RESUMEN
BACKGROUND: Carbon monoxide intoxication and smoke inhalation injury can lead to severe disorders, and the current literature has elaborated on the importance of major cardiopulmonary impairment. Exercise intolerance has seldom been discussed, particular in patient with low cardiovascular risk. CASE PRESENTATION: Two young male fire survivors who presented with exercise intolerance after CO intoxication and smoke inhalation injury. Both received bronchodilator and glucocorticoid therapy, high-flow oxygen therapy, and hyperbaric oxygen therapy for airway edema and CO intoxication during acute care. Serum carboxyhemoglobin levels improved after treatment (8.2-3.9% in Case A and 14.8-0.8% in Case B). However, subjective exercise intolerance was noted after discharge. Cardiopulmonary exercise testing revealed exercise-induced myocardial ischemia during peak exercise (significant ST-segment depression on exercise electrocardiogram). They were instructed to exercise with precaution by setting the intensity threshold according to the ischemic threshold. Their symptoms improved, and no cardiopulmonary events were reported in the 6-month follow-up. CONCLUSION: The present case report raised the attention that exercise intolerance after carbon monoxide intoxication and smoke inhalation injury in low cardiovascular risk population may be underestimated. Cardiopulmonary exercise testing help physician to discover exercise-induced myocardial ischemia and set up the cardiac rehabilitation program accordingly.
Asunto(s)
Intoxicación por Monóxido de Carbono , Enfermedad de la Arteria Coronaria , Incendios , Isquemia Miocárdica , Lesión por Inhalación de Humo , Masculino , Humanos , Lesión por Inhalación de Humo/complicaciones , Lesión por Inhalación de Humo/diagnóstico , Lesión por Inhalación de Humo/terapia , Monóxido de Carbono , Intoxicación por Monóxido de Carbono/complicaciones , Intoxicación por Monóxido de Carbono/diagnóstico , Intoxicación por Monóxido de Carbono/terapiaRESUMEN
Octopamine (OA), a biogenic monoamine, is known to mediate several immune responses. This study analyzed the effects of OA on immunological regulation in the tiger shrimp Penaeus monodon. The immune parameters including total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, superoxide dismutase activity, and phagocytic activity and clearance efficiency in response to the pathogen, Photobacterium damselae, were determined when shrimp were individually injected with saline or OA at 100 or 1000â¯pmol shrimp-1. In addition, the intracellular second messengers in haemocyte such as Ca2+ and adenosine 3',5'-cyclic monophosphate (cAMP) were examined in shrimp receiving saline or OA at 1 or 10â¯nmol shrimp-1. Results showed that all of the immune parameters significantly increased at 2-4â¯h in OA-injected shrimp except hyaline cells in 100â¯pmol shrimp-1-injected shrimp at 4â¯h, but phenoloxidase activity per granulocyte significantly decreased at 2-4â¯h. However, these had returned to saline control levels after receiving OA for 8â¯h except differential haemocyte count and phenoloxidase activity per granulocyte for 16â¯h. An injection of OA also significantly increased the survival rate of shrimp challenged with Pho. damselae. Shrimp receiving OA at 1 and 10â¯nmol shrimp-1 significantly increased the intracellular Ca2+ concentration ([Ca2+]i) at 30-60â¯min and 30â¯min, and cAMP concentration [cAMP]i) at 5-15â¯min and 15â¯min, respectively. However, [Ca2+]i at 50-60â¯min, and [cAMP]i at 30-60â¯min returned to saline control when the shrimp received OA at 10â¯nmol shrimp-1, and at 1 and 10â¯nmol shrimp-1, respectively. These results suggest that OA administration by injection at ≤1000â¯pmol shrimp-1 mediates transient upregulation of immunity together with the increased resistance of P. monodon to Pho. damselae, which are modulated through intracellular Ca2+ and cAMP second messenger pathways.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Inmunidad Innata/efectos de los fármacos , Octopamina/metabolismo , Penaeidae/genética , Penaeidae/inmunología , Transducción de Señal/inmunología , Adyuvantes Inmunológicos/farmacología , Agonistas alfa-Adrenérgicos/administración & dosificación , Agonistas alfa-Adrenérgicos/metabolismo , Animales , Calcio/metabolismo , AMP Cíclico/metabolismo , Perfilación de la Expresión Génica , Octopamina/administración & dosificación , Photobacterium/fisiología , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
Tyrosine hydroxylase (TH), the first and rate-limiting step in the synthesis of catecholamines, is required in catecholamine synthesis of the neuroendocrine regulatory network against stress in shrimp. The immunocompetence, catecholamine biosynthesis, and carbohydrate metabolites were evaluated in Litopenaeus vannamei received L. vannamei TH (LvTH) double-stranded (ds)RNA, diethyl pyrocarbonate-water, or non-targeted dsRNA for 3 days then transferred from 28 to 20 or 28 °C. The immunocompetence of LvTH-depleted shrimp held at 28 °C was promoted, and those were downregulated under hypothermal stress and revealed higher level than the other two dsRNA treatments. Meanwhile, the decrease of catecholamine biosynthesis was observed in LvTH-depleted shrimp held at 28 °C, and those were elevated under hypothermal stress and revealed lower levels, compared to two dsRNA treatments. The reduced carbohydrate metabolites was observed in LvTH-depleted shrimp held at 28 °C, and those were upregulated under hypothermal stress and showed lower levels than the other two dsRNA treatments. It was therefore concluded that LvTH-depleted shrimp revealed enhanced immunocompetence and reduced carbohydrate metabolites when exposed to a hypothermal stress condition, and in the meantime, even though catecholamine biosynthesis was downregulated, no significant difference was observed in DA or NE levels.
