 amino acids (aa). The genomic DNA sequence of AlIRF9 has nine exons and eight introns, and AlIRF9 was expressed in various tissues, containing the stomach, spleen, brain, skin, and liver, among which the highest expression was in the spleen. Moreover, AlIRF9 transcriptions in the spleen, liver, kidney, and brain were increased by S. iniae infection. By overexpression of AlIRF9, AlIRF9 is shown as a whole-cell distribution, mainly concentrated in the nucleus. Moreover, the promoter fragments of -415 to +192 bp and -311 to +196 bp were regarded as core sequences from two AlIFNa3s. The point mutation analyses verified that AlIFNa3 and AlIFNa3-like transcriptions are dependent on both M3 sites with AlIRF9. In addition, AlIRF9 could greatly reduce two AlIFNa3s and interferon signalling factors expressions. These results showed that in A. latus, both AlIFNa3 and AlIFNa3-like can mediate the regulation of AlIRF9 in the process of infection with S. iniae.
Assuntos
Doenças dos Peixes , Proteínas de Peixes , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Dourada , Infecções Estreptocócicas , Streptococcus iniae , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Infecções Estreptocócicas/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Dourada/genética , Dourada/imunologia , Dourada/microbiologia , Streptococcus iniae/fisiologia , Regiões Promotoras Genéticas/genética , Transdução de Sinais , Regulação da Expressão Gênica , Imunidade Inata/genéticaRESUMO
Silk fibroin (SF) microparticles were administered in the diet of gilthead seabream with or without experimental skin wounds to study the effects on cellular immunity and liver. A commercially available diet was enriched with varying amount of SF: 0, 50 and 100 mg kg-1 (representing the control, SF50 and SF100 diets respectively). The animals were fed for 30 days and half of them were sampled. Similar experimental wounds were then performed on the rest of fish, and they continued to be fed the same diet. At 7 days post-wounding, samples were taken from the wounded fish. Cellular immunity was studied on head kidney leucocytes (phagocytosis, respiratory and peroxidase content) and liver status (histological study and gene expression) were studied. Our results showed that experimental wounds affect both cellular immunity (by decreasing leucocyte respiratory burst and peroxidase activity) and altered liver histology (by inducing vascularisation and congestion of blood vessels). In addition, it influences the expression of genes that serve as markers of oxidative stress, endoplasmic reticulum stress and apoptosis. The highest dose of SF (SF100) increased the phagocytic capacity of leucocytes the most, as well as the expression of genes related to blood vessel formation in the liver. Furthermore, increased expression of antioxidant genes (cat and gsr) and decreased expression of genes related to reticulum endoplasmic stress (grp94 and grp170) and apoptosis (nos and jnk) were detected in these fish fed with SF100 and wounded. In conclusion, fed fish with SF100 had many beneficial effects as cellular immunostimulant and hepatoprotection in wounded fish. Its use could be of great interest for stress management in farmed fish conditions.
Assuntos
Fibroínas , Imunidade Celular , Fígado , Dourada , Animais , Fígado/efeitos dos fármacos , Dourada/imunologia , Fibroínas/química , Imunidade Celular/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/lesões , Regulação da Expressão Gênica/efeitos dos fármacos , Ração Animal/análise , Dieta/veterináriaRESUMO
The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. While the expression of most of the genes studied was upregulated, the expression of ferroportin gene (slc40a) was downregulated. In the second part of the study, seabream specimens were injected intramuscularly with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b, and ferroportin genes (hamp1, fthb, and slc40a) was upregulated in the head kidney of fish from the λ-carrageenin-injected group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was downregulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J, and Hamp2D (hamp2.5/6) genes was downregulated in the λ-carrageenin-injected group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was upregulated in the λ-carrageenin-injected group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. The primary sequence of hepcidin was conserved among the different mature peptides, although changes in specific amino acid residues were identified. These changes affected the charge, hydrophobicity, and probability of hepcidins being antimicrobial peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.
