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1.
Artigo em Inglês | MEDLINE | ID: mdl-31476362

RESUMO

Trypsinogens are the inactive precursors of trypsins (EC 3.4.21.4), which are digestive serine proteases. Despite knowing the properties of trypsins from Pacific white shrimp, Penaeus vannamei, the biochemical properties of shrimp trypsinogens including activation mechanisms and kinetics are unknown, due to difficulties isolating them from natural sources. In the present work, we describe the purification and biochemical characterization of four trypsinogen-like isoforms from recombinant P. vannamei trypsinogen, with a special emphasis on understanding its activation kinetics. The major trypsinogen-like isoform had an apparent molecular mass of 29 kDa. The other three forms of recombinant trypsinogen were: an N-glycosylated form of 32 kDa, a possibly O-glycosylated form of 41 kDa, and a likely double-chain form with a subunit of 23 kDa. The autoactivation profile of three-recombinant trypsinogen-like isoforms showed increased trypsin activity at a rate that was higher than that of bovine trypsinogen. This confirms the hypothesis proposed in the literature of a rapid trypsinogen autoactivation in the absence of aspartates in the activation peptide as it is for P. vannamei trypsinogen.


Assuntos
Proteínas de Artrópodes/química , Penaeidae/enzimologia , Tripsinogênio/química , Animais , Proteínas de Artrópodes/genética , Ativação Enzimática , Isoenzimas/química , Isoenzimas/genética , Penaeidae/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Tripsinogênio/genética
2.
Fish Shellfish Immunol ; 93: 144-152, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31326584

RESUMO

Phosphoglycerate kinase (EC 2.7.2.3, PGK) catalyses the reversible transfer of a phosphate group from 1,3-diphosphoglyceric acid and ADP to produce 3-phosphoglyceric acid and ATP, which represents the initial production of ATP during glycolysis; therefore, PGK is a key enzyme in the energy metabolism. To study the role of PGK in the resistance to WSSV infection in shrimp, the full-length cDNA of the PGK gene (LvPGK) from Litopenaeus vannamei was obtained by using homology cloning and RACE amplification. The tissue distribution of LvPGK and its expression changes in the main immune tissues after WSSV stimulation were obtained by quantitative real-time PCR. Furthermore, RNA interference (RNAi) was used to study the role of LvPGK in shrimp defending against WSSV infection. The results showed that the full-length cDNA sequence of LvPGK was 1855 bp, contained a 1248 bp open reading frame (ORF) encoding 415 amino acids, and included a conserved PGK domain. LvPGK presented ubiquitous expression in most examined tissues, with the most predominant expression in the muscle and the weakest expression in the intestine. LvPGK transcripts could be induced in the hemocytes and hepatopancreas by injection with WSSV. Both the replication of WSSV and the shrimp cumulative mortality decreased significantly after LvPGK knockdown (P < 0.01). After challenging LvPGK RNAi shrimp with WSSV, the concentration of glucose in the hepatopancreas and muscle tissue did not show significant change; however, the content of pyruvate and lactate decreased significantly (P < 0.05). Moreover, significant decreases in the expression levels of crustin, ALF1, ALF2 and ALF3 were also detected. The results suggested that LvPGK might be involved in WSSV replication by increasing host aerobic and anaerobic metabolism.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Penaeidae/enzimologia , Fosfoglicerato Quinase/química , Filogenia , Alinhamento de Sequência , Vírus da Síndrome da Mancha Branca 1/fisiologia
3.
Chemosphere ; 237: 124428, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31362133

RESUMO

The present study evaluates the enzyme activities and histopathological changes in the post larvae (PL) of shrimp (Penaeus monodon), green mussel (Perna viridis) and fingerlings of crescent perch (Terapon jarbua) exposed to sublethal gradient concentrations of Nickel (Ni). The median lethal concentration (LC50) values were 2.49, 66.03 and 43.92 mg Ni L-1 derived for the PL of shrimp, green mussel and fish fingerlings respectively. No Observed Effect Concentration (NOEC), Lowest Observed Effect Concentration (LOEC) and chronic values of the PL of shrimp were 46.5, 73.0 and 58.3 µg Ni L-1 derived for the 21-d survival endpoint. The NOEC, LOEC and chronic values for the 30-d survival endpoint of the green mussels and fish fingerlings were 4.6, 6.32, 5.4 and 1.95, 2.6, 2.25 mg Ni L-1 respectively. The isoforms of esterase, superoxide dismutase and malate dehydrogenase activities in the whole body tissues of test organisms were studied by native polyacrylamide gel electrophoresis after exposure to Ni. Histological examination of compound eye sections of shrimp revealed deformation, compression, fusion and detachement in the corneal cells from the corneal facet of the ommatidia indicating cellular anomalies due to Ni toxicity. Gill sections of the green mussel witnessed reduced haemolymph in sinuses of gill filaments, degenerative changes in interfilamentous junction and necrosis of frontal ciliated epithelial cells with vacuoles after exposure to Ni. Nickel affects the vision of shrimp and fish fingerlings, gills and byssus of green mussels.