Asunto(s)
Frío/efectos adversos , Regulación de la Expresión Génica/inmunología , Inmunocompetencia/genética , Penaeidae/genética , Penaeidae/inmunología , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/inmunología , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Perfilación de la Expresión Génica , Silenciador del Gen , Inmunidad Innata/genética , Análisis de Secuencia de ADN , Tirosina 3-Monooxigenasa/químicaRESUMEN
L-3,4-Dihydroxyphenylalanine (l-DOPA) is a precursor for dopamine (DA) synthesis. Assessments were conducted to analyze the effects of l-DOPA on mediating regulation of neuroendocrinological, immunological, and physiological parameters in the shrimp, Litopenaeus vannamei when they were individually injected with 0.01â¯N HCl or l-DOPA at 0.5 or 1.0⯵mol shrimp-1 for 60, 120, and 240â¯min. For catecholamine synthesis evaluation, tyrosine hydroxylase (TH) and DA beta hydroxylase (DBH) activities, l-DOPA, DA, and norepinephrine (NE) levels in hemolymph were determined. The total hemocyte count (THC), differential hemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, phagocytic activity, and clearance efficiency in response to the pathogen, Vibrio alginolyticus were assessed for immune responses, and plasma glucose and lactate levels were for physiological response. Results showed that the TH activity, THC, hyaline cells (HCs), and semigranular cells (SGCs) at 120 min, DA levels at 60-240 min, PO activity in hemocytes per 50 µL of hemolymph at 60-120 min, and PO activity per granulocyte (granular cells (GCs) + SGCs) at 60 min significantly increased, but TH activity, l-DOPA levels, GCs, SGCs, and respiratory bursts in hemocytes per 10⯵L of hemolymph at 60â¯min, respiratory bursts per hemocyte and SOD activity at 120â¯min, phagocytic activity at 60-240â¯min, and the clearance efficiency at 60-120â¯min significantly decreased in shrimp injected with l-DOPA at 1.0⯵mol shrimp-1. In another experiment, 60â¯min after shrimp had received l-DOPA at 0.5 or 1.0⯵mol shrimp-1, they were challenged with an injection of V. alginolyticus at 2â¯×â¯105â¯colony-forming units (cfu) shrimp-1. The injection of l-DOPA at 1.0⯵mol shrimp-1 also significantly increased the cumulative mortality of shrimp by 16.7%, compared to the HCl-challenged control after 120â¯h. These results suggest that l-DOPA administration at 1.0⯵mol shrimp-1 can mediate the transient regulation of neuroendocrinological, immunological, and physiologic responses resulting in immunosuppression, which in turn promoted the susceptibility of L. vannamei to V. alginolyticus.
Asunto(s)
Inmunidad Innata , Levodopa/farmacología , Penaeidae/fisiología , Animales , Relación Dosis-Respuesta a Droga , Inmunidad Innata/efectos de los fármacos , Penaeidae/inmunología , Penaeidae/microbiología , Vibrio alginolyticus/fisiologíaRESUMEN
White spot syndrome virus (WSSV) is one of the most devastating viral pathogens of cultured shrimp worldwide. Recently published papers show the ability of WSSV structural protein VP28 to vaccinate shrimp and raise protection against the virus. This study attempted to identify the joining proteins of the aforementioned shrimp quasi-immune response by proteomic analysis. The other envelope protein, VP36B, was used as the non-protective subunit vaccine control. Shrimp were intramuscularly injected with rVPs or PBS on day 1 and day 4 and then on day 7 their gill tissues were sampled. The two-dimensional electrophoresis (2-DE) patterns of gill proteins between vaccinated and PBS groups were compared and 20 differentially expressed proteins identified by mass spectrometry, some of which were validated in gill and hemocyte tissues using real-time quantitative RT-PCR. Many of identified proteins and their expression levels also linked with the shrimp response during WSSV infection. The list of up-regulated protein spots found exclusively in rVP28-vaccinated shrimp include calreticulin and heat shock protein 70 with chaperone properties, ubiquitin, and others. The two serine proteases, chymotrypsin and trypsin, were significantly increased in shrimp of both vaccinated groups compared to PBS controls. The information presented here should be useful for gaining insight into invertebrate immunity.