Assuntos
Proteínas de Peixes , Hepcidinas , Inflamação , Dourada , Animais , Dourada/genética , Dourada/metabolismo , Dourada/imunologia , Hepcidinas/genética , Hepcidinas/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Inflamação/genética , Inflamação/metabolismo , Fígado/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/genética , Doenças dos Peixes/metabolismo , Rim Cefálico/metabolismo , Ferro/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Leucócitos/metabolismo , Leucócitos/efeitos dos fármacos , Pele/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Regiões Promotoras GenéticasRESUMO
Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing Phdp (1 × 106 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.
Assuntos
Imunidade , Photobacterium/fisiologia , Dourada/imunologia , Dourada/microbiologia , Animais , Células Sanguíneas/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Rim Cefálico/metabolismo , Imunidade Humoral/genética , Imunidade Inata/genética , Estresse Oxidativo/genética , Dourada/sangue , Dourada/genéticaRESUMO
Stimulation of the fish immune system using immunostimulants is an environmentally friendly strategy to minimize bacterial outbreaks in aquaculture. Different biological and synthetic immunostimulants can enhance non-specific innate immune responses by directly activating immune cells. An example are Bacillus spp., known for their immunostimulatory effects, although the exact mechanisms by which Bacillus spp. offer protection against diseases remains to be elucidated. Furthermore, most studies have focused on Bacillus spp. cells, while the immunostimulant effect of their extracellular metabolome, known to harbour biologically important metabolites, including antimicrobial molecules, has been scarcely evaluated. Here, we evaluated the in vitro immune-modulatory properties of extracellular extracts of three Bacillus spp. strains (B. subtilis FI314, B. vezelensis FI436 and B. pumilus FI464), previously isolated from fish-guts and characterized for their in vitro and in vivo antimicrobial activity against a wide range of fish pathogens. Bacillus spp. extracellular extracts did not affect immune cells viability, but remarkably increased pathogens' phagocytosis when seabream head-kidney leukocytes were challenged with Vibrio anguillarum and Edwardsiella tarda. All extracts significantly increased the engulfment of bacterial pathogens 1 h post-infection. Cells stimulated with the extracellular extracts showed an up-regulation of the expression of immune-relevant genes associated with inflammation, including IL-1ß, IL-6, and COX-2. In cells challenged with E. tarda, FI314 extracellular extract significantly increased the expression of IL-1ß, IL-6, and COX-2, while FI436 and FI464 significantly increased IL-6 expression. The results of this study revealed that the extracellular molecules from Bacillus spp. fish isolates improved the in vitro response of gilthead seabream immune cells and are thus promising candidates to act as immunostimulants, helping fish fight diseases.
Assuntos
Bacillus , Doenças dos Peixes , Leucócitos/imunologia , Dourada , Adjuvantes Imunológicos , Animais , Bacillus/química , Ciclo-Oxigenase 2/genética , Interleucina-1beta/genética , Interleucina-6/genética , Dourada/imunologiaRESUMO
The protozoan parasite Cryptocaryon irritans causes marine white spot disease in a wide range of fish hosts, including gilthead seabream, a very sensitive species with great economic importance in the Mediterranean area. Thus, we aimed to evaluate the immunity of gilthead seabream after a severe natural outbreak of C. irritans. Morphological alterations and immune cell appearance in the gills were studied by light microscopy and immunohistochemical staining. The expression of several immune-related genes in the gills and head kidney were studied by qPCR, including inflammatory and immune cell markers, antimicrobial peptides (AMP), and cell-mediated cytotoxicity (CMC) molecules. Serum humoral innate immune activities were also assayed. Fish mortality reached 100% 8 days after the appearance of the C. irritans episode. Gill filaments were engrossed and packed without any space between filaments and included parasites and large numbers of undifferentiated and immune cells, namely acidophilic granulocytes. Our data suggest leukocyte mobilization from the head kidney, while the gills show the up-regulated transcription of inflammatory, AMPs, and CMC-related molecules. Meanwhile, only serum bactericidal activity was increased upon infection. A potent local innate immune response in the gills, probably orchestrated by AMPs and CMC, is triggered by a severe natural outbreak of C. irritans.
