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1.
Fish Shellfish Immunol ; 96: 122-125, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31805411

RESUMO

This study aims to investigate the effects of replacing different proportions of fishmeal with Antarctic krill (AK) on the growth performance, body composition and nonspecific immunity index of red swamp crayfish Procambarus clarkia. AK was used to replace 0 (control), 25%, 50% and 100% of the fishmeal in the basic diet of crayfish to formulate four test feeds with basically equivalent nitrogen and lipid contents; these feeds were denoted AK0, AK25, AK50 and AK100, respectively. Compared with the control group, crayfish fed diets with AK replacement showed increased body weight gain; feed efficiency; survival rate; body protein content; phenoloxidase, superoxide dismutase and glutathione peroxidase activities; total haemocyte counts; number of hyaline, semigranular and granular cells; and disease resistance against Aeromonas hydrophila. Conversely, the body lipid level of these crayfish decreased relative to that of the control. However, a high AK level (AK100) does not show improvements in efficiency compared with a moderate AK level (AK50). Based on the efficiency of AK in enhancing the growth performance and nonspecific immunity of crayfish, the optimum replacement proportion of fishmeal with AK was 50%. These results confirm that AK can promote the growth of crayfish and improve their disease resistance.


Assuntos
Astacoidea/crescimento & desenvolvimento , Astacoidea/imunologia , Euphausiacea/química , Imunidade Inata , Aeromonas hydrophila/fisiologia , Ração Animal/análise , Animais , Astacoidea/efeitos dos fármacos , Astacoidea/microbiologia , Dieta , Relação Dose-Resposta a Droga , Imunidade Inata/efeitos dos fármacos , Distribuição Aleatória
2.
Fish Shellfish Immunol ; 96: 32-40, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31786343

RESUMO

The red-swamp crayfish (Procambarus clarkii) is the most important economic shrimp species in China, and is an important model crustacean organism in many fields of research. In crustaceans, gills interface directly with the ambient environment and thus play a vital role in the toxicology. In the context of increasing environmental heavy metal pollution, the relationship between copper (Cu2+) stress and the immune response of P. clarkii has recently received considerable attention. However, impact of Cu2+ on the crayfish immune system is still not fully understood. In this study, we used Illumina sequencing technology to perform a transcriptome analysis of the gills of P. clarkii after 24 h of Cu2+ treatment. A total of 37,226,812 unigenes were assembled, and 1943 unigenes were significantly differentially expressed between the control and Cu2+ treatment groups. Functional categorization of differentially expressed genes (DEGs) revealed that genes related to antioxidant activity, detoxication, metabolic processes, biosynthetic processes, and immune system processes were differentially regulated during Cu2+ stress. In addition, DEGs in the immune system were classified as being related to the MAPK signaling pathway, purine metabolism, Toll and Imd signaling pathway, PI3K-Akt signaling pathway and Hippo signaling pathway. Five genes (CuZnSOD, CAT, IDH1, PHYH and DECR2) were significantly up-regulated in the peroxisome pathway, which plays an important role in reacting to oxidative stress. Importantly, qRT-PCR validation of the results for seven genes chosen at random (NDK, ATP6L, ATP5C1, RPS14, RPL22e, CTSF and HSP90A) confirmed the Illumina sequencing results. This study provides a valuable starting point for further studies to elucidate the molecular basis of the immune system's response to Cu2+ stress in crayfish.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Cobre/efeitos adversos , Imunidade Inata/genética , Transcriptoma/imunologia , Poluentes Químicos da Água/efeitos adversos , Animais , Astacoidea/efeitos dos fármacos , Perfilação da Expressão Gênica , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Distribuição Aleatória , Estresse Fisiológico , Transcriptoma/efeitos dos fármacos
3.
J Immunol ; 204(3): 487-497, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31852752

RESUMO

Rapid synthesis and release of active antimicrobial peptides (AMPs) is an important strategy in innate immune. Processing of the precursor into the active form is a common posttranslational modification of AMPs in mammals. However, in invertebrates, the mechanism of AMP maturation is largely unknown. In the current study, to our knowledge, a novel potential AMP, designated as PcnAMP, was identified because of its significant induction by bacterial infection in the red swamp crayfish (Procambarus clarkii). PcnAMP was cleaved into a short fragment postinfection. Using the purified native peptide, this cleavage was found to be mediated by trypsin after synthesis. Proteolysis produced an N-terminal peptide that exerted the antibacterial function. Although the N-terminal peptide did not show significant similarity to any other sequences, it was predicted to have an overall helical structure and high amphipathicity, both of which are typical features of many AMPs. The N-terminal active peptide exhibited a wide spectrum of antimicrobial activity. Atomic force microscope imaging and flow cytometry analysis showed that treatment with the active form of PcnAMP led to the collapse of the bacterial cell wall and permeabilization of the bacterial cell membrane. Thus, this study provided a new candidate for therapeutic agent development, and revealed new insights into the maturation of AMPs in invertebrates.


