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1.
Exp Appl Acarol ; 78(2): 149-172, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31190248

RESUMO

Monitoring acaricide resistance and understanding the underlying mechanisms are critically important in developing strategies for resistance management and tick control. Identification of single nucleotide polymorphisms in the acaricide-resistant associated gene of Rhipicephalus microplus has enabled the development of molecular markers for detection and monitoring of resistance against different types of acaricide. There are many molecular markers developed for resistance monitoring, including mutations on target genes such as sodium channel, acetylcholinesterase, carboxylesterase, ß-adrenergic octopamine receptor, octopamine-tyramine etc. Molecular genotyping through molecular markers can detect the presence of resistance-associated genes in a tick population before it reaches high frequency. This review aims to provide an update on the various molecular markers discovered to date from different regions of the world.


Assuntos
Acaricidas/farmacologia , Proteínas de Artrópodes/genética , Resistência a Medicamentos/genética , Polimorfismo de Nucleotídeo Único/genética , Rhipicephalus/genética , Animais , Proteínas de Artrópodes/metabolismo , Mutação , Rhipicephalus/efeitos dos fármacos , Controle de Ácaros e Carrapatos
2.
Parasit Vectors ; 12(1): 289, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31174589

RESUMO

BACKGROUND: Tick selenoproteins are involved in regulating oxidative and endoplasmic reticulum stress during prolonged tick feeding on mammalian hosts. How selenoproteins are activated upon tick-borne pathogen infection is yet to be defined. METHODS: To examine the functional role of selenoprotein K in Borrelia burgdorferi infection within the tick host Ixodes scapularis, RNA interference (RNAi)-based gene silencing was performed. RESULTS: Selenoprotein K is an endoplasmic reticulum (ER)-resident protein and a component of the ERAD complex involved in ER homeostasis. A qRT-PCR assay revealed the significant upregulation of selenogene K (selenoK) expression in B. burgdorferi-infected tick tissues. Silencing of the selenoK transcript significantly depleted B. burgdorferi copies within the infected tick tissues. Upon selenoK knockdown, another component of the ERAD complex, selenoprotein S (selenoS), was significantly upregulated, suggesting a compensatory mechanism to maintain ER homeostasis within the tick tissues. Knockdown of selenoK also upregulated ER stress-related unfolded protein response (UPR) pathway components, ATF6 and EIF2. CONCLUSIONS: The exact mechanisms that contribute to depletion of B. burgdorferi upon selenoK knockdown is yet to be determined, but this study suggests that selenoK may play a vital role in the survival of B. burgdorferi within the tick host.


Assuntos
Proteínas de Artrópodes/genética , Borrelia burgdorferi/fisiologia , Ixodes/genética , Doença de Lyme , Selenoproteínas/genética , Animais , Vetores de Doenças , Retículo Endoplasmático/química , Feminino , Ixodes/microbiologia , Masculino , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Resposta a Proteínas não Dobradas/genética , Regulação para Cima
3.
Artigo em Inglês | MEDLINE | ID: mdl-31075502

RESUMO

Cherax quadricarinatus, as one of the world's most valuable freshwater shrimp species, has received extensive attention in recent years. As males grow larger and faster than females, development of the sex control breeding techniques is of great interest, but knowledge on sex determination and differentiation in C. quadricarinatus remains poorly unknown. Sxl (Sex-lethal) is an important gene in the sexual differentiation regulatory hierarchy. It reflects the ratio of sex chromosomes to autosomes into molecule changes and directs sex-specific splicing forms of precursor mRNA. In the present study, the full-length cDNA sequences of four Sxl splice variants were identified from C. quadricarinatus, designated as CqSxl1, CqSxl2, CqSxl3 and CqSxl4, respectively. Sequence analysis determined different splicing sites near the translation termination region of four Sxl transcript isoforms. Two highly conserved classical RRM domains were found according to predicted secondary structures of Sxl proteins. mRNA expression of CqSxl in different tissues, developmental stage of embryos and testes were investigated by real-time quantitative PCR. Among four isoforms, CqSxl3 showed tissue specificity with higher expression levels in testis than in ovary. CqSxl1 and CqSxl4 were found widely expressed in various tissues and CqSxl2 was almost undetectable. In early developmental stages, the expression levels of CqSxl1/3/4 gradually increased along with embyonic development. In addition, CqSxl genes presented the higher transcript levels in the early stage of testis development. Furthermore, CqSxl3 silencing induced a significant decrease of the transcript of Cq-IAG, an androgenic hormone-encoding gene responsible for masculine development. These data indicate that CqSxl3 might be involved in male sex determination in C. quadricarinatus. Our study will contribute to understanding the mechanism of sex determination in C. quadricarinatus, and also can provide theoretical guidance for establishing a sex control technology.