Assuntos
Infecções por Cilióforos/veterinária , Cilióforos/imunologia , Imunidade Inata , Dourada/crescimento & desenvolvimento , Animais , Cilióforos/patogenicidade , Infecções por Cilióforos/genética , Infecções por Cilióforos/imunologia , Surtos de Doenças , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/parasitologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Brânquias/imunologia , Brânquias/parasitologia , Imuno-Histoquímica , Microscopia , Dourada/genética , Dourada/imunologia , Dourada/parasitologiaRESUMO
In fish culture settings, the exogenous input of steroids is a matter of concern. Recently, we unveiled that in the gilthead seabream (Sparus aurata), the G protein-coupled estrogen receptor agonist G-1 (G1) and the endocrine disruptor 17α-ethinylestradiol (EE2) are potent modulators in polyreactive antibody production. However, the integral role of the microbiota upon immunity and antibody processing in response to the effect of EE2 remains largely unexplored. Here, juvenile seabreams continuously exposed for 84 days to oral G1 or EE2 mixed in the fish food were intraperitoneally (i.p.) immune primed on day 42 with the model antigen keyhole limpet hemocyanin (KLH). A critical panel of systemic and mucosal immune markers, serum VTG, and humoral, enzymatic, and bacteriolytic activities were recorded and correlated with gut bacterial metagenomic analysis 1 day post-priming (dpp). Besides, at 15 dpp, animals received a boost to investigate the possible generation of specific anti-KLH antibodies at the systemic and mucosal interphases by the end of the trial. On day 43, EE2 but not G1 induced a significant shift in the serum VTG level of naive fish. Simultaneously, significant changes in some immune enzymatic activities in the serum and gut mucus of the EE2-treated group were recorded. In comparison, the vaccine priming immunization resulted in an attenuated profile of most enzymatic activities in the same group. The gut genes qPCR analysis exhibited a related pattern, only emphasized by a significant shift in the EE2 group's il1b expression. The gut bacterial microbiome status underwent 16S rRNA dynamic changes in alpha diversity indices, only with the exposure to oral G1, supporting functional alterations on cellular processes, signaling, and lipid metabolism in the microbiota. By the same token, the immunization elevated the relative abundance of Fusobacteria only in the control group, while this phylum was depleted in both the treated groups. Remarkably, the immunization also promoted changes in the bacterial class Betaproteobacteria and the estrogen-associated genus Novosphingobium. Furthermore, systemic and mucosal KLH-specific immunoglobulin (Ig)M and IgT levels in the fully vaccinated fish showed only slight changes 84 days post-estrogenic oral administration. In summary, our results highlight the intrinsic relationship among estrogens, their associated receptors, and immunization in the ubiquitous fish immune regulation and the subtle but significant crosstalk with the gut endobolome.
Assuntos
Etinilestradiol/toxicidade , Microbioma Gastrointestinal/imunologia , Receptores de Estrogênio/imunologia , Receptores Acoplados a Proteínas G/imunologia , Dourada/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Disruptores Endócrinos/toxicidade , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Hemocianinas/imunologia , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Dourada/metabolismo , VacinaçãoRESUMO
The present study aimed to elucidate the mechanism of dietary betaine, as a lipid-lowering substance, on the regulation of lipid metabolism and inflammation in juvenile black seabream (Acanthopagrus schlegelii) fed a high fat diet. An 8-week feeding trial was conducted in black seabream with an initial weight of 8.39 ± 0.01g fed four isonitrogenous diets including Control, medium-fat diet (11%); HFD, high-fat diet (17%); and HFD supplemented with two levels (10 and 20 g/kg) of betaine, HFD+B1 and HFD+B2, respectively. SGR and FE in fish fed HFD+B2 were significantly higher than in fish fed HFD. Liver histology revealed that vacuolar fat droplets were smaller and fewer in bream fed HFD supplemented with betaine compared to fish fed HFD. Betaine promoted the mRNA and protein expression levels of silent information regulator 1 (Sirt1), up-regulated mRNA expression and protein content of lipid peroxisome proliferator-activated receptor alpha (pparα), and down-regulated mRNA expression and protein content of sterol regulatory element-binding protein-1(srebp-1). Furthermore, the mRNA expression levels of anti-inflammatory cytokines in liver and intestine were up-regulated, while nuclear factor kB (nf-kb) and pro-inflammatory cytokines were down-regulated by dietary betaine supplementation. Likewise, in fish that received lipopolysaccharide (LPS) to stimulate inflammatory responses, the expression levels of mRNAs of anti-inflammatory cytokines in liver, intestine and kidney were up-regulated in fish fed HFD supplemented with betaine compared with fish fed HFD, while nf-kb and pro-inflammatory cytokines were down-regulated. This is the first report to suggest that dietary betaine could be an effective feed additive to alleviate hepatic steatosis and attenuate inflammatory responses in black seabream fed a high fat diet by modulating the Sirt1/Srebp-1/PparÉ pathway.