Assuntos
Aeromonas hydrophila/fisiologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/metabolismo , Astacoidea/imunologia , Parede Celular/metabolismo , Infecções por Bactérias Gram-Negativas/imunologia , Animais , Permeabilidade da Membrana Celular , Células Cultivadas , Citometria de Fluxo , Perfilação da Expressão Gênica , Imunidade Inata , Microscopia de Força Atômica , Filogenia , Proteólise
4.
Fish Shellfish Immunol ; 95: 624-634, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31698072

RESUMO

Two lipopolysaccharides (LPS) and ß-1, 3-glucan binding protein (LGBP), designated as PcLGBP isoform1 and PcLGBP isoform2, respectively, were identified from Procambarus clarkii in this study. The full-length cDNA of PcLGBP isoform1 was 1308 bp containing an open reading frame (ORF) of 1113 bp encoding a protein of 370 amino acids. The full-length cDNA of PcLGBP isoform2 was 1440 bp containing an ORF of 1245 bp encoding a protein of 414 amino acids. Predicted PcLGBP isoform1 and PcLGBP isoform 2 proteins contained a signal peptide, a glycoside hydrolase domain, and a low-complexity region. The difference between the two LGBP isoforms was that PcLGBP isoform2 had 44 more amino acids behind the signal peptide than the PcLGBP isoform1. The PcLGBP isoform1 and PcLGBP isoform2 transcripts mainly expressed in the hepatopancreas in female and male crayfish. Moreover, the expression levels of the two genes in the hepatopancreas were higher in male than that in female crayfish. Upon being challenged with Vibrio parahaemolyticus or LPS, the expression levels of PcLGBP isoform1 and PcLGBP isoform2 in the hepatopancreas of female and male crayfish were most significantly up-regulated at different time points. The transcripts of anti-lipopolysaccharide factors (ALF5, ALF6, ALF8, and ALF9) and crustins (CRU1, CRU2, CRU3, and CRU4) were evidently down-regulated in the hepatopancreas of V. parahaemolyticus-challenged total PcLGBP (including PcLGBP isoform1 and PcLGBP isoform2)-silenced male crayfish. In addition, the phenoloxidase (PO) activity in the hepatopancreas of male crayfish was evidently higher than that of female crayfish. PcLGBP knock down could significantly decrease the PO activity in the hepatopancreas lysate (HLS) in male crayfish. The PO activity of male crayfish HLS was significantly increased when incubated with a mixture of recombinant LGBP protein and LPS or ß-1, 3 glucan. We conclude that LGBP isoforms from P. clarkii function as a pattern recognition protein for recognizing and binding LPS and ß-1, 3 glucan, and thus regulate the synthesis of antimicrobial peptides and activate the prophenoloxidase system.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Artrópodes/genética , Astacoidea/genética , Astacoidea/imunologia , Proteínas de Transporte/genética , Hepatopâncreas/imunologia , Lectinas/genética , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Proteínas de Transporte/imunologia , Feminino , Imunidade Inata , Lectinas/imunologia , Lipopolissacarídeos , Masculino , Isoformas de Proteínas , Vibrio parahaemolyticus/imunologia
5.
Fish Shellfish Immunol ; 95: 140-150, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31629063

