RESUMEN
In aquatic animals, dietary protein plays a crucial role in their growth and immunity. A feeding trial was conducted on soft-shelled turtles (Pelodiscus sinensis) to assess the effects of various levels of protein on the specific growth rate (SGR), ambient water quality (total ammonia nitrogen (TAN), total nitrogen (TN) and total phosphorus (TP)), hematological parameters (respiratory burst (RB), red blood cell count (RBC), albumin content (Alb), hemoglobin level (Hb) and osmolality), plasma immunoglobulin M (IgM) levels and lysozyme activity. Soft-shelled turtles weighing about 4.02 g were fed fish meal-based diets with 14.38%, 20.41%, 26.19%, 32.23%, 37.63% and 45.23% protein for 8 weeks. SGR, RBC, Hb, Alb, RB, IgM and lysozyme activity were enhanced as the dietary protein was increased from 14.38% to 26.19%, then reached a plateau. For identical feeding times, TAN and TN were increased with elevating dietary protein levels. While, no statistically significant differences were observed among the 26.19%, 32.23% and 37.63% groups. When the turtles were cultivated for 56 days and fed with 45.23% protein, the TP in the culturing water was higher than that in the other groups. An increase in dietary protein level up to 26.19% increased the RNA/DNA ratio, which subsequently plateaued at a steady level. The levels of dietary protein had no impact on osmolality or alkaline phosphatase (AKP) activity. On the basis of broken-line analyses derived from SGR, the optimum dietary protein level for soft-shelled turtles was found to be 27.11% protein.
Asunto(s)
Tortugas , Animales , Amoníaco/metabolismo , Proteínas en la Dieta/metabolismo , ADN/metabolismo , Inmunoglobulina M/metabolismo , Muramidasa/metabolismo , Nitrógeno/metabolismo , ARN/metabolismo , Tortugas/genética , Calidad del AguaRESUMEN
The interplay between virus and host has been one of the hot spot in virology, and it is also the important aspect of revealing the mechanism of virus infection. Increasing studies revealed that several key molecules took part in the process of virus-host interaction. White spot syndrome virus (WSSV) has been proved to affect several physiological processes of the host cells, especially apoptosis. While the relationship between them still remains unclear. In this study, a IFI27 gene (LvIFI27) of Litopenaeus vannamei was cloned. It is indicated that LvIFI27 was induced upon endoplasmic reticulum (ER)-stress and unfolded protein response activator Thapsigargin. Unlike human IFI27 locating to mitochondria, LvIFI27 lied to ER, and was involved in cell apoptosis process. Moreover, results of cumulative mortality analysis showed that LvIFI27 might contributed to WSSV proliferation by promoting apoptosis during the process of viral infection. Findings in this study enriched our understanding of the relationship between WSSV infection and ER-stress mediated apoptosis.
Asunto(s)
Proteínas de Artrópodos , Infecciones por Virus ADN/veterinaria , Estrés del Retículo Endoplásmico , Proteínas de la Membrana/genética , Penaeidae , Animales , Apoptosis , Proteínas de Artrópodos/genética , Penaeidae/genética , Penaeidae/virología , Respuesta de Proteína Desplegada , Virus del Síndrome de la Mancha Blanca 1RESUMEN
Zinc (Zn) plays a role in the antioxidant capacity and immunity of aquatic animals. A twelve-week feeding experiment was performed to estimate the impact of dietary zinc on antioxidant enzyme-related gene expression, antioxidant enzyme activity and non-specific immune functions of soft-shelled turtles, Pelodiscus sinensis. Six fishmeal-based experimental diets with 32.45% protein were formulated, which contained 35.43, 46.23, 55.38, 66.74, 75.06 and 85.24 mg/kg Zn, respectively. Catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels improved with an elevation in dietary Zn from 35.43 to 55.38 mg/kg and then reduced when dietary Zn was further elevated. The expression levels of Nrf2 and antioxidant-related genes CuZnSOD, MnSOD, CAT, GPX1, GPX2, GPX3 and GPX4 escalated with elevating Zn concentration up to 55.38 mg/kg in diets and then reduced as dietary Zn elevated. The expression levels of Kelch-like ECH-associating protein 1 (keap1) showed a reverse trend with that of Nrf2. The contents of malondialdehyde (MDA) in the 55.38 and 66.74 mg/kg Zn diet-fed groups were the lowest. Alkaline phosphatase activity (AKP), superoxide anion (O2-), lysozyme activity and total antioxidant capacity (T-AOC) improved with an escalation in dietary Zn concentration up to 66.74 mg/kg. Optimal dietary Zn improved antioxidant capability, immunity, and antioxidant enzyme-related gene expression. The dietary Zn demand for soft-shelled turtles were 60.93 and 61.63 mg/kg, based on second regression analysis of SOD and T-AOC activity, respectively.