Assuntos
Betaína/administração & dosagem , Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Fígado Gorduroso/veterinária , Doenças dos Peixes/prevenção & controle , Proteínas de Peixes/metabolismo , Inflamação/veterinária , Fígado/enzimologia , PPAR alfa/metabolismo , Dourada/metabolismo , Sirtuína 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Fatores Etários , Ração Animal , Animais , Citocinas/genética , Citocinas/metabolismo , Fígado Gorduroso/enzimologia , Fígado Gorduroso/imunologia , Fígado Gorduroso/prevenção & controle , Doenças dos Peixes/enzimologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Fígado/imunologia , PPAR alfa/genética , Dourada/genética , Dourada/imunologia , Sirtuína 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genéticaRESUMO
This study investigated the acute inflammatory response induced by subcutaneous injection of carrageenin (1%) or phosphate-buffered saline (control) in gilthead seabream (Sparus aurata). Skin mucus, serum, head kidney (HK) and liver were sampled at 1.5, 3 and 6 hr post-injection (p.i.) to determine the immune and antioxidant status of this fish species. The skin mucus of the carrageenin group showed increased superoxide dismutase and peroxidase activities, lysozyme abundance, bactericidal activity against Vibrio anguillarum and Photobacterium damselae, and total immunoglobulins compared with those of the control group. However, the carrageenin-injected fish sampled at 6 hr p.i. showed decreased protease activity in the skin mucus and peroxidase activity in the HK leucocytes compared with the control. Moreover, the carrageenin injection had no effects on the systemic immune system, but it reduced the liver catalase activities at both 3 and 6 hr in the carrageenin group relative to those in the control group. The expression levels of several proinflammatory and cell marker genes in the HK and liver were also determined. In the HK, the expression levels of interleukin-1ß and prostaglandin D synthase 1 were upregulated at 1.5 and 3 hr, respectively, in the carrageenin group compared with those in the control group. Contrarily, the expression of the NADPH oxidase subunit phox40 (an acidophilic granulocyte marker) in the carrageenin group at 6 hr was downregulated compared with that in the control group. These results suggested that subcutaneous injection of κ/λ-carrageenin in gilthead seabream triggered an acute skin inflammation characterized by the rapid recruitment of acidophilic granulocytes and the release of humoral mediators into the skin mucus.
Assuntos
Antioxidantes/metabolismo , Dourada/imunologia , Pele/metabolismo , Animais , Carragenina/administração & dosagem , Regulação da Expressão Gênica , Rim Cefálico/metabolismo , Imunidade Humoral , Injeções Subcutâneas , Fígado/enzimologia , Muco/metabolismo , Photobacterium/efeitos dos fármacos , Dourada/metabolismo , Vibrio/efeitos dos fármacosRESUMO
The inclusion of a medicinal plant leaf extract (MPLE) from sage (Salvia officinalis) and lemon verbena (Lippia citriodora), rich in verbascoside and triterpenic compounds like ursolic acid, was evaluated in gilthead seabream (Sparus aurata) fed a low fishmeal-based diet (48% crude protein, 17% crude fat, 21.7 MJ kg-1, 7% fishmeal, 15% fish oil) for 92 days. In particular, the study focused on the effect of these phytogenic compounds on the gut condition by analyzing the transcriptomic profiling (microarray analysis) and histological structure of the intestinal mucosa, as well as the histochemical properties of mucins stored in goblet cells. A total number of 506 differentially expressed genes (285 up- and 221 down-regulated) were found when comparing the transcriptomic profiling of the intestine from fish fed the control and MPLE diets. The gut transcripteractome revealed an expression profile that favored biological mechanisms associated to the 1) immune system, particularly involving T cell activation and differentiation, 2) gut integrity (i.e., adherens and tight junctions) and cellular proliferation, and 3) cellular proteolytic pathways. The histological analysis showed that the MPLE dietary supplementation promoted an increase in the number of intestinal goblet cells and modified the composition of mucins' glycoproteins stored in goblet cells, with an increase in the staining intensity of neutral mucins, as well as in mucins rich in carboxylated and weakly sulfated glycoconjugates, particularly those rich in sialic acid residues. The integration of transcriptomic and histological results showed that the evaluated MPLE from sage and lemon verbena is responsible for the maintenance of intestinal health, supporting gut homeostasis and increasing the integrity of the intestinal epithelium, which suggests that this phytogenic may be considered as a promising sustainable functional additive for aquafeeds.