Assuntos
Proteínas de Artrópodes , Decápodes (Crustáceos) , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Letais , Processamento de RNA/fisiologia , RNA Mensageiro , Diferenciação Sexual/fisiologia , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Decápodes (Crustáceos)/embriologia , Decápodes (Crustáceos)/genética , Feminino , Perfilação da Expressão Gênica , Masculino , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
4.
Artigo em Inglês | MEDLINE | ID: mdl-31075503

RESUMO

The functions of Wnt signalling in crab limb regeneration are poorly understood. Herein, we isolated and characterised the full-length cDNA of WNT4 from Portunus trituberculatus, designated PtWNT4. The 4831 bp cDNA encodes 323 amino acid polypeptide. Protein structure prediction showed that PtWNT4 has a conserved WNT domain. The PtWNT4 gene was expressed in all regenerative limb stages, and was upregulated from stage I, with highest expression in stage III, and expression then declined in stages IV and V. Immunohistochemistry revealed strong localisation at loose connective tissue during limb regeneration, but PtWNT4 protein levels decreased upon formation of muscle fibers. Injection of WNT4 dsRNA into regenerative limbs significantly decreased PtWNT4 mRNA levels from 36 h to 5 days after injection, indicating successful gene silencing. RNA interference knockdown of PtWNT4 expression greatly retarded limb regeneration compared with the control group. Blastema emergence phenotype analysis revealed limb regeneration rates of 0, 31.5% and 40.5% for the dsWNT4 group at 36 h, 3 days and 5 days, respectively, compared with 29.95%, 83.0% and 92.5% for the saline-injected control groups (p <0.05). Expression analysis on the WNT4 using RNAi provide important information for understanding its functional mechanism during limb regeneration in P. trituberculatus. The results also contribute to our understanding of the role of Wnt signalling during limb regeneration in crustaceans.


Assuntos
Proteínas de Artrópodes/metabolismo , Braquiúros/metabolismo , Membro Posterior/fisiologia , Interferência de RNA , Regeneração/fisiologia , Proteína Wnt4/metabolismo , Animais , Proteínas de Artrópodes/genética , Braquiúros/genética , Proteína Wnt4/genética
5.
Exp Appl Acarol ; 77(4): 527-543, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31062204

RESUMO

Biological control of spider mites in hot and dry weather is a serious technical issue. A high-temperature adapted strain (HTAS) of the predatory mite Neoseiulus barkeri Hughes was selected from its conventional strain (CS), via long-term heat acclimation and frequent heat hardenings in our previous studies. However, the environment of high temperature is usually associated with enhanced ultraviolet (UV) radiation. In the present study, the physiological effects of UV-B radiation on survival rate and egg damage of N. barkeri were investigated, as well as the activities and expression profiles of antioxidant enzymes to UV-B radiation stress. UV-B radiation had deleterious effects on egg hatchability and survival of N. barkeri. Adults of the HTAS strain were less UV-B resistant than those of the CS strain; they also had lower levels of enzymatic activity of superoxide dismutase (SOD) and catalase against oxidative damage and weaker upregulation of SOD genes. The mRNA expression of three SOD genes of CS adult females immediately increased whereas that of HTAS showed almost no difference under UV-B stress for 1 h. The results showed the HTAS of N. barkeri had lower fitness under UV-B stress compared with the CS of N. barkeri. These results suggested that long-term heat acclimation may exert a profound impact on the developmental physiology of N. barkeri.


