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1.
Sci Total Environ ; 683: 681-689, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31150888

RESUMO

The antidepressant fluoxetine (FLX), a selective serotonin reuptake inhibitor, is widely prescribed for the treatment of depression and anxiety disorders. Nowadays, measurable quantities of FLX have been frequently detected in the aquatic ecosystems worldwide, which may pose a potential threat to aquatic organisms. Although the impacts of FLX exposure on immune responses are increasingly well documented in mammals, they remain poorly understood in aquatic invertebrates. Therefore, to gain a better understanding of the ecotoxicological effects of FLX, the impacts of waterborne FLX exposure on the immune responses of blood clam, Tegillarca granosa, were investigated in this study. Results obtained showed that both cellular and humoural immune responses in T. granosa were suppressed by exposure to waterborne FLX, as indicated by total counts of haemocytes (THC), phagocytic rate, and activities of superoxide dismutases (SOD) and catalase (CAT), suggesting that waterborne FLX renders blood clams more vulnerable to pathogen challenges. To ascertain the mechanisms explaining how waterborne FLX affects immune responses, haemocyte viabilities, intracellular Ca2+ levels, in vivo concentrations of neurotransmitters, physiological stress conditions (as indicated by in vivo concentrations of cortisol), and expressions of key regulatory genes from Ca2+ and neurotransmitter signal transduction, as well as immune-related signalling pathways, were examined after 10 days of FLX exposure by blood clams via 1, 10 and 100 µg/L waterborne FLX. The results obtained indicated that immune response suppression caused by waterborne FLX could be due to (i) inhibited haemocyte viabilities, which subsequently reduce the THC; (ii) altered intracellular Ca2+ and neurotransmitter concentrations, which lead to constrained phagocytosis; and (iii) aggravated physiological stress, which thereafter hampers immune-related NFκB signalling pathways.


Assuntos
Fluoxetina/toxicidade , Imunidade Humoral/efeitos dos fármacos , Inibidores de Captação de Serotonina/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Arcidae , Hemócitos/efeitos dos fármacos , Hemócitos/fisiologia , Testes de Toxicidade
2.
Fish Shellfish Immunol ; 89: 361-367, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974218

RESUMO

The circulating hemocytes of cultivated marine mussel (Mytilus galloprovincialis) were investigated using light microscopy and flow cytometry. In mussels two cell types, granulocytes and agranulocytes, were identified based on the existence of two subpopulations of cells differing by size and granularity level on light-scattered plots. Light microscopic observation confirmed the presence of cells with cytoplasmic granules and cells without granulation in hemolymph of mussels. The main type of cells in hemolymph were agranular cells amounting 78.4 ±â€¯8.9% in mussels. Flow cytometry showed that the agranular hemocytes of the mollusks produce significantly less reactive oxygen species compared to granulocytes. Mussel were exposed for 24 h of hypoxia and immune functions including hemocyte mortality, proliferation and reactive oxygen species (ROS) production were analysed using flow cytometric methods. Granulocyte number was higher at low oxygen concentration than that at normoxia; agranulocytes number decreased, in contrast. The ROS production after hypoxic treatment was decreased compared to normoxia level. No significant changes in hemocyte mortality and proliferation were observed.


Assuntos
Proliferação de Células , Hemócitos/fisiologia , Mytilus/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Anaerobiose , Animais , Centrifugação com Gradiente de Concentração , Citometria de Fluxo
3.
Dev Comp Immunol ; 90: 121-129, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30227217

RESUMO

Peptidoglycan is the key component forming the backbone of bacterial cell wall. It can be recognized by a group of pattern recognition receptors, known as peptidoglycan recognition proteins (PGRPs) in insects and higher animals. PGRPs may serve as immune receptors or N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28). Here, we report the characterization of a short PGRP, PGRP-S1, from the oriental armyworm, Mythimna separata. MsePGRP-S1 cDNA encodes a protein of 197 amino acids (aa) with a PGRP domain of about 150 aa. MsePGRP-S1 was expressed in several tissues of naïve larvae, including hemocytes, midgut, fat body and epidermis. Bacterial challenges caused variable changes in different tissues at the mRNA level. The recombinant protein bound strongly to Staphylococcus aureus and purified peptidoglycans from Staphylococcus aureus and Bacillus subtilis. It can inhibit the growth of gram-negative and gram-positive bacteria by disrupting bacterial surface. It can degrade peptidoglycans from Escherichia coli and Staphylococcus aureus. Taken together, these data demonstrate that M. separata PGRP-S1 is involved in defending against bacteria.