Assuntos
Imunidade nas Mucosas/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Junções Intercelulares/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Extratos Vegetais/farmacologia , Salvia officinalis , Dourada , Linfócitos T/efeitos dos fármacos , Verbenaceae , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/imunologia , Células Caliciformes/metabolismo , Fatores Imunológicos/isolamento & purificação , Junções Intercelulares/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Mucinas/metabolismo , Permeabilidade/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Folhas de Planta , Salvia officinalis/química , Dourada/genética , Dourada/imunologia , Dourada/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Transcriptoma , Verbenaceae/químicaRESUMO
Essential oils (EOs) are promising alternatives to chemotherapeutics in animal production due to their immunostimulant, antimicrobial, and antioxidant properties, without associated environmental or hazardous side effects. In the present study, the modulation of the transcriptional immune response (microarray analysis) and microbiota [16S Ribosomal RNA (rRNA) sequencing] in the intestine of the euryhaline fish gilthead seabream (Sparus aurata) fed a dietary supplementation of garlic, carvacrol, and thymol EOs was evaluated. The transcriptomic functional analysis showed the regulation of genes related to processes of proteolysis and inflammatory modulation, immunity, transport and secretion, response to cyclic compounds, symbiosis, and RNA metabolism in fish fed the EOs-supplemented diet. Particularly, the activation of leukocytes, such as acidophilic granulocytes, was suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the gut. Fish growth performance and gut microbiota alpha diversity indices were not affected, while dietary EOs promoted alterations in bacterial abundances in terms of phylum, class, and genus. Subtle, but significant alterations in microbiota composition, such as the decrease in Bacteroidia and Clostridia classes, were suggested to participate in the modulation of the intestine transcriptional immune profile observed in fish fed the EOs diet. Moreover, regarding microbiota functionality, increased bacterial sequences associated with glutathione and lipid metabolisms, among others, detected in fish fed the EOs supported the metabolic alterations suggested to potentially affect the observed immune-related transcriptional response. The overall results indicated that the tested dietary EOs may promote intestinal local immunity through the impact of the EOs on the host-microbial co-metabolism and consequent regulation of significant biological processes, evidencing the crosstalk between gut and microbiota in the inflammatory regulation upon administration of immunostimulant feed additives.
Assuntos
Bactérias/efeitos dos fármacos , Suplementos Nutricionais , Microbioma Gastrointestinal/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Óleos Voláteis/administração & dosagem , Dourada , Transcriptoma/efeitos dos fármacos , Compostos Alílicos/administração & dosagem , Ração Animal , Animais , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Cimenos/administração & dosagem , Dieta , Combinação de Medicamentos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Imunidade Inata/genética , Imunidade nas Mucosas/genética , Intestinos/imunologia , Intestinos/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos , Ribotipagem , Dourada/genética , Dourada/imunologia , Dourada/metabolismo , Dourada/microbiologia , Sulfetos/administração & dosagem , Timol/administração & dosagemRESUMO
One of the main targets for the use of phytogenics in aquafeeds is the mucosal tissues as they constitute a physical and biochemical shield against environmental and pathogenic threats, comprising elements from both the innate and acquired immunity. In the present study, the modulation of the skin transcriptional immune response, the bacterial growth capacity in skin mucus, and the overall health condition of gilthead seabream (Sparus aurata) juveniles fed a dietary supplementation of garlic essential oil, carvacrol, and thymol were assessed. The enrichment analysis of the skin transcriptional profile of fish fed the phytogenic-supplemented diet revealed the regulation of genes associated to cellular components involved in the secretory pathway, suggesting the stimulation, and recruitment of phagocytic cells. Genes recognized by their involvement in non-specific immune response were also identified in the analysis. The promotion of the secretion of non-specific immune molecules into the skin mucus was proposed to be involved in the in vitro decreased growth capacity of pathogenic bacteria in the mucus of fish fed the phytogenic-supplemented diet. Although the mucus antioxidant capacity was not affected by the phytogenics supplementation, the regulation of genes coding for oxidative stress enzymes suggested the reduction of the skin oxidative stress. Additionally, the decreased levels of cortisol in mucus indicated a reduction in the fish allostatic load due to the properties of the tested additive. Altogether, the dietary garlic, carvacrol, and thymol appear to promote the gilthead seabream skin innate immunity and the mucus protective capacity, decreasing its susceptibility to be colonized by pathogenic bacteria.
