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1.
Comp Biochem Physiol B Biochem Mol Biol ; 240: 110373, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31678268

RESUMO

The Japanese eel Anguilla japonica is a catadromous fish species with considerable farming scale. Previous studies showed that dietary α-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) satisfied essential fatty acid requirements in eel, which suggested that Japanese eel should have a complete pathway for the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA). However, existing knowledge was insufficient to explain the molecular basis of LC-PUFA biosynthetic capacity in eel. In order to further characterize this pathway in eel, a full-length cDNA of a putative fatty acyl elongase was isolated, with the ORF encoding a protein with 294 amino acids. The putative elongase displayed high homology to Elovl2 of other teleosts. Functional characterization by heterologous expression in yeast showed the protein product of the cDNA had high activity towards C20 and C22 PUFA substrates and low activity towards C18 PUFA substrates, characteristic of Elovl2 elongases. Tissue distribution of the elovl2 mRNA showed highest expression in brain and eyes, which was different from freshwater and anadromous species. This may reflect an important role for this enzyme in the in situ endogenous biosynthesis of docosahexaenoic acid (DHA) in neural tissues in eel. This is the first report of an Elovl2 in a catadromous teleost and demonstrates that Japanese eel has a complete enzyme repertoire required for the endogenous biosynthesis of DHA via the Sprecher pathway. These data have increased our knowledge of the diversity of LC-PUFA biosynthesis in vertebrates, and provided further insight into the regulatory mechanisms of LC-PUFA biosynthesis in teleost fish.

2.
J Biol Chem ; 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619521

RESUMO

Streptococcus suis is a globally distributed zoonotic pathogen associated with meningitis and septicemia in humans, posing a serious threat to public health. To successfully invade and disseminate within its host, this bacterium must overcome the innate immune system. The antimicrobial peptide LL-37 impedes invading pathogens by directly perforating bacterial membranes and stimulating the immune function of neutrophils, which are the major effector cells against S. suis However, little is known about the biological relationship between S. suis and LL-37 and how this bacterium adapts to and evades LL-37-mediated immune responses. In the present study, using an array of approaches, including enzyme, chemotaxis, and cytokine assays; quantitative RT-PCR; and CD spectroscopy, we found that the cysteine protease ApdS from S. suis cleaves LL-37 and thereby plays a key role in the interaction between S. suis and human neutrophils. S. suis infection stimulated LL-37 production in human neutrophils, and S. suis exposure to LL-37 up-regulated ApdS protease expression in the bacterium. We observed that ApdS targets and rapidly cleaves LL-37, impairing its bactericidal activity against S. suis We attributed this effect to the decreased helical content of the secondary structure in the truncated peptide. Moreover, ApdS rescued S. suis from killing by human neutrophils and neutrophil extracellular traps because LL-37 truncation attenuated neutrophil chemotaxis and inhibited the formation of extracellular traps and production of reactive oxygen species. Altogether, our findings reveal an immunosuppressive strategy of S. suis whereby the bacterium blunts the innate host defenses via ApdS protease-mediated LL-37 cleavage.

3.
Fish Shellfish Immunol ; 94: 852-860, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31600594

RESUMO

Bcl-2 associated athanogene-1 (BAG1) is involved in various signalling pathways including apoptosis, cell proliferation, gene transcriptional regulation and signal transduction in animals. However the functions of BAG1 during the antiviral response of mud crab Scylla paramamosain is still unclear. In this study, the mud crab BAG1 (SpBAG1) was characterized to consist of 1761 nucleotides, containing an opening frame of 630bp encoding 209 amino acids with an ubiquitin domain and a BAG1 domain. SpBAG1 was found to be significantly up-regulated at 6 h-24 h, but down-regulated from 48 h-72 h in the hemocytes of mud crab after challenge with white spot syndrome virus (WSSV). RNAi knock-down of SpBAG1 significantly reduced the copies of WSSV and increased the apoptotic rate in mud crabs. The finding from this study suggested that SpBAG1 could promote the WSSV infection by inhibiting apoptosis in mud crab. Therefore, to the best of our knowledge, this is the first study demonstrating the role of SpBAG1 as a novel apoptosis inhibitor to promote virus infection in mud crab.

