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1.
Gene ; 763: 145115, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32891773

RESUMO

Dopamine (DA) is a crucial neuroendocrine-immune factor regulating the stress response of Litopenaeus vannamei. To understand the regulatory mechanisms of DA in L. vannamei, the eyestalks of L. vannamei with injection of DA (10-6 mol/shrimp) at 3 and 12 h were chosen to perform transcriptome analysis in this study. Furthermore, quantitative real-time PCR (RT-PCR) method was used to validate the accuracy of transcriptome data and analyze the expression pattern of candidate differentially expressed genes (DEGs) at different time points (0, 3, 6, and 12 h) after DA injection. The transcriptome data showed that 79,434 unigenes were generated. Therein 204 and 434 DEGs were obtained at 3 and 12 h respectively. Besides, the results of enriched pathways showed that the DEGs were involved in GnRH signaling pathway (ko04912) dopaminergic synapse (ko04728), glutamatergic synapse (ko04724), synapse (GO:0045202), synaptic vesicle transport (GO:0048489). Moreover, the Pearson's correlation coefficient (R) of 13 candidate DEGs between transcriptome sequencing and RT-PCR was 0.948, which confirmed the reliability and the accuracy of the transcriptome sequencing results. Furthermore, the results of interaction analysis uncovered 4 pairs of DEGs between eyestalks and hemocytes. Therefore, these results revealed that DA promoted the sensitivity of eyestalk to gonadal related hormones, induced the expression of neuroendocrine factor, enhanced the synaptic behavior and neural signal transduction, regulated immune systems and antioxidation, inhibited the visual function, and promoted the molting. These findings will benefit to foster the understanding on the effects of biogenic amines on neuroendocrine-immune (NEI) networks of crustacean, and supply a substantial material and foundation for further researching of the NEI response.


Assuntos
Dopamina/metabolismo , Hormônios/metabolismo , Penaeidae/genética , Transmissão Sináptica , Transcriptoma , Animais , Dopamina/farmacologia , Olho/metabolismo , Hemócitos/metabolismo , Penaeidae/efeitos dos fármacos , Penaeidae/metabolismo , Pró-Proteína Convertase 2/genética , Pró-Proteína Convertase 2/metabolismo , Estresse Fisiológico
2.
Gene ; 763: 144956, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32739586

RESUMO

Sox transcription factors play essential roles in a variety of critical physiological processes. Still, members of the sox gene family have not yet been genome-wide identified in shrimps. In this study, a total of five members of the sox gene family were identified from the genome of Pacific white shrimp Litopenaeus vannamei and classified into three subgroups based on the conserved HMG-box domain. Among them, three belong to the SoxB subgroup (one in B1 and two in B2), one in the SoxC subgroup, and one in the SoxE subgroup. The five sox genes had different sex-biased expression in some tissues. Sox21, soxB1, and sox14 had a higher expression in ovary than in testis. In comparison, sox4 had a male-biased specific expression in the gonad, hepatopancreas, gill, and eyestalk. There was no difference in soxE gene expression between testis and ovary. During embryonic development, the expression level of three sox genes (soxB1, sox21, and soxE) was higher in gastrulation stage compared to previous stages, declined in limb bud stage and then increased in intramembrane nauplius stage; the expression of sox4 was detected in blastula stage and continued to increase in the following two stages and then surged in intramembrane nauplius stage; the highest expression of sox14 was in the fertilized egg stage, and the expression level decreased with the development of the embryo. These results suggest that the shrimp sox gene family may be involved in gametogenesis, tridermogenesis, and neurogenesis.


Assuntos
Proteínas de Artrópodes/genética , Penaeidae/genética , Fatores de Transcrição SOX/genética , Animais , Proteínas de Artrópodes/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Brânquias/embriologia , Brânquias/metabolismo , Hepatopâncreas/embriologia , Hepatopâncreas/metabolismo , Masculino , Especificidade de Órgãos , Ovário/embriologia , Ovário/metabolismo , Penaeidae/embriologia , Fatores de Transcrição SOX/metabolismo , Testículo/embriologia , Testículo/metabolismo
3.
Gene ; 758: 144986, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32711100