RESUMO

To learn more about red swamp crayfish related genes in response to bacterial infections, we investigated immune-related genes induced by lipopolysaccharide (LPS) in the hepatopancreas using high-throughput sequencing method. In present the study, a total of 55,107 unigenes were identified, with an average length of 678 bp. A total of 2215 differentially expressed genes (DEGs) were found, including 669 up-regulated genes and 1546 down-regulated genes. The result of Gene ontology (GO) analysis revealed that 3017 DEGs were enriched in 19 biological process subcategories, 17 cellular component subcategories and 15 molecular function subcategories. The top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that "ribosome" was the most abundant group, which had 34 DEGs. KEGG enrichment analysis identified several immune response pathways. Real-time quantitative reverse transcription-PCR (qRT-PCR) results exhibited that several immune responsive genes were greatly up-regulated following LPS stimulation as observed in the results of high-throughput sequencing. Overall, this study provides new insight into the immune defense mechanisms of P. clarkii against LPS infection.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Lipopolissacarídeos/administração & dosagem , Transcriptoma , Animais , Astacoidea/efeitos dos fármacos , Perfilação da Expressão Gênica , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala
6.
Fish Shellfish Immunol ; 94: 934-943, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31600596

RESUMO

Really Interesting New Gene (RING) finger proteins are highly conserved molecules that participate in a variety of biological processes such as regulation of development, apoptosis and antiviral immunity in vertebrates. However, the functions of RING finger proteins are still poorly understood in crustaceans. Previously, we found that the transcript of a homolog of RING finger protein 152 (CqRNF152-like) was up-regulated in a differentially expressed transcriptome library of the haematopietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus upon white spot syndrome virus (WSSV) infection, which is one of the most devastating viral diseases for crustaceans like shrimp and crayfish. The full-length cDNA sequence of CqRNF152-like was then identified with 975 bp, including an ORF of 685 bp that encoded a 195 amino acids protein, a 5'- UTR of 180 bp, and a 3'-UTR with a poly (A) tail of 207 bp. The conserved domain prediction showed that CqRNF152-like contained a conserved RING-finger domain. Gene expression analysis showed that CqRNF152-like was distributed in all tissues examined and the transcript is significantly up-regulated after WSSV challenge both in vivo in Hpt tissue and in vitro in cultured Hpt cells. Furthermore, the transcripts of both an immediate early gene ie1 and a late envelope protein gene vp28 of WSSV were clearly increased in the Hpt tissues, hemocytes and cultured Hpt cells after gene silencing of CqRNF152-like, which were further proved to be significantly decreased after overloading of recombinant CqRNF152-like protein in Hpt cell cultures. Meanwhile, CqRNF152-like was found to bind with WSSV envelope protein VP28 by proteins pull-down assay. Similar to most of RNF proteins, CqRNF152-like protein sequence contained a conserved RING-finger domain and showed self-ubiquitination activity in a RING finger domain dependent manner. Taken together, CqRNF152-like is likely to function as an antiviral molecular against WSSV infection through interaction with the envelope protein VP28 in a crustacean C. quadricarinatus. This is the first report that a RING finger protein with directly antiviral functions via interaction with viral protein and self-ubiquitination activity in crustacean, which sheds new light on the molecular mechanism of WSSV infection and the control of white spot disease.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência , Ubiquitina-Proteína Ligases/química
7.
Fish Shellfish Immunol ; 94: 792-799, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31585244

RESUMO

The caspase is an essential module in the Drosophila immune deficiency (IMD) pathway, which plays a crucial role in countering pathogen infection. In this study, a gene named PcCaspase-3C was found in Procambarus clarkia with a full-length of 4684 bp, including a 1572 bp opening reading frame, which encoded a putative protein of 523 amino acids. PcCaspase-3C contained a CASc domain constituted of 237 amino acids. The PcCaspase-3C gene was primarily expressed in heart, stomach, and intestine, while less in gonad, hepatopancreas, gills, and hemocytes, with the least expression in muscle. Infection with Staphyloccocus aureus, Vibrio parahaemolyticus or white spot syndrome virus (WSSV) induced an up-regulated expression of PcCaspase-3C in intestine or stomach to varying degrees. When PcCaspase-3C was silenced by double-stranded RNA, the expression of some antimicrobial peptides such as ALF2, ALF5, ALF6, Cru3, Cru4, and Lys was significantly inhibited. In addition, silencing of PcCaspase-3C accelerated infection with WSSV in vivo. According to these results, we suggest that PcCaspase-3C might play a crucial role in the immune response of P. clarkia against pathogenic bacterial and viral infections.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Caspase 3/genética , Caspase 3/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Caspase 3/química , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência
8.
Fish Shellfish Immunol ; 94: 861-870, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31585246