Assuntos
Imunidade Inata/efeitos dos fármacos , Muco/metabolismo , Óleos Voláteis/farmacologia , Dourada/imunologia , Via Secretória/efeitos dos fármacos , Pele/efeitos dos fármacos , Ração Animal/análise , Animais , Aquicultura , Cimenos/química , Cimenos/farmacologia , Suplementos Nutricionais/análise , Alho/química , Imunidade Inata/genética , Imunidade nas Mucosas/efeitos dos fármacos , Muco/efeitos dos fármacos , Muco/microbiologia , Óleos Voláteis/classificação , Dourada/genética , Via Secretória/imunologia , Timol/química , Timol/farmacologiaRESUMO
The present study was designed to test if the damage caused by scale loss provokes a change in other innate immune barriers such as the intestine and how chronic stress affects this response. Sea bream (Sparus aurata) were kept in tanks at low density (16 kg m-3, LD) or exposed to a chronic high density (45 kg m-3, HD) stress for 4 weeks. Scales were then removed (approximately 50%) from the left flank in the LD and HD fish. Intestine samples (n = 8/group) were examined before and at 12 h, 3 days and 7 days after scale removal. Changes in the morphology of the intestine revealed that chronic stress and scale loss was associated with intestinal inflammation. Specifically, enterocyte height and the width of the lamina propria, submucosa and muscle layer were significantly increased (p < 0.05) 3 days after skin damage in fish under chronic stress (HD) compared to other treatments (LDWgut3d or HDgut0h). This was associated with a significant up-regulation (p < 0.05) in the intestine of gene transcripts for cell proliferation (pcna) and anti-inflammatory cytokine tgfß1 and down-regulation of gene transcripts for the pro-inflammatory cytokines tnf-α and il1ß (p < 0.05) in HD and LD fish 3 days after scale removal compared to the undamaged control (LDgut0h). Furthermore, a significant up-regulation of kit, a marker of mast cells, in the intestine of HDWgut3d and LDWgut3d fish suggests they may mediate the crosstalk between immune barriers. Skin damage induced an increase in cortisol levels in the anterior intestine in HDWgut12 h fish and significant (p < 0.05) down-regulation of mr expression, irrespective of stress. These results suggest glucocorticoid levels and signalling in the intestine of fish are modified by superficial cutaneous wounds and it likely modulates intestine inflammation.