Asunto(s)
Inmunidad Innata , Penaeidae/inmunología , Proteínas Estructurales Virales/inmunología , Vacunas Virales/inmunología , Virus del Síndrome de la Mancha Blanca 1/inmunología , Animales , Branquias/inmunología , Branquias/virología , Penaeidae/genética , Penaeidae/metabolismo , Proteómica , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
White spot syndrome virus (WSSV) is a serious shrimp pathogen that has spread globally to all major shrimp farming areas, causing enormous economic losses. Here we investigate the role of hermit crabs in transmitting WSSV to Penaeus monodon brooders used in hatcheries in Vietnam. WSSV-free brooders became PCR-positive for WSSV within 2 to 14 d, and the source of infection was traced to hermit crabs being used as live feed. Challenging hermit crabs with WSSV confirmed their susceptibility to infection, but they remained tolerant to disease even at virus loads equivalent to those causing acute disease in shrimp. As PCR screening also suggests that WSSV infection occurs commonly in hermit crab populations in both Vietnam and Taiwan, their use as live feed for shrimp brooders is not recommended.
Asunto(s)
Alimentación Animal , Anomuros , Dieta , Penaeidae/virología , Virus del Síndrome de la Mancha Blanca 1/fisiología , Animales , Acuicultura , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de RiesgoRESUMEN
Automatic bird detection in ornithological analyses is limited by the accuracy of existing models, due to the lack of training data and the difficulties in extracting the fine-grained features required to distinguish bird species. Here we apply the domain randomization strategy to enhance the accuracy of the deep learning models in bird detection. Trained with virtual birds of sufficient variations in different environments, the model tends to focus on the fine-grained features of birds and achieves higher accuracies. Based on the 100 terabytes of 2-month continuous monitoring data of egrets, our results cover the findings using conventional manual observations, e.g., vertical stratification of egrets according to body size, and also open up opportunities of long-term bird surveys requiring intensive monitoring that is impractical using conventional methods, e.g., the weather influences on egrets, and the relationship of the migration schedules between the great egrets and little egrets.
Asunto(s)
Migración Animal/fisiología , Aves/fisiología , Aprendizaje Profundo , Distribución Aleatoria , Animales , Ambiente , Tiempo (Meteorología)RESUMEN
In this study, the probiotic, Bacillus subtilis E20, isolated from the human health food, natto, was used for white shrimp, Litopenaeus vannamei, larvae breeding to improve the larval survival rate and development by adding probiotic to the rearing water at (control), 10(8), and 10(9) cfu L(-1) salt water once every 3 days during the 14 days of breeding experiment. Thereafter, stress tolerance and immune status of postlarvae were evaluated. Shrimp larval development was significantly accelerated after adding the probiotic to the larval rearing water at a level of 10(9) cfu L(-1). The survival rate of larvae was significantly higher in the treatment with 10(9) cfu L(-1) compared to the control and the treatment with 10(8) cfu L(-1) after all larvae had metamorphosed to postlarvae. Adding the probiotic to the shrimp larvae rearing water produced a weak inhibition of bacterial growth by an analysis of the total bacterial count and presumptive Vibrio count. For stress tests, no postlarvae died when they were reared in water in which the temperature was decreased from 30 to 2 degrees C at a rate of 0.1 degrees C min(-1). Postlarvae had significantly lower cumulate mortality in the treatments with 10(8) and 10(9) cfu L(-1) compared to the control when they were suddenly exposed to fresh water and 60 per thousand salt water. A significant decrease in the cumulative mortality of postlarvae treated with the probiotic at a level of 10(9) cfu L(-1) was recorded after the sudden transfer to 300 mg L(-1) nitrite-N compared to the control and treatment with 10(8) cfu L(-1). The analysis of immune-related gene expressions showed that the gene expression of prophenoloxidase I, prophenoloxidase II, and lysozyme of larvae were significantly increased after being reared in probiotic-containing water at the levels of 10(8) and 10(9) cfu L(-1). However, no significant difference in serine proteinase or glutathione peroxidase gene expressions was recorded in this study. It is therefore suggested that 10(9) cfu L(-1) of probiotic, B. subtilis E20 adding to rearing water for shrimp larva breeding.