Asunto(s)
Antioxidantes , Tortugas , Alimentación Animal/análisis , Animales , Dieta/veterinaria , Suplementos Dietéticos , Expresión Génica , Proteína 1 Asociada A ECH Tipo Kelch , Factor 2 Relacionado con NF-E2/genética , Superóxido Dismutasa/metabolismo , Tortugas/genética , Tortugas/metabolismo , ZincRESUMEN
Lectins are a group of carbohydrate-binding proteins, which play an important role in innate immune system against pathogen infection. In this study, a B-type mannose-binding lectin (OnBML) was identified from Nile tilapia (Oreochromis niloticus), and characterized at expression patterns against bacterial infection and capability to promote phagocytosis by macrophages. The open reading frame of OnBML is 354 bp of nucleotide sequence encoding polypeptides of 117 amino acids. The deduced protein is highly homologous to other teleost BMLs, containing two repeats of the conserved mannose-binding motif QXDXNXVXY. Expression of OnBML was widely exhibited in all examined tissues, with the most abundance in spleen and following gill, peripheral blood, and head kidney. The OnBML expressions were significantly up-regulated following two major bacterial infections including a Gram-positive bacterium (Streptococcus agalactiae) and a Gram-negative bacterium (Aeromonas hydrophila) in vivo and in vitro. Recombinant OnBML protein possessed capacities of mannose-binding and calcium-dependent agglutination to S. agalactiae and A. hydrophila, and promoted the phagocytosis by macrophages. Taken together, the present study indicated that OnBML is likely to get involved in host defense against bacterial infection in Nile tilapia.
Asunto(s)
Cíclidos/inmunología , Enfermedades de los Peces/inmunología , Proteínas de Peces/inmunología , Lectina de Unión a Manosa/inmunología , Infecciones Estreptocócicas/inmunología , Aeromonas hydrophila , Animales , Cíclidos/microbiología , Proteínas de Peces/genética , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Macrófagos/inmunología , Lectina de Unión a Manosa/genética , Monocitos/inmunología , Infecciones Estreptocócicas/veterinaria , Streptococcus agalactiaeRESUMEN
Water temperature is an important environmental factor in aquaculture farming that affects the survival and growth of organisms. The change in culture water temperature may not only modify various chemical and biological processes but also affect the status of fish populations. In previous studies, high temperature induced apoptosis and oxidative stress. However, the precise mechanism and the pathways that are activated in fish are still unclear. In the present study, we investigated the effects of high temperature (34°C) on the induction of apoptosis and oxidative stress in pufferfish (Takifugu obscurus) blood cells. The data showed that high temperature exposure increased oxygen species (ROS), cytoplasmic free-Ca(2+) concentration and cell apoptosis. To test the apoptotic pathway, the expression pattern of some key apoptotic related genes including P53, Bax, caspase 9 and caspase 3 were examined. The results showed that acute high temperature stress induced up-regulation of these genes, suggesting that the p53-Bax pathway and the caspase-dependent apoptotic pathway could be involved in apoptosis induced by high temperature stress. Furthermore, the gene expression of antioxidant enzymes (Cu/Zn-SOD, Mn-SOD, CAT, GPx, and GR) and heat shock proteins (HSP90 and HSP70) in the blood cells were induced by high temperature stress. Taken together, our results showed that high temperature-induced oxidative stress may cause pufferfish blood cells apoptosis, and cooperatively activated p53-Bax and caspase-dependent apoptotic pathway.