Assuntos
Proteínas de Artrópodes/genética , Aptidão Genética/efeitos da radiação , Ácaros/efeitos da radiação , Comportamento Predatório/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Adaptação Biológica , Animais , Antioxidantes/metabolismo , Proteínas de Artrópodes/metabolismo , Feminino , Temperatura Alta , Longevidade/efeitos da radiação , Ácaros/enzimologia , Ácaros/genética , Ácaros/fisiologia , Óvulo/fisiologia , Óvulo/efeitos da radiação , Controle Biológico de Vetores , Transcrição Genética/efeitos da radiação
6.
Gene ; 710: 1-8, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31078655

RESUMO

Investigation of sex determination system in Eriocheir sinensis is important because of sex-dimorphism in its growth traits. However, little information about the sex-related genes in embryonic development stages were exposed. To obtain more information of sex determination in Chinese mitten crab, we performed the transcriptome analysis in embryonic development stage (fertilized egg stage, cleavage stage, blastula stage, gastrula stage and heartbeat stage) of Chinese mitten crab using nextgeneration sequencing technology. Thirty-one of 32,088 annotated unigenes were identified as sex-related genes including 16 genes involved in primary sex determination in model organisms and 8 genes of SOX family and 7 genes of DMRT gene family. Heatmap based on the RPKM value in five embryonic development stages indicated that these genes were clustered into two branches. Analysis of the differentially expressed 12 genes, including 3 genes of SOX family, 3 genes of DMRT gene family and 6 genes involved in primary sex determination in model organisms, showed significantly difference between the first three periods (fertilized egg stage-cleavage stage-blastula stage) and the last two periods (gastrula stage-heartbeat stage) and all 12 genes were up-regulated after blastula stage. In conclusion, we inferred that sex determination might be initiated after blastula stage in E. sinensis. Transcriptome analysis from embryonic development stage could provide a background information for further investigation in sex determination of Eriocheir sinensis.


Assuntos
Proteínas de Artrópodes/genética , Braquiúros/embriologia , Perfilação da Expressão Gênica/métodos , Caracteres Sexuais , Animais , Braquiúros/genética , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Família Multigênica , Análise de Sequência de RNA
7.
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
8.
Chem Biol Interact ; 307: 29-36, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30991043

RESUMO

Carbonyl reductases (CRs) represent a fundamental enzymatic defense mechanism against oxidative stress. While commonly two carbonyl reductases (CBR1 and CBR3) are found in mammalian genomes, invertebrate model organisms like Drosophila melanogaster express no CR but a functional homolog to human CBR1, termed sniffer. The importance of sniffer could be demonstrated in D. melanogaster where it protected against age-dependent neurodegeneration. Interestingly, the microcrustacean Daphnia harbors four copies of the CR gene (CR1, CR2, CR3, CR4) in addition to one sniffer gene. Due to this unique equipment Daphnia is an ideal model organism to investigate the function of sniffer. Recombinant sniffer from D. magna und D. pules were produced in E. coli, purified by Ni-affinity chromatography and tested with a variety of aliphatic and aromatic diketones, reactive aldehydes and precursors of advanced glycation end products (AGE). The highest catalytic activities were determined for sniffer from D. pulex with the aromatic dicarbonyls 9,10-phenanthrenequinone (kcat/Km = 2.6 s-1 x µM-1) and isatin (kcat/Km = 1.5 s-1 x µM-1). While sniffer from D. magna displayed preference for the same two substances, the respective catalytic activities were noticeably lower. Kinetic constants with aliphatic diketones were generally lower than those with aromatic dicarbonyls for both sniffer enzymes. The best aliphatic diketone as substrate for sniffer from D. magna and D. pulex was hexane-3,4-dione with kcat/Km = 0.23 s-1 µM-1 and kcat/Km = 0.35 s-1 µM-1, respectively. Poor or no detectable activity of the two sniffer enzymes was seen with the aliphatic diketones 2,5-hexanedione and 3,5-heptanedione, the aldehydes butanal, hexanal, decanal, crotonaldehyde, acrolein, trans-2-hexenal, and the AGE precursors glyoxal, methylglyoxal, furfural and glyceraldehyde, indicating no physiological function in the metabolism of short-chain aldehydes. Substrate inhibition for both sniffer enzymes was observed with the quinone substrates 1,4-naphthoquinone and 2-methyl-1,4-benzoquinone. From a variety of pesticides endosulfan turned out as an effective inhibitor of the sniffer enzymes (Ki = 9.2 µM for sniffer from D. magna, Ki = 12.0 µM for sniffer from D. pulex). In conclusion, the present results on sniffer from the protein superfamily of the short-chain dehydrogenases/reductases (SDR) in Daphnia ssp. complement earlier studies on carbonyl reductases in the same species and indicate that Daphnia is an interesting model to study the overall response to carbonyl stress.


Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Artrópodes/metabolismo , Clonagem Molecular , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/genética , Animais , Proteínas de Artrópodes/antagonistas & inibidores , Proteínas de Artrópodes/genética , Biocatálise , Daphnia/enzimologia , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Endossulfano/química , Endossulfano/metabolismo , Cinética , Fenantrenos/química , Fenantrenos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
9.
Arthropod Struct Dev ; 50: 43-52, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30974153

RESUMO

Naupliar development in copepods includes the generation of usually five pairs of post-mandibular segments. Since copepod nauplii show no outer body articulation, the only indication of larval segmentation is the expression of limb buds. Yet, in copepods the timing and sequence of limb bud expression in larval development varies to a large degree. In harpacticoid nauplii for instance, the 1st maxillae are formed at an early naupliar stage. By contrast, the four remaining pairs of limb buds frequently appear simultaneously with the last naupliar stage. The complete process of larval segment formation takes place under the body surface and has never been described in detail. To broaden our knowledge of early segmentation in copepods, we here describe the segmentation of the harpacticoid nauplius Tigriopus californicus by analysing the expression of the segment marker Engrailed and uncover the sequential addition of seven post-mandibular segments. The stripe formation and arrangement of labelled cells corresponds largely to those of other crustaceans studied in this respect. Together with a morphological approach using histology, SEM, and 3D-reconstructions based on CLSM we solve the so far controversial identity of the external limb buds in the final naupliar stage. In contrast to previous studies, we can show that all limb pairs from the 1st maxillae to the 3rd thoracopods are formed. Yet, the anlage of the maxilliped (1st thoracopod) remains hidden underneath the cuticle being never externally expressed in the nauplius.


Assuntos
Padronização Corporal , Copépodes/crescimento & desenvolvimento , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Padronização Corporal/genética , Copépodes/genética , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Microscopia Confocal , Microscopia Eletrônica de Varredura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
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
11.
Fish Shellfish Immunol ; 89: 458-467, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30954523

RESUMO

White spot disease (WSD) is a highly virulent viral disease in shrimps. Clinical signs and high mortality of WSD is generally observed after a few days of infection by White Spot Syndrome virus (WSSV). Mud crabs are the major carrier and persistent host for the WSSV. However, an elucidation of viral interaction and persistent mode of WSSV infection in mud crab is still limited. We investigated the defensive role of mud crab (Scylla olivacea) hemocytes against WSSV infection by using comparative proteomic analysis coupled with electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS). The proteomic maps of expressed proteins obtained from WSSV infected hemocytes revealed differential proteins related to various biological functions, including immune response, anti-apoptosis, endocytosis, phosphorylation signaling, stress response, oxygen transport, molting, metabolism, and biosynthesis. Four distinctive cell types of crab hemocytes: hyaline cells (HC), small granular cells (SGC), large granular cells (LGC) and mixed granular cells (MGC) were found susceptible to WSSV. However, immunohistochemistry analysis demonstrated a complete replication of WSSV only in SGC and LGC. WSSV induced apoptosis was also observed in HC, SGC and MGC except for LGC. These results suggested that HC and MGC may undergo apoptosis prior to a complete assembly of virion, while SGC is more susceptible showing higher amplification and releasing of virion. In contrast, WSSV may inhibit apoptosis in infected LGC to stay in latency. This present finding provides an insight for the responsive roles of crustacean hemocyte cells involved in molecular interaction and defense mechanism against WSSV.