Asunto(s)
Bacillus subtilis , Penaeidae/fisiología , Probióticos , Estrés Fisiológico , Animales , Catecol Oxidasa/metabolismo , Precursores Enzimáticos/metabolismo , Glutatión Peroxidasa/metabolismo , Larva , Muramidasa/metabolismo , Penaeidae/crecimiento & desarrollo , Penaeidae/inmunología , Penaeidae/microbiología , Salinidad , Agua de Mar/química , Agua de Mar/microbiología , Serina Proteasas/metabolismo , Análisis de SupervivenciaRESUMEN
Viral glycoproteins are expressed by many viruses, and during infection they usually play very important roles, such as receptor attachment or membrane fusion. The mature virion of the white spot syndrome virus (WSSV) is unusual in that it contains no glycosylated proteins, and there are currently no reports of any glycosylation mechanisms in the pathogenesis of this virus. In this study, we cloned a glycosylase, mannosyl-glycoprotein endo-ß-N-acetylglucosaminidase (ENGase, EC 3.2.1.96), from Penaeus monodon and found that it was significantly up-regulated in WSSV-infected shrimp. A yeast two-hybrid assay showed that PmENGase interacted with both structural and non-structural proteins, and GST-pull down and co-immunoprecipitation (Co-IP) assays confirmed its interaction with the envelope protein VP41B. In the WSSV challenge tests, the cumulative mortality and viral copy number were significantly decreased in the PmEngase-silenced shrimp, from which we conclude that shrimp glycosylase interacts with WSSV in a way that benefits the virus. Lastly, we speculate that the deglycosylation activity of PmENGase might account for the absence of glycosylated proteins in the WSSV virion.
Asunto(s)
Proteínas de Artrópodos/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Penaeidae/virología , Proteínas del Envoltorio Viral/metabolismo , Virus del Síndrome de la Mancha Blanca 1/patogenicidad , Animales , Acuicultura , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/aislamiento & purificación , Línea Celular , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/aislamiento & purificación , Penaeidae/inmunología , Unión Proteica/inmunología , Interferencia de ARN , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleasas/metabolismo , Técnicas del Sistema de Dos Híbridos , Regulación hacia Arriba/inmunología , Virus del Síndrome de la Mancha Blanca 1/inmunología , Virus del Síndrome de la Mancha Blanca 1/metabolismoRESUMEN
A selenium dependent glutathione peroxidase (Se-GPx) cDNA was cloned from haemocyte by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA (RACE). The 913 bp cDNA contained an open reading frame (ORF) of 558 bp encoded a deduced amino acid sequence of 186 amino acids. The prawn Se-GPx sequence contains a selenocysteine (Sec) residue which is encoded by the unusual stop codon, (115)TGA(117). According to the molecular modeling analysis, the active site Sec residue, located in the loop between beta3 and alpha2 in a pocket on the protein surface, and hydrogen bonded to Gln(73) and Trp(141). A GPx signature motif 2, (63)LAFPCNQF(70) and active site motif, (151)WNFEKF(156), two arginine (R) residues, R(89) and R(167) contribute to the electrostatic architecture that directs the glutathione donor substrate, and two putative N-glycosylation site, (75)NNT(77) and (107)NGS(109) were observed in the prawn Se-GPx sequence. In addition, the eukaryotic selenocysteine insertion sequence element is conserved in the 3'-UTR. Comparison of amino acid sequences showed that prawn Se-GPx is more closely related to vertebrate GPx 1. The prawn Se-GPx was synthesized in haemocyte, hepatopancreas, muscle, stomach, gill, intestine, eyestalk, heart, epidermis, lymph organ, ventral nerve cord, testis and ovary. The increase of respiratory burst in haemocyte was observed in pathogen, Debaryomyces hansenii-injected prawn in order to kill the pathogen, and the up-regulation in SOD and GPx acitivity, and prawn Se-GPx mRNA transcription were involved with the protection against damage from oxidation.
Asunto(s)
Glutatión Peroxidasa/genética , Palaemonidae/enzimología , Palaemonidae/genética , Selenio/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Debaryomyces/fisiología , Regulación Enzimológica de la Expresión Génica , Glutatión Peroxidasa/química , Glutatión Peroxidasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Palaemonidae/inmunología , Palaemonidae/microbiología , Filogenia , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Although the JAK/STAT signaling pathway is usually involved in antiviral defense, a recent study suggested that STAT might be annexed by WSSV (white spot syndrome virus) to enhance the expression of a viral immediate early gene in infected shrimps. In the present study, we clone and report the first full-length cDNA sequence for a crustacean STAT from Penaeus monodon. Alignment and comparison with the deduced amino acid sequences of other STATs identified several important conserved residues and functional domains, including the DNA binding domain, SH2 domain and C-terminal transactivation domain. Based on these conserved sequences, a phylogenetic analysis suggested that shrimp STAT belongs to the ancient STAT family, while the presence of the functional domains suggested that shrimp STAT might share similar functions and regulating mechanisms with the well-known STATs isolated from model organisms. Real-time PCR showed a decreased transcription level of shrimp STAT after WSSV infection, but a Western blot analysis using anti-phosphorylated STAT antibody showed an increased level of phosphorylated (activated) STAT in the lymphoid organ of shrimp after WSSV infection. We further show that a primary culture of lymphoid organ cells from WSSV-infected shrimp resulted in activated STAT being translocated from the cytoplasm to the nucleus. This report provides experimental evidence that shrimp STAT is activated in response to WSSV infection. Our results support an earlier finding that WSSV does not disrupt JAK/STAT pathway, but on the contrary benefits from STAT activation in the shrimp host.