RESUMO

The main advantage of antimicrobial peptides (AMPs) used as the effectors in the innate immunity system of invertebrates is that the high specificity is not indispensable. And they play important roles in the systemic defenses against microbial invasion. In this study, a new full-length cDNA of the crustins molecule was identified in red swamp crayfish, P. clarkii (named Pc-crustin 4). The ORF of Pc-crustin 4 contained 369 bp which encoded a protein of 122 amino acids, with a 20-amino-acid signal peptide sequence. On the base of the classification method established by Smith et al., Pc-crustin 4 belonged to Type Ⅰ crustin molecule. The Pc-crustin 4 transcripts were expressed in hemocytes at relatively high level, and relatively low level in hepatopancreas, gills, and intestine in normal crayfish. After respectively challenged with S. aureus or E. ictaluri, the expression levels of Pc-crustin 4 showed up-regulation trends at different degrees in the hemocytes, hepatopancreas, gills, and intestine tissues. Besides, the results of liquid antibacterial assay showed that rPc-crustin 4 inhibited obviously the growth of S. aureus and E. ictaluri. The results of bacteria binding assay showed that rPc-crustin 4 could bind strongly to S. aureus and E. ictaluri. Finally, RNAi assay was performed to study the immunity roles of Pc-crustin 4 in crayfish in vivo. Taken together, Pc-crustin 4 is an important immunity effector molecule, which plays crucial roles in defending against bacterial infection in crayfish.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Astacoidea/genética , Astacoidea/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Edwardsiella ictaluri/fisiologia , Perfilação da Expressão Gênica , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Alinhamento de Sequência , Staphylococcus aureus/fisiologia
9.
Fish Shellfish Immunol ; 94: 592-598, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31518688

RESUMO

Whey acidic protein domain (WAPD) is a usual motif in crustaceans, and is found mainly in the immune-related proteins. In the present study, a protein containing three tandem WAPDs was identified in red swamp crayfish Procambarus clarkii and designated as PcTWD. This is the first report of a protein of such domain architecture in crustaceans. Introducing the WAPDs of PcTWD into phylogenetic analysis led to the classification of crustacean WAP proteins into classical crustins and proteins containing solely WAPDs. PcTWD was widely expressed in multiple tissues, including hemocytes, gills, hepatopancreas, heart, stomach and intestine. Its expression could be significantly induced by Staphylococcus aureus or Aeromonas hydrophila challenge. Knockdown PcTWD expression by RNAi suppressed host resistance against A. hydrophila, while exogenous recombinant PcTWD could enhance the host immunity. The three WAPDs showed a labor division. The first two domains were responsible for the protease inhibitory activity, and the third domain contributed to the antimicrobial activity. Thus PcTWD was found as an important protein in crayfish antibacterial immunity.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Evolução Molecular , Imunidade Inata/genética , Proteínas do Leite/genética , Proteínas do Leite/imunologia , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Proteínas do Leite/química , Filogenia , Domínios Proteicos/genética , Alinhamento de Sequência , Staphylococcus aureus/fisiologia
10.
Fish Shellfish Immunol ; 93: 796-800, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31422177

RESUMO

This study aims to investigate the effects of Rhodiola rosea polysaccharide (RRP) on the growth performance and nonspecific immunity of red swamp crayfish Procambarus clarkia. RRP was prepared by hot water extraction and partly characterised by high-performance liquid chromatography and sugar composition analyses. Three diets supplemented with three different levels of RRP (0.2, 0.6 and 1 g kg diet-1) were formulated and tested for growth performance and nonspecific immunity of red swamp crayfish Procambarus clarkii, while a diet without any RRP supplementation served as control. After 8 weeks of feeding, body weight gain, feed efficiency, survival rate, phenoloxidase activity, superoxide dismutase activity, glutathione peroxidase level, total haemocyte count and number of hyaline cells, semigranular cells and granular cells and resistance to Aeromonas hydrophila were higher than those of the control. Moreover, based on the efficiency of RRP on the growth performance and nonspecific immunity of crayfish, the optimum dose of RRP was found to be 0.6 g kg diet-1. Hence, intake of diets containing RRP could enhance the growth performance, immune responses and improve resistance of crayfish to infection by A. hydrophila.


