RESUMO
Hemocytes are the circulating cells of the hemolymph of oysters and are responsible for numerous physiological functions, including immune defense. The oyster Crassostrea gasar is a native species inhabiting mangrove habitat and is of great commercial interest, cultured throughout the Brazilian coast, mainly in the north and northeast. Despite its commercial importance, little is known about its immunological aspects and defense cells, the hemocytes. This work aimed to morphologically characterize hemocytes of the oyster C. gasar and to study one of the main cellular defense response, phagocytosis, using light microscopy and flow cytometry. The results showed the presence of six hemocyte populations in C. gasar hemolymph. These comprise of large and small granulocytes, large and small hyalinocytes, blast-like cells and a rare type classified as vesicular or serous hemocytes. Hyalinocytes were highly abundant and the most heterogeneous cell population, while small granulocytes, along with vesicular hemocytes were the less abundant population. Hemocytes of C. gasar oysters demonstrated capabilities to phagocytose three different types of particles tested: zymosan A, latex particles and Escherichia coli, indicating a broad defense capacity. The zymosan A were the most engulfed particles, followed by beads, mainly phagocytized by granulocytes, the most phagocytic cells, and finally E. coli, which were the least phagocytized. This study is the first characterization of C. gasar oyster hemocytes and will support future studies that aim to understand the participation of different hemocyte types in defense responses against pathogens and/or environmental changes.
RESUMO
Oyster production in Brazil has been highlighted as an important economic activity and is directly impacted by the quality of the environment, which is largely the result of human interference and climate change. Harmful algal blooms occur in aquatic ecosystems worldwide, including coastal marine environments which have been increasing over the last decades as a result of global change and anthropogenic activities. In this study, the native oysters Crassostrea gasar from Northeast of Brazil were exposed to two toxic benthic dinoflagellate species, Prorocentrum lima and Ostreopsis cf. ovata. Their respective effects on C. gasar physiology and defense mechanisms were investigated. Oyster hemocytes were first exposed in vitro to different concentrations of both dinoflagellate species to assess their effects on hemocyte functions, such as phagocytosis, production of reactive oxygen species, as well as mortality. Results highlighted an alteration of hemocyte phagocytosis and viability in presence of O. cf. ovata, whereas P. lima did not affect the measured hemocyte functions. In a second experiment, oysters were exposed for 4 days in vivo to toxic culture of O. cf. ovata to assess its effects on hemocyte parameters, tissues damages and pathogenic Perkinsus spp. infection. An increase in hemocyte mortality was also observed in vivo, associated with a decrease of ROS production. Histopathological analyses demonstrated a thinning of the epithelium of the digestive tubules of the digestive gland, inflammatory reaction and a significant increase in the level of infection by Perkinsus spp. in oysters exposed to O. cf. ovata. These results indicate that oysters C. gasar seem to be pretty resilient to an exposure to P. lima and may be more susceptible to O. cf. ovata. Furthermore, the latter clearly impaired oyster physiology and defense mechanisms, thus highlighting that harmful algal blooms of O. cf. ovata could potentially lead to increased susceptibility of C. gasar oysters to parasite infections.
Assuntos
Crassostrea/imunologia , Dinoflagellida/fisiologia , Proliferação Nociva de Algas , Animais , Brasil , Crassostrea/efeitos dos fármacos , Ecossistema , Hemócitos/imunologia , Humanos , Imunidade , FagocitoseRESUMO
Disseminated neoplasia (DN) is a disease that affects bivalves worldwide and can lead to mass mortalities. In the present study, a pathological survey conducted from December 2011 to August 2012 in Crassostrea gasar, an oyster of commercial interest in northeast Brazil, revealed the occurrence of DN in oysters reared in the Mamanguape estuary, Paraíba State, Brazil. The present work describes the pathological and functional aspects of the disease in C. gasar by light microscopy (haemolymph cell monolayer and histological section) and flow cytometry analyses. The prevalence of the disease was low (7.1% of 182 oysters examined). Enlarged (neoplastic) cells showed reniform, ovoid or circular-shaped nuclei, with prominent nucleoli and predominantly short filipodia. They were found in the haemolymph and infiltrated the connective tissues of different organs, including the digestive system, gills and gonads, as well as in the sinuses and vessels. Three levels of progression of DN in tissues were observed, light (61.5%), moderate (15.4%) and advanced (23.1%). The viability of neoplastic cells circulating in the haemolymph (97.4%) was similar to that in the haemocytes (95.7%). The neoplastic cells showed low phagocytic ability (3.9%) compared with that of haemocytes (42.4%). Conversely, reactive oxygen species production (679 A.U.) and the total haemocyte count (3.9â¯×â¯106â¯cellsâ¯mL-1) were higher in the affected oysters than in unaffected oysters (268 A.U. and 1.5â¯×â¯106â¯cellsâ¯mL-1, respectively). The low prevalence and primarily mild intensity found in the sampled oysters does not preclude an impact at the population level. A timely survey of DN is thus recommended in order to assess the severity and impact of this disease in wild and cultured populations of C. gasar oysters.