RESUMO

Myostatin (Mstn) inhibits muscle growth in vertebrates with endoskeleton, but it is still inconclusive that Mstn is a positive or negative regulator in crustacean with exoskeleton, and little information was available for its function on myogenesis. In this study, we identified and characterized the Mstn from Fenneropenaeus chinensis (FcMstn), and investigated its function on myogenesis and muscle growth. Two different cDNA sequences (2628 bp and 2604 bp) encoding for slightly different sizes of proteins were obtained for FcMstn, containing 86 bp of 5' untranslated regions (UTR) and 1258 bp of 3' UTR. The open reading frame of the long sequence and the short sequence contain 1284 bp and 1260 bp cDNA, encoding 427 and 419 amino acid sequence, respectively. Sequence analysis revealed that the overall protein sequence and specific functional sites of FcMstn were highly conserved with those in other crustacean species. In the early development stage, the muscle firstly appeared in nauplius stage and developed gradually until post larval, but the expression of FcMstn at mRNA and protein levels decreased from nauplius stage to post larval stage, indicating that Mstn involved in myogenesis as a negative regulator in shrimp. In the adult shrimp, the expression of FcMstn at mRNA and protein levels in muscle were significantly lower in the larger group than in the smaller group, and the diameter and number of muscle fiber of the muscle were significantly different between the two groups. Moreover, the shrimp with reduced level of FcMstn by RNAi displayed a dramatic faster growth rate compared with the control group. The present study demonstrates that FcMstn involved in myogenesis and muscle growth probably also as a negative regulator in shrimp like in vertebrates.


Assuntos
Desenvolvimento Muscular/genética , Músculos/metabolismo , Miostatina/genética , Miostatina/metabolismo , Penaeidae/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica , Fases de Leitura Aberta/genética , Filogenia , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Alinhamento de Sequência
4.
PLoS One ; 15(7): e0236343, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730349

RESUMO

Using pooled DNA genotyping to estimate the proportional contributions from multiple families in a pooled sample is of particular interest for selective breeding in aquaculture. We compared different pooled libraries with separate 2b-RAD sequencing of Litopenaeus vannamei individuals to assess the effect of different population structures (different numbers of individuals and families) on pooled DNA sequencing, the accuracy of parent sequencing of the DNA pools and the effect of SNP numbers on pooled DNA sequencing. We demonstrated that small pooled DNA genotyping of up to 53 individuals by 2b-RAD sequencing could provide a highly accurate assessment of population allele frequencies. The accuracy increased as the number of individuals and families increased. The allele frequencies of the parents from each pool were highly correlated with those of the pools or the corresponding individuals in the pool. We chose 500-28,000 SNPs to test the effect of SNP number on the accuracy of pooled sequencing, and no linear relationship was found between them. When the SNP number was fixed, increasing the number of individuals in the mixed pool resulted in higher accuracy of each pooled genotyping. Our data confirmed that pooled DNA genotyping by 2b-RAD sequencing could achieve higher accuracy than that of individual-based genotyping. The results will provide important information for shrimp breeding programs.


Assuntos
DNA/genética , Técnicas de Genotipagem , Sequenciamento de Nucleotídeos em Larga Escala , Penaeidae/genética , Mapeamento por Restrição , Alelos , Animais , Frequência do Gene/genética , Genética Populacional , Polimorfismo de Nucleotídeo Único/genética , Reprodutibilidade dos Testes
5.
Food Chem ; 332: 127389, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32645674

RESUMO

Food allergens that cause anaphylactic reactions have become an important health problem worldwide. Among them, shrimp is a popular seafood in many cuisines. The best way to avoid allergic reactions is to mitigate the intake of food allergens. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of shrimp DNA. Using LAMP primers, the identification of shrimp DNA by the LAMP assay was specific and rapid (within 30 min). It exhibited no cross-reaction with the DNA of other Crustacea, including crabs and lobster, and at least 0.01% shrimp DNA existed in the test sample. Additionally, the sensitivity of LAMP for detecting shrimp DNA was 100-fold greater than that of conventional PCR. LAMP for the detection of shrimp DNA was reproducible regardless of whether the genomic DNA was extracted from boiled, steamed or roasted shrimp samples. In summary, the LAMP assay established herein not only could be potentially used for diagnosing shrimp DNA but could also be applicable for identifying shrimp allergens in commercial food products in marketplaces.