Asunto(s)
Apoptosis , Células Sanguíneas/metabolismo , Proteínas de Peces/metabolismo , Respuesta al Choque Térmico , Estrés Oxidativo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Calcio/metabolismo , Catalasa/genética , Catalasa/metabolismo , Proteínas de Peces/genética , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , TakifuguRESUMEN
Waterborne ammonia has become a persistent pollutant of aquatic habitats. The exposure to ammonia stress can reduce growth in a wide range of aquatic organisms. To assess the effect of ammonia exposure on the growth hormone/insulin-like growth factors (GH/IGF) axis, we identified and characterized GHR1, GHR2 and IGF-1 from pufferfish. Comparative analysis showed that these genes shared high identity and similarity with corresponding genes in other fish species. The transcripts of these genes were widely expressed in all tested tissues. The highest level of GHR1 mRNA was found in the brain, whereas GHR2 and IGF-1 mRNA levels were the highest in the liver. Following acute ammonia exposure (100 mg/L total ammonia-nitrogen), GHR2 expression in the liver did not change at 6 h and then significantly decreased at 12, 24 and 48 h, whereas GHR1 and IGF-1 expressions were significantly down-regulated at 6, 12, 24 and 48 h, respectively. These results indicated that ammonia stress decreased the expression of GH/IGF axis genes, which might have negative effect on the growth and development of pufferfish.
Asunto(s)
Amoníaco/toxicidad , Exposición a Riesgos Ambientales/efectos adversos , Regulación de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Receptores de Somatotropina/metabolismo , Takifugu/metabolismo , Contaminantes Químicos del Agua/toxicidad , Análisis de Varianza , Animales , Secuencia de Bases , Encéfalo/metabolismo , Clonación Molecular , Cartilla de ADN/genética , Hígado/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Análisis de Secuencia de ADN/veterinariaRESUMEN
Apoptosis plays a crucial role in many biological processes, including development, cellular homeostasis and immune responses. The BCL-2 family is a key regulator of the mitochondrial response to apoptotic signals in the intrinsic pathway. In this study, we identified and characterized the cDNA and expression pattern of pufferfish BCL-2 (PfBCL-2). The full-length cDNA of PfBCL-2 was 1412 bp with an open reading frame of 657 bp encoding a putative protein of 219 amino acids (Accession no: KP898414). The calculated molecular mass of the PfBCL-2 was 24.2 kDa with a predicted isoelectric point of 5.27. The deduced PfBCL-2 protein exhibited four highly conserved BCL-2 homology domains, suggesting that PfBCL-2 may play a similar role in the apoptotic-signaling pathway as in other species. Real-time PCR results showed that PfBCL-2 transcript was expressed in a wide range of tissues but exhibited the greatest level of expression in blood. Transcriptional responses of PfBCL-2 exhibited different spatial and temporal expression profiles in liver and blood after bacterial infection. PfBcl-2 transcript was significantly up-regulated in liver at 6, 12, 24 and 48 h (with maximum induction at 48 h) and was up-regulated in blood at 3, 6, 12 and 24 h (with maximum induction at 12 h). Meanwhile, recombinant PfBCL-2 fused with His6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified using Ni-nitrilotriacetic acid resin. Western blot analysis indicated that its protein level appeared to be elevated during the initial bacterial infection. These results suggest that PfBCL-2 plays important roles in immune responses against bacteria challenge.
Asunto(s)
Proteínas Proto-Oncogénicas c-bcl-2 , Aeromonas hydrophila , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , ADN Complementario/genética , Enfermedades de los Peces/metabolismo , Branquias/metabolismo , Infecciones por Bacterias Gramnegativas/metabolismo , Infecciones por Bacterias Gramnegativas/veterinaria , Riñón Cefálico/metabolismo , Hígado/metabolismo , Datos de Secuencia Molecular , Músculos/metabolismo , Miocardio/metabolismo , Conformación Proteica , Proteínas Proto-Oncogénicas c-bcl-2/sangre , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Bazo/metabolismo , TakifuguRESUMEN
This study evaluated the expression level of antioxidant enzymes Cu, Zn-superoxide dismutase (Cu, Zn-SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) in hemocytes of Macrobrachium rosenbergii exposed to copper by real-time PCR (qRT-PCR). Results showed that the mRNA expression of Cu, Zn-SOD increased to reach a peak at 6 h, then recovered to its normal level at 48 h. CAT expression level was significantly increased at 12 h and reached a peak at 24 h, but recovered to its normal level later. GPx expression level was significantly increased at 6 h and reached the peak at 12 h. GST expression level was significantly induced from 12 to 24 h and then dropped to its normal level at 48 h. These results indicated that antioxidant enzymes were inducible, possibly for removing excessive reactive oxygen species to protect prawn from oxidative stress.