Assuntos
Astacoidea/imunologia , Carboidratos da Dieta/farmacologia , Imunidade Inata/efeitos dos fármacos , Polissacarídeos/farmacologia , Rhodiola/química , Ração Animal/análise , Animais , Astacoidea/crescimento & desenvolvimento , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Distribuição Aleatória
11.
PLoS One ; 14(8): e0219223, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31393870

RESUMO

Crayfish plague, caused by the pathogen Aphanomyces astaci, is one of the main factors responsible for the decimation of the native European crayfish species Austropotamobius pallipes. In Spain, two North American freshwater crayfish species, Procambarus clarkii and Pacifastacus leniusculus, were intentionally introduced during the 1970s for aquaculture and fishery purposes. Since then, incidences of crayfish plague have been continually reported. In this work, we evaluated more than 50 diagnosed cases of crayfish plague that have occurred in the Iberian Peninsula since 2004 by performing a microscopic examination of infected specimens and by molecularly identifying and haplotyping the pathogen. Our results showed that (i) the pathogen A. astaci has been active 45 years since the first introductions of the invasive North American crayfish species in the Iberian Peninsula, and (ii) P. clarkii and P. leniusculus are chronic reservoirs of the crayfish plague pathogen. Moreover, our data confirmed a correspondence between pathogen origin and spread and the specific haplotypes carried by the North American invasive crayfish located in the vicinity of each outbreak. We generated a crayfish plague incidence map of the Iberian Peninsula that shows (i) a northern area, mainly inhabited by alien P. leniusculus, where crayfish plague cases are associated with the b-haplotype specific to P. leniusculus, and (ii) southern, central and eastern areas that are basically inhabited by alien P. clarkii, where crayfish plague cases are associated with the d1- and d2-haplotypes specific to P. clarkii. The results presented here are evidence of the long standing and negative impact of the two invasive crayfish species on the native species, indicating the need for more extensive control measures.


Assuntos
Aphanomyces/patogenicidade , Astacoidea/imunologia , Astacoidea/microbiologia , Animais , Aphanomyces/metabolismo , Surtos de Doenças , Água Doce , Haplótipos/imunologia , Espécies Introduzidas/economia , Peste/epidemiologia , Peste/veterinária , Portugal , Espanha
12.
Fish Shellfish Immunol ; 94: 10-16, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31465869

RESUMO

In crustaceans, it has been suggested that specific protection against pathogens could be triggered by vaccines and biological response modifiers; although the specific mechanisms of this protection have not been clarified yet. In the crayfish Cherax quadricarinatus, a humoral lectin (CqL) binds its own granular hemocytes through a specific receptor (CqLR) and increases the production of reactive oxygen species (ROS). In the present study, we challenged in vivo crayfishes with immunostimulants, ß-glucan (200 µg/kg) or LPS (20 µg/kg), and identified the participation of cellular and humoral mechanisms. The stimulants generated a complex modification in the total hemocytes count (THC), as well as in the proportion of hemocyte subsets. At 2 h after the challenge, the largest value in THC was observed in either challenged crayfishes. Furthermore, at the same time, hyaline hemocytes were the most abundant subset in the hemolymph; after 6 h, granular hemocytes (GH) were the most abundant hemocyte subset. It has been observed that a specific subset of GH possesses a CqLR that has been related to ROS production. After 2 and 6 h of the ß-glucan challenge, a significant increase in CqLR expression was observed in the three circulating hemocyte subsets; also, an increased expression of CqL was detected in a granular hemocytes sub-population. After 2 and 6 h of stimulation, the specific activity of the serum lectin challenged with ß-glucan was 250% and 160% higher than in the LPS-treated-group, respectively (P < 0.05). Hemocytes from challenged crayfishes were stimulated ex vivo with CqL, ROS production was 180% higher in hemocytes treated with ß-glucan + CqL than in hemocytes treated with LPS + CqL (P < 0.05). The results evidence the effectivity of immune stimulators to activate specific crayfish defense mechanisms, the participation of CqL and its receptor (CqLR) could play an important role in the regulation of immune cellular functions, like ROS production, in Cherax quadricarinatus.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Expressão Gênica/efeitos dos fármacos , Imunidade Celular/genética , Imunidade Humoral/genética , Lectinas/genética , Receptores Mitogênicos/genética , Adjuvantes Imunológicos/farmacologia , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Astacoidea/efeitos dos fármacos , Hemócitos , Hemolinfa/metabolismo , Lectinas/metabolismo , Lipopolissacarídeos/farmacologia , Receptores Mitogênicos/metabolismo , beta-Glucanas/farmacologia
13.
Fish Shellfish Immunol ; 94: 66-71, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31465872

