RESUMO
Invertebrates' immune priming or innate immune memory is an analogous response to the vertebrates' adaptive memory. We investigated if honey bees have immune memory. We compared survival and immune response between bees that were: 1) manipulated (Naïve), 2) challenged twice with the same pathogen Escherichia coli (Memory), 3) challenged twice with different pathogens (Staphylococcus aureus versus E. coli, Micrococcus lysodeikticus versus E. coli), or 4) with PBS (the diluent of bacteria) versus E. coli (heterologous challenge; Control). Results indicate better survival in the Memory than the Control group, and the Memory group showed a similar survival than Naïve insects. The Memory group had higher lytic activity but lower prophenoloxidase, phenoloxidase activity, and hemocyte count than the Control and Naïve groups. No differences were found in relative expression of defensin-1. This first demonstration of immune memory opens the questions about its molecular mechanisms and whether, immune memory could be used against natural parasites that affect honey bees, hence, if they could be "vaccinated" against some natural parasites.
Assuntos
Escherichia coli , Monofenol Mono-Oxigenase , Animais , Abelhas , Defensinas , Escherichia coli/metabolismo , Hemócitos/metabolismo , Memória Imunológica , Monofenol Mono-Oxigenase/metabolismoRESUMO
The white shrimp Litopenaeus vannamei is exposed to hypoxic conditions in natural habitats and in shrimp farms. Hypoxia can retard growth, development and affect survival in shrimp. The hypoxia-inducible factor 1 (HIF-1) regulates many genes involved in glucose metabolism, antioxidant proteins, including metallothionein (MT) and apoptosis. In previous studies we found that the L. vannamei MT gene expression changed during hypoxia, and MT silencing altered cell apoptosis; in this study we investigated whether the silencing of HIF-1 affected MT expression and apoptosis. Double-stranded RNA (dsRNA) was used to silence HIF-1α and HIF-1ß under normoxia, hypoxia, and hypoxia plus reoxygenation. Expression of HIF-1α, HIF-1ß and MT, and apoptosis in hemocytes or caspase-3 expression in gills, were measured at 0, 3, 24 and 48 h of hypoxia and hypoxia followed by 1 h of reoxygenation. The results showed that hemocytes HIF-1α expression was induced during hypoxia and reoxygenation at 3 h, while HIF-1ß decreased at 24 and 48 h. In normoxia, HIF-1 silencing in hemocytes increased apoptosis at 3 h and decreased at 48 h; while in gills, caspase-3 increased at 3, 24 and 48 h. In hypoxia, HIF-1 silencing decreased apoptosis in hemocytes at 3 h, but caspase-3 increased in gills. During reoxygenation, apoptosis in hemocytes and caspase-3 in gills increased. During normoxia in hemocytes, silencing of HIF-1 decreased MT expression, but in gills, MT increased. During hypoxia and reoxygenation, silencing induced MT in hemocytes and gills. These results indicate HIF-1 differential participation in MT expression regulation and apoptosis during different oxygen conditions.
Assuntos
Apoptose , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Proteínas de Peixes/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Metalotioneína/metabolismo , Oxigênio/metabolismo , Penaeidae/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Brânquias/metabolismo , Brânquias/patologia , Hemócitos/metabolismo , Hemócitos/patologia , Hipóxia/genética , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Metalotioneína/genética , Penaeidae/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Harmful effects caused by the exposure to paralytic shellfish toxins (PSTs) and bioactive extracellular compounds (BECs) on bivalves are frequently difficult to attribute to one or the other compound group. We evaluate and compare the distinct effects of PSTs extracted from Alexandrium catenella (Alex5) cells and extracellular lytic compounds (LCs) produced by A. tamarense (NX-57-08) on Mytilus edulis hemocytes. We used a 4 h dose-response in vitro approach and analyzed how these effects correlate with those observed in a previous in vivo feeding assay. Both bioactive compounds caused moderated cell death (10-15%), being dose-dependent for PST-exposed hemocytes. PSTs stimulated phagocytic activity at low doses, with a moderate incidence in lysosomal damage (30-50%) at all tested doses. LCs caused a dose-dependent impairment of phagocytic activity (up to 80%) and damage to lysosomal membranes (up to 90%). PSTs and LCs suppressed cellular ROS production and scavenged H2O2 in in vitro assays. Neither PSTs nor LCs affected the mitochondrial membrane potential in hemocytes. In vitro effects of PST extracts on M. edulis hemocytes were consistent with our previous study on in vivo exposure to PST-producing algae, while for LCs, in vivo and in vitro results were not as consistent.