Assuntos
Bivalves/efeitos dos fármacos , Níquel/toxicidade , Penaeidae/efeitos dos fármacos , Percas/crescimento & desenvolvimento , Perna (Organismo)/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Bivalves/enzimologia , Bivalves/crescimento & desenvolvimento , Esterases/química , Olho/efeitos dos fármacos , Olho/patologia , Brânquias/efeitos dos fármacos , Brânquias/patologia , Malato Desidrogenase/química , Níquel/farmacologia , Penaeidae/enzimologia , Penaeidae/crescimento & desenvolvimento , Perna (Organismo)/enzimologia , Perna (Organismo)/crescimento & desenvolvimento , Superóxido Dismutase/química
4.
Fish Shellfish Immunol ; 92: 528-535, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31252042

RESUMO

The present study investigates the effects of the dietary microbial lysozyme (ML) as an immunostimulant, on the growth performance, some immune parameters and digestive enzyme of Pacific white shrimp, Penaeus vannamei. Six hundred shrimps were obtained and randomly allocated into four groups as follows with three replicates. The shrimps were fed diets supplemented with 0 (control), 0.5, 1, and 2 g kg-1 ML for 4 months. The results indicated that dietary supplementation of ML significantly improved final weight, weight gain, average daily weight gain rate (ADG), feed conversion rate (FCR), and feed efficiency rate (FER) compared to the control (P ˂ 0.05). However, weight gain specific growth rate (SGR) and survival rate were not significantly affected by dietary ML (P ˃ 0.05). Dietary ML had a progressive effects on some immune parameters status including total antioxidant capacity (TAOC), superoxide dismutase (SOD), glutathione peroxidase (GPX), lysozyme (LYZ), alkaline phosphatase (ALP), phenoloxidase (PO) and acid phosphatase (ACP) activity as well as differential haemocyte count (DHC) and total haemocyte count (THC), in shrimps treated with the lysozyme than untreated shrimps (P ˂ 0.05). However, feeding with ML had no significant effect on plasma malondialdehyde (MDA) level (P ˃ 0.05). Furthermore, intestinal digestive enzymes (lipase, protease, and amylase) in shrimp fed with dietary ML were significantly (P ˂ 0.05) higher than those fed with non-supplemented control basal diet. Thus, the results indicate that oral administration of ML can be recommended for shrimp feed to improve immune response as well digestive enzymes activity modulation.


Assuntos
Imunidade Inata/efeitos dos fármacos , Muramidase/metabolismo , Penaeidae/crescimento & desenvolvimento , Penaeidae/imunologia , Ração Animal/análise , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Digestão/efeitos dos fármacos , Muramidase/administração & dosagem , Penaeidae/enzimologia , Distribuição Aleatória
5.
Fish Shellfish Immunol ; 90: 118-125, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31054358

RESUMO

The present study reveals purification and characterization of the lectin from the haemolymph of Metapenaeus dobsoni. The Md-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 68 kDa in SDS-PAGE. Furthermore, the molecular mass was confirmed by MALDI-TOF and functional groups present were analysed by FTIR. The surface morphology of purified Md-Lec displays the homogeneous nature of protein. The X-ray diffraction (XRD) analysis expresses three peaks at 10.7716̊, 21.6258̊ and 31.7523̊which indicate the crystalline nature of the protein and the retention time of 3.068 min evident from HPLC reveals the purity of the sample. Functional analysis of purified Md-Lec exhibits yeast agglutination activity against Saccharomyces cerevisiae and has the ability to agglutinate the human erythrocytes, which was observed by light microscopy. It also exhibited phenoloxidase activation, encapsulation and phagocytic activities. In addition, purified Md-Lec showed the broad spectrum of bacterial agglutination activity against Gram negative Vibrio parahaemolyticus and Aeromonas hydrophila, important fish pathogens. Antiviral potential and anticancer activity of purified Md-Lec against CyHV-2 virus and MDA-MB-231 breast cancer cell lines were also evaluated in this study.