Assuntos
Proteínas de Artrópodes/genética , Braquiúros/genética , Hemócitos/imunologia , Proteoma/imunologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Braquiúros/imunologia , Cromatografia Líquida , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
12.
Fish Shellfish Immunol ; 89: 448-457, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974220

RESUMO

Mannose-binding lectin (MBL) is a pattern recognition receptor (PRR) that plays an important role in the innate immune response. In this study, a novel mannose-binding lectin was cloned from the swimmimg crab Portunus trituberculatus (designated as PtMBL). The complete cDNA of PtMBL gene was 1208 bp in length with an open reading frame (ORF) of 732 bp that encoded 244 amino acid proteins. PtMBL shared lower amino acid similarity with other MBLs, yet it contained the conserved carbohydrate-recognition domain (CRD) with QPD motif and was clearly member of the collectin family. PtMBL transcripts were mainly detected in eyestalk and gill with sexually dimorphic expression. The temporal expression of PtMBL in hemocytes showed different activation times after challenged with Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris. The recombinant PtMBL protein revealed antimicrobial activity against the tested Gram-negative and Gram-positive bacteria. It could also bind and agglutinate (Ca2+-dependent) both bacteria and yeast. Furthermore, the agglutinating activity could be inhibited by both d-galactose and d-mannose, suggesting the broader pathogen-associated molecular patterns (PAMPs) recognition spectrum of PtMBL. These results together indicate that PtMBL could serve as not only a PRR in immune recognition but also a potential antibacterial protein in the innate immune response of crab.


Assuntos
Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Feminino , Perfilação da Expressão Gênica , Masculino , Lectina de Ligação a Manose/química , Micrococcus luteus/fisiologia , Filogenia , Pichia/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Vibrio alginolyticus/fisiologia
13.
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
14.
Fish Shellfish Immunol ; 89: 574-585, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30995541

RESUMO

Alpha-2 macroglobulin (A2M) is a ubiquitous protease inhibitor involved in the innate host defense system. Herein, two distinct A2M genes (designated as PtA2M-1 and PtA2M-2, respectively) were isolated from the swimming crab Portunus trituberculatus. PtA2M-1 and PtA2M-2 encoded proteins with 1541 or 1516 amino acids, respectively, containing the typically functional domains of A2M. Unlike highly expressed in hemocytes of most arthropods, PtA2M-1 and PtA2M-2 were predominantly detected in gill, eyestalk and digestive tracks. During the embryonic stages, PtA2Ms were found to be expressed most highly in fertilized eggs, suggesting their maternal origin. After challenged with Vibrio alginolyticus, the transcripts of PtA2Ms showed similar time-dependent response expression pattern, while PtA2M-1 was more sensitive to Micrococcus luteus and Pichia pastoris infection than PtA2M-2. Knockdown of PtA2M-1 or PtA2M-2 could significantly enhance the expression of prophenoloxidase (proPO) associated genes (PtproPO and PtPPAF) and serine protease related genes (PtcSP1-3 and PtSPH), however, PtLSZ and the phagocytosis-related genes (PtMyosin and PtRab5) were effectively inhibited. These results were further supported by the PO and lysozyme activities in hemolymph of the PtA2M-1- or PtA2M-2-silenced crabs. In addition, PtA2M-1 and PtA2M-2 could regulate the expression of antimicrobial peptide (AMP) genes (PtALF1-3, PtCrustin1 and PtCrustin3) through the Toll and NF-κB pathways. Our findings together suggest that PtA2Ms might function in crab host defense via regulating the proPO system, phagocytosis and the expression of AMP genes.


