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1.
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
2.
Fish Shellfish Immunol ; 90: 456-465, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31075403

RESUMO

This study was conducted to elucidate the effects of dietary mixed probiotics on growth, non-specific immunity, intestinal morphology and microbiota of juvenile pacific white shrimp, Litopenaeus vannamei. Juvenile shrimp (initial body weight 1.21 ±â€¯0.01 g) were fed diets containing graded probiotics (F1: 0 mg/kg probiotics; F2: 1000 mg/kg probiotics; F3: 2000 mg/kg probiotics; F4: 4000 mg/kg compound probiotics; F5: 6000 mg/kg probiotics; F6: 8000 mg/kg probiotics) for 8 weeks. The result of this trial showed that the growth performance (SGR, WG, FBW) of shrimp fed diets containing probiotics (F2∼F6) were significantly higher than that of shrimp fed diet without supplemental probiotics (F1) (P < 0.05), and the highest values of the growth performance (SGR, WG, FBW) and lowest FCR were found in shrimp fed the diet containing 2000 mg/kg probiotics. Total antioxidant capacity of shrimp fed diet F2 and F3 were significantly higher than that of shrimp fed the basal diets (P < 0.05). Superoxide dismutase in F4 treatment was significantly higher than that of basal treatment (P < 0.05). Catalase of shrimp in all probiotics supplemented (F2∼F6) treatments were significantly higher than that of the control one (F1) (P < 0.05). Malondialdehyde in F5 groups was significantly lower than that of F1 groups (P < 0.05). Alkline phosphatase and acid phosphatase in F3 treatments were significantly higher than those of the basal one (P < 0.05). Lysozyme of shrimp fed F2∼F6 were significantly higher than that of shrimp fed F1 diet (P < 0.05). The lipase and amylase activities in 2000 mg/kg probiotics groups showed the highest activities and were significantly higher than that of control one (P < 0.05). Intestinal villi height in F3∼F6 treatments were significantly higher than that of control one (P < 0.05). Alpha diversity indices including observed species, chao1, ACE and shannon indices showed that F2 and F3 groups had higher microbial diversity in their intestines, both richness and evenness. PCA plot showed that there was a clear shift of F2 and F3 groups from the control groups in microbial community structure. The dominant phyla in pacific white shrimp are proteobacteria, bacteroidetes and actinobacteria, the dominant genus were algoriphagus and vibrio. As the probiotics increased, the gemmatimonadetes, acidobacteria, deltaproteobacteria and xanthomonadales firstly increased and then decreased, with the highest content in F2 group, which was no significant difference to F3 group (P > 0.05) while significantly higher than other groups (P < 0.05). In conclusion, the supplement of mixed species probiotics can promote growth performance, enhance the non-specific immunity, influence the microbiota of the pacific white shrimps and the recommended optimum dosage in diet of Litopenaeus vannamei was 2000 mg/kg.


Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Penaeidae/imunologia , Probióticos/metabolismo , Ração Animal/análise , Animais , Dieta/veterinária , Relação Dose-Resposta a Droga , Intestinos/anatomia & histologia , Intestinos/microbiologia , Penaeidae/anatomia & histologia , Penaeidae/crescimento & desenvolvimento , Penaeidae/microbiologia , Probióticos/administração & dosagem , Distribuição Aleatória
3.
Fish Shellfish Immunol ; 90: 126-133, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059814

RESUMO

To investigate the role of the Rab7 effector RILP (Rab-interacting lysosomal protein) in white spot syndrome virus (WSSV) infection, the full-length cDNA of RILP (LvRILP) was cloned in Litopenaeus vannamei, which consists of 1595 bp and encodes a polypeptide of 411 amino acids. Sequence analysis and multiple sequence alignment displayed that LvRILP contained a conserved RILP region from 277 amino acid to 325 amino acid. Both the LvRILP and Rab7 mRNA were most highly expressed in stomach and most lowly expressed in hemocyte, which were significantly up-regulated and exhibited similar kinetics post WSSV infection. The interaction of Rab7 with LvRILP was verified by both GST Pull-down and ELISA. Meanwhile, the results of Pull-down assays showed that the GST-tagged VP28 (GST-VP28), His-tagged Rab7 (His-Rab7) and His-RILP formed a tripartite complex. After silencing by specific LvRILP dsRNA, the LvRILP mRNA level exhibited a significant reduction, and the expression levels of three WSSV genes ie1, wsv477 and vp28 all exhibited decreases at 24, 36 and 48 h post WSSV infection. These results suggested that the Rab7 effector RILP was involved in WSSV infection.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência , Vírus da Síndrome da Mancha Branca 1/fisiologia
4.
Fish Shellfish Immunol ; 89: 384-392, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30951853