Asunto(s)
Antioxidantes/metabolismo , Cobre/toxicidad , Hemocitos/efectos de los fármacos , Palaemonidae/efectos de los fármacos , Animales , Catalasa/metabolismo , Agua Dulce/química , Expresión Génica/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Glutatión Transferasa/metabolismo , Hemocitos/metabolismo , Estrés Oxidativo , Palaemonidae/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Superóxido Dismutasa/metabolismoRESUMEN
This study investigated the effect of ambient Cadmium (Cd) on haemocyte apoptosis of the shrimp, Penaeus monodon. Cellular response was determined in Cd-exposed (0, 0.05, 0.5 and 5 mg L(-1)) shrimp. Results showed that 0.05 mg L(-1) Cd(2+) had no significant effect on the haemocyte parameters during the 48 h exposure. Cadmium at doses of 0.5 and 5 mg L(-1) depressed the total haemocyte count (THC), and increased reactive oxygen species (ROS) production and apoptosis ratio in haemocytes. Esterase activity increased in shrimp exposed to 0.5 mg L(-1) Cd(2+) for 6 h, and decreased to the initial level later. Depressed esterase activity could be observed in shrimp after 24 and 48 h exposure to 5 mg L(-1) Cd(2+). These results demonstrated that Cd(2+) modified esterase activity and induced ROS generation, which led to haemocyte apoptosis and THC reduction. Oxidative stress is one of the induction mechanisms for Cd-caused apoptosis of shrimp haemocytes.
Asunto(s)
Cadmio/toxicidad , Hemocitos/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Apoptosis , Hemocitos/patología , Hemocitos/fisiología , Estrés Oxidativo , Penaeidae , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Five feeding trials based on the isonitrogenous and isoenergetic experimental diets containing 34% protein, 6%, 8%, 10%, 12% or 14% lipid respectively in the circulating water culture system for both 30 and 60 days were conducted to investigate the effect of the dietary lipid level on the growth and immunity in white shirmp, Litopenaeus vannamei adults. The body weight and specific growth rate of white shrimp in different treatments indicated that shrimps fed the diet of 12% lipid level for 30d and 10% lipid level for 60d had the best developmental status. The ability of respiratory burst in hemocytes was improved as the increase of dietary lipid level. The transcripts of LGBP and pPO were sensitive to the dietary lipid in hemocyte and hepatopancreas respectively. The activities of CAT, GPx and AKP were increased to a certain extend according to dietary lipid level. Qualification of MDA showed the lowest level in the sample subjected to 12% lipid level diet, indicating an optimal utilization of the dietary lipid and an efficient clearance of MDA in vivo. These results suggested that dietary lipid level of 10-12% significantly tunes the growth and enhance the immune abilities mainly via ROS pathway of L. vannamei.
Asunto(s)
Acuicultura , Lípidos/administración & dosificación , Penaeidae/fisiología , Alimentación Animal , Animales , Dieta/veterinaria , Regulación de la Expresión Génica , Hemocitos/citología , Inmunidad Innata , Longevidad , Malondialdehído/metabolismo , Penaeidae/genética , Penaeidae/crecimiento & desarrollo , Penaeidae/inmunología , Reacción en Cadena de la Polimerasa/veterinaria , Estallido RespiratorioRESUMEN
In the present study, transcriptome of nitrite-exposed Litopenaeus vannamei was performed using a newly developed high-throughput sequencing technology (Illumina RNA-seq). As many as 42,336 unigenes were generated with 561 bp of average length and 736 bp of unigene N50 after filtering and assembly. These unigenes from the de novo assembly were further annotated using BLAST and BLAST2GO softwares. A total of 23,532 unigenes were unambiguous alignments to the reference when BLAST against non-redundant protein sequence (Nr), non-redundant nucleotide (Nt), Swiss-Prot, Gene Ontology database (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases available at NCBI. Numerous candidate genes associated with immune response, detoxification, apoptosis pathway were identified. Ten candidate genes related to immune responses and apoptosis were selected for validating the results of assembly and annotation by real-time quantitative PCR. Results revealed that the expressions of all these ten genes were up-regulated after nitrite exposure. Combining to our previous study, we speculate that all these selected genes may be involved in the response to nitrite stress. The study shows a systematic overview of the transcriptome analysis in L. vannamei, and provides valuable gene information for studying molecular mechanisms under nitrite exposure.