4.
Fish Shellfish Immunol ; 94: 643-653, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31563555

RESUMO

Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase with multiple cellular functions in vertebrates including apoptosis, differentiation, proliferation, survival, tumorigenesis, signal transduction, immune regulation and inflammation. In the current study, the catalytic and regulatory subunit homologs of Litopenaeus vannamei protein kinase CK2 (LvCK2α and LvCK2ß) were cloned and characterized. LvCK2α has a full-length cDNA sequence of 1764 bp with a 1053 bp open reading frame (ORF) encoding a putative protein of 351 amino acids, which contains a typical serine/threonine kinase domain. On the other hand, LvCK2ß has a 1394 bp full-length cDNA with an ORF of 663 bp encoding a protein with 221 amino acids, which contains a Casein kinase II regulatory subunit domain. Sequence and phylogenetic analysis revealed that LvCK2 was evolutionary related with the CK2 of invertebrates. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that LvCK2α and LvCK2ß transcripts were widely expressed in all shrimp tissues tested, and were both induced in hemocytes and hepatopancreas upon challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting their involvement in shrimp immune response. Moreover, RNA interference (RNAi) of LvCK2α resulted in increased hemocytes apoptosis, shown by high caspase 3/7 activity, increased number of apoptotic cells, coupled with an elevation in transcript levels of pro-apoptotic LvCaspase3 and LvCytochrome C, and a reduction in mRNA levels of pro-survival LvBcl2, LvIAP1, and LvIAP2. In addition, LvCK2α knockdown followed by V. parahaemolyticus challenge resulted in higher cumulative mortality of shrimp. Taken together, our current findings suggest that LvCK2 modulates shrimp hemocytes apoptosis as part of the innate immune response to pathogens.

5.
Fish Shellfish Immunol ; 93: 135-143, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31326583

RESUMO

Mud crabs (Scylla paramamosain), a commercially important cultured species in the southeastern region of China, is usually infected by Vibriosis or parasites, causing great economic losses in cultured farms. Previous studies have demonstrated that probiotics benefited in enhancing the immune response against invading pathogens in aquatic animals. In this study, the effects of dietary administration of lactic acid bacteria (LAB) (Enterococcus faecalis Y17 and Pediococcus pentosaceus G11) on growth performance and immune responses of mud crab were assessed. Both strains (Y17 and G11) showed an inhibitory activity against bacterial pathogens (Aeromonas hydrophila, Vibrio parahaemolyticus, Vibrio alginolyticus, Staphylococcus aureus, and ß Streptococcus), and a wide pH tolerance range of 2-10. In vivo, mud crabs were fed a control diet and experimental diets supplemented with 109 cfu g-1 diet either Y17 or G11 for 6 weeks before subjecting to a challenge test with V. parahaemolyticus for 12 h. The probiotic-supplemented diets had significant effects on weight gain and specific growth rate during the feeding trial. Increased serum enzyme activities of phenoloxidase, lysozyme, and SOD were observed in the hemolymph of mud crab in Y17 and G11-supplemented groups compared to that in the controls (P < 0.01). The significantly up-regulated expression of gene CAT, LYS, proPO, and SOD could be seen in hepatopancreas in G11-supplemented groups. After the pathogenicity test, the survival rate of Y17 + and G11 + V. parahaemolyticus groups was 66.67% and 80.00%, respectively, compared with 53.33% for the control groups. Taken together, dietary supplementation of Y17 and G11 strains were beneficial in mud crab, which could increase growth performance, modulate immune system and protect the host against V. parahaemolyticus infection.

6.
Fish Shellfish Immunol ; 93: 313-321, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31351111

RESUMO

The Src family kinases (SFK) are involved in signaling transductions that regulate numerous biological activities including host-virus interaction. These features of SFK have been well explored in vertebrates, however, in shrimp, the invertebrate SFK family member Src64B, has not been characterized and therefore its role in shrimp-virus interaction remains unknown. In this study, two Litopenaeus vannamei Src64B isoforms (designated LvSrc64B1 and LvSrc64B2) were first cloned and their role in white spot syndrome virus (WSSV) infection was explored. Bioinformatics analysis revealed that LvSrc64B1 and LvSrc64B2 were similar to other Src64B family members, with high homology in primary and tertiary structures, and contained the conserved SFK functional domains, as well as the putative myristylation and phosphorylation sites. Tissue distribution analysis showed that both LvSrc64B isoforms were ubiquitously expressed, albeit distinctively in the tested tissues. In addition, transcript levels of LvSrc64B1 and LvSrc64B2 were significantly induced following WSSV challenge and had similar expression patterns. Furthermore, siRNA-mediated knockdown of LvSrc64B1 and LvSrc64B2 followed by WSSV infection resulted in increased expression of viral genes, enhanced viral DNA replication, and elevation of hemocytes apoptosis. Depletion of LvSrc64B1 and LvSrc64B2 also reduced shrimp survival upon WSSV infection. In conclusion, the current data strongly suggest that Src64B is a host factor that inhibits WSSV replication by modulating apoptosis in shrimp.