RESUMO

Antimicrobial peptides (AMPs) are an essential component of innate immunity of invertebrates. Anti-lipopolysaccharide factor (ALF), as a main type of AMPs in crustaceans, attends in the disease prevention in general. In this research, a novel Group D ALF was identified and characterized from Penaeus monodon, named PenmonALF8. It was an anionic peptide, with both the full-length peptide and lipopolysaccharide binding domain (LBD) a low isoelectric point. PenmonALF8, composed of a signal peptide of 26 amino acids and a mature peptide of 98 amino acids, probably contained three alpha helixes and four beta sheets. Moreover, PenmonALF8 was detected in all tested tissues of P. monodon, and the expression level in hemocyte and intestine was relatively high. When challenged by Vibrio parahaemolyticus, PenmonALF8 showed 30-100 times higher expression level in all the tissues except in hemocyte and intestine, indicating that PenmonALF8 played a very important role in the immune response of P. monodon. By fusing to a SUMO protein, PenmonALF8 was successfully over-expressed in E. coli and purified by affinity chromatography. Additionally, the reconstituted PenmonALF8 and its LBD region displayed modest antimicrobial activity. This is the first research about the Group D ALF in P. monodon, which provides more information for humoral immunity study of shrimps.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência , Vibrio/imunologia
5.
Fish Shellfish Immunol ; 89: 623-631, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30991151

RESUMO

Antimicrobial peptides (AMPs) participate in immune defenses of invertebrate, vertebrate and plant species. As a kind of AMPs, penaeidins play important roles in innate immunity of shrimp. In this study, two penaeidin homologues termed FmPEN3 and FmPEN5 were cloned and identified from Fenneropenaeus merguiensis for the first time. The complete open reading frames (ORFs) of FmPEN3 and FmPEN5 were 216 bp and 240 bp, encoding 71 and 79 amino acids, respectively. Both FmPEN3 and FmPEN5 contain an N-terminal proline-rich domain (PRD) and a C-terminal cysteine-rich domain (CRD). The genome structure of FmPEN3 and FmPEN5 genes both consist of 2 exons and 1 intron. qPCR analysis showed that FmPEN3 was constitutively expressed but FmPEN5 transcripts were found only in hemocytes, gills, epidermis, nerve and pyloric cecum. The FmPEN3 and FmPEN5 expression were responsive to Vibrio parahaemolyticus and Micrococcus lysodeikticus infection and their transcription levels were downregulated by RNAi silencing of the transcription factors FmDorsal and FmRelish. In addition, recombinant proteins of FmPEN3 (rFmPEN3) and FmPEN5 (rFmPEN5) were successfully expressed in E. coli. The antibacterial assays revealed that rFmPEN3 and rFmPEN5 could inhibit the growth of M. lysodeikticus but only rFmPEN5 could inhibit the growth of V. parahaemolyticus in vitro. In summary, the results presented in this study indicated the functions of FmPEN3 and FmPEN5 played in anti-bacterial immunity of F. merguiensis, providing some insights into the function of AMPs in shrimp.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Peptídeos/genética , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Micrococcus/fisiologia , Peptídeos/química , Filogenia , Alinhamento de Sequência , Vibrio parahaemolyticus/fisiologia
6.
Fish Shellfish Immunol ; 89: 555-563, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30999041