Asunto(s)
Penaeidae/metabolismo , Penaeidae/virología , Factores de Transcripción STAT/metabolismo , Virus del Síndrome de la Mancha Blanca 1/fisiología , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Humanos , Datos de Secuencia Molecular , Penaeidae/química , Penaeidae/genética , Filogenia , ARN Mensajero , Factores de Transcripción STAT/química , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/inmunología , Alineación de Secuencia , Transcripción Genética/genéticaRESUMEN
The core of the Vibrio Harveyi clade contains V. harveyi, V. campbellii, V. owensii, V. jasicida, and V. rotiferianus. They are well recognized aquatic animal pathogens, but misclassification has been common due to similarities in their rDNA sequences and phenotypes. To better understand their evolutionary relationships and functional features, we sequenced a shrimp pathogen strain V. harveyi 1114GL, reclassified it as V. campbellii and compared this and 47 other sequenced Vibrio genomes in the Harveryi clade. A phylogeny based on 1,775 genes revealed that both V. owensii and V. jasicida were closer to V. campbellii than to V. harveyi and that V. campbellii strains can be divided into two distinct groups. Species-specific genes such as intimin and iron acquisition genes were identified in V. campbellii. In particular, the 1114GL strain contains two bacterial immunoglobulin-like genes for cell adhesion with 22 Big_2 domains that have been extensively reshuffled and are by far the most expanded among all species surveyed in this study. The 1114GL strain differed from ATCC BAA-1116 by ~9% at the synonymous sites, indicating high diversity within V. campbellii. Our study revealed the characteristics of V. campbellii in the Harveyi clade and the genetic basis for their wide-spread pathogenicity.
Asunto(s)
Genoma Bacteriano , Genómica , Filogenia , Vibrio/genética , Secuencia de Bases , Elementos Transponibles de ADN/genética , Dosificación de Gen , Genes Bacterianos , Selección Genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Sintenía/genéticaRESUMEN
Transglutaminases (TGs) play critical roles in blood coagulation, immune responses, and other biochemical functions, which undergo post-translational remodeling such as acetylation, phosphorylation and fatty acylation. Two types of TG have been identified in white shrimp, Litopenaeus vannamei, and further investigation on their potential function was conducted by gene silencing in the present study. Total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, transglutaminase (TG) activity, haemolymph clotting time, and phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when shrimps were individually injected with diethyl pyrocarbonate-water (DEPC-H2O) or TG dsRNAs. In addition, haemolymph glucose and lactate, and haemocytes crustin, lysozyme, crustacean hyperglycemic hormone (CHH), transglutaminaseI (TGI), transglutaminaseII (TGII) and clotting protein (CP) mRNA expression were determined in the dsRNA injected shrimp under hypothermal stress. Results showed that TG activity, phagocytic activity and clearance efficiency were significantly decreased, but THC, hyaline cells (HCs) and haemolymph clotting time were significantly increased in the shrimp which received LvTGI dsRNA and LvTGI + LvTGII dsRNA after 3 days. However, respiratory burst per haemocyte was significantly decreased in only LvTGI + LvTGII silenced shrimp. In hypothermal stress studies, elevation of haemolymph glucose and lactate was observed in all treated groups, and were advanced in LvTGI and LvTGI + LvTGII silenced shrimp following exposure to 22 °C. LvCHH mRNA expression was significantly up-regulated, but crustin and lysozyme mRNA expressions were significantly down-regulated in LvTGI and LvTGI + LvTGII silenced shrimp; moreover, LvTGII was significantly increased, but LvTGI was significantly decreased in LvTGI silenced shrimp following exposure to 28 and 22 °C. Knockdown of LvTGI and LvTGI + LvTGII also significantly increased the mortality of L. vannamei challenged with the pathogen V. alginolyticus. The same consequences have been confirmed in LvTGII silenced shrimp in our previous study. These results indicate that LvTGI and LvTGII not only reveal a complementary effect in gene expression levels but also play a key function in the immune defence mechanism of shrimp, by regulating the haemolymph coagulation, immune parameters and immune related gene expression, and in the regulation of carbohydrate metabolism.