Assuntos
Hemócitos/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Mytilus edulis , Animais , Sobrevivência Celular/efeitos dos fármacos , Dinoflagellida , Hemócitos/metabolismo , Hemócitos/fisiologia , Lisossomos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Intoxicação por Frutos do MarRESUMO
Programmed cell death (PCD) is an essential process for the immune system's development and homeostasis, enabling the remotion of infected or unnecessary cells. There are several PCD's types, depending on the molecular mechanisms, such as non-inflammatory or pro-inflammatory. Hemocytes are the main component of cellular immunity in bivalve mollusks. Numerous infectious microorganisms produce toxins that impair hemocytes functions, but there is little knowledge on the role of PCD in these cells. This study aims to evaluate in vitro whether marine toxins induce a particular type of PCD in hemocytes of the bivalve mollusk Crassostrea gigas during 4 h at 25°C. Hemocytes were incubated with two types of marine toxins: non-proteinaceous toxins from microalgae (saxitoxin, STX; gonyautoxins 2 and 3, GTX2/3; okadaic acid/dynophysistoxin-1, OA/DTX-1; brevetoxins 2 and 3, PbTx-2,-3; brevetoxin 2, PbTx-2), and proteinaceous extracts from bacteria (Vibrio parahaemolyticus, Vp; V. campbellii, Vc). Also, we used the apoptosis inducers, staurosporine (STP), and camptothecin (CPT). STP, CPT, STX, and GTX 2/3, provoked high hemocyte mortality characterized by apoptosis hallmarks such as phosphatidylserine translocation into the outer leaflet of the cell membrane, exacerbated chromatin condensation, DNA oligonucleosomal fragments, and variation in gene expression levels of apoptotic caspases 2, 3, 7, and 8. The mixture of PbTx-2,-3 also showed many apoptosis features; however, they did not show apoptotic DNA oligonucleosomal fragments. Likewise, PbTx-2, OA/DTX-1, and proteinaceous extracts from bacteria Vp, and Vc, induced a minor degree of cell death with high gene expression of the pro-inflammatory initiator caspase-1, which could indicate a process of pyroptosis-like PCD. Hemocytes could carry out both PCD types simultaneously. Therefore, marine toxins trigger PCD's signaling pathways in C. gigas hemocytes, depending on the toxin's nature, which appears to be highly conserved both structurally and functionally.
Assuntos
Apoptose/efeitos dos fármacos , Toxinas Bacterianas/toxicidade , Crassostrea/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Animais , Toxinas Bacterianas/isolamento & purificação , Caspases/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Crassostrea/imunologia , Crassostrea/metabolismo , Quebras de DNA de Cadeia Dupla , Hemócitos/imunologia , Hemócitos/metabolismo , Hemócitos/patologia , Fosfatidilserinas/metabolismo , Vibrio/metabolismo , Vibrio parahaemolyticus/metabolismoRESUMO
Bivalves survive to biotoxin consumption but their metabolism could be affected. The objective of this work was to study the oxidative and nitrosative changes in the hemocytes of the mussel Mytilus edulis platensis in different seasons, including spring, characterized by the appearance of harmful algal blooms (HAB). Reactive species generation rate (measured as the 2',7'dichlorofluorescein diacetate oxidation rate) was increased by 2.5- and 8.3-fold in hemocytes from spring and summer, respectively, as compared to winter samples. Neither total Fe nor labile Fe pool content was changed in the three seasons. Superoxide anion generation rate was 3-fold higher in spring as compared to winter and summer samples. Catalase content in spring cells were significantly higher as compared to winter (60%) and summer (3-fold increase) but glutathione-S-transferase activity only increased compared to summer season (125% increase). Lipid radical content in spring samples was 140 and 50% higher as compared to cells from winter and summer, respectively. Nitric oxide and nitro-tyrosine content were significantly higher in samples from spring as compared to values obtained either in winter or summer cells. Considering the aspects that influence metabolism, changes in temperature seem to mainly affect the oxidative over the nitrosative condition of the hemocytes. Nevertheless, HAB biotoxins seem as a contributing factor to affect not only reactive oxygen species generation, antioxidant activity and protein/lipid damage, but also the nitrosative metabolism. In this regard, the changes in the nitric oxide content are new and critical evidence that HAB-related toxins could affect reactive nitrogen species metabolism.