Assuntos
Anti-Infecciosos/farmacologia , Proteínas de Artrópodes/imunologia , Lectinas/imunologia , Monofenol Mono-Oxigenase/metabolismo , Penaeidae/imunologia , Aeromonas hydrophila/imunologia , Aglutinação/fisiologia , Animais , Lectinas/metabolismo , Penaeidae/enzimologia , Penaeidae/metabolismo , Saccharomyces cerevisiae/imunologia , Vibrio parahaemolyticus/imunologia
6.
J Food Sci ; 84(5): 1078-1086, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30958915

RESUMO

Distribution of polyphenoloxidase (PPO) from different anatomical parts of Pacific white shrimp was examined. Among all parts, cephalothorax possessed the maximal PPO activity (P < 0.05), followed by pereopods, telson, pleopods, carapace, cuticle, and muscle, respectively. The higher PPO activity in cephalothorax was in line with the greater melanosis in this part during chilled storage. According to activity-staining toward 3,4-dihydroxy-ʟ-phenylalanine (ʟ-DOPA), PPO exhibited an activity band with a molecular weight (MW) of 210 kDa. When cephalothorax PPO was purified using ammonium sulfate precipitation and a series of chromatographic techniques, involving DEAE-Sepharose anion exchange and Sephadex G-75 gel filtration columns, homogeneity was obtained. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE, the Sephadex G-75 fraction showed a single band. The MW band on SDS-PAGE and gel filtration was estimated as 210 kDa, suggesting a monomeric molecule. For the inhibitor study, cysteine and 4-hexylresorcinol showed competitive inhibition toward PPO, while epigallocatechin gallate and kojic acid demonstrated mixed-type inhibition toward PPO. PRACTICAL APPLICATION: Melanosis (black spot formation) triggered by polyphenoloxidase (PPO) drastically reduces the shelf-life of shrimp. PPO was localized in several anatomical parts of Pacific white shrimp with varying activities. Certain compounds, including cysteine, 4-hexylresorcinol, epigallocatechin gallate, and kojic acid, showed PPO inhibitory activity with different modes of inhibition. The obtained information provided a promising method for manufacturers to keep the prime eating quality of Pacific white shrimp throughout postmortem transportation and storage using selected PPO inhibitors.


Assuntos
Catecol Oxidase , Penaeidae/enzimologia , Alimentos Marinhos/análise , Animais , Catecol Oxidase/análise , Catecol Oxidase/química , Catecol Oxidase/metabolismo , Distribuição Tecidual
7.
Int J Biol Macromol ; 130: 564-572, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30831167

RESUMO

In this study, we report the synthesis of graphene oxide nanosheets (GON) by a modified Hummers method. Then, a protease purified from the Penaeus vannamei shrimp was immobilized on the GON activated with glutaraldehyde. Several techniques such as SEM, DLS and FTIR were applied to characterize the different nano-structures at the different levels. The immobilization of the protease on the GON activated with glutaraldehyde did not affect the optimum pH, but significantly improved thermal stability and stability at extreme pH values, as well as activity at 90 °C. After 24 h of incubation at 90 °C, the free enzyme retained less than 10% of the activity, while the immobilized enzyme kept more than 90% of its original activity. The apparent Km and Vmax for Penaeus vannamei protease remained fairly similar after immobilization, a very relevant data considering the large size of the substrate (casein).In the hydrolysis of casein at 70 °C and in the presence of 2 M urea, the immobilized enzyme exhibited a higher activity than the free enzyme. The results indicate that the immobilization of the enzyme Penaeus vannamei protease on GON activated with glutaraldehyde increases its already high stability against environmental stresses and makes it suitable for biotechnological and industrial applications.


Assuntos
Enzimas Imobilizadas , Glutaral/química , Grafite , Nanoestruturas/química , Óxidos , Penaeidae/enzimologia , Peptídeo Hidrolases/química , Animais , Estabilidade Enzimática , Grafite/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Nanoestruturas/ultraestrutura , Termodinâmica
8.
Fish Shellfish Immunol ; 88: 284-292, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30849500

RESUMO

SAHH is an enzyme, playing a significant role in the catalyzation of the S-adenosyl homocysteine (SAH) into homocysteine (Hcy) and adenosine (Ado). However, little is known information of the enzyme in crustaceans. In the present study, SAHH cDNA was cloned from Litopenaeus vannamei (LvSAHH). The full length of the LvSAHH was found, containing a 5' UTR of 119 bp, an ORF of 1236 bp and a 3' UTR of 549 bp. The LvSAHH gene encoded a polypeptide of 411 amino acids with an estimated molecular mass of 45.55 kD and a predicted isoelectronic point (pI) of 5.63. Comparison of the deduced amino acid sequence showed that LvSAHH has high identity (70 %-82%) with other known species. qRT-PCR analysis revealed that LvSAHH mRNA was broadly expressed in all of the examined tissues, while the highest expression level was observed in muscle, followed by the expression in stomach, gill, pleopod, hepatopancreas, heart, eye and intestine. Subcellular localization analysis revealed that LvSAHH was predominantly localized in the cytoplasm and nucleus. LvSAHH mRNA expression levels in hepatopancreas and gill were significantly up-regulated from 6 to 48 h after V. alginolyticus injection and reached the highest level (15-fold and 8-fold, p < 0.01) at 24 h, respectively. Additionally, the Toll-like receptors (TLR) and interleukins-16 (IL-16) were detected in hepatopancreas and gill of LvSAHH-knockdown SAHH. LvRack1, LvToll1, LvToll2, LvToll3 and LvIL-16 transcripts were decreased significantly in LvSAHH-knockdown shrimp at 24 h post V. alginolyticus stimulation in hepatopancreas and gill. But LvToll3 was no significant difference in gill. In summary, these results indicated that LvSAHH may play a regulatory role in the invertebrate innate immune defense by regulating TLR and IL-16 expression.