Asunto(s)
Hemocitos/citología , Monofenol Monooxigenasa/genética , Penaeidae/inmunología , Fagocitosis/inmunología , Transglutaminasas/genética , Vibriosis/inmunología , Vibrio alginolyticus/inmunología , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Metabolismo de los Hidratos de Carbono/genética , Dietil Pirocarbonato/farmacología , Regulación de la Expresión Génica/inmunología , Hemolinfa/química , Hemolinfa/inmunología , Inmunidad Innata , Hormonas de Invertebrados/genética , Hormonas de Invertebrados/metabolismo , Monofenol Monooxigenasa/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Estallido Respiratorio/inmunología , Estrés Fisiológico/inmunología , Superóxido Dismutasa/metabolismo , Transglutaminasas/metabolismo , Vibriosis/microbiologíaRESUMEN
By microarray screening, we identified a white spot syndrome virus (WSSV)-strongly induced novel gene in gills of Penaeus monodon. The gene, PmERP15, encodes a putative transmembrane protein of 15 kDa, which only showed some degree of similarity (54-59%) to several unknown insect proteins, but had no hits to shrimp proteins. RT-PCR showed that PmERP15 was highly expressed in the hemocytes, heart and lymphoid organs, and that WSSV-induced strong expression of PmERP15 was evident in all tissues examined. Western blot analysis likewise showed that WSSV strongly up-regulated PmERP15 protein levels. In WSSV-infected hemocytes, immunofluorescence staining showed that PmERP15 protein was colocalized with an ER enzyme, protein disulfide isomerase, and in Sf9 insect cells, PmERP15-EGFP fusion protein colocalized with ER -Tracker™ Red dye as well. GRP78, an ER stress marker, was found to be up-regulated in WSSV-infected P. monodon, and both PmERP15 and GRP78 were up-regulated in shrimp injected with ER stress inducers tunicamycin and dithiothreitol. Silencing experiments showed that although PmERP15 dsRNA-injected shrimp succumbed to WSSV infection more rapidly, the WSSV copy number had no significant changes. These results suggest that PmERP15 is an ER stress-induced, ER resident protein, and its induction in WSSV-infected shrimp is caused by the ER stress triggered by WSSV infection. Furthermore, although PmERP15 has no role in WSSV multiplication, its presence is essential for the survival of WSSV-infected shrimp.
Asunto(s)
Estrés del Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Penaeidae/virología , Virus del Síndrome de la Mancha Blanca 1/patogenicidad , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Ditiotreitol/farmacología , Chaperón BiP del Retículo Endoplásmico , Técnicas de Silenciamiento del Gen , Branquias/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Penaeidae/genética , Penaeidae/metabolismo , Análisis de Secuencia de ADN , Células Sf9 , Tunicamicina/farmacologíaRESUMEN
We cloned the complementary deoxyribonucleic acid (cDNA) of the heat shock cognate 70 (hsc70) gene of tiger shrimp (Penaeus monodon). It was 2207 bp long and included a 1959-bp coding region, a 40-bp flanking region at the 5' end, and a 208-bp flanking region at the 3' end. The deduced, 652-amino acid sequence had a molecular mass of 71 481 Da and an estimated isoelectric point (pI) of 5.2. Based on phylogenetic analysis, the gene is clustered with the hsc70 proteins of invertebrates and vertebrates. In native gel electrophoresis, recombinant P. monodon hsc70 expressed in an Escherichia coli system is tightly associated with carboxymethylated alpha-lactalbumin (CMLA), which indicates that hsc70 probably functions as a chaperone. In an in vitro adenosine triphosphatase assay, recombinant hsc70 hydrolyzed adenosine triphosphate to adenosine-5'-diphosphate and increased hydrolysis activity by binding to unfolded peptide, CMLA. In situ hybridization using an antisense riboprobe revealed that the hsc70 gene was active in most tissues of unstressed shrimp. The expression of hsc70 messenger ribonucleic acid (mRNA) in hemocytes increased 2- to 3-fold at the first hour after shrimp experienced heat shock and 0.5-hour recovery. Hsc70 mRNA decreased gradually to the background level. Cloning and characterizing the P. monodon hsc70 gene is the first, crucial step in studying the relationship of heat shock proteins with the stress or immune responses of shrimp.
Asunto(s)
Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Penaeidae/genética , Penaeidae/metabolismo , Adenosina Trifosfatasas/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Proteínas del Choque Térmico HSC70 , Hemocitos/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Positive results were obtained with nested white spot syndrome virus (WSSV) diagnostic PCR performed on 5 commercial brands of dry-packed Artemia cysts using several WSSV genomic sequence-specific primers. In 2 brands, PCR and nucleotide sequence analysis found C-->T and C-->G point mutations in the pms 146 WSSV amplicon, but in all 5 brands, the nucleotide sequences that were successfully amplified by the rrl, rr2 and tk-tmk gene-specific primer sets were identical to those of Penaeus monodon WSSV. However, despite the inarguable presence of WSSV or WSSV-like template DNA, we were unable to detect WSSV by PCR in hatched nauplii derived from PCR-positive cysts or in P. monodon postlarvae fed Artemia nauplii hatched from such cysts. Most simply, these results suggested that the cysts were externally contaminated with WSSV or WSSV-like template material that was removed during hatching and washing of the nauplii. Given the small sequence variations found, it may also have been a variety of WSSV non-infectious for P. monodon or Artemia and derived from other crustaceans or arthropods in the Artemia environment. However, we could not establish this conclusively and a small possibility remained that the PCR template in these tests was derived from WSSV template present internally in the cysts and derived from infected Artemia adults. However small, this possibility must be vigorously tested, given the impact that a positive outcome could have on the shrimp industry.