RESUMO

In shrimp, the JAK-STAT pathway is essentially implicated in both antiviral and antibacterial responses. However, few regulatory target genes of the JAK-STAT pathway in shrimp have been reported so far. In this study, a novel single WAP domain-containing peptide (LvSWD4) was identified from Pacific white shrimp Litopenaeus vannamei. The promoter of LvSWD4 was predicted to harbor multiple STAT-binding DNA motifs. Over-expression of the JAK-STAT pathway components STAT, JAK and Domeless in vitro significantly enhanced the transcriptional activity of the LvSWD4 promoter, and in vivo silencing of STAT and the the JAK-STAT pathway upstream regulator IRF down-regulated the expression of LvSWD4, suggesting that LvSWD4 could be a target gene of the JAK-STAT pathway. The expression of LvSWD4 was significantly increased after infection with Gram-negative and positive bacteria, fungi and virus, and silencing of LvSWD4 increased the susceptibility of shrimp to V. parahaemolyticus and WSSV infections. In vitro experiments also demonstrated that the recombinant LvSWD4 protein had significant inhibitory activities against Gram negative bacteria V. parahaemolyticus and E. coli and Gram positive bacteria S. aureus and B. subtilis. Furthermore, silencing of LvSWD4 in vivo significantly affected expression of various immune functional genes and attenuated the phagocytic activity of hemocytes. These suggested that as a target gene of STAT, LvSWD4 was essentially implicated in shrimp immunity, which could constitute part of the mechanism underlying the immune function of the shrimp JAK-STAT pathway.


Assuntos
Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Sequência de Bases , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência , Transdução de Sinais , Vibrio parahaemolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
7.
Fish Shellfish Immunol ; 89: 701-709, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31004801

RESUMO

Based on the transcriptome database, we screened out four ferritin subunit genes (MnFer2-5) from the oriental river prawn Macrobrachium nipponense, which encode two non-secretory and two secretory peptides. MnFer2 and 4 possess a strictly conserved ferroxidase site, and MnFer3 has a non-typical ferroxidase site. MnFer5 seems to be a number of ferritin families, which has a distinct dinuclear metal binding motif, but lacks an iron ion channel, a ferroxidase site and a nucleation site. Diverse tissue-specific transcriptions of the four genes indicate their functional diversity in the prawn. Among them, MnFer2 is mainly expressed in hepatopancreas and intestines, MnFer3 and 4 are predominantly expressed in gills, and MnFer5 is widely expressed in various tissues with high presence in intestines, hepatopancreas and haemocytes. The transcription of all the four MnFer genes can be strongly induced by doxorubicin, indicating the involvement of these ferritin subunits in protection from oxidative stress. Upon Aeromonas hydrophila infection, only MnFer5 is persistently up-regulated, while other subunits including MnFer2-4 are down-regulated during the early stage, followed by recovery and even a slight increase at 48 h post bacterial challenge. Moreover, the iron binding capacity of recombinant MnFer2 is also demonstrated in vitro. The E. coli expressing MnFer2 displays increased resistance to hydrogen peroxidase cytotoxicity. These results suggest a protective role of ferritins from M. nipponense in iron homeostasis, redox biology and antibacterial immunity and shed light on the molecule evolution of crustacean ferritin subunits.


Assuntos
Ferritinas/genética , Ferritinas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Estresse Oxidativo/imunologia , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Ferritinas/química , Perfilação da Expressão Gênica , Homeostase/imunologia , Oxirredução , Palaemonidae , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Distribuição Aleatória , Alinhamento de Sequência
8.
Artigo em Inglês | MEDLINE | ID: mdl-30981908

RESUMO

Aflatoxin B1 (AFB1) is a mycotoxin mainly produced by Aspergillus flavus and Aspergillus parasiticus contaminating food, feed ingredients and products of animal origin. In mammals, this toxin causes widespread organ-specific damage; it is immunotoxicity and could promote hepatotoxicity, alter intestinal functions and so on. In this study, we conducted transcriptome and histomorphology analyses of hepatopancreas and intestinal in Litopenaeus vannamei (L. vannamei) challenged with AFB1. Totally 12,014 and 1387 differentially expression genes (DEGs) were identified in the hepatopancreas and intestine, respectively. In hepatopancreas, a total of 1995 DEGs were mainly annotated and grouped into 18 processes or pathways related to animal immune system. With respect to intestine, a total of 152 DEGs were mainly annotated to 7 processes or pathways related to animal immune system. Meanwhile, we determined the relative mRNA expression of several crucial representative immune genes including Toll, immune deficiency (IMD), prophenoloxidase (proPO), Rab and glutathione S-transferase (GST) in the hepatopancreas and intestines of shrimp at 3-, 6-, 12-, 18-, 24- and 30-d after challenged by AFB1. Exposure to AFB1 increased mortality, decrease weight gain rate, severely destroyed the histomorphology of hepatopancreas and intestine, and resulted in the damaged of immune system of shrimp. The present data reveals the different roles between hepatopancreas and intestine of L. vannamei in immune response to AFB1 challenge, and provides insight into the molecular basis of the relationship between hepatopancreas and intestinal immunity during either homeostasis or inflammation.


