RESUMO
Temperature fluctuations, particularly elevated temperatures, can significantly affect immune responses. These fluctuations can influence the immune system and alter its response to infection signals, such as lipopolysaccharide (LPS). Therefore, this study was designed to investigate how high temperatures and LPS injections collectively influence the immune system of the crab Neohelice granulata. Two groups were exposed to 20 °C (control) or 33 °C for four days. Subsequently, half were injected with 10 µL of physiological crustacean (PS), while the rest received 10 µL of LPS [0.1 mg.kg-1]. After 30 min, the hemolymph samples were collected. Hemocytes were then isolated and assessed for various parameters using flow cytometry, including cell integrity, DNA fragmentation, total hemocyte count (THC), differential hemocyte count (DHC), reactive oxygen species (ROS) level, lipid peroxidation (LPO), and phagocytosis. Results showed lower cell viability at 20 °C, with more DNA damage in the same LPS-injected animals. There was no significant difference in THC, but DHC indicated a decrease in hyaline cells (HC) at 20 °C following LPS administration. In granular cells (GC), an increase was observed after both PS and LPS were injected at the same temperature. In semi-granular cells (SGC), there was a decrease at 20 °C with the injection of LPS, while at a temperature of 33 °C, the SGC there was a decrease only in SGC injected with LPS. Crabs injected with PS and LPS at 20 °C exhibited higher levels of ROS in GC and SGC, while at 33 °C, the increase was observed only in GC and SGC cells injected with LPS. A significant increase in LPO was observed only in SGC cells injected with PS and LPS at 20 °C and 33 °C. Phagocytosis decreased in animals at 20 °C with both injections and exposed to 33 °C only in those injected with LPS. These results suggest that elevated temperatures induce changes in immune system parameters and attenuate the immune responses triggered by LPS.
Assuntos
Braquiúros , Hemócitos , Temperatura Alta , Lipopolissacarídeos , Animais , Hemócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Braquiúros/imunologia , Braquiúros/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , 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
This study aimed to investigate the acute effects of chlorpyrifos on biomarkers related to neurotoxicity and immunotoxicity in two allopatric freshwater gastropod species belonging to the family Planorbidae. For this purpose, Planorbarius corneus and Biomphalaria glabrata were exposed to chlorpyrifos (active ingredient or commercial formulation) for 48 h at environmentally realistic concentrations (1 and 7.5 µg L-1). Basal acetylcholinesterase activity in soft tissues and hemolymph was almost one order of magnitude higher in P. corneus than in B. glabrata. However, upon chlorpyrifos exposure, statistically significant inhibition of enzymatic activity was registered in both species. Acetylcholinesterase was more sensitive to inhibition in soft tissues than in hemolymph. The highest inhibition was observed in the B. glabrata soft tissues exposed to the commercial formulation (88 % at 1 µg L-1 and 93 % at 7.5 µg L-1). Hemocyte number and lysosomal membrane stability did not show significant changes with respect to controls in any of the exposed groups. Superoxide anion generation was diminished (21-46 %) in P. corneus hemocytes exposed to the active ingredient and in B. glabrata hemocytes exposed to the active ingredient or the formulation. In contrast, hemocyte phagocytic activity increased in all exposed groups. Phagocytosis was most stimulated (89 %) in hemocytes sampled from B. glabrata treated with 7.5 µg L-1 chlorpyrifos. Altogether the results suggest that the freshwater gastropods P. corneus and B. glabrata are suitable model animals for environmental monitoring studies in the Northern Hemisphere and Latin America, respectively. Furthermore, these results add information on the relevance of testing pesticide formulations and on the usefulness of acetylcholinesterase inhibition and immunological parameters as biomarkers of the acute effects of chlorpyrifos in these species.