Asunto(s)
Artemia/virología , Virus ADN/aislamiento & purificación , Decápodos/virología , Secuencia de Aminoácidos , Animales , Acuicultura , Secuencia de Bases , Cartilla de ADN , Virus ADN/genética , ADN Viral/química , Amplificación de Genes , Larva/virología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Prophenoloxidase (proPO) and cytosolic manganese superoxide dismutase (cytMnSOD) play crucial roles in crustacean innate immunity. In the present study, both of the above genes were cloned from hemocytes of the red claw crayfish Cherax quadricarinatus. A phylogenetic analysis of the amino acid sequences showed that C. quadricarinatus proPO and cytMnSOD were more closely related to the proPO and cytMnSOD of other crayfish than to those of penaeids, crabs, lobsters, or freshwater prawns. A tissue distribution analysis revealed that proPO was primarily expressed in hemocytes, gills, and the heart, while cytMnSOD was detected in all tissues examined. All of the crayfish artificially infected with white spot syndrome virus (WSSV) died within 4 days. According to a non-lethal dose, there was no mortality in crayfish when infected deliberately with Aeromonas hydrophila. Total hemocyte counts (THCs) had significantly decreased in crayfish at 48 and 72 h after infection with WSSV compared to the control group. In contrast, THCs of crayfish after A. hydrophila challenge had recovered by 48 and 72 h from a lower level at 24 h. There were similar responses in enzyme activities toward WSSV and A. hydrophila infection. Phenoloxidase (PO) and superoxide dismutase (SOD) activities per hemocyte significantly increased from 48 to 72 h compared to the control group. After WSSV challenge, expressions of proPO and cytMnSOD transcripts in hemocytes significantly decreased at 12h, then had respectively recovered and increased at 24 h. At 48-72 h, transcript levels were finally downregulated. No significant differences in the expression profiles of proPO and cytMnSOD were observed between the A. hydrophila-infected and control groups, besides the significant upregulation at 24h post-infection. These results implicate proPO and cytMnSOD in the immune response, and they presented similar expression patterns, although different defense mechanisms may exist for crayfish induced by WSSV and A. hydrophila.
Asunto(s)
Aeromonas hydrophila/inmunología , Astacoidea/inmunología , Catecol Oxidasa/inmunología , Precursores Enzimáticos/inmunología , Superóxido Dismutasa/inmunología , Virus del Síndrome de la Mancha Blanca 1/inmunología , Aeromonas hydrophila/fisiología , Secuencia de Aminoácidos , Animales , Astacoidea/microbiología , Astacoidea/virología , Secuencia de Bases , Catecol Oxidasa/genética , Catecol Oxidasa/metabolismo , Clonación Molecular , Citosol/enzimología , Citosol/inmunología , ADN Complementario/química , ADN Complementario/genética , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Agua Dulce , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica/inmunología , Hemocitos/inmunología , Hemocitos/metabolismo , Interacciones Huésped-Patógeno/inmunología , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Análisis de Supervivencia , Factores de Tiempo , Virus del Síndrome de la Mancha Blanca 1/fisiologíaRESUMEN
Complementary (c)DNA encoding selenophosphate synthetase (SPS) messenger (m)RNA of the tiger shrimp Penaeus monodon, designated PmSPS, was obtained from the hepatopancreas by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The 1582-bp cDNA contained an open reading frame (ORF) of 1248 bp, a 103-bp 5'-untranslated region (UTR), and a 231-bp 3'-UTR, which contained a conserved selenocysteine insertion sequence (SECIS) element, a conventional polyadenylation signal, and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (416 aa) was 45.5 kDa with an estimated pI of 4.85. It contained a putative selenocysteine residue which was encoded by the unusual stop codon, (275)TGA(277), which formed at the active site with residues Sec(58) and Lys(61). A comparison of amino acid sequences showed that PmSPS was more closely related to invertebrate SPS1, such as those of Heliothis virescens and Drosophila melanogaster a and b. PmSPS cDNA was synthesized in all tested tissues, especially in the hepatopancreas. PmSPS in the hepatopancreas of shrimp significantly increased after an injection with either Photobacterium damsela or white spot syndrome virus (WSSV) in order to protect cells against damage from oxidation, and enhance the recycling of selenocysteine or selenium metabolism, indicating that PmSPS is involved in the disease-resistance response. The PmSPS expression by hemocytes significantly increased in stage C, and then gradually decreased until stage A, suggesting that the cloned PmSPS in hemocytes might play a role in viability by renewing hemocytes and antioxidative stress response for new exoskeleton synthesis during the molt cycle of shrimp.