Assuntos
Aflatoxina B1/toxicidade , Perfilação da Expressão Gênica , Hepatopâncreas/fisiologia , Intestinos/fisiologia , Penaeidae/imunologia , Penaeidae/fisiologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Hepatopâncreas/efeitos dos fármacos , Hepatopâncreas/imunologia , Intestinos/efeitos dos fármacos , Intestinos/imunologia
9.
Fish Shellfish Immunol ; 90: 188-198, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31028898

RESUMO

C-type lectins (CTLs) are pattern recognition receptors (PRRs) that are important in invertebrate innate immunity for the recognition and elimination of pathogens. Although they were reported in many shrimp, C-type lectins subfamily contain a large number of members with different functions that need to research in deep. In this present study, a new type of CTL, PmCL1 with 861 bp long full-length cDNA, that encodes a protein with 164-amino acid from a 495-bp open reading frame, was isolated and characterized from tiger shrimp (Penaeus monodon). The mRNA transcript of PmCL1 showed the highest expression in the hepatopancreas, whereas it was barely detected in the ovary. After the shrimp were stimulated by Vibrio harveyi and Vibrio anguillarum, PmCL1 expression in the hepatopancreas and gill was significantly upregulated. A carbohydrate-binding assay revealed the specificity of PmCL1 for pathogen-associated molecular patterns (PAMPs) that included peptidoglycan (PGN) and lipopolysaccharide (LPS), and saccharides that included d-glucose, galactosamine, α-lactose, treholose, and d-mannose. Recombinant PmCL1 agglutinated gram-positive (Staphylococcus aureus) and gram-negative bacteria (V. harveyi, V. anguillarum, Vibrio alginolyticus, Vibrio parahemolyticus, Vibrio vulnificus, and Aeromonas hydrophila) in the presence of calcium ions and enhanced the efficiency of clearing the invading bacteria. Collectively, our results suggested that PmCL1 might play an important role as a pattern recognition receptor (PRR) in the immune response towards pathogen infections, as well as the response towards ammonia nitrogen stress.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Penaeidae/genética , Penaeidae/imunologia , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Amônia/efeitos adversos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Lectinas Tipo C/química , Dose Letal Mediana , Nitrogênio/efeitos adversos , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Staphylococcus aureus/fisiologia , Estresse Fisiológico , Vibrio/fisiologia
10.
Fish Shellfish Immunol ; 89: 248-256, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30951852

RESUMO

The effect of acute ammonia challenge on survival, immune response and antioxidant status of Litopenaeus vannamei pretreated with diets containing different inositol levels was investigated. Shrimp (initial mean weight 0.40 ±â€¯0.00 g) were randomly allocated in 18 tanks (30 shrimp per tank) and triplicate tanks were fed with a control diet without myo-inositol (MI) supplementation (242.6 mg inositol kg-1 diet) or diets containing diverse levels of inositol (368.8, 459.7, 673.1, 993.8 and 1674.4  mg kg-1 diet) as treatment groups for 8-week. Randomly selected 10 shrimp per tank (final mean weight approximately 11.1-13.8g) were exposed to ammonia stress (total ammonia-nitrogen, 60.21  mg L-1) for 24 h after feeding trial. The results showed that after exposed to ammonia stress, survival rates of MI-supplemented groups were enhanced by 31-77% when compared with the control group. MI supplementation increased activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in plasma, and reduced its activities in hepatopancreas. It also enhanced activities of total antioxidant capacity (T-AOC), glutathione S-transferase (GST) and glutathione peroxidase (GPX) and content of reduced glutathione (GSH), and lowered malondialdehyde (MDA) and protein carbonyl (PC) content in plasma or hepatopancreas. In addition, mRNA expression levels of ferritin (FT), arginine kinase (AK), thioredoxin (Trx), heat shock protein 70 (Hsp70), catalase (CAT) and peroxiredoxin (Prx) were significantly differentially regulated in hepatopancreas owing to MI supplementation. Therefore, it suggested that L. vannamei pretreated with higher dietary inositol content may have better ammonia stress tolerance and antioxidant status after ammonia stress, and the optimum levels ranged from 459.7 to 993.8 mg inositol kg-1 when total ammonia-nitrogen concentration was 60.21  mg L-1.