Assuntos
Proteínas de Peixes/metabolismo , Mucosa Intestinal/metabolismo , Dourada/imunologia , Pele/lesões , Estresse Fisiológico/imunologia , Escamas de Animais/imunologia , Animais , Regulação para Baixo/imunologia , Imunidade Inata , Mucosa Intestinal/imunologia , Dourada/metabolismo , Pele/imunologia , Regulação para Cima/imunologiaRESUMO
Prostaglandin E2 (PGE2) plays an important role in immune activities in teleost fish, including seabream. However, receptors involved in PGE2 signaling, as well as the pathways activated downstream, are largely unknown. In this study, one ortholog of mammalian PTGER1, PTGER3 and PTGER4, and two of PTGER2 (Ptger2a and Ptger2b) were identified and characterized in gilthead seabream. In silico analysis showed that all these receptors possessed the organization domain of G protein-coupled receptors, with the exception of Ptger2b. The corresponding in vivo studies revealed that they were expressed in all the tissues examined, the highest mRNA levels of ptger1 and ptger3 being observed in the spleen and of ptger2a and ptger4 in the blood. Bacterial infection induced higher mRNA levels of ptger2a, ptger3 and ptger4 in peritoneal exudate (the site of bacterial injection). In addition, head kidney acidophilic granulocytes and macrophages displayed different ptger1, ptger2a, ptger3 and ptger4 expression profiles. Furthermore, in macrophages the expression of the receptors was weakly affected by stimulation with bacterial DNA or with PGE2, while in acidophilic granulocytes stimulation resulted in the upregulation of ptger2a and ptger4. Taken together, these results suggest different roles for seabream PGE2 receptors in the regulation of the immune responses.
Assuntos
Proteínas de Peixes/genética , Neutrófilos/imunologia , Receptores de Prostaglandina/genética , Dourada/imunologia , Baço/metabolismo , Vibrio/fisiologia , Animais , Clonagem Molecular , Dinoprostona/metabolismo , Proteínas de Peixes/metabolismo , Imunidade Inata , Mamíferos , Filogenia , Domínios Proteicos/genética , Receptores de Prostaglandina/metabolismo , Transcriptoma , Regulação para Cima , Vibrioses/imunologiaRESUMO
Chemical contaminants such as industrial and urban by-products, pharmaceuticals, drugs metabolites and, plastics, are continuously found in the oceans, affecting its quality and organism's welfare. Although these compounds are found at concentrations ranged ng L-1, there is an increasing concern about the potential adverse effects of the interactions among those substances present, simultaneously, in a mixture. In the present study, specimens of sea bream (Sparus aurata) were exposed, by food, to rising concentrations of a mixture of carbamazepine, polybrominated diphenyl ether-47 and cadmium chloride, for 15 days and then, maintained, with the same control diet, without contaminants, for other 15 days. Samples of skin mucus, serum, head-kidney, liver and intestine were sampled at 0, 15 and 30 days. Cellular immune parameters were evaluated on head-kidney, as well as humoral parameters were determined on skin mucus and serum. In addition, the expression of some genes, related to immunity, was analysed on liver and intestine. Both cellular and humoral response were affected at 15 days, showing slightly signs of recovery at 30 days. Besides, the expression of immune-related genes was highly affected, suggesting the development of inflammatory processes, as well as a reduction of immune parameters. Overall, the mixture of compounds severally affected the immune system of sea bream, suggesting a lower degree of recovery. The prolonged exposure to a mixture of these compounds could entail serious change on population immunity and, eventually, promote changes on marine biota.
Assuntos
Doenças dos Peixes/imunologia , Imunidade Celular , Imunidade Humoral , Inflamação/veterinária , Dourada/imunologia , Poluentes Químicos da Água/efeitos adversos , Animais , Cloreto de Cádmio/efeitos adversos , Carbamazepina/efeitos adversos , Doenças dos Peixes/induzido quimicamente , Éteres Difenil Halogenados/efeitos adversos , Inflamação/induzido quimicamente , Inflamação/imunologiaRESUMO
The complement system comprises a large family of plasma proteins that play a central role in innate and adaptive immunity. To better understand the evolution of the complement system in vertebrates and the contribution of complement to fish immunity comprehensive in silico and expression analysis of the gene repertoire was made. Particular attention was given to C3 and the evolutionary related proteins C4 and C5 and to one of the main regulatory factors of C3b, factor H (Cfh). Phylogenetic and gene linkage analysis confirmed the standing hypothesis that the ancestral c3/c4/c5 gene duplicated early. The duplication of C3 (C3.1 and C3.2) and C4 (C4.1 and C4.2) was likely a consequence of the (1R and 2R) genome tetraploidization events at the origin of the vertebrates. In fish, gene number was not conserved and multiple c3 and cfh sequence related genes were encountered, and phylogenetic analysis of each gene generated two main clusters. Duplication of c3 and cfh genes occurred across the teleosts in a species-specific manner. In common, with other immune gene families the c3 gene expansion in fish emerged through a process of tandem gene duplication. Gilthead sea bream (Sparus aurata), had nine c3 gene transcripts highly expressed in liver although as reported in other fish, extra-hepatic expression also occurs. Differences in the sequence and protein domains of the nine deduced C3 proteins in the gilthead sea bream and the presence of specific cysteine and N-glycosylation residues within each isoform was indicative of functional diversity associated with structure. The diversity of C3 and other complement proteins as well as Cfh in teleosts suggests they may have an enhanced capacity to activate complement through direct interaction of C3 isoforms with pathogenic agents.