Assuntos
Bacillus subtilis/fisiologia , Proteínas de Transporte/genética , Hemócitos/fisiologia , Proteínas de Insetos/genética , Receptores de Reconhecimento de Padrão/genética , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Animais , Antibacterianos/metabolismo , Proteínas de Transporte/metabolismo , Clonagem Molecular , Imunidade Inata , Proteínas de Insetos/metabolismo , Lepidópteros/imunologia , Peptidoglicano/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Proteínas Recombinantes/genética
4.
Dev Comp Immunol ; 90: 138-146, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30236881

RESUMO

Heat shock protein 70 (HSP70) acts as a molecular chaperone and a stress protein, but also plays important roles in innate and adaptive immune responses. Previous studies have reported that non-lethal heat shock (NLHS) could enhance the resistance of Pacific white shrimp Litopenaeus vannamei to a specific strain of Vibrio parahaemolyticus, which carried a toxin-producing plasmid (VPAHPND), via the induction of LvHSP70 transcription. Here, we further investigated the specific function of LvHSP70 in shrimp immunity. The upregulation of LvHSP70 at the protein level was detected during recovery time after NLHS treatment, using both western blot analysis and immunofluorescence microscopy. We found that NLHS immediately activated the production of LvHSP70 in shrimp hemocytes and that such induction was observed in all three types of hemocytes: hyaline; granular and semi-granular cells. Furthermore, the role of LvHSP70 in bacterial defense was investigated using the heterologous expression of recombinant LvHSP70 (rLvHSP70) in Escherichia coli. Shrimp receiving rLvHSP70 by injection showed an increased survival rate (75%) to VPAHPND infection compared to just 20% survival in the control group injected with bovine serum albumin (BSA). We also demonstrated that the injected rLvHSP70 accumulated in shrimp hemocytes and was detected in the intracellular space of hemocyte cells leading to the induced expression (P<0.05) of several immune-related genes (LvMyD88, LvIKKß, LvIKKε, LvCrustin I, LvPEN2, LvPEN3, LvproPO1, LvproPO2 and LvTG1). Collectively, these results suggest that LvHSP70 plays a crucial role in bacterial defense by activating the shrimp immune system.


Assuntos
Proteínas de Choque Térmico HSP70/genética , Hemócitos/fisiologia , Hepatopâncreas/imunologia , Penaeidae/imunologia , Vibrioses/imunologia , Vibrio parahaemolyticus/fisiologia , Animais , Anti-Infecciosos/metabolismo , Proteínas de Artrópodes/metabolismo , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Hepatopâncreas/microbiologia , Hepatopâncreas/patologia , Imunidade Inata/genética , Necrose , Penaeidae/microbiologia
5.
Dev Comp Immunol ; 91: 1-7, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30287242

RESUMO

Pattern recognition receptors (PRRs) are essential in recognizing specific pathogen-associated molecular patterns (PAMPs) on microbes and triggering responses to eliminate the invading pathogens. Previous genomic studies have revealed a great number of PRR genes in the Pacific oyster Crassostrea gigas, a sessile and filter-feeder marine bivalve belonging to the phylum Mollusca. On the survey of PRRs in the assembly oyster reference genome version 9, a total of 1084 PRRs were identified, which were composed of at least 12 gene families. Some of the gene families were significantly expanded, including C-type lectins (CTLs), fibrinogen-related proteins (FREPs), scavenger receptor cysteine-rich repeat protein (SRCRs), leucine-rich repeat (LRR)-only proteins (LRRops), and especially C1q domain-containing proteins (C1qDCs). The transcriptomic profiles of these abundant PRRs in response to PAMP treatments were investigated by RNA-Seq using the SOLiD EZ BeadTM system. Compared to the control library, there were 6,655, 7,273, 7,593, 6,830, 6687 and 8250 differentially expressed genes in the haemocytes of oysters in response to lipopolysaccharide (LPS) stimulation for 6 h, 12 h and 24 h, and peptidoglycan (PGN), glucan (GLU) and poly I:C (IC) stimulation for 12 h, respectively. After stimulation for 12 h, there were 134, 97, 114 and 159 genes up-regulated in the LPS, PGN, GLU and IC library, respectively. Most of the gene families involved in immune response towards PAMPs were C1qDCs, CTLs and FREPs, while only a few members of LRR and immunoglobin-containing proteins (LRRIGs), retinoic acid-inducible gene I [RIG-I]-like receptors (RLRs) and Toll like receptors (TLRs) were up-regulated. After LPS stimulation, the expression level of 258 non-redundant PRR genes in oyster haemocytes increased significantly with different expression pattern, and most of them were C1qDCs, CTLs, LRRops and FREPs. The transcriptomic analyses indicated that there was a dynamic and orchestrated specific expression regulation of numerous PRR genes in response to pathogen invasion. The expanded PRR gene family members were differentiated with more specific functional responses to certain PAMPs rather than the versatile ones. Based on the different expression pattern during the LPS stimulation, the oyster PRRs could be assigned into three consecutive steps in the response against pathogen invading. All the results would provide useful information for future studies of oyster PRRs and deep insight into the researches on invertebrate innate immunity.