Assuntos
Biomphalaria/fisiologia , Clorpirifos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Biomphalaria/efeitos dos fármacos , Inibidores da Colinesterase , Monitoramento Ambiental , Água Doce , Gastrópodes/fisiologia , Hemócitos/efeitos dos fármacos , Hemolinfa/efeitos dos fármacos , PraguicidasRESUMO
Bivalve molluscs rely only on an innate immune system to execute cellular and humoral processes. Haemocytes, the haemolymph circulating cells, play a major role in this type of immunity, principally regarding cellular defences. Considering that environmental pollutants can affect the immune system of invertebrates, this work evaluated the effects of the antifouling biocide 4,5-dicloro-2-n-octil-4-isotiazolin-3-ona (DCOIT) on the haemocytes of mussels Perna perna. Individuals were exposed to 0 (control), 0.1 µg L-1 and 10 µg L-1 of DCOIT for up to 96 h. The analysed parameters included: total (THC) and differential (DHC) haemocyte count, cellular viability, adhesion capacity, phagocytic activity, levels of reactive oxygen species and DNA damage. Moreover, the stress on stress (SOS) response of mussels was analysed as a general stress index. The results show that DCOIT increased the haemocyte adhesion capacity and caused a decrease in THC and in the haemocyte viability after 24 h of exposure. After 96 h of exposure, DCOIT only affected the haemocyte adhesion capacity, which was decreased by biocide exposure. Moreover, exposure to DCOIT for 96 h did not affect the capacity for air survival of mussels. These results indicate that DCOIT interferes in important parameters associated with the innate immunity of P. perna, mainly after 24 h of exposure. It is suggested that the animals were able to develop some compensatory response strategy, making them more resistant to the biocide.
Assuntos
Hemócitos/imunologia , Imunidade Inata , Perna (Organismo)/imunologia , Fagócitos/imunologia , Tiazóis/toxicidade , Animais , Hemócitos/efeitos dos fármacos , Hemócitos/fisiologia , Perna (Organismo)/efeitos dos fármacos , Perna (Organismo)/fisiologia , Fagócitos/efeitos dos fármacos , Fagócitos/fisiologia , Poluentes Químicos da Água/toxicidadeRESUMO
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
Cellular and humoral responses were evaluated in Litopenaeus vannamei juveniles when exposed to malathion, endosulfan, and their mixture. Each experiment was performed in the hemolymph collected at each exposure time (5 and 96â¯h) in duplicate; total hemocyte count, coagulation time, hemocyanin concentration, phenoloxidase (PO) and superoxide dismutase (SOD) activities were quantified. Survival was not affected by pesticides applied individually and mixed. Clotting time did not show significant differences concerning increase of concentration percentage of the pesticides tested. In organisms exposed to the pesticide mixture, hemocyanin decreased at 5â¯h of exposure as the concentration increased. Only in the malathion experiment did exposed shrimp to 10 and 50% of the LC50-96â¯h show significantly (pâ¯<â¯0.05) higher hemocyte contents. For malathion, significantly (pâ¯<â¯0.05) lower PO values in shrimp exposed to higher concentrations (10 and 50% of the LC50-96â¯h) were determined. While for the mixture treatment, high SOD value was determined at high exposure time and concentration. Malathion was the pesticide that showed an effect on some variables even at sublethal concentrations. The Continuous Concentration Criteria of the United States Environmental Protection Agency did not represent effects on the variables when they were compared with the averages of the control group.
Assuntos
Endossulfano/toxicidade , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Malation/toxicidade , Penaeidae/efeitos dos fármacos , Animais , Sinergismo Farmacológico , Hemocianinas/sangue , Hemócitos/efeitos dos fármacos , Hemócitos/imunologia , Hemolinfa/efeitos dos fármacos , Hemolinfa/imunologia , Monofenol Mono-Oxigenase/metabolismo , Penaeidae/imunologiaRESUMO
The latex of Euphorbia tirucalli L. (LET) has great etnopharmacological relevance for several traditional communities. In this study, the in vitro and in vivo (using Tenebrio molitor larvae) antimicrobial effects of LET were evaluated. LET did not inhibit the growth of S. aureus, however, a reduction on staphyloxanthin production (an important virulence factor of S. aureus) was observed. LET (at 10 µL/kg) was also able to enhance the survival of larvae infected with a lethal dose of S. aureus, an effect associated with reduction in the numbers of haemocytes. Furthermore, haemocytes from LET-treated larvae exhibited dysfunctional lysosome activity. These results indicate the effectiveness of LET as an anti-infective agent which could be useful as source of lead molecules for the development of new therapies against S. aureus-induced infections.
Assuntos
Antibacterianos/farmacologia , Euphorbia/química , Látex/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Tenebrio/microbiologia , Animais , Modelos Animais de Doenças , Hemócitos/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/patogenicidade , Fatores de Virulência/metabolismo , Xantofilas/metabolismoRESUMO
Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.