Assuntos
Alérgenos/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Penaeidae/genética , Alimentos Marinhos/análise , Alérgenos/genética , Animais , Sequência de Bases , Braquiúros/genética , Primers do DNA/metabolismo , Nephropidae/genética , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Alinhamento de Sequência
6.
Gene ; 752: 144765, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32413480

RESUMO

The natural flight response in shrimp is powered by rapid contractions of the abdominal muscle fibres to propel themselves backwards away from perceived danger. This muscle contraction is dependent on repetitive depolarization of muscle plasma membrane, triggering tightly spaced cytoplasmic [Ca2+] transients and rapidly rising tetanic force responses. To achieve such high amplitude and high frequency of Ca2+ transients requires a high abundance of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) to rapidly clear cytoplasmic Ca2+ between each transient and an efficient Ca2+ release system consisting of the Ryanodine Receptor (RyR), and voltage gated Ca2+ channels (CaVs). With the aim to expand our knowledge of muscle gene function and identify orthologous genes regulating muscle excitation-contraction (EC) coupling, this study assembled nine Penaeid shrimp muscle transcriptomes. On average, the nine transcriptomes contained 27,000 contigs, with an annotation rate of 36% and a BUSCO completeness of 70%. Despite maintaining their function, the crustacean RyR and CaV proteins showed evidence of significant diversification from mammalian orthologs, while SERCA remained more conserved. Several key components of protein interaction were conserved, while others showed distinct crustacean specific evolutionary adaptations. Lastly, this study revealed approximately 1,000 orthologous genes involved in muscle specific processes present across all nine species.


Assuntos
Acoplamento Excitação-Contração/genética , Penaeidae/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Evolução Biológica , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/fisiologia , Citosol/metabolismo , Evolução Molecular , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Especificidade da Espécie , Transcriptoma/genética
7.
PLoS One ; 15(3): e0229512, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163430

RESUMO

Seafood mislabeling occurs in a wide range of seafood products worldwide, resulting in public distrust, economic fraud, and health risks for consumers. We quantified the extent of shrimp mislabeling in coastal and inland North Carolina. We used standard DNA barcoding procedures to determine the species identity of 106 shrimp sold as "local" by 60 vendors across North Carolina. Thirty-four percent of the purchased shrimp was mislabeled, and surprisingly the percentage did not differ significantly between coastal and inland counties. One third of product incorrectly marketed as "local" was in fact whiteleg shrimp: an imported and globally farmed species native to the eastern Pacific, not found in North Carolina waters. In addition to the negative ecosystem consequences of shrimp farming (e.g., the loss of mangrove forests and the coastal buffering they provide), North Carolina fishers-as with local fishers elsewhere-are negatively impacted when vendors label farmed, frozen, and imported shrimp as local, fresh, and wild-caught.


Assuntos
Aquicultura/ética , Aquicultura/métodos , Penaeidae/genética , Animais , Conservação dos Recursos Naturais/métodos , Código de Barras de DNA Taxonômico/métodos , Ecossistema , North Carolina , Penaeidae/classificação , Alimentos Marinhos/análise , Alimentos Marinhos/economia , Frutos do Mar/análise , Frutos do Mar/classificação
8.
Mol Immunol ; 120: 113-121, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32113131

RESUMO

Litopenaeus vannamei, as an important marine aquaculture species, has attracted more and more attentions in past several years. More recently people got its genome fine mapping, which unveiled a gene treasure. In this study, we have identified a novel trypsin-like protein which came from previous WSSV-infected shrimp plasma iTRAQ data. This protein is a 39 kDa protein with 363 amino acids. It contains a conserved trypsin-domain and could be strongly induced with WSSV infection. Interestingly, knockdown of this protein made shrimps vulnerable to WSSV infection. Further exploration unveiled that this fragility was probably due to the fact that knockdown of this protein could cause shrimp hemocytes apoptosis, which indicated that this protein played key roles in preventing shrimp hemocytes from apoptosis. To further explore how LvTLAP protected shrimp hemocytes from apoptosis, GST pull down assay was applied to screen LvTLAP interacting protein in shrimp plasma. L. vannamei growth and transformation-dependent-like protein (LvGTD-like protein) was identified as a LvTLAP interacting protein, which played proapoptotic roles in cells. Thus, a possible explanation for LvTLAP anti-apoptosis activity was that this protein could block LvGTD-like protein proapoptotic activity to protect shrimp hemocytes from death. In general, our study has uncovered a novel WSSV responsive shrimp plasma protein, which played key roles in shrimp hemocytes anti-apoptosis and shrimp against WSSV infection.