Assuntos
Adenosil-Homocisteinase/metabolismo , Penaeidae/imunologia , Vibrio alginolyticus , Adenosil-Homocisteinase/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Expressão Gênica , Técnicas de Silenciamento de Genes , Imunidade Inata/genética , Interleucina-16/metabolismo , Penaeidae/enzimologia , Penaeidae/microbiologia , RNA Mensageiro/metabolismo , Receptores Toll-Like/metabolismo
9.
Fish Shellfish Immunol ; 89: 35-42, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30890430

RESUMO

C-type lectin has received widespread attention in animal immunomodulation functions since it was discovered, but it is still limited in crustaceans. The present study is to explore effects of one recombinant C-type lectin (LvLec protein) on haemocyte immune response in Litopenaeus vannamei (L. vannamei). The methods of keeping haemocyte immune activity were optimised by the Key Laboratory of Mariculture. The experiment was divided into four groups: control group, recombinant protein group (LvLec protein, 1.0 mg mL-1), Lipopolysaccharide group (LPS, 1.0 mg mL-1), and LPS combine with LvLec protein group (LPS + LvLec protein, 1.0 mg mL-1 + 1.0 mg mL-1), while each group processes 0, 3, 6, 9, 12, and 24 h respectively. The results showed that the haemocyte count reduced, while the exocytosis PO activity, hemagglutinating activity and phagocytic activity promoted, and the concentration of cGMP and PKA increased after LvLec protein treatment. However, the levels of antibacterial activity and bacteriolytic activity as well as the concentrations of cAMP and PKG did not change significantly after treating with LvLec protein, LPS or LPS + LvLec protein. Therefore, these results suggest that LvLec protein can stimulate the exocytosis PO activity through cGMP-PKA pathway to affect the phagocytic activity and hemagglutinating activity of L. vannamei haemocytes in vitro.


Assuntos
Hemócitos/imunologia , Imunidade Inata/genética , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Penaeidae/genética , Penaeidae/imunologia , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Catecol Oxidase/genética , Catecol Oxidase/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Hemócitos/enzimologia , Imunomodulação/genética , Lipopolissacarídeos/fisiologia , Penaeidae/enzimologia , Fagocitose/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
10.
Artigo em Inglês | MEDLINE | ID: mdl-30880278

RESUMO

Methyl farnesoate (MF), a sesquiterpenoid synthesized in the mandibular organ, regulates many physiological processes in crustaceans including growth and reproduction. In the present study, farnesoic acid O-methyltransferase (FAMeT), the key enzyme responsible for final step conversion of farnesoic acid (FA) to methyl farnesoate (MF), was cloned and characterized from the nervous tissues of Penaeus indicus. Multiple sequence alignment, prediction of conserved domain regions, phosphorylation sites identification and phylogenetic analysis indicated that putative FAMeT fragment from P. indicus (PiFAMeT), shares a high degree of sequence identity to FAMeT proteins isolated from other crustaceans species. Quantitative real-time PCR analysis revealed ubiquitous expression of PiFAMeT in all the tissues examined, with comparative higher mRNA levels in nervous tissue and ovary. Additionally, the levels of PiFAMeT also showed gradual increase of expression correlating with the advancement in ovarian maturation. Further to support their role in promoting ovarian development, serotonin treatment (5HT, 50 µg/g body weight) was given to eyestalk intact and unilaterally eyestalk ablated females which resulted in significant increase in PiFAMeT transcript levels at day 7 and day 14. The relatively higher levels of PiFAMeT, reflecting higher levels of MF, suggest a role during secondary vitellogenesis thereby regulating ovarian development in P. indicus. Further research is required to understand the synergistic interaction of MF pathways with serotonergic and other regulatory pathways in regulating ovarian maturation in penaeid shrimps.