Assuntos
Crassostrea/imunologia , Hemócitos/fisiologia , Receptores de Reconhecimento de Padrão/genética , Transcriptoma/genética , Animais , Clonagem Molecular , Complemento C1q/genética , Fibrinogênio/genética , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Imunidade Inata , Lectinas Tipo C/genética , Lipopolissacarídeos/imunologia , Estrutura Molecular , Padrões Moleculares Associados a Patógenos/imunologia , Análise de Sequência de RNA , Receptores Toll-Like/metabolismo
6.
Dev Comp Immunol ; 91: 37-49, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30336173

RESUMO

To elucidate the proteomic responses of shrimp hemocytes to white spot syndrome virus (WSSV) infection at the proteome level, a quantitative shotgun proteomic analysis was performed to detect differentially synthesized proteins in infected hemocytes of white shrimp (Litopenaeus vannamei). We identified 1528 proteins associated to 203 gene ontology (GO) categories. The most representative GO categories were regulation of cellular processes, organic substance metabolic processes and nitrogen compound metabolic processes. Most of the 83 detected up-regulated proteins are involved in DNA regulation and organization and cell signaling. In contrast, most of the 40 down-regulated proteins were related to immune defense processes, protein folding, and development. Differentially induced proteins were further analyzed at the transcript level by RT-qPCR to validate the results. This work provides new insights into the alterations of L. vannamei hemocytes at the protein level at 12 h post-infection with WSSV. Interestingly, several of the up-regulated proteins are allergy-related proteins in humans. Based on our results, we suggest a deeper analysis of the effects of this interaction on the regulation of allergy related-proteins as their up-regulation during WSSV could represent a threat to human health.


Assuntos
Proteínas de Artrópodes/metabolismo , Infecções por Vírus de DNA/imunologia , Hemócitos/fisiologia , Hipersensibilidade/metabolismo , Penaeidae/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia , Animais , Proteínas de Artrópodes/genética , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Hipersensibilidade/genética , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/virologia , Proteoma
7.
Dev Comp Immunol ; 91: 108-114, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30385315

RESUMO

P38 mitogen-activated protein kinases are serine/threonine protein kinases reportedly involved in the innate immunity of vertebrates and invertebrates. In the present study, a P38 homolog (CgP38) was characterized from the Pacific oyster Crassostrea gigas. The full-length cDNA of CgP38 was of 1515 bp containing a 1101 bp open reading frame. A serine/threonine protein kinase (S_TKc) domain with a conserved Thr-Gly-Tyr motif and an ATRW substrate-binding site was found in the deduced amino acid sequence of CgP38. CgP38 shared a close evolutionary relationship with ChP38 from the Hong Kong oyster Crassostrea hongkongensis. The transcript levels of CgP38 in hemocytes increased significantly from 12 h to 48 h after lipopolysaccharide (LPS) stimulation and from 12 h to 24 h after Vibrio splendidus stimulation. The phosphorylation level of CgP38 in oyster hemocytes increased significantly at 2 h after LPS stimulation. CgP38 positively regulated the expression of interleukins, such as CgIL17-1, CgIL17-2, CgIL17-3, CgIL17-4 and CgIL17-6, and tumor necrosis factor CgTNF after LPS or V. splendidus stimulation. These results suggested that CgP38 participated in oyster immune response by regulating the expressions of inflammatory cytokines.


Assuntos
Crassostrea/imunologia , Hemócitos/fisiologia , Inflamação/imunologia , Vibrioses/imunologia , Vibrio/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Células Cultivadas , Clonagem Molecular , Crassostrea/microbiologia , Citocinas/metabolismo , Evolução Molecular , Imunidade Inata , Lipopolissacarídeos/imunologia , Fosforilação , Filogenia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Dev Comp Immunol ; 91: 101-107, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30385317

RESUMO

Caspase, an aspartate specific proteinase mediating apoptosis, plays a key role in immune response. In our previous study, the expression of a caspase gene was up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To further reveal the effect of caspase on WSSV infection, we cloned this caspase gene (denominated as CqCaspase) with an open reading frame of 1062 bp, which encoded 353 amino acids with a caspase domain (CASc) containing a p20 subunit and a p10 subunit. Tissue distribution analysis indicated that the mRNA transcript of CqCaspase was widely expressed in all tested tissues with the highest expression in Hpt, while the lowest expression in muscle. To further explore the effect of CqCaspase on WSSV replication, recombinant protein of CqCaspase (rCqCaspase) was delivered into Hpt cells followed by WSSV infection, which resulted in a significantly decreased expression of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV, suggesting that CqCaspase, possibly by the enhanced apoptotic activity, had a strong negative effect on the WSSV replication. These data together indicated that CqCaspase was likely to play a vital role in immune defense against WSSV infection in a crustacean C. quadricarinatus, which shed a new light on the mechanism study of WSSV infection in crustaceans.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/imunologia , Caspases/genética , Infecções por Vírus de DNA/imunologia , Hemócitos/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/metabolismo , Astacoidea/virologia , Caspases/metabolismo , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Imunidade Inata/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral
9.
Dev Comp Immunol ; 91: 132-142, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389518