Asunto(s)
Nitritos/toxicidad , Penaeidae/efectos de los fármacos , Penaeidae/genética , Contaminantes Químicos del Agua/toxicidad , Animales , Apoptosis/efectos de los fármacos , Perfilación de la Expresión Génica , Inmunidad Innata/efectos de los fármacos , Inactivación Metabólica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARNRESUMEN
A flow cytometric method to measure the production of intracellular nitric oxide (NO) was adapted for use with shrimp haemocytes. We applied fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) for NO detection in haemocytes from the tiger shrimp Penaeus monodon, and used flow cytometry to quantify fluorescence intensity in individual haemocyte. The optimized protocol for intracellular NO analysis consists to incubate haemocytes with DAF-FM DA at 10 µM for 60 min to determine the mean fluorescence intensity. Result showed that NO was also produced in the untreated shrimp haemocytes. NO level in granular cells and semigranular cells were much higher than that in hyaline cells. Deï¬ned by different characteristic of NO content, three subsets of haemocytes were observed. Zymosan A at dose of 10 or 100 particles per haemocyte triggered higher DAF-FM fluorescence intensity in granular and semigranular cells, than PMA that had no significant impact on all three cell types. These results indicate that granular and semigranular cells are the primary cells for NO generation. Cytochalasin B significantly inhibited the NO level induced by zymosan A. NG-Monomethyl-L-arginine (L-NMMA) and diphenylene iodonium chloride (DPI) significantly suppressed the DAF-FM fluorescence in haemocytes, but apocynin could not modulate it, indicating that the DAF-FM fluorescence was closely related to the activity of NO-synthase pathway. The NO donor sodium nitroprusside (SNP) improved the DAF-FM fluorescence in haemocytes, while the NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) significantly decreased the fluorescence, demonstrating that the fluorescence intensity of DAF-FM is mainly dependent on the intracellular NO level.
Asunto(s)
Citometría de Flujo/métodos , Hemocitos/metabolismo , Óxido Nítrico/metabolismo , Penaeidae/metabolismo , Animales , Hemocitos/efectos de los fármacos , Penaeidae/citología , Acetato de Tetradecanoilforbol/farmacología , Zimosan/farmacologíaRESUMEN
This study was aimed at investigating the toxicity mechanism of lipopolysaccharide (LPS) on Penaeus monodon haemocytes at a cellular level. Reactive oxygen species (ROS) production, nitric oxide (NO) production, non-specific esterase activity, cytoplasmic free-Ca(2+) (CF-Ca(2+)) concentration, DNA damaged cell ratio and apoptotic cell ratio of in vitro LPS-treated haemocytes were measured by flow cytometry. Two concentrations of Escherichia coli LPS (5 and 10 µg mL(-1)) were used. Results showed that ROS production, NO production and CF-Ca(2+) concentration were significantly induced in the LPS-treated haemocytes. Ratio of DNA damaged cell and apoptotic cell increased caused by LPS, while esterase activity increased at the initial 60 min and dropped later. The initial increase in esterase activity suggested that LPS activated the release of esterase, and the later decrease might result from apoptosis. These results indicated that LPS would induce oxidative stress on shrimp haemocytes, and cause Ca(2+) release, DNA damage and subsequently cell apoptosis. This process of ROS/RNS-induced Ca(2+)-mediated apoptosis might be one of the toxicity mechanisms of LPS on shrimp haemocytes.
Asunto(s)
Apoptosis/efectos de los fármacos , Lipopolisacáridos/farmacología , Penaeidae , Animales , Daño del ADN/efectos de los fármacos , Escherichia coli/química , Hemocitos/efectos de los fármacos , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Penaeidae/citología , Penaeidae/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
This study investigated the toxic effects of cadmium on viability, reactive oxygen species (ROS) production and non-specific esterase activity of Penaeus monodon haemocytes in vitro, using a flow cytometric assay. After 6 h in vitro exposure with 10(-9)-10(-3) M Cd(2+), cell viability, ROS production and esterase activity of haemocytes from P. monodon were determined. Results showed that at the lowest exposures (10(-9)-10(-6 )M), Cd(2+) induced no effect on cell viability, ROS production and esterase activity. At a higher level (10(-5) M) of exposure, production of ROS was stimulated while Cd(2+) had no effect on cell viability and esterase activity. At the two highest concentrations (10(-4) and 10(-3) M), Cd(2+) caused increased ROS production, cell death and inhibited esterase activity. These results showed a relationship between Cd(2+) exposure dose and its cytotoxicity on shrimp haemocytes. Cadmium was cytotoxic and immunotoxic for P. monodon haemocytes in vitro when the dose reached 10(-4) M. The study also suggested that flow cytometry could be used as a tool for cytotoxic research of aquatic contamination on shrimp.