7.
Mol Immunol ; 112: 360-368, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31261021

RESUMO

Hemocyanin is primarily a respiratory copper-containing glycoprotein present in the hemolymph of mollusks and arthropods. Recently, hemocyanin has attracted huge research interest due to its multifunctionality and polymorphism. Most previous immune-related studies on shrimp hemocyanin have focused on the C-terminal. Moreover, we previously reported that the C-terminal domain of Litopenaeus vannamei hemocyanin possesses single nucleotide polymorphisms (SNPs), but little is known about the molecular diversity of the N-terminal domain. In the current study, diversity within the N-terminal domain of L. vannamei hemocyanin (LvHMC-N) was explored using bioinformatics and molecular biology techniques as well as immune challenge. Twenty-five LvHMC-N variants were identified using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing, with multiple sequence alignment showing that the 25 variants shared 87%-99 % sequence homology with LvHMC (AJ250830.1). In different shrimp individuals and different shrimp tissues (i.e., hemocytes, stomach, muscle and hepatopancreas), the LvHMC-N variants were expressed differently. Pathogen challenge could modulate the molecular diversity of LvHMC-N, as three LvHMC-Nr variants (LvHMC-Nr1, LvHMC-Nr2 and LvHMC-Nr3) were identified by sequencing following Vibrio parahaemolyticus challenge. Most importantly, recombinant proteins of these three variants (rLvHMC-Nr1, rLvHMC-Nr2 and rLvHMC- Nr3) had relatively high in vitro agglutinative activities against V. parahaemolyticus, Vibrio alginolyticus and Streptoccocus iniae. Our present data indicates that the N-terminus of L. vannamei hemocyanin also possess molecular diversity, which seems to be associated with immune resistance to pathogenic infections.


Assuntos
Hemocianinas/genética , Hemocianinas/imunologia , Imunidade/genética , Imunidade/imunologia , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Clonagem Molecular/métodos , Biologia Computacional/métodos , Hemócitos/imunologia , Hemócitos/microbiologia , Hemolinfa/imunologia , Hemolinfa/microbiologia , Hepatopâncreas/imunologia , Hepatopâncreas/microbiologia , Alinhamento de Sequência , Análise de Sequência de DNA , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrio parahaemolyticus/imunologia
8.
Neurochem Res ; 44(8): 1840, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31201675

RESUMO

The article titled "TREM2 Attenuates Aß1­42­Mediated Neuroinflammation in BV­2 Cells by Downregulating TLR Signaling", written by Huiping Long, Gang Zhong, Chengzhi Wang, Jian Zhang, Yueling Zhang, Jinglian Luo, Shengliang Shi, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 27 May 2019 with open access.

9.
Dev Comp Immunol ; 100: 103421, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31254562

RESUMO

White spot syndrome virus (WSSV) is one of the most virulent and widespread pathogens that infect almost all marine crustaceans and therefore cause huge economic losses in aquaculture. The Bcl2 protein plays a key role in the mitochondrial apoptosis pathway, which is a crucial immune response in invertebrates. However, the role of Bcl2 in apoptosis and immunoregulation in mud crab, Scylla paramamosain, is poorly understood. Here, the Bcl2 homolog (SpBcl2) in S. paramamosain was cloned and its role in WSSV infection explored. The expression of SpBcl2 increased at both the transcriptional level and post-transcriptional level after WSSV infection, while the hemocytes apoptosis decreased significantly. Furthermore, there was increase in the level of cytochrome c coupled with an upregulation in the expression of SpBcl2. These results indicated that SpBcl2 suppressed apoptosis by preventing the release of cytochrome c from mitochondria, thereby promoting WSSV replication in mud crab. The findings here therefore provide novel insight into the immune response of mud crabs to WSSV infection.