RESUMO

As an important post-translational protein modification, ubiquitination has been demonstrated to play a vital role in immune response of vertebrates. Ubiquitin (Ub)-conjugating enzyme E2 is the "heart" of ubiquitination, which is responsible for Ub cellular signaling and substrate modification. In the present study, an Ub-conjugating enzyme E2 (designed as CgUbe2g1) was identified from oyster Crassostrea gigas, and its regulation in the immune response against lipopolysaccharide (LPS) stimulation was investigated. CgUbe2g1 encoded a polypeptide of 168 amino acids with the predicted molecular mass of 19.20 kDa and contained conserved catalytic 'Ubc' domains. It shared a higher similarity with the known UBC2G1 type E2s and was closely clustered with the type E2s identified from invertebrates in the phylogenetic assay. The mRNA transcripts of CgUbe2g1 were mainly distributed in hemocyte, mantle, hepatopancreas and male gonad of C. gigas. CgUbe2g1 protein was found to be colocalized with Ub around the nucleus of oyster hemocyte. The recombinant CgUbe2g1 protein (rCgUbe2g1) could activate the ubiquitination in vitro by binding both activated and un-activated Ub. The expressions of inflammation-related factors TNF-α and NF-κB in CgUbe2g1 transfected cells were both significantly up-regulated after LPS stimulation, which were 12.9-fold at 3 h (p < 0.01) and 2.3-fold at 6 h (p < 0.01) of that in negative control group, respectively. The phagocytic rate of hemocyte and the ROS level in hemocyte were both significantly decreased (p < 0.01), while the apoptosis rate was significantly increased (p < 0.01) after CgUbe2g1 mRNA was interfered. These results demonstrated that Ub-conjugating enzyme CgUbe2g1 was involved in the innate immune response of oyster against invading pathogen, which might play important roles in the activation of inflammatory response and regulation of cellular immune response.


Assuntos
Crassostrea/imunologia , Hemócitos/fisiologia , Enzimas de Conjugação de Ubiquitina/genética , Animais , Apoptose , Células Cultivadas , Clonagem Molecular , Humanos , Imunidade Inata , NF-kappa B/metabolismo , Filogenia , Transcriptoma , Fator de Necrose Tumoral alfa/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
10.
Environ Toxicol Pharmacol ; 62: 177-180, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30044999

RESUMO

Potential immunotoxicity and genotoxicity of as-produced and purified single walled carbon nanotubes (SWCNT, 500 µg L-¹) with or without cadmium (20 µg L-¹) was investigated in hemocytes of the freshwater mussel, Elliptio complanata. Our results showed a decrease in hemocyte viability after 3, and 8 days of exposure and an increase of hemocyte phagocytic efficiency for organisms exposed to Cd. No modification of the cyclo-oxygenase (COX) activity was measured. An increase in DNA damage was measured after 1 day of exposure to Cd and a potentiating effect of combined exposures was observed.


Assuntos
Cádmio/toxicidade , Mutagênicos/toxicidade , Nanotubos de Carbono/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Bivalves , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA , Hemócitos/efeitos dos fármacos , Hemócitos/fisiologia , Fagocitose/efeitos dos fármacos
11.
Fish Shellfish Immunol ; 80: 115-123, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29864586

RESUMO

The mussel Perna perna is an intertidal bivalve that is widely distributed, cultivated and consumed in South Africa, Brazil and Venezuela. Among marine resources, bivalve mollusks are one of the most impacted by anthropogenic pollution, as they can accumulate pathogenic bacteria and water pollutants. Hemocytes are molluscan defense cells, and their abundance and functions can be affected in response to contaminants, such as bacterial load. However, no previous study has investigated the immune response of P. perna hemocytes. The aim of this study was to evaluate several immune parameters in P. perna as indicators of fecal pollution in mussel hemolymph and in seawater. We collected mussels and adjacent seawater from beaches with different levels of fecal contamination in Rio de Janeiro state (Brazil): Vermelha Beach (VB); Icaraí Beach (IB); Urca Beach (UB); and Jurujuba Beach (JB). Hemocyte parameters (density, morphology, phagocytic activity and production of Reactive Oxygen Species - ROS) were evaluated using flow cytometry. We quantified Fecal Indicator Bacteria (FIB) in seawater by the multiple tubes technique for each beach and for hemolymph by the spread-plate technique. In agreement with historical evaluation of fecal contamination levels, UB presented the highest FIB abundance in seawater (thermotolerant coliforms, TEC = 1600 NMP 100 mL-1), whereas VB exhibited the lowest (TEC = 17 NMP 100 mL-1). UB mussels had six and eight times higher hemocyte density and phagocytic activity, respectively, than mussels from VB. Mussels from VB and IB presented a significantly lower number of total coliforms in hemolymph and a significantly higher relative internal complexity of hemocytes than those from UB and JB (p ≤ 0.01, PERMANOVA). ROS production by hemocytes was significantly lower in mussels from VB compared to those from JB (p = 0.04, ANOVA). Our results indicate a significant relationship between the level of fecal contamination in aquatic environments and the immune response of mussel hemocytes. Immune-related parameters may therefore be useful as indicators of bivalve health and environmental quality. Our flow cytometric analysis of P. perna hemocytes represents a new approach for studying Perna perna biology and might represent a novel tool for measuring organic pollution and water quality.