Assuntos
Proteínas de Artrópodes , Regulação Enzimológica da Expressão Gênica/fisiologia , Metiltransferases , Ovário/enzimologia , Penaeidae , Vitelogênese/fisiologia , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Clonagem Molecular , Feminino , Metiltransferases/biossíntese , Metiltransferases/genética , Ovário/citologia , Penaeidae/enzimologia , Penaeidae/genética
11.
Fish Shellfish Immunol ; 88: 142-149, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30807860

RESUMO

pH of water environment affects the survival of aquatic animals. Intestine barrier function influences the health of animals, which is related to its mucosa structure, immune components, and microbial communities. In this study, we investigated the histological structure, digestive and metabolic capacity, immune responses, and microbial composition in the intestine of Litopenaeus vanmei under three different conditions: control (pH 8.3), low pH stress (pH 6.9), and high pH stress (pH 9.7) for 72 h. The results showed both low and high pH stress disrupted the intestine morphological structure, and induced variations in the activities of digestive (AMS, LPS, Tryp, and Pep) and metabolic (HK, PK, CCO, and LDH) enzymes. Low and high pH stress also increased oxidative stress (MDA, LPO, PC, and ·O2- generation), and decreased the antioxidant enzyme activities (T-AOC, SOD, and GST); shrimp enhanced CAT activity and HSP70, Trx, MT and Fer gene transcripts as defense mechanism. Additionally, Immune confusion was also found in the shrimp intestine in response to low and high pH stress, including the antibacterial ability (T-NOS, PO, proPO, ALF, and Lys), pathogen recognition (TLR and Lec), apoptosis (Casp, IAP and p53), and mucus homeostasis (Muc-1, Muc-2, Muc-5AC, Muc-5B, and Muc-19). pH exposure also decreased the diversity of the intestine bacterial, disturbed the composition of microbiota, and decreased the microbial metabolite SCFA contents. Our results indicated that acute pH stress can impair the intestine barrier function of white shrimp, probably via destroying mucosa structure, confusing digestion and metabolism, inducing oxidative stress, disordering immunity, and disrupting the microbial composition.


Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Intestinos/fisiologia , Penaeidae/fisiologia , Estresse Fisiológico , Animais , Aquicultura , Bactérias/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Imunidade Inata , Intestinos/enzimologia , Intestinos/imunologia , Intestinos/microbiologia , Penaeidae/enzimologia , Penaeidae/imunologia , Penaeidae/microbiologia , Poluição da Água/efeitos adversos
12.
Fish Shellfish Immunol ; 86: 160-168, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30391532

RESUMO

In this experiment, 426 strains were isolated from the intestinal tract of Litopenaeus vannamei, and 11 strains showed strong digestive enzyme production activity and antagonistic effect against common bacterial pathogens of shrimp. After hemolysis activity test and drug sensitivity test, 2 candidate probiotics with good bacteriostatic activity, strong enzyme production ability and relatively sensitive to antibiotics were screened out, and were identified by 16s rDNA molecular identification and Biolog-System as Enterobacter hominis (E3) and lactobacillus (L3). First, the biological characteristics of 2 candidate probiotics were studied. The optimum growth conditions of E3: temperature, 30 °C; pH, 8.0; NaCl, 2.5%; bovine bile salt, 0.15%; and the optimum growth conditions of L3: temperature, 40 °C; pH, 6.0; NaCl, 0.5%; bovine bile salt, 0.0015%. Secondly, a 28-day feeding experiment was conducted using probiotic concentration of 107 CFU g-1 to determine the changes of the activities of blood related immune enzymes (SOD, PPO, ACP, POD, CAT, LZM) and intestinal digestive enzymes (NP, AL, LPS) during the feeding process of shrimp, the results showed that during the course of feeding, the activities of immune enzyme and digestive enzyme of shrimp fed with probiotics showed an increasing trend, and the growth rate of body weight of shrimp was higher than that of control group. After feeding, the cumulative mortality of probiotics groups were significantly lower than that of the control group after WSSV infection. And the mid-gut of L. vannamei was observed by electron microscope, the results showed that the intestinal mucosa was tight and the epithelium cells showed an active secretory state in probiotics group. Finally, the intestinal microbial communities of shrimp were compared and analyzed by using Biolog-ECO method in the later period of feeding, the results showed: compared with the control group, the average color change rate of the experimental group fed with probiotics increased significantly, indicating that probiotics enhanced the intestinal microorganism activity; The ability of intestinal microorganism to utilize carbon source was significantly enhanced in the experimental group, which indicated that the digestive enzyme secreted by probiotics could improve the digestion and absorption rate of prawn feed, thus promoting the rapid growth of shrimp; The Shannon index, Simpson index and McIntosh index of probiotics groups showed significant difference in 1st and 5th days, but tended to be the same in the 10th day, the results showed that probiotics could maintain in L. vannamei intestines at least 5 days.