RESUMO

Astakine 1 is a small cytokine-like peptide which is directly involved in hematopoiesis in crustaceans. Astakines are present in many different invertebrate groups primarily in arthropods. In this study we found that astakine1 was present as a high molecular weight (HMW) complex in plasma. It is known that calcium concentration are fluctuating in several crustaceans especially during the molting process. This HMW-complex was formed under low calcium concentrations in plasma and could be partially reversed provided calcium was added. The biological function of the naïve astakine1 and that in the HMW complex was about the same, but if the protein is to be isolated or studied for its function it is important to know about this property of astakine1 which may previously have hampered isolation and functional studies in other animals than freshwater crayfish.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/genética , Astacoidea/imunologia , Cálcio/metabolismo , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/genética , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/imunologia , Animais , Proteínas de Artrópodes/imunologia , Plasma/química
14.
Aquat Toxicol ; 214: 105262, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31351400

RESUMO

To understand the toxic effects of nitrite exposure on crayfish, expression of genes involved in the immune system, the antioxidant defense, and the heat shock protein 70 (HSP70) was measured after 12, 24, and 48 h of different nitrite concentrations exposure in the hepatopancreas and hemocytes of Procambarus clarkii. Nitrite exposure up-regulated mRNA levels of cytoplasmic Mn superoxide dismutase (cMn-SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase (GST), after 24 h nitrite exposure. At 48 h, nitrite exposure decreased the mRNA levels of mitochondrial MnSOD (mMn-SOD), CAT, and GPx. High concentrations of nitrite at 48 h of exposure decreased expression of ß-1,3-glucan-bingding protein in the hepatopancreas, and lysozyme expression in hemocytes. Nitrite exposure caused little effect on the heat shock protein 70 (HSP70) in hemocytes. Through overall clustering analysis, we found that 24 h of nitrite exposure caused stronger transcriptional responses. Our study indicated that the response of P. clarkii to acute nitrite exposure was exposure time-dependent. These results will help to understand the dynamic response pattern of crustaceans to nitrite pollution, and improve our understanding of the toxicological mechanisms of nitrite in crustaceans.


Assuntos
Antioxidantes/metabolismo , Astacoidea/genética , Astacoidea/imunologia , Exposição Ambiental , Nitritos/toxicidade , Transcrição Genética , Animais , Astacoidea/efeitos dos fármacos , Catalase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Hemócitos/efeitos dos fármacos , Hemócitos/enzimologia , Hepatopâncreas/efeitos dos fármacos , Hepatopâncreas/enzimologia , Superóxido Dismutase/metabolismo , Fatores de Tempo , Transcrição Genética/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade
15.
Fish Shellfish Immunol ; 93: 116-123, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31302287

RESUMO

Hesperetin is a natural flavanone compound, which mainly exists in lemons and oranges, and has potential antiviral and anticancer activities. In this study, hesperetin was used in a crayfish pathogen challenge to discover its effects on the innate immune system of invertebrates. The crayfish Procambarus clarkii was used as an experimental model and challenged with white spot syndrome virus (WSSV). Pathogen challenge experiments showed that hesperetin treatment significantly reduced the mortality caused by WSSV infection, while the VP28 copies of WSSV were also reduced. Quantitative reverse transcriptase polymerase chain reaction revealed that hesperetin increased the expression of several innate immune-related genes, including NF-kappaB and C-type lectin. Further analysis showed that hesperetin treatment plays a positive effects on three immune parameters like total hemocyte count, phenoloxidase and superoxide dismutase activity. Nevertheless, whether or not infected with WSSV, hesperetin treatment would significantly increase the hemocyte apoptosis rates in crayfish. These results indicated that hesperetin could regulate the innate immunity of crayfish, and delaying and reducing the mortality after WSSV challenge. Therefore, the present study provided novel insights into the potential therapeutic or preventive functions associated with hesperetin to regulate crayfish immunity and protect crayfish against WSSV infection, provide certain theoretical basis for production practice.