Asunto(s)
Cadmio/toxicidad , Exposición a Riesgos Ambientales , Monitoreo del Ambiente/métodos , Citometría de Flujo/métodos , Penaeidae/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Esterasas/metabolismo , Hemocitos/efectos de los fármacos , Hemocitos/metabolismo , Penaeidae/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
An 8-week feeding trial was performed to assess the influence of a gradient of protein levels (14.38-45.23%) on flesh quality, skin color, amino acid profile, collagen, antioxidant capability, and antioxidant-related signaling molecule expression of the softshell turtle (Pelodiscus sinensis). Hardness, gumminess, chewiness, and yellowness values in the plastron and carapace, along with collagen, superoxide dismutase, catalase, total antioxidant capacity, and glutathione peroxidase, all improved with elevating dietary protein up to 26.19%, after which they leveled off. Additionally, total amino acids, flavor amino acids, essential amino acids, and non-essential amino acids in the muscle, as well as the expression of copper/zinc superoxide dismutase, glutathione peroxidase, catalase, manganese superoxide dismutase, NF-E2-related factor 2 were all enhanced by increasing the dietary protein level but not changed by higher protein levels. When dietary protein levels were less than 26.19%, the mRNA expression of Kelch-like ECH-associated protein 1, malondialdehyde, and redness values in the carapace and plastron were reduced, as was the lightness values of the carapace, all of which plateaued at higher protein levels. Using catalase activity and malondialdehyde as the indicators and applying a broken-line analysis, the optimal dietary protein level for P. sinensis was inferred to be 26.07 and 26.06% protein, respectively. In summary, an optimal protein input improved turtle flesh quality by strengthening antioxidant capacity in muscle tissue and by regulating the expression of antioxidant-related enzymes via the Nrf2/keap1 signaling pathway.
RESUMEN
In this article, the complete 16,439 bp mitochondrial genome of Thamnaconus septentrionalis was determined from a specimen collected in China. It contains 13 protein-coding genes, 22 transfer RNA genes, 1 D-loop region, 2 rRNA genes, the base composition of A 27.74%, G 17.09%, T 26.04%, and C 29.13%, resulting in a G + C content of 46.22%. Phylogenetic analysis showed that Thamnaconus septentrionalis was the nearest sister to Thamnaconus modestus. These presented data will be significant for evolution relationships study among fish species.
RESUMEN
Laccases (Lacs) are copper-containing oxidase enzymes that are found in various plants, fungi, and microorganisms. For invertebrates, particularly insects and crustaceans, Lacs have been shown to be involved in immune responses. In shrimp, a Lac gene has been cloned and functionally characterized, which revealed that it is involved in shrimp anti-pathogen infection. In the present study, a novel Lac gene (LvLac2) was cloned from Litopenaeus vannamei. Real-time RT-PCR analysis showed that LvLac2 is induced by white spot syndrome virus (WSSV)- or Vibrio alginolyticus infection. In addition, the downregulated expression of LvLac2 decreased the cumulative mortality of WSSV- or V. alginolyticus infected shrimps. Moreover, LvLac2 is also induced by oxidative stress. Knocking down the expression of LvLac2 decreased the severity of hepatopancreatic injury caused by oxidative stress, as well as reduced the cumulative shrimp mortality during oxidative stress. Furthermore, gene reporter assays showed that the expression of LvLac2 is regulated by NF-E2-related factor 2, which is the key transcription factor of the oxidative stress response signaling pathway. Our study revealed that LvLac2 not only participates in immune responses against infections in L. vannamei but is also involved in oxidative stress responses.