Assuntos
Amônia/efeitos adversos , Antioxidantes/metabolismo , Imunidade Inata/efeitos dos fármacos , Inositol/farmacologia , Penaeidae/imunologia , Substâncias Protetoras/farmacologia , Animais , Relação Dose-Resposta a Droga , Inositol/administração & dosagem , Longevidade/efeitos dos fármacos , Penaeidae/efeitos dos fármacos , Penaeidae/fisiologia , Substâncias Protetoras/administração & dosagem , Estresse Fisiológico
11.
Fish Shellfish Immunol ; 89: 326-336, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974215

RESUMO

Transglutaminase (TGase) is important in blood coagulation, a conserved immunological defense mechanism among invertebrates. This study is the first report of the TGase in mud crab (Scylla paramamosain) (SpTGase) with a 2304 bp ORF encoding 767 amino acids (molecular weight 85.88 kDa). SpTGase is acidic, hydrophilic, stable and thermostable, containing three transglutaminase domains, one TGase/protease-like homolog domain (TGc), one integrin-binding motif (Arg270, Gly271, Asp272) and three catalytic sites (Cys333, His401, Asp424) within the TGc. Neither a signal peptide nor a transmembrane domain was found, and the random coil is dominant in the secondary structure of SpTGase. Phylogenetic analysis revealed a close relation between SpTGase to its homolog EsTGase 1 from Chinese mitten crab (Eriocheir sinensis). Expression of SpTGase was investigated using qRT-PCR (1) in eight tissues from healthy mud crabs, with the highest expression in hemocytes, and (2) in response to various immune challenges (Vibrio parahaemolyticus, lipopolysaccharide (LPS) or Poly I:C infection), revealing a major up-regulation in hemocytes, skin, and hepatopancreas during the 96-h post injection. The recombinant SpTGase showed a capacity of agglutination activities on both Gram-negative bacteria and yeast. SpTGase was found to directly interact with another important blood coagulation component clip domain serine protease (SpcSP). Moreover, knockdown of SpTGase resulted in a decreased expression of both clotting protein precursor (SppreCP) and SpcSP and an increase of duration time in the blood coagulation. Taken together, the findings of this study suggest SpTGase play an important role in the hemolymph clotting in mud crab S. paramamosain.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Transglutaminases/genética , Transglutaminases/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Braquiúros , Perfilação da Expressão Gênica , Lipopolissacarídeos/farmacologia , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência , Transglutaminases/química , Vibrio parahaemolyticus/fisiologia
12.
BMC Genomics ; 20(1): 247, 2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30922216

RESUMO

BACKGROUND: Functional communications between nervous, endocrine and immune systems are well established in both vertebrates and invertebrates. Circulating hemocytes act as fundamental players in this crosstalk, whose functions are conserved during the evolution of the main groups of metazoans. However, the roles of the neuroendocrine-immune (NEI) system in shrimp hemocytes during pathogen infection remain largely unknown. RESULTS: In this study, we sequenced six cDNA libraries prepared with hemocytes from Litopenaeus vannamei which were injected by WSSV (white spot syndrome virus) or PBS for 6 h using Illumina Hiseq 4000 platform. As a result, 3444 differentially expressed genes (DEGs), including 3240 up-regulated genes and 204 down-regulated genes, were identified from hemocytes after WSSV infection. Among these genes, 349 DEGs were correlated with innate immunity and categorized into seven groups based on their predictive function. Interestingly, 18 genes encoded putative neuropeptide precursors were induced significantly by WSSV infection. Furthermore, some genes were mapped to several typical processes in the NEI system, including proteolytic processing of prohormones, amino acid neurotransmitter pathways, biogenic amine biosynthesis and acetylcholine signaling pathway. CONCLUSIONS: The data suggested that WSSV infection triggers the activation of NEI in shrimp, which throws a light on the pivotal roles of NEI system mediated by hemocytes in shrimp antiviral immunity.