Assuntos
Monitoramento Ambiental/métodos , Fezes , Perna (Organismo)/imunologia , Poluição da Água/análise , Animais , Brasil , Enterobacteriaceae/isolamento & purificação , Fezes/microbiologia , Hemócitos/imunologia , Hemócitos/fisiologia , Hemolinfa/microbiologia , Perna (Organismo)/microbiologia , Fagocitose , Espécies Reativas de Oxigênio/imunologia , Explosão Respiratória , Água do Mar , Poluentes da Água/análise
12.
Pestic Biochem Physiol ; 148: 151-158, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29891366

RESUMO

Pedunsaponin A, a novel molluscicidal compound isolated from Pueraria peduncularis, exhibits strong toxicity against Pomacea canaliculata. To determine the mechanisms of Pedunsaponin A toxicity, its effects on the organs and hemocytes of P. canaliculata were examined in this study. The results showed that Pedunsaponin A had significant toxic effects on different organs of the snail, including the lungs, gills, mantle, siphon tube, ventricle, pericardial cavity, hepatopancreas, kidneys, and the major symptom of this toxicity was the loss of cilia in the lungs and gills. Additionally, in further studies on the effects of Pedunsaponin A treatment, we found that the hemocyte count was changed and hemocyte morphology was damaged, which was primarily reflected by cytoplasm leakage, nuclei deformation, and significant reductions in the number of ribosomes and granulocyte mitochondria. Based on these results and considering that blood vessels are distributed in the lungs and gills, we hypothesized that Pedunsaponin A would first destroy the cilia, which disrupt physiological activities such as respiration, excretion and feeding, and then enter the hemolymph through blood vessels, disrupt the normal function of the hemocytes and destroy the snail immune system, eventually resulting in the death of the snail.


Assuntos
Cílios/efeitos dos fármacos , Gastrópodes/efeitos dos fármacos , Brânquias/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Moluscocidas/toxicidade , Saponinas/toxicidade , Triterpenos/toxicidade , Animais , Vasos Sanguíneos/efeitos dos fármacos , Cílios/patologia , Gastrópodes/imunologia , Gastrópodes/fisiologia , Brânquias/irrigação sanguínea , Brânquias/patologia , Hemócitos/efeitos dos fármacos , Hemócitos/fisiologia , Hemolinfa/efeitos dos fármacos , Hepatopâncreas/efeitos dos fármacos , Hepatopâncreas/patologia , Rim/efeitos dos fármacos , Rim/patologia , Pulmão/irrigação sanguínea , Pulmão/patologia , Mitocôndrias/efeitos dos fármacos , Pericárdio/efeitos dos fármacos , Pericárdio/patologia , Ribossomos/efeitos dos fármacos
13.
Environ Pollut ; 235: 1006-1014, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29751396

RESUMO

The problem of effective assessment of risk posed by complex mixtures of toxic chemicals in the environment is a major challenge for government regulators and industry. The biological effect of the individual contaminants, where these are known, can be measured; but the problem lies in relating toxicity to the multiple constituents of contaminant cocktails. The objective of this study was to test the hypothesis that diverse contaminant mixtures may cause a greater toxicity than the sum of their individual parts, due to synergistic interactions between contaminants with different intracellular targets. Lysosomal membrane stability in hemocytes from marine mussels was used for in vitro toxicity tests; and was coupled with analysis using the isobole method and a linear additive statistical model. The findings from both methods have shown significant emergent synergistic interactions between environmentally relevant chemicals (i.e., polycyclic aromatic hydrocarbons, pesticides, biocides and a surfactant) when exposed to isolated hemocytes as a mixture of 3 & 7 constituents. The results support the complexity-based hypothesis that emergent toxicity occurs with increasing contaminant diversity, and raises questions about the validity of estimating toxicity of contaminant mixtures based on the additive toxicity of single components. Further experimentation is required to investigate the potential for interactive effects in mixtures with more constituents (e.g., 50-100) at more environmentally realistic concentrations in order to test other regions of the model, namely, very low concentrations and high diversity. Estimated toxicant diversity coupled with tests for lysosomal damage may provide a potential tool for determining the toxicity of estuarine sediments, dredge spoil or contaminated soil.