Assuntos
Proteínas Sanguíneas/imunologia , Penaeidae/imunologia , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/patogenicidade , Sequência de Aminoácidos , Animais , Apoptose/genética , Apoptose/imunologia , Apoptose/fisiologia , Sequência de Bases , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , DNA/genética , Técnicas de Silenciamento de Genes , Hemócitos/metabolismo , Hemócitos/patologia , Hemócitos/virologia , Penaeidae/genética
9.
Gene ; 736: 144421, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32018014

RESUMO

5-Aminolevulinic acid synthase (ALAS) is the rate-limiting enzyme in the biosynthesis of heme, a prosthetic group that is found in hemoproteins, including those involved in molting. To better understand the roles of ALAS in L. vannamei (LvALAS), we analyzed its sequence and tissue distribution, the effects of age and bacterial infection on its gene expression, and the effects of LvALAS gene silencing. We also examined the expressions of three hemoproteins, the cytochrome oxidase subunit I (COX I) and subunit IV (COX IV) and catalase. Three LvALAS splicing variants were found in the hepatopancreas, with the main splicing variant having an open reading frame that encodes 532 aa. LvALAS transcripts were found in each of the eleven tissues tested in this study, with the highest gene expression in the intestine. The transcript abundances of LvALAS, COX I and COX IV in the hepatopancreas and stomach tended to decrease with age. LvALAS and catalase gene expressions significantly increased in the stomach after V. parahaemolyticus infection. LvALAS gene expression in the hepatopancreas, stomach and intestine (12- and 24-hours post-injection) was relatively lower in dsALAS-injected shrimp than in PBS-injected shrimp. All the PBS-injected shrimp molted after 8-10 days while no molting activity was observed in the dsALAS-injected shrimp group within the 14 days post-injection period. Our results provide evidence that (1) only the housekeeping form of ALAS exists in L. vannamei; LvALAS gene expression (2) decreases with age and (3) increases after bacterial infection; and (4) an ALAS-dependent pathway is necessary for proper molting in L. vannamei.


Assuntos
5-Aminolevulinato Sintetase/genética , Proteínas de Artrópodes/genética , Expressão Gênica/genética , Penaeidae/genética , Sequência de Aminoácidos , Ácido Aminolevulínico/metabolismo , Animais , Clonagem Molecular/métodos , Hepatopâncreas/metabolismo , Hepatopâncreas/patologia , Intestinos/patologia , Penaeidae/patogenicidade , Filogenia , Alinhamento de Sequência , Estômago/patologia
10.
Chemosphere ; 249: 126157, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32062217

RESUMO

Waterborne metals may be hazardous to aquatic organisms and trigger stress responses. The present study aimed to assess the effect of exposure to 100 µg/L cadmium (Cd) or copper (Cu) for 48 h on juvenile Marsupenaeus japonicus, in terms of bioaccumulation and the whole body transcriptome. The results demonstrated that Cu accumulation in M. japonicas was much higher than that of Cd. Meanwhile, transcriptome analysis identified 1802 and 2670 differentially expressed genes (DEGs) after 48 h exposure to 100 µg/L Cd and Cu, respectively. Among them, 851 DEGs responded to both metals. Cd and Cu stress shared genes were related to the cytoskeleton, immunity, antioxidation, and detoxification. Metallothionein 1 (MT1) was specifically induced in the Cd-stress response, while glycometabolism, heat shock protein 90 (HSP90), metallothionein 2 (MT2), apoptosis, and iron transport-related genes were changed specifically in response to Cu stress. In addition, real-time PCR was used to verify the expression patterns of 28 randomly selected DEGs. The sequencing and real-time PCR results were consistent. Moreover, based on the number of significantly modulated genes and their expression levels, we deduced that Cu acts as a stronger stress inducer than Cd in M. japonicus. The identified Cd and Cu stress related genes and pathways will provide new insights into the common and different molecular mechanisms underlying Cd and Cu toxicity effects in M. japonicus.