10.
Neurochem Res ; 44(8): 1830-1839, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31134514

RESUMO

The pathogenesis of late-onset Alzheimer's disease (LOAD) mainly involves abnormal accumulation of extracellular ß-amyloid (Aß) and the consequent neurotoxic effects. The triggering receptor expressed on myeloid cells 2 (TREM2) gene is associated with the pathogenesis of LOAD and plays important roles in mediating the phagocytosis of Aß by microglia and regulating inflammation in central nervous system. However, the exact mechanisms of these processes have not yet been clarified. In this study, we investigated the mechanism by which TREM2 regulates neuroinflammation and promotes Aß1-42 clearance by BV-2 cells and further elucidated the underlying molecular mechanisms. We either silenced or overexpressed TREM2 in BV-2 cells and evaluated the cell viability, Aß1-42 content, and expression of inflammatory markers (IL-1ß, IL-6, and TNF-α). TREM2 overexpression up-regulated cell activity, promoted clearance of Aß1-42 by BV-2 cells, and down-regulated expression of the inflammatory factors. In addition, TREM2 overexpression downregulation the expression of the TLR family (TLR2, TLR4 and TLR6) in BV-2 cells. Moreover, LPS, as an agonist of the TLR family, up-regulated the expression of inflammatory cytokines (IL-1ß, TNF-α, and IL-6) in BV-2 cells overexpressing TREM2. In conclusion, TREM2 promoted clearance of Aß1-42 by BV-2 cells and restored BV-2 cell viability from Aß1-42-mediated neuroinflammation by downregulating TLRs. These findings suggest that TREM2 may be a target for LOAD therapy.

11.
Dev Comp Immunol ; 98: 99-107, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31051195

RESUMO

White spot syndrome, which is caused by white spot syndrome virus (WSSV), is a highly contagious disease of penaeid shrimp. However, there is currently incomplete understanding of the infection mechanism and pathogenesis of WSSV. In this study, a novel gene of a previously uncharacterized WSSV response protein (LvWRP) in Litopenaeus vannamei was identified and characterized. The LvWRP gene has an open reading frame (ORF) of 879 bp encoding a putative protein of 292 amino acids. Sequence analysis revealed that LvWRP shared 24.9% identity with an uncharacterized protein of Penaeus monodon nudivirus. Real-time qPCR analysis showed that LvWRP was ubiquitously expressed in shrimp tissues, with transcript levels induced in hemocytes upon immune challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and WSSV. In addition, RNA interference-mediated knockdown of LvWRP followed by WSSV challenge revealed significant decrease in the transcript levels of WSSV IE1 and VP28 genes coupled with a reduction in WSSV copies in shrimp hemocytes. Moreover, depletion of LvWRP followed by WSSV challenge significantly increased the transcript levels of Vago4 and Vago5 as well as increased the phosphorylation of STAT, while hemocytes apoptosis in terms of caspase 3/7 activity was decreased. These results suggest that LvWRP is important for WSSV replication in shrimp, and therefore one of the vital host factors in WSSV infection.

12.
Am J Orthod Dentofacial Orthop ; 155(5): 642-649, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31053279

RESUMO

INTRODUCTION: The purpose of this study was to investigate the effect of systemic delivery of Substance P (SP) on experimental tooth movement. METHODS: Forty-eight adult Sprague-Dawley rats were randomly divided into 2 groups and their maxillary first molars were mesially moved with the use of closed-coil springs. The experiment group received systemic injection of SP and the control group received phosphate-buffered saline solution. Transportation distances of first molars were measured. Hematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, and immunohistochemistry staining were performed to evaluate alveolar bone remodeling. Then the interferon (IFN) γ and tumor necrosis factor (TNF) α concentrations in peripheral blood and local periodontal tissue were measured. Finally, the effects of SP on bone marrow-derived stem cell (BMSC) proliferation and migration were tested in vitro. RESULTS: Systemic delivery of SP significantly increased the distance of tooth movement and stimulated both osteoclast and osteoblast activities. The concentrations of IFN-γ and TNF-α increased in peripheral blood at early phases of the experiment and decreased in periodontal tissue at late phases. In vitro, the proliferation and migration of BMSCs were promoted by SP. CONCLUSIONS: Systemic delivery of SP can accelerate orthodontic tooth movement and promote alveolar bone remodeling potentially through immunomodulation and mobilizing endogenous mesenchymal stem cells.