Assuntos
Enterobacter/fisiologia , Intestinos/microbiologia , Lactobacillus/fisiologia , Penaeidae/crescimento & desenvolvimento , Probióticos , Animais , Aquicultura , Digestão/fisiologia , Penaeidae/enzimologia , Penaeidae/imunologia , Penaeidae/microbiologia
13.
Artigo em Inglês | MEDLINE | ID: mdl-30594527

RESUMO

The cell cycle comprises a series of steps necessary for cell growth until cell division. The participation of proteins responsible for cell cycle regulation, known as cyclin dependent kinases or Cdks, is necessary for cycle progression. Cyclin dependent kinase 2 (Cdk-2) is one of the most studied Cdks. This kinase regulates the passage through the G1/S phase and is involved in DNA replication in the S phase. Cdks have been extensively studied in mammals, but there is little information about these proteins in crustaceans. In the present work, the nucleotide and amino acid sequence of Cdk-2 from the white shrimp (Cdk-2) and its expression during hypoxia and reoxygenation are reported. Cdk-2 is a highly conserved protein and contains the serine/threonine catalytic domain, an ATP binding site and the PSTAIRE sequence. The predicted Cdk-2 structure showed the two-lobed structure characteristic of kinases. Expression of Cdk-2 was detected in hepatopancreas, gills and muscle, with hepatopancreas having the highest expression during normoxic conditions. Cdk-2 expression was significantly induced after hypoxia for 24 h in muscle cells, but in hypoxia exposure for 24 followed by 1 h of reoxygenation, the expression levels returned to the levels found in normoxic conditions, suggesting induction of cell cycle progression in muscular cells during hypoxia. No significant changes in expression of Cdk-2 were detected in these conditions in hepatopancreas and gills.


Assuntos
Proteínas de Artrópodes/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Hipóxia/enzimologia , Oxigênio/metabolismo , Penaeidae/enzimologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Sequência de Bases , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/genética , Brânquias/enzimologia , Hepatopâncreas/enzimologia , Músculos/enzimologia , Penaeidae/metabolismo , Filogenia
14.
Aquat Toxicol ; 208: 1-11, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30592983

RESUMO

Proline (Pro) metabolism is intimately associated with stress adaptation. The catabolism of Pro includes two dehydrogenation reactions catalyzed by proline dehydrogenase (ProDH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDh). P5CDh is a mitochondrial matrix NAD+-dependent dehydrogenase that is critical in preventing P5C-Pro intensive cycling and avoiding ROS production from electron run-off. Little is known about the roles of P5CDh in invertebrates, however. We cloned the P5CDh sequence in the Pacific white shrimp, Litopenaeus vannamei, and found that LvP5CDh is expressed predominantly in pleopod, hepatopancreas and gill. Subcellular localization analysis revealed that LvP5CDh protein was mainly found in the cytoplasm. In addition, overexpressing LvP5CDh in cells reduced ROS formation and inhibited apoptosis induced by LC50 Cd2+. Shrimp were exposed to various stress factors including infection with Vibrio alginolyticus, (½ LC50 and LC50) Cd2+, acid (pH 5.6) and alkali stress (pH 9.3). Both biotic and abiotic stress resulted in increased LvP5CDh expression and Pro accumulation; V. alginolyticus infection, pH 9.3 and LC50 Cd2+ stress apparently stimulated the Glu pathway of Pro synthesis, while pH 5.6 and ½ LC50 Cd2+ stress promoted the Orn pathway of Pro synthesis. Silencing of Lvp53 in shrimp attenuated LvP5CDh expression during Cd2+ stress, but had no effect on LvP5CDh mRNA levels if no Cd2+ stress was imposed. Our study contributes to the functional characterization of LvP5CDh in biotic and abiotic stress and reveals it to protect against ROS generation, damage to the cell, including the mitochondria, and apoptosis. Thus, LvP5CDh plays a critical role in immune defense and antioxidant responses.


Assuntos
1-Pirrolina-5-Carboxilato Desidrogenase/metabolismo , Penaeidae/enzimologia , Penaeidae/fisiologia , Estresse Fisiológico , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Apoptose , DNA Complementar/genética , Inativação Gênica , Ácido Glutâmico/metabolismo , Mitocôndrias/metabolismo , Especificidade de Órgãos , Penaeidae/virologia , Peptídeos/química , Prolina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Proteína Supressora de Tumor p53/metabolismo
15.
Front Immunol ; 9: 2184, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30337920

RESUMO

MicroRNAs (miRNAs), the small non-coding RNAs, play a pivotal role in post-transcriptional gene regulation in various cellular processes. However, the miRNA function in shrimp antiviral response is not clearly understood. This research aims to uncover the function of pmo-miR-315, a white spot syndrome virus (WSSV)-responsive miRNAs identified from Penaeus monodon hemocytes during WSSV infection. The expression of the predicted pmo-miR-315 target mRNA, a novel PmPPAE gene called PmPPAE3, was negatively correlated with that of the pmo-miR-315. Furthermore, the luciferase assay indicated that the pmo-miR-315 directly interacted with the target site in PmPPAE3 suggesting the regulatory role of pmo-miR-315 on PmPPAE3 gene expression. Introducing the pmo-miR-315 into the WSSV-infected shrimp caused the reduction of the PmPPAE3 transcript level and, hence, the PO activity activated by the PmPPAE3 whereas the WSSV copy number in the shrimp hemocytes was increased. Taken together, our findings state a crucial role of pmo-miR-315 in attenuating proPO activation via PPAE3 gene suppression and facilitating the WSSV propagation in shrimp WSSV infection.