Assuntos
Bivalves/fisiologia , Poluentes Ambientais/toxicidade , Hemócitos/efeitos dos fármacos , Testes de Toxicidade/métodos , Animais , Hemócitos/fisiologia , Compostos Orgânicos/toxicidade , Praguicidas , Hidrocarbonetos Policíclicos Aromáticos/toxicidade
14.
Fish Shellfish Immunol ; 78: 131-139, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29684604

RESUMO

The ability to successfully prepare and preserve haemocyte cells for microscopy and flow cytometry is critical for the investigation of animal immune systems. In this study, we observed the total cell count, in vitro viability and stability of New Zealand black-footed abalone (Haliotis iris) haemocytes with different antiaggregants and handling protocols. Haemocyte stability was evaluated by direct observation of haemocytes under the microscope and calculating the aggregation index. Haemocyte counts and viability were measured via flow cytometry and tested for the effect of different antiaggregants (Alsever's solution at three concentrations, and specialised blood collection tubes containing lithium heparin and K2EDTA) at different temperatures and storage times. Results showed that Alsever's solution is an effective antiaggregant at haemolymph:antiaggregant dilution ratios of 1:1, 1:2 and 1:3. Lithium heparin was ineffective as an antiaggregant, whereas K2EDTA was similarly as effective as Alsever's solution. The influence of different mixing techniques (vortex, pipetting and flipping) were subsequently tested using the K2EDTA Microtainer® tubes, revealing that proper mixing should be performed immediately. High cell viability can be achieved by mixing samples by either 10 s of vortexing (1000 rpm), 10 times pipetting or 20 times flipping. The in vitro storage of abalone haemocytes in AS and K2EDTA as antiaggregants at ambient room temperature was highly effective for up to 24 h (75-85% viability; 0.05-0.15 aggregation index) and is recommended for haemocyte studies in H. iris. Utilization of K2EDTA Microtainer® tubes were advantageous since they are more cost effective compared to Alsever's solution, and samples can be prepared more efficiently.


Assuntos
Citometria de Fluxo/métodos , Gastrópodes/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Gastrópodes/fisiologia , Hemócitos/fisiologia , Técnicas In Vitro
15.
Fish Shellfish Immunol ; 77: 208-213, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29609026

RESUMO

Exposure to heavy metals such as Cadmium (Cd) may exert detrimental impacts on the immune responses of marine bivalve species. However, the immunotoxicity of Cd on blood clams remains unknown to date. Furthermore, though Cd2+ is known to compete with calcium (Ca2+) ions for their binding sites in cells and inhibit Ca2+ influx, whether Cd2+ weakens the immune responses of marine bivalves through inducing intracellular Ca2+ disorders still remains unclear. Therefore, the immunotoxicity of Cd2+ at different waterborne Ca2+ concentrations on blood clam, Tegillarca granosa, were investigated in the present study. Results obtained demonstrated that the total number, phagocytic activity, and red granulocytes ratio of the haemocytes were all significantly reduced after 10 days exposure of individuals to 25 µg/L Cd2+. However, when the waterborne Ca2+ concentrations were elevated by 10% and 20% (approximately 370 and 410 mg/L, respectively), mitigation effects on the immune responses of individuals were detected. In addition, though the expressions of genes from the Ca2+ signaling and Ca2+-related apoptosis pathways were significantly altered by Cd2+ exposure, the expression patterns of these genes were similar to that of the control when the waterborne Ca2+ concentrations were elevated, suggesting a relieving effect of waterborne Ca2+ on Cd2+ induced toxicity to haemocytes. The results obtained in the present study revealed that waterborne Cd2+ may hamper the immune responses of T. granosa through influencing Ca2+ signaling and Ca2+-related apoptosis pathways, which can be partially mitigated by elevating the waterborne Ca2+ concentrations.


Assuntos
Bivalves/imunologia , Cádmio/efeitos adversos , Sinalização do Cálcio/genética , Imunidade Inata/efeitos dos fármacos , Poluentes Químicos da Água/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Bivalves/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Hemócitos/fisiologia , Imunidade Inata/genética , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Distribuição Aleatória
16.
Fish Shellfish Immunol ; 78: 18-25, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29635064

RESUMO

Hemocytes associated with the mucus lining of pallial (mantle, gill) surfaces of the oyster Crassostrea virginica have been recently suggested to facilitate infection by the Alveolate parasite Perkinsus marinus by mediating the uptake and dispersion of parasite cells. These "pallial hemocytes", which are directly exposed to microbes present in surrounding seawater, are able to migrate bi-directionally between mucosal surfaces and the circulatory system, potentially playing a sentinel role. Interestingly, P. marinus was shown to increase trans-epithelial migration of hemocytes suggesting it may regulate cell motility to favor infection establishment. The purpose of this study was to investigate the effect of P. marinus on hemocyte motility and identify specific molecular mechanisms potentially used by the parasite to regulate hemocyte migration. In a first series of experiments, various components of P. marinus (live P. marinus cells, extracellular products, fragments of P. marinus cell membrane, membrane-modified live P. marinus cells, heat-killed P. marinus) along with components of the opportunistic bacterial pathogen Vibrio alginolyticus (bacterial cells and extracellular products) were investigated for their effects on hemocyte motility. In a second series of experiments, inhibitors of specific molecular pathways involved in motility regulation (Y-27632: inhibitor of Rho-associated protein kinase, RGDS: integrin inhibitor, CK-666: Arp2/3 inhibitor) were used in conjunction with qPCR gene expression experiments to identify pathways regulated by P. marinus exposure. Results showed a specific increase in hemocyte motility following exposure to live P. marinus cells. The increase in motility induced by P. marinus was suppressed by RGDS and CK-666 implicating the involvement of integrins and Arp2/3 in cell activation. Gene expression data suggest that Arp2/3 is possibly regulated directly by an effector produced by P. marinus. The implications of increased hemocyte motility prompted by P. marinus during the early stage of the infection process are discussed.