Assuntos
Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , alfa 2-Macroglobulinas Associadas à Gravidez/genética , alfa 2-Macroglobulinas Associadas à Gravidez/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Braquiúros/enzimologia , Catecol Oxidase/metabolismo , Precursores Enzimáticos/metabolismo , Perfilação da Expressão Gênica , Fagocitose/genética , Filogenia , alfa 2-Macroglobulinas Associadas à Gravidez/química , Alinhamento de Sequência
15.
Fish Shellfish Immunol ; 89: 632-640, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30995542

RESUMO

Iron homeostasis is vital to organismal health; it is maintained by the iron regulatory protein (IRP)-iron-responsive element (IRE) signaling pathway. In the Chinese mitten crab Eriocheir sinensis, EsFer-1 and EsFer-2 reportedly have a putative IRE, but an IRP has not yet been identified. In this study, we successfully amplified the full-length cDNA of EsIRP using gene cloning and rapid amplification of cDNA ends techniques. The length of this cDNA was 4474 bp, and it included a 2682-bp open reading frame encoding 893 amino acids. Using quantitative real-time PCR, mRNA transcripts of EsIRP were detected in various tissues. The highest and lowest expression level was detected in the muscle and gills, respectively. In response to Staphylococcus aureus and Vibrio parahaemolyticus challenge, the transcription level of EsIRP was downregulated and that of EsFer-1 and EsFer-2 was upregulated in hemocytes. EsIRP knockdown resulted in increased expression of both EsFer-1 and EsFer-2. After EsFer-1 and EsFer-2 knockdown, the bacterial clearance ability of E. sinensis against S. aureus and V. parahaemolyticus was impaired. In conclusion, our results suggest that the IRP-IRE signaling pathway plays an important role in the innate immune system response in E. sinensis.


Assuntos
Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteínas Reguladoras do Ferro/genética , Proteínas Reguladoras do Ferro/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 Reguladoras do Ferro/química , Masculino , Filogenia , Alinhamento de Sequência , Staphylococcus aureus/fisiologia , Vibrio parahaemolyticus/fisiologia
16.
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
17.
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
18.
J Agric Food Chem ; 67(17): 4958-4966, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30966750

RESUMO

The mud crab ( Scylla paramamosain) is widely consumed but can cause a severe food allergic reaction. To reduce allergenicity to arginine kinase (AK), site-directed mutagenesis was used to destroy disulfide bonds or mutate critical amino acids of conformational epitopes. Three hypoallergenic mutant AKs (mAK1, mAK2, and mAK3) were generated, with the immunoreactivity decreasing by 54.2, 40.1, and 71.4%, respectively. In comparison to recombinant AK (rAK), the structure of mAKs was clearly changed. Additionally, antisense peptides were designed on the basis of linear epitopes and pepsin-cutting sites of AK. Five peptide aptamers were screened by molecular docking and then analyzed by the immunoglobulin E inhibition enzyme-linked immunosorbent assay and human Laboratory of Allergic Diseases 2 mast cell degranulation assay. The peptide aptamers could significantly inhibit allergenicity of rAK and mAKs, and the inhibitory effect of peptide aptamer 3 was slightly better than the others. These results provide synergistic methods to reduce allergenicity to AK, which could be applied to other shellfish allergens.


Assuntos
Aptâmeros de Peptídeos/genética , Arginina Quinase/genética , Arginina Quinase/imunologia , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Braquiúros/imunologia , Hipersensibilidade a Frutos do Mar/imunologia , Adolescente , Adulto , Alérgenos/química , Alérgenos/genética , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Aptâmeros de Peptídeos/imunologia , Arginina Quinase/química , Proteínas de Artrópodes/química , Braquiúros/enzimologia , Braquiúros/genética , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina E/imunologia , Masculino , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Adulto Jovem
19.
Fish Shellfish Immunol ; 89: 108-116, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30928665