Assuntos
Complemento C3/genética , Fator H do Complemento/genética , Proteínas de Peixes/genética , Dourada/genética , Animais , Complemento C4/genética , Complemento C5/genética , Evolução Molecular , Filogenia , Dourada/imunologia , Pele/imunologia , TranscriptomaRESUMO
The interaction between diet and intestinal health has been widely discussed, although in vivo approaches have reported limitations. The intestine explant culture system developed provides an advantage since it reduces the number of experimental fish and increases the time of incubation compared to similar methods, becoming a valuable tool in the study of the interactions between pathogenic bacteria, rearing conditions, or dietary components and fish gut immune response. The objective of this study was to determine the influence of the total substitution of fish meal by plants on the immune intestinal status of seabream using an ex vivo bacterial challenge. For this aim, two growth stages of fish were assayed (12 g): phase I (90 days), up to 68 g, and phase II (305 days), up to 250 g. Additionally, in phase II, the effects of long term and short term exposure (15 days) to a plant protein (PP) diet were determined. PP diet altered the mucosal immune homeostasis, the younger fish being more sensitive, and the intestine from fish fed short-term plant diets showed a higher immune response than with long-term feeding. Vibrio alginolyticus (V. alginolyticus) triggered the highest immune and inflammatory response, while COX-2 expression was significantly induced by Photobacterium damselae subsp. Piscicida (P. damselae subsp. Piscicida), showing a positive high correlation between the pro-inflammatory genes encoding interleukin 1ß (IL1-ß), interleukin 6 (IL-6) and cyclooxygenase 2(COX-2).
Assuntos
Dieta , Microbioma Gastrointestinal , Mucosa Intestinal/imunologia , Dourada/microbiologia , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Imunidade Inata , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Mucosa Intestinal/microbiologia , Photobacterium/patogenicidade , Proteínas de Vegetais Comestíveis , Dourada/imunologia , Dourada/fisiologia , Técnicas de Cultura de Tecidos/métodos , Vibrio alginolyticus/patogenicidadeRESUMO
Passive immunization constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. Enteromyxum leei is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. We have previously demonstrated that gilthead sea bream (GSB, Sparus aurata) that survive E. leei infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific IgM in serum. Thus, the current work was aimed to determine if passive immunization could help to prevent enteromyxosis in GSB and to study in detail the nature of these protective antibodies. Serum from a pool of resistant (SUR) or naïve (NAI) animals was intracoelomically injected 24 h prior to the E. leei-effluent challenge and at 9 days post-challenge (dpc). Effluent challenge lasted for 23 days, and then the injected groups were allocated in separate tanks with clean water. A non-lethal parasite diagnosis was performed at 56 dpc. At the final sampling (100 dpc), blood, serum and tissues were collected for histology, molecular diagnosis and the detection of circulating antibodies. In parallel, we performed an immunoglobulin repertoire analysis of the fish generating SUR and NAI sera. The results showed that, fish injected with parasite-specific antibodies (spAbs) became infected with the parasite, but showed lower disease signs and intensity of infection than the other groups, indicating a later establishment of the parasite. Repertoire analysis revealed that E. leei induced a polyclonal expansion of diverse IgM and IgT subsets that could be in part an evasion strategy of the parasite. Nonetheless, GSB was able to produce sufficient levels of parasite-spAbs to avoid re-infection of surviving animals and confer certain degree of protection upon passive transfer of antibodies. These results highlight the crucial role of spAb responses against E. leei and set the basis for the development of effective treatment or prophylactic methods for aquaculture.