Asunto(s)
Proteínas de Artrópodos/genética , Infecciones por Virus ADN/inmunología , Hepatopáncreas/inmunología , Lacasa/genética , Penaeidae/inmunología , Vibriosis/inmunología , Vibrio alginolyticus/fisiología , Virus del Síndrome de la Mancha Blanca 1/fisiología , Animales , Técnicas de Silenciamiento del Gen , Inmunidad , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Transducción de Señal , Regulación hacia ArribaRESUMEN
Ammonia is one of major environmental pollutants in the freshwater aquatic system that affects the survival and growth of organisms. In the present study, we investigated the effects of ammonia exposure on apoptosis, oxidative stress and immune response in pufferfish (Takifugu obscurus). Fish were exposed to various concentrations of ammonia (0, 1.43, 3.57, 7.14mM) for 72h. The date showed that ammonia exposure could induce intracellular reactive oxygen species (ROS), interrupt intracellular Ca(2+) (cf-Ca(2+)) homeostasis, and subsequently lead to DNA damage and cell apoptosis. To test the apoptotic pathway, the expression patterns of some key apoptotic related genes including P53, Bax Bcl2, Caspase 9, Caspase 8 and Caspase 3 in the liver were examined. The results showed that ammonia stress could change these genes transcription, associated with increasing of cell apoptosis, suggesting that the P53-Bax-Bcl2 pathway and caspase-dependent apoptotic pathway could be involved in cell apoptosis induced by ammonia stress. In addition, ammonia stress could induced up-regulation of inflammatory cytokines (BAFF, TNF-α, IL-6 and IL-12) transcription, indicating that innate immune system play important roles in ammonia-induced toxicity in fish. Furthermore, the gene expressions of antioxidant enzymes (Mn-SOD, CAT, GPx, and GR) and heat shock proteins (HSP90 and HSP70) in the liver were induced by ammonia stress, suggesting that antioxidant system and heat shock proteins tried to protect cells from oxidative stress and apoptosis induced by ammonia stress. Our results will be helpful to understand the mechanism of aquatic toxicology induced by ammonia in fish.
Asunto(s)
Amoníaco/toxicidad , Apoptosis/efectos de los fármacos , Sistema Inmunológico/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Takifugu/fisiología , Animales , Daño del ADN/efectos de los fármacos , Proteínas de Peces/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidadRESUMEN
Apoptotic cell ratio and mRNA expression of caspase-3, cathepsin B (CTSB), heat shock protein 70 (HSP70), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx) in hemocytes of white shrimp Litopenaeus vannamei exposed to nitrite-N (20 mg/L) was investigated at different stress time (0, 4, 8, 12, 24, 48 and 72 h). The apoptotic cell ratio and mRNA expression level of CTSB were significantly increased in shrimp exposed to nitrite-N for 48 and 72 h. Caspase-3 mRNA expression level significantly increased by 766.50% and 1811.16% for 24 and 48 h exposure, respectively. HSP70 expression level significantly increased at 8 and 72 h exposure. MnSOD mRNA expression in hemocytes up-regulated at 8 and 48 h, while CAT mRNA expression level increased at 24 and 48 h. GPx expression showed a trend that increased first and then decreased. Significant increases of GPx expression were observed at 8 and 12 h exposure. Expression level of TRx reached its highest level after 48 h exposure. These results suggest that nitrite exposure induces expression of apoptosis-related genes in hemocytes, and subsequently caused hemocyte apoptosis. Meanwhile, expression levels of HSP70 and antioxidant enzymes up-regulated to protect the hemocyte against nitrite stress.
Asunto(s)
Antioxidantes/metabolismo , Apoptosis , Regulación Enzimológica de la Expresión Génica , Hemocitos/enzimología , Nitritos/farmacología , Penaeidae/efectos de los fármacos , Penaeidae/enzimología , Animales , Caspasa 3/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Exposición a Riesgos Ambientales/análisis , Glutatión Peroxidasa/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Hemocitos/efectos de los fármacos , Estrés Oxidativo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Superóxido Dismutasa/metabolismo , Factores de TiempoRESUMEN
This study investigated the in vitro effects of nitrite on reactive oxygen species (ROS) production, NO production, esterase activity and cell apoptosis of Penaeus monodon haemocytes. Haemocytes were in vitro exposed to different dose of nitrite (0, 0.1, 0.5, 1, 5 and 10 µM). Cellular responses of nitrite-treated haemocytes were determined by flow cytometry. The results revealed that haemocytes treated by nitrite in vitro showed conspicuous time- and dose-dependent decreases in ROS and NO production as well as esterase activity. Additionally, 0.1 and 0.5 µM nitrite did not affect the apoptotic cell ratio during the 3h experimental time, while significant increases in apoptotic cells were observed after haemocyte exposure to nitrite at 1 µM for 3h, and at 5 or 10 µM for 1h. These results indicated that nitrite suppresses cellular functions, including production of ROS and NO, and activity of esterase. Cell apoptosis of haemocytes would be induced by extracellular nitrite as doses exceed 1 µM.