Assuntos
Astacoidea/efeitos dos fármacos , Hesperidina/metabolismo , Imunidade Inata/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Ração Animal/análise , Animais , Astacoidea/imunologia , Astacoidea/virologia , Dieta , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Hesperidina/administração & dosagem , Longevidade/efeitos dos fármacos , Vírus da Síndrome da Mancha Branca 1/efeitos dos fármacos , Vírus da Síndrome da Mancha Branca 1/fisiologia
16.
Dev Comp Immunol ; 99: 103405, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31145913

RESUMO

The Gamma interferon inducible lysosomal thiol reductase (GILT) plays a key biological role in the immune responses and involves in the processing of class II MHC-restricted antigen by stimulating disulfide bond reduction in mammals. To determine the biological function of GILT in the innate immune system of crustaceans, we sequenced and cloned GILT gene from red swamp crayfish, Procambarus clarkii (Pc-GILT). The deduced amino acid sequence of Pc-GILT contained the putative conserved structures of the GILT family proteins: the GILT signature (CQHGX2ECX2NX4C) sequence and the active site (CXXS) motif. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis suggested that a recombinant Pc-GILT protein was successfully expressed in Escherichia coli (E. coli). Quantitative real-time PCR analysis showed that Pc-GILT transcript level was highest in the hepatopancreas followed by the gut, heart and muscles. Additionally, we analyzed the transcription level of Pc-GILT gene in hepatopancreas of red swamp crayfish under biotic stress conditions. The expression of Pc-GILT gene upregulated after viral (poly I:C) and bacterial (peptidoglycan, lipopolysaccharide) infection. The suppression of Pc-GILT by double stranded RNA influenced the transcript levels of various immune-related genes. These observations indicate that the Pc-GILT probably plays a key biological role in the innate immune responses of red swamp crayfish, since it modulates the expression of genes associated with immune pathways.


Assuntos
Proteínas de Artrópodes/imunologia , Astacoidea/imunologia , Imunidade Inata/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/antagonistas & inibidores , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Astacoidea/classificação , Astacoidea/genética , Sequência de Bases , Expressão Gênica , Regulação da Expressão Gênica/imunologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Filogenia , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Estresse Fisiológico/genética , Estresse Fisiológico/imunologia , Distribuição Tecidual
17.
Fish Shellfish Immunol ; 92: 83-90, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31059813

RESUMO

Prophenoloxidase (proPO) is the zymogen form of phenoloxidase (PO), a key enzyme in melanization cascade that has been co-opted in invertebrate immune reactions. There have been reported that proPO plays many essential roles in the crustacean immune system. However, little is known about the function of proPO from red swamp crayfish (Procambarus clarkii) which is an important cultured species worldwide. Here, we cloned and expressed proPO gene from red swamp crayfish (PcproPO). Subsequently, specific antibody against PcproPO was generated. The immune function of PcproPO was further characterized in vitro and in vivo. The results showed that the expression of PcproPO mRNA could be significantly up-regulated during the challenge of Gram-positive-negative (Vibrio parahaemolyticus) and Gram-positive-positive bacterial (Staphylococcus aureus). Furthermore, the purified recombinant PcproPO protein had a strong affinity binding to both bacteria and polysaccharides. In vivo knockdown of PcproPO could significantly reduce the crayfish bacterial clearance ability, resulting in the higher mortality of the crayfish during V. parahaemolyticus infection. In addition, in vitro knockdown of PcproPO in the hemocytes significantly reduced the phenoloxidase (PO) activity and the bacterial clearance ability, indicating that PcproPO might involve in hemocyte-mediated melanization. Our results will shed a new light on the immune function of PcproPO in the crayfish.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Catecol Oxidase/genética , Catecol Oxidase/imunologia , Precursores Enzimáticos/genética , Precursores Enzimáticos/imunologia , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Astacoidea/microbiologia , Técnicas de Silenciamento de Genes , Lipopolissacarídeos/farmacologia , Peptidoglicano/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Staphylococcus aureus/fisiologia , Ácidos Teicoicos/farmacologia , Vibrio parahaemolyticus/fisiologia
18.
Fish Shellfish Immunol ; 89: 170-178, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30928663