Assuntos
Processo Alveolar/efeitos dos fármacos , Remodelação Óssea/efeitos dos fármacos , Substância P/farmacologia , Técnicas de Movimentação Dentária , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Interferon gama/metabolismo , Maxila , Dente Molar , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem , Fator de Necrose Tumoral alfa/metabolismo
13.
Arch Virol ; 164(7): 1889-1895, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31087191

RESUMO

Singapore grouper iridovirus (SGIV) is a lethal grouper virus containing 162 predicted ORFs. Previous proteomic studies led to identification of 73 SGIV structural proteins. Here, SDS-assisted tube-gel digestion and DOC-assisted in-solution digestion coupled with LC-ESI-MS/MS were applied to further profile the SGIV structural proteome. We identified a total of 90 SGIV structural proteins including 24 newly reported proteins. Additionally, several PTMs were identified, including 26 N-terminal acetylated proteins, three phosphorylated proteins, and one myristoylated protein. Importantly, 47 of the proteins that were identified are predicted to contain conserved domains. Our work greatly expands the repertoire of the SGIV structural proteome and provides more insight into the biology of SGIV.


Assuntos
Bass/virologia , Doenças dos Peixes/virologia , Iridovirus/genética , Iridovirus/isolamento & purificação , Proteínas Estruturais Virais/genética , Animais , Perfilação da Expressão Gênica , Fases de Leitura Aberta/genética , Proteoma/genética , Proteômica , Espectrometria de Massas em Tandem
14.
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
15.
J Proteomics ; 201: 57-64, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991158

RESUMO

Hemocyanin is the main component of hemolymph plasma proteins and possesses diverse immunological properties and immunomodulatory functions. However, the interacting networks of hemocyanin in shrimp immune response remain poorly understood. In this study, 39 potential hemocyanin interacting partners were identified from Litopenaeus vannamei plasma by co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Gene Ontology (GO) analysis showed that most of the identified interactors were cell proteins involved in metabolic process and binding. Among these identified proteins, transglutaminase (TGase), a crucial regulator in hemolymph clotting cascade, was chosen for further studies. Far-Western blot and His-pull down assays revealed that hemocyanin directly interacted with TGase. Further analysis demonstrated that hemocyanin and TGase followed similar expression patterns upon pathogen infection. Moreover, in vivo knockdown of hemocyanin led to a significant decrease in TGase expression, as well as inhibited hemolymph clotting. Taken together, these data suggest that hemocyanin might positively regulate hemolymph clotting by modulating TGase in shrimp. SIGNIFICANCE: The interaction networks among immune-related factors is critical for the innate immune response in invertebrates. We report for the first time, proteins that potentially interact with hemocyanin, which led to the identification of 39 possible hemocyanin-proteins including the clotting-related factor TGase. Further studies demonstrated that hemocyanin directly interacted with TGase and modulated its expression, therefore affecting the formation of hemolymph clotting. These findings not only extend our knowledge of the immune interaction networks but also contribute to shrimp disease control and prevention.

16.
Mar Biotechnol (NY) ; 21(3): 320-334, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30835008

RESUMO

Although the sexual dimorphism in terms of gonadal development and gametogenesis of mud crab has been described, the internal regulating mechanism and sex differentiation process remain unclear. A comparative gonadal miRNA transcriptomic study was conducted to identify miRNAs that are differentially expressed between testes and ovaries, and potentially uncover miRNAs that might be involved in sex differentiation and gonadal maturation mechanisms of mud crabs (Scylla paramamosain). A total of 10 known miRNAs and 130 novel miRNAs were identified, among which 54 were differentially expressed. Target gene prediction revealed a significant enrichment in 30 KEGG pathways, including some reproduction-related pathways, e.g. phosphatidylinositol signalling system and inositol phosphate metabolism pathways. Further analysis on six differentially expressed known miRNAs, six differentially expressed novel miRNAs and their reproduction-related putative target genes shows that both miRNAs and putative target genes showed stage-specific expression during gonadal maturation, suggesting their potential regulatory roles in sex differentiation and reproductive development. This study reveals the sex-biased miRNA profile and establishes a solid foundation for understanding the sex differentiation and gonadal maturation mechanisms of S. paramamosain.


Assuntos
Braquiúros/crescimento & desenvolvimento , Braquiúros/genética , Gônadas/crescimento & desenvolvimento , MicroRNAs/genética , MicroRNAs/metabolismo , Diferenciação Sexual/genética , Maturidade Sexual/genética , Animais , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino
17.
Genomics ; 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30851358

RESUMO

In this study, we first identified male-specific SNP markers using restriction site-associated DNA sequencing, and further developed a PCR-based sex identification technique for Charybdis feriatus. A total of 296.96 million clean reads were obtained, with 114.95 and 182.01 million from females and males. After assembly and alignment, 10 SNP markers were identified being heterozygous in males but homozygous in females. Five markers were further confirmed to be male-specific in a large number of individuals. Moreover, two male-specific sense primers and a common antisense primer were designed, using which, a PCR-based genetic sex identification method was successfully developed and used to identify the sex of 103 individuals, with a result of 49 females and 54 males. The presence of male-specific SNP markers suggests an XX/XY sex determination system for C. feriatus. These findings should be helpful for better understanding sex determination mechanism, and drafting artificial breeding program in crustaceans.