Assuntos
Catecol Oxidase/genética , Precursores Enzimáticos/genética , Proteínas de Insetos/genética , MicroRNAs/metabolismo , Penaeidae/genética , Viroses/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia , Animais , Catecol Oxidase/imunologia , Precursores Enzimáticos/imunologia , Regulação da Expressão Gênica/imunologia , Hemócitos/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Proteínas de Insetos/imunologia , Penaeidae/enzimologia , Penaeidae/imunologia , Penaeidae/virologia , Viroses/enzimologia , Viroses/virologia
16.
Dev Comp Immunol ; 89: 54-65, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30092318

RESUMO

Lactic acid bacteria (LAB) are group of beneficial bacteria that have been proposed as relevant probiotics with immunomodulatory functions. In this study, we initially isolated and identified host-derived LAB from the gut of the Pacific white shrimp Litopenaeus vannamei. Analysis of the bacterial 16S rRNA gene sequence revealed two candidate LAB, the Lactobacillus plantarum strain SGLAB01 and the Lactococcus lactis strain SGLAB02, which exhibited 99% identity to the L. plantarum strain LB1-2 and the L. lactis strain R-53658, which were isolated from bee gut, respectively. The two LAB displayed antimicrobial activities against gram-positive and gram-negative bacteria, including the virulent acute hepatopancreatic necrosis disease (AHPND)-causing strain of Vibrio parahaemolyticus (VPAHPND). Viable colony count and SEM analysis showed that the two candidate LAB, administered via oral route as feed supplement, could reside and adhere in the shrimp gut. Double-stranded RNA-mediated gene silencing of LvproPO1 and LvproPO2 revealed a significant role of two LvproPOs in the proPO system as well as in the immune response against VPAHPND infection in L. vannamei shrimp. The effect of LAB supplementation on modulation of the shrimp proPO system was investigated in vivo, and the results showed that administration of the two candidate LAB significantly increased hemolymph PO activity, the relative mRNA expression of LvproPO1 and LvproPO2, and resistance to VPAHPND infection. These findings suggest that administration of L. plantarum and L. lactis could modulate the immune system and increase shrimp resistance to VPAHPND infection presumably via upregulation of the two LvproPO transcripts.


Assuntos
Proteínas de Artrópodes/imunologia , Catecol Oxidase/imunologia , Precursores Enzimáticos/imunologia , Lactobacillales/imunologia , Penaeidae/imunologia , Penaeidae/microbiologia , Vibrio parahaemolyticus/patogenicidade , Animais , Aquicultura , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Catecol Oxidase/genética , Catecol Oxidase/metabolismo , Ativação Enzimática , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Lactobacillales/genética , Lactobacillus plantarum/genética , Lactobacillus plantarum/imunologia , Penaeidae/enzimologia , Filogenia , Probióticos , Alimentos Marinhos , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio parahaemolyticus/imunologia
17.
Fish Shellfish Immunol ; 81: 168-175, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30010014

RESUMO

Shrimps, which mainly rely on their innate immune system to response to infectious pathogens, have clottable proteins as an important component of this system. While transglutaminases (TGase) are found in Litopenaeus vannamei and constitute part of the coagulation system, the specific immune-related roles played by its functional domains in the immunoregulation of shrimp has not been well understood. In the present study, we report that the Ig-like domain of L. vannamei transglutaminase (TGase-C) is the main immune-related domain among the three functional domains, as it had higher bacterial agglutinative activity against Vibrio parahaemolyticus and Streptococcus iniae. Using Co-immunoprecipitation and LC-MS/MS analysis, TGase-C was shown to interact with 474 proteins, of which 52 proteins were annotated to L. vannamei. More than half of the L. vannamei annotated proteins have immune-related functions, including apoptosis. Further analysis using pull-down assay revealed that TGase-C interacted with CAP-3 (a homologue of caspase 3). In addition, siRNA-mediated knockdown of LvTGase significantly (p < 0.01) increased the expression level of LvCAP-3 coupled with a significant (p < 0.01) increase in caspase 3/7 activity, suggesting that probably LvTGase participates in shrimp immune response by modulating the activity of LvCAP-3. These findings thus suggest the Ig-like functional domain of L. vannamei's transglutaminase is the domain that is involved in immunoregulation in shrimp.