Asunto(s)
Hemocitos/metabolismo , Infecciones/metabolismo , Penaeidae , Fosfotransferasas/metabolismo , Photobacterium/inmunología , Virus del Síndrome de la Mancha Blanca 1/inmunología , Animales , Secuencia de Bases , Clonación Molecular , Drosophila melanogaster/genética , Evolución Molecular , Hemocitos/inmunología , Hemocitos/microbiología , Hemocitos/patología , Hemocitos/virología , Inmunidad/genética , Infecciones/genética , Infecciones/inmunología , Datos de Secuencia Molecular , Muda/genética , Estrés Oxidativo/genética , Fosfotransferasas/genética , Fosfotransferasas/inmunología , Photobacterium/patogenicidad , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Activación Transcripcional , Virus del Síndrome de la Mancha Blanca 1/patogenicidadRESUMEN
Although adaptive immunity is believed to exist only in higher vertebrates, recent studies showed the ability to vaccinate shrimp and other crayfish against white spot syndrome virus (WSSV). This study attempted to establish parameters of vaccination coordinated with subsequent viral challenge to gain insights into the mechanisms of the protective response of penaeid shrimp. Two WSSV envelope proteins, VP28 and VP36B, were used as subunit vaccines expressed in Escherichia coli followed by histidine-tag affinity chromatographic purification. Shrimp vaccinated with the recombinant WSSV proteins and challenged with diluted WSSV inocula were intramuscularly injected in order to give a precise load. Results of the viral challenge trials showed complete survival in the rVP28 group in contrast to the rVP36B and PBS groups which exhibited 100% mortality. But this effective protection was exclusively induced from a combination of a higher dosage of rVP28 and a lower viral challenge pressure. The innate immune parameters analyzed among the three groups revealed that rVP28-treated shrimp showed the highest activity level (p<0.05) of phenoloxidase and superoxide dismutase during the entire period of 7 days post-vaccination. But there were no significant differences (p>0.05) in mRNA abundances of the Down syndrome cell adhesion molecule (Dscam) among all groups. In addition, total hemocyte counts significantly decreased in shrimp treated with the recombinant viral proteins compared to the PBS group. These results indicated that the existence of structure- and dose-dependent protective responses and the elevated innate immunity in shrimp following a protein-based vaccination might be responsible for conferring resistance against WSSV.
Asunto(s)
Inmunidad/inmunología , Penaeidae/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Virus del Síndrome de la Mancha Blanca 1/inmunología , Inmunidad Adaptativa/inmunología , Animales , Recuento de Células , Resistencia a la Enfermedad/inmunología , Electroforesis en Gel de Poliacrilamida , Expresión Génica/inmunología , Hemocitos/citología , Hemocitos/inmunología , Hemolinfa/citología , Hemolinfa/inmunología , Hemolinfa/metabolismo , Inmunidad Innata/inmunología , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/inmunología , Monofenol Monooxigenasa/metabolismo , Penaeidae/metabolismo , Penaeidae/virología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/genética , Superóxido Dismutasa/inmunología , Superóxido Dismutasa/metabolismo , Vacunación/métodos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/administración & dosificación , Virus del Síndrome de la Mancha Blanca 1/genética , Virus del Síndrome de la Mancha Blanca 1/metabolismoRESUMEN
In the early days of shrimp aquaculture, wild-captured brooders usually spawned repeatedly once every 2-4days. However, since the first outbreaks of white spot disease (WSD) nearly 20years ago, captured female brooders often died soon after a single spawning. Although these deaths were clearly attributable to WSD, it has always been unclear how spawning stress could lead to an outbreak of the disease. Using real-time qPCR, we show here that while replication of the white spot syndrome virus (WSSV; the causative agent of WSD) is triggered by spawning, there was no such increase in the levels of another shrimp DNA virus, IHHNV (infectious hypodermal and hematopoietic necrosis virus). We also show that levels of activated STAT are increased in brooders during and after spawning, which is important because shrimp STAT is known to transactivate the expression of the WSSV immediate early gene ie1. Lastly, we used dsRNA silencing experiment to show that both WSSV ie1 gene expression and WSSV genome copy number were reduced significantly after shrimp STAT was knocked-down. This is the first report to demonstrate in vivo that shrimp STAT is important for WSSV replication and that spawning stress increases activated STAT, which in turn triggers WSSV replication in WSSV-infected brooders.