Assuntos
Proteínas de Artrópodes/genética , Perfilação da Expressão Gênica/veterinária , Hemócitos/imunologia , Penaeidae/virologia , Animais , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Anotação de Sequência Molecular , Sistemas Neurossecretores/imunologia , Penaeidae/genética , Penaeidae/imunologia , Análise de Sequência de RNA/veterinária , Vírus da Síndrome da Mancha Branca 1/imunologia , Vírus da Síndrome da Mancha Branca 1/patogenicidade
13.
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
14.
Fish Shellfish Immunol ; 88: 528-539, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30885745

RESUMO

The high concentration of ammonia resulting from intensive culture system and environmental pollution could cause disease occurrence in shrimp, but little information is available on its molecular mechanisms. In this study, we performed comparative transcriptome analysis among WSSV-infected shrimp under ammonia stress (LAV), WSSV-infected shrimp under normal water (LV), and normal shrimp under ammonia stress (LA) groups to identify the key genes and pathways involved in immunosuppression and increasing pathogen infection severity caused by ammonia toxicity in Litopenaeus vannamei. Totally, 526 significantly differential expressed genes (DEGs) were identified in LAV group compared to LV and LA groups, among which 270 genes were lost expressed and 67 genes uniquely expressed in the LAV group. According to the public functional reports for the annotated DEGs, they potentially involved in the following functions: (1) accelerating pathogen adhesion, invasion and multiplication; (2) reducing the ability for pathogen defense and immune response; (3) inhibiting positive regulation of apoptotic and antioxidant defense for host homeostasis; (4) inhibiting transcription and protein transport; (5) and increasing protein methylation and ubiquitination, etc. A total of 13 pathways were obtained mainly involving in this process, which mainly led to the following changes: (1) increasing the immunosuppression, anemia, endocrine dysfunction, neurotoxic effect and neuroinvasion, atherosclerosis and thrombogenesis, blood-brain barrier penetration, thyroid disorder, necrosis, inflammation, and circadian disturbance; (2) reducing the ability of vascular remodeling, angiogenesis, cell survival, migration, apoptosis, and lymph transferred to blood stream; (3) leading to cell hypertrophy, cellular shape changes, and mesangial matrix expansion. The present results firstly supplied molecular mechanisms for the ammonia toxicity inhibiting the immune system and increasing pathogen infection severity in shrimp, which is a prerequisite for better understanding the pathogenesis caused by ammonia toxicity.


Assuntos
Amônia/toxicidade , Imunossupressão , Penaeidae/efeitos dos fármacos , Penaeidae/genética , Viroses/veterinária , Vírus da Síndrome da Mancha Branca 1/patogenicidade , Animais , Expressão Gênica , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Imunidade Inata/genética , Penaeidae/imunologia , Penaeidae/virologia , Transcriptoma , Viroses/imunologia
15.
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
16.
Fish Shellfish Immunol ; 88: 47-52, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30831245

RESUMO

Herein, we evaluated the immunomodulatory and the antiviral protective properties of a cyanobacteria-enriched diet on the immune responses of the Pacific white shrimp Litopenaeus vannamei challenged with the White spot syndrome virus (WSSV). Shrimp were fed with an Arthrospira platensis supplemented feed during 20 days, and its effects were examined by evaluating well-known standardized shrimp immune parameters (total hemocyte counts, total protein concentration, phenoloxidase activity, and serum agglutination titer). Additionally, we assessed the expression of crucial genes involved in both hemolymph- and gut-based immunities related to the shrimp capacity to circumvent viral and microbial infections. Dietary supplementation improved shrimp survival rates after challenge with a median lethal dose of WSSV. From all immune parameters tested, only the serum agglutination titer was higher in treated animals. On the other hand, the expression of some representative marker genes from different immune response pathways was only modulated in the midgut and not in the circulating hemocytes, suggesting that this feed supplementation can be used as an attractive strategy to enhance immunity in shrimp gut. Altogether, our results evidence the immunomodulatory properties of A. platensis supplemented feed in shrimp humoral and intestinal defenses and highlight the potential use of cyanobacteria-based immunostimulants in shrimp farming for protection against infectious diseases.