Assuntos
Cádmio/toxicidade , Cobre/toxicidade , Penaeidae/fisiologia , Poluentes Químicos da Água/toxicidade , Adolescente , Animais , Antioxidantes , Perfilação da Expressão Gênica , Humanos , Metalotioneína/metabolismo , Metais , Penaeidae/genética , Transcriptoma
11.
Mar Genomics ; 52: 100751, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32033920

RESUMO

World production of farmed crustaceans was 7.8 million tons in 2016. While only making up approximately 10% of world aquaculture production, crustaceans are generally high-value species and can earn significant export income for producing countries. Viet Nam is a major seafood producing country earning USD 7.3 billion in 2016 in export income with shrimp as a major commodity. However, there is a general lack of genomic resources available for shrimp species, which is challenging to obtain due to the need to deal with large repetitive genomes, which characterize many decapod crustaceans. The first tiger prawn (P. monodon) genome assembly was assembled in 2016 using the standard Illumina PCR-based pair-end reads and a computationally-efficient but relatively suboptimal assembler, SOAPdenovo v2. As a result, the current P. monodon draft genome is highly fragmented (> 2 million scaffolds with N50 length of <1000 bp), exhibiting only moderate genome completeness (< 35% BUSCO complete single-copy genes). We sought to improve upon the recently published P. monodon genome assembly and completeness by generating Illumina PCR-free pair-end sequencing reads to eliminate genomic gaps associated with PCR-bias and performing de novo assembly using the updated MaSurCA de novo assembler. Furthermore, we scaffolded the assembly with low coverage Nanopore long reads and several recently published deep Illumina transcriptome paired-end sequencing data, producing a final genome assembly of 1.6 Gbp (1,211,364 scaffolds; N50 length of 1982 bp) with an Arthropod BUSCO completeness of 96.8%. Compared to the previously published P. monodon genome assembly from China (NCBI Accession Code: NIUS01), this represents an almost 20% increase in the overall BUSCO genome completeness that now consists of more than 90% of Arthropod BUSCO single-copy genes. The revised P. monodon genome assembly (NCBI Accession Code: VIGR01) will be a valuable resource to support ongoing functional genomics and molecular-based breeding studies in Vietnam.


Assuntos
Genoma , Penaeidae/genética , Transcriptoma , Animais , Aquicultura , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia
12.
Fish Shellfish Immunol ; 96: 319-329, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31805414

RESUMO

Viral immediate early (IE) genes encode regulatory proteins that are critical for viral replication. WSV056 is an IE protein of white spot syndrome virus (WSSV), an important pathogen of farmed shrimp. It targets the host Rb protein(s) and, according to a previous study, may enhance the replication of the viral genome. However, the ectopic expression of WSV056 in transgenic Drosophila melanogaster exerted an inhibitory effect on the replication of Drosophila C virus (DCV). Transcriptome study using Affymetrix GeneChip suggested that the enrichment of serine proteases (SPs) likely accounts for DCV inhibition in WSV056-overexpressing Drosophila. Injection of recombinant WSV056 to the WSSV natural host Litopenaeus vannamei enhanced the expression of the SP family member prophenoloxidase-activating enzyme 2 (LvPPAE2) and conferred shrimp with more resistance to WSSV infection. LvPPAE2 knockdown contributed to decreased expression of antimicrobial peptides LvAlf1 and LvLyz1, reduced hemolymph phenoloxidase activity, and increased virus load, suggesting that LvPPAE2 is involved in the host defense against WSSV infection. Taken together, these results suggest that wsv056 plays a role in restricting viral replication by inducing the SP-mediated immune responses in the host.


Assuntos
Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/imunologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Análise Serial de Proteínas
13.
Fish Shellfish Immunol ; 96: 53-61, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31801694