Assuntos
Alveolados/fisiologia , Movimento Celular , Crassostrea/parasitologia , Hemócitos/fisiologia , Interações Hospedeiro-Parasita , Animais , Crassostrea/fisiologia , Hemócitos/parasitologia , Vibrio alginolyticus/fisiologia
17.
Fish Shellfish Immunol ; 78: 248-258, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29702235

RESUMO

The inositol-requiring enzyme 1 (IRE1), one of the primary endoplasmic reticulum (ER) transmembrane receptor proteins, is involved in regulating unfolded protein response (UPR) signaling pathway and plays an import role in maintaining cell homeostasis. In the present study, an IRE1 homologue was identified from Patinopecten yessoensis (designated as PyIRE1). The cDNA of PyIRE1 was of 3314 bp with a 2646 bp open reading frame (ORF) of IRE1 encoding a polypeptide of 881 amino acids. There was a signal peptide, four pyrrolo-quinoline quinine (PPQ) domains, a transmembrane helix region, a Serine/Threonine protein kinases domain (S_TKc) and a protein kinases or N-glycanases containing protein domain (PUG) in the deduced amino acid sequence of PyIRE1. The PyIRE1 mRNA was constitutively expressed in all the tested tissues, with the highest expression level in gills. PyIRE1 protein was mainly located in the ER of P. yessoensis hemocytes. The expression profiles of PyIRE1, glucose-regulated protein 94 (designated as PyGRP94) and glucose-regulated protein 78 (designated as PyGRP78) were determined by SYBR Green qRT-PCR after heat shock treatment. The mRNA expression levels of all these three genes were significantly up-regulated and reached their peak values at 2 h (3.97-fold, p < 0.05), 8 h (19.67-fold, p < 0.05) and 4 h (27.37-fold, p < 0.05) in hemocytes, 2 h (3.55-fold, p < 0.05), 12 h (8.58-fold, p < 0.05) and 8 h (35.31-fold, p < 0.05) in gills after heat shock treatment, respectively. After the injection with PyIRE1 dsRNA, the mRNA expression of pro-apoptotic B-cell lymphoma-2 (Bcl-2) family member PyBax and the activity of caspase-3 significantly decreased in comparison with the control group (p < 0.05) after heat shock treatment. These results collectively suggested that PyIRE1, as an ER stress sensor, was potentially involved in the response upon heat stress by regulating the expression of PyBax and apoptosis of hemocytes in P. yessoensis.


Assuntos
Apoptose , Endorribonucleases/genética , Hemócitos/fisiologia , Pectinidae/fisiologia , Animais , Endorribonucleases/metabolismo , Temperatura Alta/efeitos adversos , Pectinidae/genética , Estresse Fisiológico
18.
Dev Comp Immunol ; 84: 343-352, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29530548

RESUMO

Iron-sulphur clusters (ISCs), one of the oldest and most versatile cofactors of proteins, are involved in catalysis reactions, electron transport reactions, regulation processes as well as sensing of ambient conditions. Iron-sulphur cluster assembly protein (IscA) is a scaffold protein member of ISC formation system, which plays a significant role in the assembly and maturation process of ISC proteins. In the present study, the cDNA sequence of iron-sulphur cluster assembly protein 2 (designated as EsIscA2) was cloned from Eriocheir sinensis. The open reading frame (ORF) of EsIscA2 was of 507 bp, encoding a peptide of 168 amino acids with a typically conserved Fe-S domain. A tetrameric form was predicated by the SWISS-MODEL prediction algorithm, and three conserved cysteine residues (Cys-93, Cys-158, Cys-160) from each IscA monomer were predicted to form a 'cysteine pocket'. The deduced amino acid sequence of EsIscA2 shared over 50% similarity with that of other IscAs. EsIscA2 was clustered with IscA2 proteins from invertebrates and vertebrates, indicating that the protein was highly conservative in the evolution. rEsIscA2 exhibited a high iron binding affinity in the concentration ranging from 2 to 200 µM. EsIscA2 transcripts were detected in all the tested tissues including gonad, hemocytes, gill, muscle, heart, hepatopancreas and eyestalk, and EsIscA2 protein was detected in the mitochondria of hemocytes. The highest mRNA expression level of EsIscA2 was detected in muscle and hepatopancreas, which was about 34.66-fold (p < 0.05) and 27.07-fold (p < 0.05) of that in hemocytes, respectively. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulations, the mRNA expression of EsIscA2 in hemocytes was down-regulated and reached the lowest level at 24 h (0.31-fold, p < 0.05) and 48 h (0.29-fold, p < 0.05) compared to control group, respectively. And the expression of EsIscA2 mRNA in hepatopancreas was repressed from 6 h to 48 h post stimulation (p < 0.05). When the primary cultured crab hemocytes were incubated with different concentrations of H2O2 for 15 min, the expression level of EsIscA2 mRNA was significantly repressed to the 0.34-0.44-fold of that in the control group. After A. hydrophila stimulation, the mRNA expression of EsGrx2 was up-regulated at 3 h (3.22-fold compared to control group, p < 0.05) and reached the peak at 12 h (4.88-fold, p < 0.05). All these results suggested that EsIscA2 had iron-binding capabilities as observed in IscA proteins from other organisms, supporting the role of EsIscA2 as a mitochondrial iron donor for ISC synthesis in Chinese mitten crab. Its differential mRNA expression after immune and oxidative stress challenges suggested the adaptations of ISC synthesis rates to these stress conditions.