Assuntos
Antioxidantes/metabolismo , Proliferação Nociva de Algas , Hemócitos/metabolismo , Mytilus/metabolismo , Óxido Nítrico/metabolismo , Estresse Nitrosativo , Estresse Oxidativo , Animais , Catalase/metabolismo , Hemócitos/efeitos dos fármacos , Mytilus/efeitos dos fármacos , Oxirredução , Estações do Ano , Superóxido Dismutase/metabolismoRESUMO
An in-situ experiment was performed to study metabolic responses of the freshwater mussel Diplodon chilensis to water contaminated by leachates from an open dump and cattle activity, in order to analyze both the effects of those contaminants on aquatic environments and the potential use of a native bivalve to evaluate the effects of anthropic influence and eutrophication. Bivalves from a reference site were cage-transplanted to a control site (site A) and to a temporal water pond (site B) over 30 and 60 periods. Water quality analyses revealed that the site B was affected by anthropogenic influence. Mussel's hemocytes from site B showed 50% lower reactive oxygen species production and 130% higher lysosomal membrane stability in the site B mussels. In addition, no oxidative stress was evident in gills, despite the elevated copper and iron concentrations recorded in the site B water samples (CuB = 0.3350 ± 0.0636 mg. L-1vs. CuA = 0.0045 ± 0.0007 mg. L-1; FeB = 3.8650 ± 0.4031 mg. L-1vs. FeA = 0.0365 ± 0.0049 mg. L-1). In contrast, the adductor muscle accumulated more Fe (~10-20-fold) than the gills and showed signs of oxidative stress, e.g. superoxide dismutase activity and TBARS levels were increased by 10% were 34%, respectively, in the site B compared with the site A after 60 days of exposure. Additionally, the adductor muscle showed signs of anaerobic metabolism activation. Cu is accumulated in gills from both sites' individuals, at 60 days, in concordance with the increase in the activity of the cu-containing enzyme cytochrome-c-oxidase. There was a reduction in the overall condition and digestive gland index in bivalves exposed at site B, associated with diminished levels of lipid and protein contents. Metal-pollution and eutrophication affects D. chilensis metabolism and is associated to tissue-specific exposure, anaerobic metabolism and general energetic condition depletion.
Assuntos
Bivalves/efeitos dos fármacos , Eutrofização , Metais Pesados/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Bivalves/enzimologia , Bivalves/metabolismo , Bovinos , Cobre/metabolismo , Água Doce , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Metais Pesados/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Poluentes Químicos da Água/metabolismo , Qualidade da ÁguaRESUMO
Phagocytes use their actomyosin cytoskeleton to migrate as well as to probe their environment by phagocytosis or macropinocytosis. Although migration and extracellular material uptake have been shown to be coupled in some immune cells, the mechanisms involved in such coupling are largely unknown. By combining time-lapse imaging with genetics, we here identify the lysosomal Ca2+ channel Trpml as an essential player in the coupling of cell locomotion and phagocytosis in hemocytes, the Drosophila macrophage-like immune cells. Trpml is needed for both hemocyte migration and phagocytic processing at distinct subcellular localizations: Trpml regulates hemocyte migration by controlling actomyosin contractility at the cell rear, whereas its role in phagocytic processing lies near the phagocytic cup in a myosin-independent fashion. We further highlight that Vamp7 also regulates phagocytic processing and locomotion but uses pathways distinct from those of Trpml. Our results suggest that multiple mechanisms may have emerged during evolution to couple phagocytic processing to cell migration and facilitate space exploration by immune cells.