RESUMO

Target of rapamycin (TOR) is an atypical of Ser/Thr protein kinase that plays an important role in many aspects such as cell growth, reproduction, differentiation, cell cycle regulation, autophagy and apoptosis. However, little information is known about the enzyme in crustaceans. Here, a novel TOR was identified from shrimp Penaeus vannamei (PvTOR) and its biological functions were investigated in response low temperature stress. The PvTOR gene encoded a polypeptide of 2464 amino acids with an estimated molecular mass of 279.4 kD and a predicted isoelectronic point (pI) of 7.30. Phylogenetic analysis revealed that PvTOR shared high similarity with other known species. PvTOR mRNA was detected in all the tested tissues and highest transcription in muscle and hepatopancreas. PvTOR transcriptional level was up-regulated significantly at 1.5 h and 3 h, and down-regulated at 12 h and 24 h after low temperature stress. TEM and autophagy indicator system GFP-PvLC3 suggested that low temperature induced autophagy generation. ROS, Ca2+ concentration and apoptosis rate were increased significantly in TOR-knockdown shrimp after low temperature stress. The autophagy associated gene ATG8II/I, PvBeclin-1, PvATG14, apoptosis gene PvPARP, Pvcasp-3, PvBAX and Pvp53 transcripts, and casp-3/8 activity in hemocyte were increased significantly in TOR-knockdown group shrimp at 3 h after low temperature stress. Additionally, THC counts of TOR-knockdown group were significantly higher than the dsGFP group. In summary, these results suggested that PvTOR plays an important role in the adaptation mechanisms of shrimp at low temperature by regulating autophagy and apoptosis.


Assuntos
Proteínas de Artrópodes/genética , Temperatura Baixa/efeitos adversos , Penaeidae/genética , Penaeidae/imunologia , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Animais , Apoptose/genética , Proteínas de Artrópodes/metabolismo , Autofagia/genética , Filogenia , Análise de Sequência de DNA , Estresse Fisiológico , Serina-Treonina Quinases TOR/metabolismo
14.
Fish Shellfish Immunol ; 96: 245-253, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31830564

RESUMO

RNA polymerase (RNAP) II (DNA-directed) (POLR2) genes are essential for cell viability under environmental stress and for the transfer of biological information from DNA to RNA. However, the function and characteristics of POLR2 genes in crustaceans are still unknown. In the present study, a POLR2H cDNA was isolated from Pacific white shrimp (Litopenaeus vannamei) and designated as Lv-POLR2H. The full-length Lv-POLR2H cDNA is 772 bp in length and contains a 32-bp 5'- untranslated region (UTR), a 284-bp 3'- UTR with a poly (A) sequence, and an open reading frame (ORF) of 456 bp encoding an Lv-POLR2H protein of 151 amino acids with a deduced molecular weight of 17.21 kDa. The Lv-POLR2H protein only contains one functional domain, harbors no transmembrane domains and mainly locates in the nucleus. The expression of the Lv-POLR2H mRNA was ubiquitously detected in all selected tissues, with the highest level in the gills. In situ hybridization (ISH) analysis showed that Lv-POLR2H was mainly located in the secondary gill filaments, the transcript levels of Lv-POLR2H in the gills were found to be significantly affected after challenge by pH, low salinity and high concentrations of NO2- and NH4+, indicating that Lv-POLR2H in gill tissues might play roles under various physical stresses. Specifically, under high-pH stress, knockdown of Lv-POLR2H via siRNA significantly decreased the survival rate of the shrimp, indicating its key roles in the response to high-pH stress. Our study may provide the first evidence of the role of POLR2H in shrimp responding to high-pH stress and provides new insight into molecular regulation in response to high pH in crustaceans.


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 , Brânquias/metabolismo , Concentração de Íons de Hidrogênio , Peptídeos/química , Filogenia , Estresse Fisiológico
15.
Protein Expr Purif ; 166: 105511, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31622664

RESUMO

Metallothioneins (MTs) are cysteine rich proteins with antioxidant capacity that participate in the homeostasis and detoxification of metals and other cellular processes, and help to counteract the oxidative stress produced by Reactive Oxygen Species (ROS). The production of ROS increases during several stress conditions, including metal intoxication and hypoxia (oxygen deficiency). During hypoxia the expression of the MT gene is induced in the shrimp Litopenaeus vannamei; however, the MT protein coded by this gene has not been purified nor characterized. In this work, the coding sequence of L. vannamei MT was cloned and overexpressed in Escherichia coli as a fusion protein, containing an intein and a chitin binding domain (CBD). The MT was purified by chitin affinity chromatography and its antioxidant capacity and ability to bind cadmium (Cd) and copper (Cu) were evaluated. This MT has an antioxidant capacity of 27.23 µM equivalent to Trolox in a 100 µg/mL solution. Addition of CdCl2 to the culture media augments 273-fold the Cd content, while addition of CuCl2 increases Cu content 569-fold in the purified MT. Thus, the shrimp MT gene codes for a functional protein that has antioxidant capacity and binds Cu and Cd.