Assuntos
Aeromonas hydrophila/imunologia , Proteínas de Artrópodes/metabolismo , Braquiúros/fisiologia , Infecções por Bactérias Gram-Negativas/imunologia , Hemócitos/fisiologia , Proteínas com Ferro-Enxofre/metabolismo , Mitocôndrias/fisiologia , Animais , Proteínas de Artrópodes/genética , Células Cultivadas , Clonagem Molecular , Regulação da Expressão Gênica , Imunidade Inata/genética , Invertebrados , Ferro/metabolismo , Proteínas com Ferro-Enxofre/genética , Lipopolissacarídeos/imunologia , Estresse Oxidativo/genética , Ligação Proteica , Alinhamento de Sequência
19.
Fish Shellfish Immunol ; 77: 112-119, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29578050

RESUMO

Crustacean hemocytes are known to remove invading pathogens by phagocytosis. In this study, we investigated how the semigranular cells (SGCs) and granular cells (GCs) of crayfish Cherax quadricarinatus participated in this process. By injecting the animals with excessive amounts of fluorescent microspheres (FMs), we showed that only a small portion of the circulating hemocytes were phagocytic cells, and they took up FMs in a size-dependent manner. The 0.2 µm FMs were internalized almost entirely by SGCs, while GCs and SGCs both contributed to the uptake of 2 µm FMs. Further analysis of the hemocytes from the animals injected with a mixture of FMs suggested that there were a subpopulation of SGCs specifically ingesting 0.2 µm FMs. The size-dependent manner was also applied to biological particles. Escherichia coli was internalized by both SGCs and GCs, whereas white spot syndrome virus (WSSV) was mostly ingested by SGCs. However, the bacterial cells were rapidly taken and cleared from the circulation by the hemocytes, while the WSSV virions were gradually internalized and remained in the cells for a relatively longer period of time. These findings provide basic information of the phagocytic hemocytes of crayfish and how they respond to different foreign particles.


Assuntos
Astacoidea/citologia , Escherichia coli/fisiologia , Hemócitos/fisiologia , Imunidade Inata , Fagocitose , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Astacoidea/imunologia , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Hemócitos/ultraestrutura , Hemócitos/virologia , Masculino , Microscopia Eletrônica de Transmissão , Internalização do Vírus
20.
Dev Comp Immunol ; 84: 264-272, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29510164

RESUMO

Influenza A virus non-structural-1A binding protein (named as Ns1abp) was originally identified as a host protein from human that bound to the viral NS-1 protein. In our previous study, the expression of an Ns1abp-like gene (denoted as CqNs1abp-like gene) was found to be up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To elucidate the role of CqNs1abp-like gene involved in WSSV infection, we cloned the CqNs1abp-like gene in which the open reading frame was 2232 bp, encoding 743 amino acids with two typical domains of one BTB (Broad-Complex, Tramtrack and Bric a brac) domain at N-terminal and six Kelch domains at C-terminal. The gene expression profile showed that the mRNA transcript of CqNs1abp-like gene was widely expressed in all the tested tissues with highest expression in nerve, relatively high expression in Hpt and lowest expression in eyestalk. Importantly, both the WSSV entry and the viral replication were significantly reduced in Hpt cells after gene silencing of CqNs1abp-like gene. By using protein pull-down assay, we found that the recombinant BTB domain, six Kelch domains and CqNs1abp-like intact protein were all bound to the WSSV envelope protein VP28, respectively, in which the BTB domain showed slightly less binding affinity than that of the six Kelch domains or the recombinant intact protein. Besides, the WSSV entry into Hpt cells was clearly decreased when the virus was pre-incubated with the recombinant BTB domain, six Kelch domains, or the recombinant CqNs1abp-like intact protein, respectively, suggesting that the CqNs1abp-like gene was likely to function as a putative recognition molecular towards WSSV infection in a crustacean C. quadricarinatus. Taken together, these data shed new light on the mechanism of WSSV infection and a putatively novel target on anti-WSSV infection in crustacean farming.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/imunologia , Infecções por Vírus de DNA/imunologia , Hemócitos/fisiologia , Tecido Nervoso/fisiologia , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/metabolismo , Células Cultivadas , Clonagem Molecular , Humanos , Vírus da Influenza A/fisiologia , Proteínas Nucleares/metabolismo , Domínios Proteicos/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Proteínas do Envelope Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
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