Assuntos
Ração Animal/análise , Penaeidae/imunologia , Spirulina , Adjuvantes Imunológicos , Animais , Aquicultura/métodos , Dieta/veterinária , Expressão Gênica , Hemolinfa/imunologia , Intestinos/imunologia , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
17.
Fish Shellfish Immunol ; 87: 590-599, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30738864

RESUMO

Complementary (c)DNA encoding novel protein kinase C (PKC) messenger (m)RNA of the white shrimp Litopenaeus vannamei, consisted of 2454-bp cDNA containing an open reading frame (ORF) of 2232 bp, belonging to the novel (n)PKC family of proteins characterized by their containing two phorbol ester/diacylglycerol-binding domains (C1 domain), a C2 domain, and a catalytic domain of the serine/threonine kinase, designated LvnPKC. A comparison of amino acid sequences showed that LvnPKC was closely related to arthropod nPKC. LvnPKC cDNA was detected in all tested tissues with a real-time PCR including the hepatopancreas, gills, muscles, subcuticular epithelium, abdominal nerve, thoracic nerve, brain, the stomach, heart, and especially in hemocytes and the intestines. Moreover, significantly upregulated LvnPKC expression was only observed in the eyestalk, brain, and hepatopancreas of shrimp transferred from 28 °C to 18 °C for 30 min. Induction of LvnPKC expression in hemocytes of L. vannamei injected with Vibrio alginolyticus at 105 cfu shrimp-1 was detected earlier than in those injected with 103 cfu shrimp-1. Shrimp received LvnPKC-dsRNA for 1 days specifically depleted the expression of LvnPKC mRNA in hemocytes compared those of diethylpyrocarbonate water treatment. After that, significantly decreased expressions of lipopolysaccharide - and ß-1,3-glucan-binding protein, prophenoloxidase-activating enzyme, peroxinectin, prophenoloxidase I, and prophenoloxidase II in the prophenoloxidase-activating system; lysozyme and cytosolic manganese superoxide dismutase and mitochondrial manganese superoxide dismutase in the antioxidant system were observed. We therefore concluded that LvnPKC is involved in immune defense of L. vannamei exposed to hypothermal stress or infected with V. alginolyticus.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Proteína Quinase C/genética , Proteína Quinase C/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Imunocompetência , Filogenia , Proteína Quinase C/química , Vibrio alginolyticus/fisiologia
18.
Fish Shellfish Immunol ; 87: 534-545, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30721776

RESUMO

Enterocytozoon hepatopenaei (EHP) causes hepatopancreatic microsporidiosis (HPM) in shrimp. HPM is not normally associated with shrimp mortality, but is associated with significant growth retardation. In this study, the responses induced by EHP were investigated in hepatopancreas of shrimp Litopenaeus vannamei using proteomics and metabolomics. Among differential proteins identified, several (e.g., peritrophin-44-like protein, alpha2 macroglobulin isoform 2, prophenoloxidase-activating enzymes, ferritin, Rab11A and cathepsin C) were related to pathogen infection and host immunity. Other proteomic biomarkers (i.e., farnesoic acid o-methyltransferase, juvenile hormone esterase-like carboxylesterase 1 and ecdysteroid-regulated protein) resulted in a growth hormone disorder that prevented the shrimp from molting. Both proteomic KEGG pathway (e.g., "Glycolysis/gluconeogenesis" and "Glyoxylate and dicarboxylate metabolism") and metabolomic KEGG pathway (e.g., "Galactose metabolism" and "Biosynthesis of unsaturated fatty acids") data indicated that energy metabolism pathway was down-regulated in the hepatopancreas when infected by EHP. More importantly, the changes of hormone regulation and energy metabolism could provide much-needed insight into the underlying mechanisms of stunted growth in shrimp after EHP infection. Altogether, this study demonstrated that proteomics and metabolomics could provide an insightful view into the effects of microsporidial infection in the shrimp L. vannamei.