Assuntos
Proteínas de Artrópodes/imunologia , Imunidade Inata , Penaeidae/enzimologia , Penaeidae/imunologia , Transglutaminases/imunologia , Animais , Apoptose , Regulação da Expressão Gênica , Hemócitos , Filogenia , Alinhamento de Sequência , Streptococcus iniae , Vibrio parahaemolyticus
18.
Int J Biol Macromol ; 118(Pt A): 92-98, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-29913192

RESUMO

The activity, stability, and kinetics of immobilized enzymes are influenced by the nature of the support or carrier material. In this research, zinc oxide nanoparticles were synthesized by chemical precipitation, and the purified protease from shrimp Penaeus vanamei was immobilized on the nanoparticles. Size, structure, and morphology of the ZnO nanoparticles, and the immobilization of the protease were studied by transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, UV-Vis spectroscopy, and dynamic light scattering (DLS). The immobilization of protease on ZnO nanoparticles improved the long-term and thermal stability, plus its stability at extreme pH values, and it increased the optimum functional temperature of the enzyme. The optimum pH value of the immobilized protease was shifted from 7.0 to 8.0 upon immobilization. Additionally, and due to the immobilization an increased Km was observed, whereas its catalytic efficiency was estimated a little less as compared to that of free enzyme. These results show that the immobilization of Penaeus vanamei protease on zinc oxide nanoparticles enhanced its appropriateness for a future use in various biotechnological and industrial applications.


Assuntos
Enzimas Imobilizadas/química , Nanopartículas de Magnetita/química , Óxido de Zinco/química , Animais , Estabilidade Enzimática , Cinética , Microscopia Eletrônica de Transmissão , Penaeidae/enzimologia , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura Ambiente , Difração de Raios X
19.
Food Chem ; 260: 166-173, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-29699658

RESUMO

Phenoloxidases (POs) play a crucial role in melanization of crustaceans. There are at least two types of POs characterized in crustaceans: the conventional type (POα here) that is expressed in hemocytes and POß, a secreted protein synthesized in the hepatopancreas. We investigated the source of PO activity in the hemolymph of a lobster and determined the kinetic parameters of mono- and di-PO activities. In the lobster hemolymph, POα, which formed a hexamer similar to both POß and hemocyanin, contributed to PO activity, whereas the amount of POß was low. Kinetic analyses using purified prophenoloxidase of crustaceans showed that lobster POα has a higher rate constant, while shrimp POß has higher specificity in both mono- and di-PO reactions, when tyramine and dopamine were employed as substrates. There should be at least two types of PO molecules in crustacean hemolymph, but the dominant PO molecule type varies among species.


Assuntos
Hemolinfa/enzimologia , Monofenol Mono-Oxigenase/metabolismo , Palinuridae/enzimologia , Animais , Catecol Oxidase , Dopamina/metabolismo , Precursores Enzimáticos , Hemocianinas/metabolismo , Hemócitos/enzimologia , Cinética , Penaeidae/enzimologia , Especificidade da Espécie , Especificidade por Substrato , Tiramina/metabolismo
20.
Fish Shellfish Immunol ; 78: 1-9, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29656126

RESUMO

Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea and exhibits potential antibacterial and anticancer activities. In this study, EGCG was used in pathogen-challenge experiments in shrimp to discover its effect on the innate immune system of an invertebrate. Kuruma shrimp Marsupeneaus japonicus was used as an experimental model and challenged with white spot syndrome virus (WSSV) and the Gram-negative bacterium Vibrio alginolyticus. Pathogen-challenge experiments showed that EGCG pretreatment significantly delayed and reduced mortality upon WSSV and V. alginolyticus infection, with VP-28 copies of WSSV also reduced. Quantitative reverse transcription polymerase chain reaction revealed the positive influence of EGCG on several innate immune-related genes, including IMD, proPO, QM, myosin, Rho, Rab7, p53, TNF-alpha, MAPK, and NOS, and we observed positive influences on three immune parameters, including total hemocyte count and phenoloxidase and superoxide dismutase activities, by EGCG treatment. Additionally, results showed that EGCG treatment significantly reduced apoptosis upon V. alginolyticus challenge. These results indicated the positive role of EGCG in the shrimp innate immune system as an enhancer of immune parameters and an inhibitor of apoptosis, thereby delaying and reducing mortality upon pathogen challenge. Our findings provide insight into potential therapeutic or preventive functions associated with EGCG to enhance shrimp immunity and protect shrimp from pathogen infection.


Assuntos
Catequina/análogos & derivados , Imunidade Inata/efeitos dos fármacos , Penaeidae/efeitos dos fármacos , Penaeidae/imunologia , Substâncias Protetoras/farmacologia , Vibrio alginolyticus/efeitos dos fármacos , Vírus da Síndrome da Mancha Branca 1/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Antivirais/farmacologia , Catequina/farmacologia , Penaeidae/enzimologia , Penaeidae/genética , Distribuição Aleatória , Vibrio alginolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
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