Assuntos
Actomiosina/metabolismo , Movimento Celular , Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Hemócitos/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Fagocitose , Canais de Potencial de Receptor Transitório/metabolismo , Actomiosina/genética , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Sinalização do Cálcio , Citoesqueleto/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Hemócitos/imunologia , Lisossomos/genética , Macrófagos/imunologia , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Fatores de Tempo , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Decapod crustaceans, like mammals, retain the ability to make new neurons throughout life. In mammals, immune cells are closely associated with stem cells that generate adult-born neurons. In crayfish, evidence suggests that immune cells (hemocytes) originating in the immune system travel to neurogenic regions and transform into neural progenitor cells. This nontraditional immune activity takes place continuously under normal physiological conditions, but little is known under pathological conditions (neurodegeneration). In this study, the immune system and its relationship with neurogenesis were investigated during neurodegeneration (unilateral antennular ablation) in adult crayfish. Our experiments show that after ablation (1) Proliferating cells decrease in neurogenic areas of the adult crayfish brain; (2) The immune response, but not neurogenesis, is ablation-side dependent; (3) Inducible nitric oxide synthase (iNOS) plays a crucial role in the neurogenic niche containing neural progenitors during the immune response; (4) Brain areas targeted by antennular projections respond acutely (15 min) to the lesion, increasing the number of local immune cells; (5) Immune cells are recruited to the area surrounding the ipsilateral neurogenic niche; and (6) The vasculature in the niche responds acutely by dilation and possibly also neovascularization. We conclude that immune cells are important in both neurodegeneration and neurogenesis by contributing in physiological conditions to the maintenance of the number of neural precursor cells in the neurogenic niche (neurogenesis), and in pathological conditions (neurodegeneration) by coordinating NO release and vascular responses associated with the neurogenic niche. Our data suggest that neural damage and recovery participate in a balance between these competing immune cell roles.
Assuntos
Astacoidea/imunologia , Sistema Imunitário/imunologia , Degeneração Neural/imunologia , Neurogênese/imunologia , Animais , Astacoidea/ultraestrutura , Vasos Sanguíneos/metabolismo , Encéfalo/patologia , Bromodesoxiuridina/metabolismo , Contagem de Células , Proliferação de Células , Feminino , Glutamato-Amônia Ligase/metabolismo , Hemócitos/metabolismo , Masculino , Neurópilo/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Nicho de Células-TroncoRESUMO
Crustins are cysteine-rich antimicrobial peptides (AMPs) widely distributed across crustaceans. From the four described crustin Types (I to IV), crustins from the subtype IIa are the most abundant and diverse members found in penaeid shrimp. Despite the critical role of Type IIa crustins in shrimp antimicrobial defenses, there is still limited information about their synthesis and antimicrobial properties. Here, we report the subcellular localization and the antibacterial spectrum of crusFpau, a Type IIa crustin from the pink shrimp Farfantepenaeus paulensis. The recombinantly expressed crusFpau showed antimicrobial activity against both Gram-positive and Gram-negative bacteria at low concentrations. Results from immunofluorescence using anti-rcrusFpau antiserum revealed that crusFpau is synthetized and stored by both granular and semigranular hemocytes, but not by hyaline cells. Interestingly, not all granular and semigranular hemocytes stained for crusFpau, revealing that this crustin is produced by specific granule-containing hemocyte subpopulations. Finally, we showed that the granule-stored peptides are not constitutively secreted into the plasma of healthy animals.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/biossíntese , Proteínas de Artrópodes/biossíntese , Hemócitos/metabolismo , Penaeidae/imunologia , Animais , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Penaeidae/metabolismo , Penaeidae/microbiologiaRESUMO
Quantum dots have generated great interest because of their optical properties, both to life sciences and electronics applications. However, possible risks to the environment associated with these nanoparticles are still under investigation. The present study aimed to evaluate the toxicity of suspensions of cadmium telluride (CdTe) quantum dots to Biomphalaria glabrata mollusks, a very sensitive aquatic environmental bioindicator for physical and chemical agents. Toxicity was examined by using embryos and adult mollusks as well as hemocytes. The distribution of cadmium in the organs of adults was also assessed. Effects of the stabilizing agent of the quantum dots were also evaluated. Animals were exposed to suspensions of quantum dots for 24 h, at concentrations varying from 1.2 to 20 nM for embryos and from 50 to 400 nM for adult mollusks. Results showed that suspensions of quantum dots induced malformations and mortality in embryos and mortality in adults, depending on the concentration applied. In the cytotoxicity study, hemocyte apoptosis was observed in adults exposed to the highest concentration of quantum dots applied as well as to the stabilizing agent. Cell binucleation and micronucleus frequencies were not significative. Bioaccumulation evaluation revealed that quantum dots targeted the digestive gland (hepatopancreas). Taken together, outcomes suggested that specific nano-effects related directly not only to composition but also to the aggregation of quantum dots may be mediating the observed toxicity. Thus B. glabrata was determined to be a very sensitive species for interpreting possible nano-effects in aquatic environments. Environ Toxicol Chem 2019;38:2128-2136. © 2019 SETAC.