18.
FEBS Lett ; 593(8): 820-830, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30901486

RESUMO

The respiratory glycoprotein hemocyanin has been implicated in immune-related functions. Using lectin blotting, we show that the binding of shrimp (Litopenaeus vannamei) hemocyanin to concanavalin A decreases markedly with O-glycosidase treatment but not with PNGase F. Twelve O-glycosylation sites, three on the large hemocyanin subunit and nine on the small hemocyanin subunit (HMCs), were identified by LC-MS/MS. Importantly, when the glycosylation sites at Thr-537, Ser-539, and Thr-542 on the C terminus of HMCs were replaced with alanine, the resultant mutant hemocyanin had reduced carbohydrate content, coupled with a fourfold reduction in bacterial agglutination and 0.2-fold reduction in antibacterial activities toward Vibrio parahaemolyticus and Staphylococcus aureus. These results suggest that the glycosylation sites on shrimp hemocyanin are closely related to its immunological functions.

19.
Dev Comp Immunol ; 95: 38-49, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30738078

RESUMO

The p38 mitogen-activated protein kinases (MAPKs) are evolutionally conserved from yeasts to mammals, and are involved in the regulation of cells response to various extracellular stimuli. In this study, the p38 MAPK gene (designated as Spp38) of mud crab (Scylla paramamosain) was identified and studied. Spp38 contained the conserved Thr-Gly-Tyr (TGY) motif and a Ala-Thr-Arg-Trp (ATRW) substrate-binding site. Spp38 transcript was ubiquitously expressed in all tissues examined, with the highest expression found in muscle and hepatopancras. Quantitative real-time PCR revealed that Spp38 was upregulated in hemocytes and hepatopancras after infection with Vibrio parahemolyticus and Lipopolysaccharides (LPS). Reporter gene assays indicated that Spp38 activated the expression of anti-lipopolysaccharides (SpALF1 - SpALF6) in S. paramamosian. RNA interference (RNAi)-mediated knockdown of Spp38 or inhibition of Spp38 by SB203580 decreased the expression levels of SpALF1-6 and dual oxidase (SpDuox1 and SpDuox2) in S. paramamosian, which consequently reduced reactive oxygen species (ROS) production thereby significantly increasing the bacterial count in the hemolymph of mud crabs. Similarly, there was a significant reduction in bacterial clearance ability of hemolymph after Spp38 knockdown followed by V. parahemolyticus infection. Taken together, the current data indicated that Spp38 could play a vital role in maintaining the homeostasis of hemolymph microbiota in S. paramamosain.


Assuntos
Proteínas de Artrópodes/imunologia , Braquiúros/imunologia , Hemolinfa/microbiologia , Microbiota/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Proteínas de Artrópodes/metabolismo , Braquiúros/microbiologia , Hemócitos/imunologia , Hemócitos/metabolismo , Hemolinfa/citologia , Hemolinfa/imunologia , Hepatopâncreas/metabolismo , Homeostase , Lipopolissacarídeos/imunologia , Músculos/metabolismo , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrio parahaemolyticus/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
Genomics ; 2019 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-30807818

RESUMO

PIWI-interacting RNAs (piRNAs) are abundantly found in germ cells and involved in gametogenesis and gonadal development. Information on the regulatory roles of piRNAs in crustacean reproduction, however, is scarce. Thus, we identified gonadal piRNAs of mud crab Scylla paramamosain. Of the 115,491 novel piRNAs, 596 were differentially expressed. Subsequently, 389,887 potential piRNA-target genes were predicted. The expression of 4 piRNAs and 9 genes with high piRNA interactions were validated with the inclusion of additional immature specimens, including LRP2 that is involved in growth and reproduction, MDN1 in ribosome biogenesis pathway and gametogenesis, and PRKDC, a DNA repair gene involved in gonadal differentiation and maturation. KEGG analysis further revealed the involvement of predicted piRNA target genes in gametogenesis- and reproduction-related pathways. Our findings provide baseline information of mud crab piRNAs and their differential expression between testes and ovaries suggests that piRNAs play an essential role in regulating gametogenesis and gonadal development.

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