Assuntos
Metalotioneína/química , Metalotioneína/genética , Penaeidae , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Animais , Cádmio/química , Quitina/química , Cromatografia de Afinidade , Clonagem Molecular , Cobre/química , Escherichia coli , Vetores Genéticos , Penaeidae/enzimologia , Penaeidae/genética
16.
J Sci Food Agric ; 100(1): 119-128, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31441054

RESUMO

BACKGROUND: T-2 toxin (T-2) is a potent mycotoxin and a common contaminant of aquatic animal feed, posing a serious risk to health and aquatic animals. We investigated the effect of T-2 on shrimp muscle proteins using proteomics and conventional biochemical methods. Shrimp were fed a diet containing T-2 at 0-12.2 mg kg-1 for 20 days, and changes to the muscle protein composition, ATPase activities, and the sulfhydryl (SH) content and hydrophobicity of actomyosin (AM) were determined. A proteomics study of the proteins was conducted with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional (2D) electrophoresis, and matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF/TOF MS). RESULTS: Exposure to T-2 markedly affected the muscle protein composition of shrimp in a concentration-responsive manner that displayed a diphasic effect. At a low T-2 concentration (1.2 mg kg-1 ), the levels of three major muscle proteins (myofibrillar, sarcoplasmic, and stroma) increased but at higher concentrations they declined progressively. T-2 exposure also led to a breakdown of muscle proteins as evidenced by increases in alkali-soluble protein and the surface hydrophobicity (SoANS) of AM. Thirty differentially expressed proteins were detected, 12 of which showed a concentration-response relationship with T-2 exposure. Among them, 11 homologous proteins were identified by mass spectrometry (MS), with several being key enzymes in energy metabolism. CONCLUSION: This study demonstrated that T-2 exposure at medium to high concentrations could significantly affect the protein composition and quality of shrimp muscle, and potentially some of its key metabolisms. One of the arginine kinases (spot 27) was particularly responsive to T-2 and could potentially be used as a biomarker protein for T-2 intoxication by shrimp. © 2019 Society of Chemical Industry.


Assuntos
Proteínas Musculares/química , Penaeidae/efeitos dos fármacos , Frutos do Mar/análise , Toxina T-2/toxicidade , Ração Animal/análise , Animais , Eletroforese em Gel de Poliacrilamida , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculos/química , Músculos/efeitos dos fármacos , Músculos/metabolismo , Penaeidae/química , Penaeidae/genética , Penaeidae/metabolismo , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
Food Funct ; 10(11): 7042-7051, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31580362

RESUMO

Tropomyosin is the most potent allergen of shrimp that can cause severe food allergy. However, to date, an effective approach to eliminate this allergenicity is still lacking. Glycation is a promising approach that can reduce the allergenicity of shrimp tropomyosin by destroying the epitopes; however, advanced glycation end products (AGEs) are also generated during glycation, which can function as neoallergens to strengthen the allergenicity; therefore, it is hard to tell how the glycation of an allergen with different saccharides affects the allergenicity via epitope loss and neoallergen generation. This study was aimed at the elucidation of how the glycation of tropomyosin (TM) with saccharides of different molecular sizes (glucose, maltose, and maltotriose) affected the allergenicity through epitope loss and the generation of neoallergns that belonged to advanced glycation end products (AGEs). Saccharides of higher molecular sizes (maltotriose) could lead to higher glycated TM than saccharides of smaller molecular sizes (glucose and maltose). Compared with TM, the TM glycated by glucose (TM-G) and maltotriose (TM-MTS) had lower allergenicity and contributed to weaker mouse allergy symptoms; on the other hand, the TM glycated by maltose (TM-M) had no significant impact on the allergenicity due to the generation of AGE-related neoallergens, which might offset the glycation-induced epitope loss. The glycation of TM by maltotriose led to lower generation of AGE neoallergens (e.g. CML) than that in the cases of glucose and maltose; therefore, maltotriose could be applied to desensitize TM-induced food allergy through glycation, and this could be a potential immunotherapy for shrimp allergy patients.