RESUMO

To identify molecules involved in Macrobrachium rosenbergii nodavirus (MrNV) entry into hemocytes of the giant freshwater prawn M. rosenbergii, biotinylated prawn hemocyte membrane proteins were prepared, purified and separated by SDS-PAGE. The proteins were blotted on the nitrocellulose membrane before incubation with the MrNV capsid protein (MrNV-CP) by a VOPBA technique. Subsequent mass spectrometry and analysis of immune-reactive bands represent putative binding partners including transglutaminase (TG), actin, α2-macroglobulin, α1-tubulin, F1-ATP synthase ß-subunit and a currently uncharacterized protein. The sequence of TG has been characterized and found 5 amino acids differences to a previously reported MrTG (ADX99580), mainly at its N-terminal part and thus, we named it MrTGII (KM008611). Recombinant MrTGII was prepared to produce a polyclonal antibody against it, which was successfully revealed the presence of MrTGII (100 kDa) in prawn hemocyte lysates. Using the pentylamine-biotin incorporation assay, an acyl transfer reaction was observed when hemocyte lysates were added to solutions containing MrNV-CP, suggesting that hemocyte MrTG could use MrNV-CP as the substrate. The expression levels of MrTGII were changed during the course of MrNV infection. By using immunostaining technique, location of MrTGII on the hemocyte surface was confirmed. Specific interaction between MrTGII with MrNV-CP in a dose-dependent manner was confirmed by in vitro ELISA assay. The highest binding activity of MrNV-CP was found with the N-terminal portion of the protein. In vitro neutralization using anti-MrTGII antibody resulted in inhibition of MrNV attachment to the hemocyte surface, accompanied by a dramatic reduction in viral replication. This is the first time that crustacean TG has been shown to be involved in viral entry, in addition to its roles in blood clotting and haematopoiesis.


Assuntos
Hemócitos/enzimologia , Nodaviridae/fisiologia , Palaemonidae/imunologia , Transglutaminases/genética , Replicação Viral , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Hemócitos/virologia , Microscopia de Fluorescência , Transglutaminases/química , Transglutaminases/metabolismo
20.
Parasit Vectors ; 12(1): 169, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987665

RESUMO

BACKGROUND: Ticks, as blood-feeding arthropod vectors, have evolved their own unique mechanism to suppress host immune responses and evade immune defenses in order to complete blood-feeding. The immunoregulatory effect of tick bioactive molecules on hosts has been widely reported, and the cystatin family has been identified as one of the major immunomodulators. In previous studies, we obtained a novel tick salivary bioactive protein named RHcyst-1, which belongs to the type 1 cystatin family. Here, we demonstrated the effects of RHcyst-1 on the host immune response mainly on dendritic cell (DC) function. Understanding the function of tick-derived bioactive molecule may help to clarify the mechanisms of how ticks escape the host immune response and help to control ticks and tick-borne disease transmission. METHODS: Bone marrow-derived DCs (BMDCs) were generated and induced by GM-CSF and IL-4 with or without RHcyst-1 addition. Flow cytometry was used to analyze the differentiation and maturation of BMDCs and T cell cytokine production. Quantitative real-time PCR (qRT-PCR) and western blot were used to measure changes in expression within STAT and p38 MAPK signaling pathways. RESULTS: Flow cytometry analysis revealed that RHcyst-1 inhibited the differentiation of BMDCs, but had no effect on the maturation of BMDCs. T cells co-cultured with DCs treated with RHcyst-1 produced significantly less TNF-α, IFN-γ and IL-2 than the control group. Further analysis showed that the mRNA level and phosphorylation of p38, ERK and STAT were significantly changed after RHcyst-1 added to bone marrow monocytes during the differentiation stage. CONCLUSIONS: Our results suggest that RHcyst-1 is one of the major immunosuppressive proteins of BMDC function from blood-feeding ticks.


Assuntos
Proteínas de Artrópodes/imunologia , Células Dendríticas/imunologia , Imunossupressores/imunologia , Carrapatos/imunologia , Animais , Proteínas de Artrópodes/genética , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Transcriptoma
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