RESUMO

Peroxiredoxin 6 (Prx6) is an important member of the peroxiredoxin family that plays critical roles in protecting host against the toxicity of oxidative stress and participates in cell signaling. Herein, we report Prx6 gene from red swamp crayfish, Procambarus clarkii. The cDNA fragment of PcPrx6 was 660 bp, encoding a 219 amino acid residues protein. The quantitative real time PCR analysis showed ubiquitous expression of PcPrx6 mRNA in the tested tissues. The challenge with peptidoglycan and Poly I:C remarkably suppressed the mRNA level of PcPrx6 in hepatopancreas at 3, 12, 48 h compared with the PBS control. However, the expression level significantly increased after 36 h of their treatment. The knockdown of PcPrx6 by small interference RNA significantly enhanced the transcript levels of Toll pathway-responsive genes at 24 h. Recombinant PcPrx6 protein was purified using affinity chromatography and analyzed for its biological role. The results revealed that the recombinant PcPrx6 protein manifested the ability to protect supercoiled DNA damage from oxidative stress elicited by mixed function oxidative assay. Altogether, PcPrx6 may have multiple functional roles in the physiology of P. clarkii, since it negatively regulates the Toll signaling transduction and protects supercoiled DNA damage from oxidative stress.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Peroxirredoxina VI/genética , Peroxirredoxina VI/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Cromatografia de Afinidade , Dano ao DNA , DNA Super-Helicoidal/fisiologia , Perfilação da Expressão Gênica , Estresse Oxidativo , Peptidoglicano/farmacologia , Peroxirredoxina VI/química , Filogenia , Poli I-C/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência
19.
Immunol Invest ; 48(7): 682-690, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30794007

RESUMO

Five different serine protease homologs (SPH) transcripts presumably or possibly resulting from alternative splicing were cloned from the hemocytes of crayfish (Procambarus clarkii) in this paper. Although different deletions of cDNA of SPH-2 and SPH-4 were found in the 5' untranslated regions, they shared the same open reading frame and encoded a 424 amino acids protein with a calculated molecular weight of 45.84 kDa compared with SPH-5. The predicted cutting site of the signal peptide was located between Ala22 and Glu23; a clip domain and a trypsin-like serine protease domain were located in the N-terminal and the C-terminal, respectively. Large deletions were found in the SPH-1 and SPH-3. Both of them lacked the clip domain. The 22 amino acids signal peptide existed in the SPH-1 coding protein, and a low complexity region (LCR) was formed in the N-terminal of it. The deduced protein of SPH-1 contained 358 amino acids with a molecular weight of 38.80 kDa. There was only one trypsin-like serine protease domain found in the C-terminal of the SPH-3 coding protein. The deduced protein of SPH-3 contained 250 amino acids with a molecular weight of 26.90 kDa. The amino acid Ser (S) of the catalytic triad in trypsin-like serine protease domain of the proteins analyzed in this paper was replaced by Gly (G), suggesting that the SPH-1, SPH-2, SPH-3, SPH-4, and SPH-5 were serine protease homologs.


Assuntos
Astacoidea/enzimologia , Serina Proteases/genética , Transcriptoma , Processamento Alternativo , Sequência de Aminoácidos , Animais , Astacoidea/genética , Astacoidea/imunologia , Sequência de Bases , Clonagem Molecular , Hemócitos/enzimologia , Isoenzimas , Peso Molecular , Domínios Proteicos , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Serina Proteases/química
20.
Fish Shellfish Immunol ; 87: 178-183, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30639478

RESUMO

Ras-related C3 botulinum toxin substrate 1 (Rac1) participates in many biological processes. In this study, a Rac1 gene was identified in the crayfish Procambarus clarkii with an open reading frame of 579 bp that encoded 192 amino acids. This predicted 21.4 kDa protein was highly homologous to those in other invertebrates. Real-time PCR analysis revealed that Pc-Rac1 was expressed in all examined tissues with the highest expression level in hemocytes. The transcriptional expression level of Pc-Rac1 was significantly upregulated in hemocytes and hepatopancreas after lipopolysaccharide (LPS) or polyinosinic: polycytidylic acid (poly I: C) induction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis suggested that a recombinant Pc-Rac1 protein was successfully expressed in E. coli. Far-western blot analysis demonstrated that Rac1 can interact with the PBD domain of p21-activated kinase 1 (PAK1). RNA interference of Pc-Rac1 affected the mRNA expression levels of immune-related genes lectin, Toll, crustin, TNF, ALF and cactus. These results suggest that Pc-Rac1 is involved in the innate immune responses in P. clarkii.


Assuntos
Astacoidea/imunologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/genética , Animais , Astacoidea/genética , Astacoidea/metabolismo , Escherichia coli , Expressão Gênica , Hemócitos/metabolismo , Hepatopâncreas/metabolismo , Imunidade Inata/genética , Lipopolissacarídeos/farmacologia , Proteínas Monoméricas de Ligação ao GTP/química , Poli I-C/farmacologia , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Quinases Ativadas por p21/metabolismo
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