Assuntos
Enterocytozoon/fisiologia , Metaboloma/imunologia , Penaeidae/genética , Penaeidae/imunologia , Proteoma/imunologia , Animais , Hepatopâncreas/imunologia , Penaeidae/metabolismo
19.
PLoS Pathog ; 15(2): e1007558, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30726286

RESUMO

Viral entry into the host cell is the first step towards successful infection. Viral entry starts with virion attachment, and binding to receptors. Receptor binding viruses either directly release their genome into the cell, or enter cells through endocytosis. For DNA viruses and a few RNA viruses, the endocytosed viruses will transport from cytoplasm into the nucleus followed by gene expression. Receptors on the cell membrane play a crucial role in viral infection. Although several attachment factors, or candidate receptors, for the infection of white spot syndrome virus (WSSV) were identified in shrimp, the authentic entry receptors for WSSV infection and the intracellular signaling triggering by interaction of WSSV with receptors remain unclear. In the present study, a receptor for WSSV infection in kuruma shrimp, Marsupenaeus japonicus, was identified. It is a member of the immunoglobulin superfamily (IgSF) with a transmembrane region, and is similar to the vertebrate polymeric immunoglobulin receptor (pIgR); therefore, it was designated as a pIgR-like protein (MjpIgR for short). MjpIgR was detected in all tissues tested, and its expression was significantly induced by WSSV infection at the mRNA and protein levels. Knockdown of MjpIgR, and blocking MjpIgR with its antibody inhibited WSSV infection in shrimp and overexpression of MjpIgR facilitated the invasion of WSSV. Further analyses indicated that MjpIgR could independently render non-permissive cells susceptible to WSSV infection. The extracellular domain of MjpIgR interacts with envelope protein VP24 of WSSV and the intracellular domain interacts with calmodulin (MjCaM). MjpIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV. The viral internalization facilitating ability of MjpIgR could be blocked using chlorpromazine, an inhibitor of clathrin dependent endocytosis. Knockdown of Mjclathrin and its adaptor protein AP-2 also inhibited WSSV internalization. All the results indicated that MjpIgR-mediated WSSV endocytosis was clathrin dependent. The results suggested that MjpIgR is a WSSV receptor, and that WSSV enters shrimp cells via the pIgR-CaM-Clathrin endocytosis pathway.


Assuntos
Penaeidae/imunologia , Receptores de Imunoglobulina Polimérica/imunologia , Vírus da Síndrome da Mancha Branca 1/metabolismo , Animais , Aquicultura/métodos , Vírus de DNA , Endocitose , Penaeidae/metabolismo , Penaeidae/patogenicidade , Ligação Proteica , Receptores de Imunoglobulina Polimérica/metabolismo , Proteínas do Envelope Viral , Internalização do Vírus , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/patogenicidade
20.
Vet Immunol Immunopathol ; 208: 25-33, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30712789

RESUMO

Intestine is not only the nutrients digestion and absorption centers, but also an important place of microbial infection. Therefore, intestine immunity plays a key defense means for the host against the invasion of pathogenic microorganisms. In this study, we use kuruma shrimp (Marsupenaeus japonicus) as a model to study the intestine immune characteristics of shrimp against WSSV through next-generation sequencing technique. A total of 63,458 and 44,350 unigenes were generated from the control sample and the WSSV infection sample, respectively. Based on homology searches, KEGG, GO, and COG analysis, 39,520 unigenes were annotated. Among them, 12,920 differentially expressed genes were identified. Some of them, including mucin, peritrophin, chitinase, et al., are involved in immune response. These results contribute to a better understanding of the intestine immune response of the shrimp to WSSV.


Assuntos
Intestinos/imunologia , Penaeidae/imunologia , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1 , Animais , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Penaeidae/anatomia & histologia , Filogenia , Análise de Sequência de RNA
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