Assuntos
Produtos Finais de Glicação Avançada/imunologia , Penaeidae/imunologia , Hipersensibilidade a Frutos do Mar/imunologia , Tropomiosina/imunologia , Sequência de Aminoácidos , Animais , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Glucose/química , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/genética , Glicosilação , Humanos , Imunoglobulina E/imunologia , Maltose/química , Camundongos , Camundongos Endogâmicos BALB C , Penaeidae/química , Penaeidae/genética , Trissacarídeos/química , Tropomiosina/química , Tropomiosina/genética
18.
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
19.
Fish Shellfish Immunol ; 94: 497-509, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31541775

RESUMO

As a crucial neuroendocrine-immune factor, dopamine (DA) could regulate the immune system of Litopenaeus vannamei. To understand the immune mechanisms and regulatory pathways of DA in L. vannamei, the transcriptome analysis of hemocytes of L. vannamei with injection of DA (10-6 mol/shrimp) at 3 and 12 h were performed in this study. Moreover, quantitative real-time PCR (qPCR) method was applied to validate the accuracy of transcriptome sequencing and analyze the expression pattern of candidate differentially expressed genes (DEGs) at different time points (0, 3, 6, 12, and 24 h) after DA injection. The results showed that a total of 51382 unigenes with a N50 length of 2341 bp were generated. And 1397 and 457 DEGs were obtained by comparative transcriptome at 3 and 12h respectively. Moreover, the results of functional annotation and enriched pathway showed that the DEGs were involved in phagosome (ko04145), lysosome (ko04142), Endocytosis (ko04144), and NOD-like receptor signaling pathway (ko04621). Besides, the Pearson's correlation coefficient (R) between transcriptome sequencing and qPCR was 0.845, which confirmed the reliability of the transcriptome sequencing results and the accuracy of assembly. Furthermore, the expression pattern of 15 candidate DEGs, containing 9 up-regulated and 6 down-regulated DEGs at 3 h, indicated the regulation of DA in physiological functions especially in the immune system. Therefore, these results revealed that DA induced the expressions of membrane receptors or proteins, activated intracellular signaling pathways, regulated cellular and humoral immune systems, controlled antioxidation and apoptosis, and was involved in the regulation of neuroendocrine system. These findings are helpful to promote the understanding on the effects of biogenic amines on physiological functions and regulatory networks of crustacean, and offer a substantial material and foundation for researching the immune response of crustacean.


Assuntos
Dopaminérgicos/metabolismo , Dopamina/metabolismo , Hemócitos/imunologia , Imunidade Inata/genética , Penaeidae/imunologia , Transcriptoma/efeitos dos fármacos , Animais , Dopamina/administração & dosagem , Dopaminérgicos/administração & dosagem , Perfilação da Expressão Gênica , Imunidade Inata/efeitos dos fármacos , Penaeidae/genética
20.
Fish Shellfish Immunol ; 94: 607-616, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31541777

RESUMO

Akirin, which are members of the NF-κB signaling pathway, play critical roles in regulating the expression of antimicrobial peptides. In the present study, the Akirin gene from Penaeus monodon was identified from a transcriptome database and designated as PmAkirin. The complete sequence of the PmAkirin cDNA was 1508 bp, encoding a protein of 213 amino acids, and it showed 99% amino acid identity to the Litopenaeus vannamei Akirin. Two predicted nuclear localization signals (NLSs) were found, and the amino acid sequence alignments showed that PmAkirin was highly conserved at the N-terminus and C-terminus. PmAkirin expression was found to be the highest in the hemolymph, followed by the heart, gill, stomach, hepatopancreas, intestine, and muscle. When challenged with Vibrio parahaemolyticus infection, the PmAkirin mRNA and three antimicrobial peptides (AMPs: PmALF2, PmALF3, and PmCrus4) were upregulated. However, another five AMPs (PmALF6, PmCrus1, PmPEN3a, PmPEN3b, and PmPEN5) were downregulated by V. parahaemolyticus infection. Silencing PmAkirin by dsRNA significantly decreased the expression of the eight AMPs, which lead to an increase in the blood concentration of V. parahaemolyticus and higher mortality in the shrimp. In contrast, the overexpression of PmAkirin significantly increased the expression of the eight AMPs, which led to a reduction in the blood concentration of V. parahaemolyticus and promoted the survival of the shrimp. Taken together, we concluded that PmAkirin plays an important role in regulating the expression of AMPs in black tiger shrimp to defend against V. parahaemolyticus infection.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Proteínas Nucleares/química , Filogenia , Domínios Proteicos , Alinhamento de Sequência , Vibrio parahaemolyticus/fisiologia
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