Assuntos
Crassostrea , Frutos do Mar , Animais , Brasil/epidemiologia , PrevalênciaRESUMO
Shrimp farm effluents are one of the principal causes of eutrophication in coastal environments. Integrated processes of bioremediation involving the culturing of purifying organisms have been suggested, but very few studies have focused on microalgae. For that purpose evaluated the growth potential of Amphora sp. in the residual waters of shrimp farm activity fulfilled on the Paraíba State, Brazil. The experiments were performed using Conway medium as the control and wastewaters from shrimp farm at 100% concentrations. Amphora sp. demonstrated good growth in the shrimp farm effluents under test conditions, although less than that observed in the control medium. This diatom was found to removed 73.357 and 72.572% of PO4- and NO3-, respectively, demonstrating a high mitigation potential for this type of effluent. Comparisons of the physiological responses measured by flow cytometry demonstrated higher cell densities of Amphora sp. in the control medium, but a higher lipid content was observed in Amphora cultured in shrimp farm effluents.
Assuntos
Crustáceos/química , Diatomáceas/química , Microalgas/química , Águas Residuárias/análise , Animais , Biodegradação Ambiental , Brasil , Fenômenos Fisiológicos Celulares , Eutrofização , Fazendas , NutrientesRESUMO
Perkinsosis is a disease caused by protozoan parasites from the Perkinsus genus. In Brazil, two species, P. beihaiensis and P. marinus, are frequently found infecting native oysters (Crassostrea gasar and C. rhizophorae) from cultured and wild populations in several states of the Northeast region. The impacts of this disease in bivalves from Brazil, as well as the interactions with environmental factors, are poorly studied. In the present work, we evaluated the in vitro effects of the cyanobacteria Synechocystis spp. on trophozoites of P. marinus and haemocytes of C. gasar. Four cyanobacteria strains isolated from the Northeast Brazilian coast were used as whole cultures (WCs) and extracellular products (ECPs). Trophozoites of P. marinus were exposed for short (4h) and long (48h and 7days, the latter only for ECPs) periods, while haemocytes were exposed for a short period (4h). Cellular and immune parameters, i.e. cell viability, cell count, reactive oxygen species production (ROS) and phagocytosis of inert (latex beads) and biological particles (zymosan and trophozoites of P. marinus) were measured by flow cytometry. The viability of P. marinus trophozoites was improved in response to WCs of Synechocystis spp., which could be a beneficial effect of the cyanobacteria providing nutrients and reducing reactive oxygen species. Long-term exposure of trophozoites to ECPs of cyanobacteria did not modify in vitro cell proliferation nor viability. In contrast, C. gasar haemocytes showed a reduction in cell viability when exposed to WCs, but not to ECPs. However, ROS production was not altered. Haemocyte ability to engulf latex particles was reduced when exposed mainly to ECPs of cyanobacteria; while neither the WCs nor the ECPs modified phagocytosis of the biological particles, zymosan and P. marinus. Our results suggest a negative effect of cyanobacteria from the Synechocystis genus on host immune cells, in contrast to a more beneficial effect on the parasite cell, which could together disrupt the balance of the host-parasite interaction and make oysters more susceptible to P. marinus as well as opportunistic infections.
Assuntos
Alveolados/crescimento & desenvolvimento , Crassostrea/parasitologia , Interações Hospedeiro-Parasita , Modelos Biológicos , Synechocystis/crescimento & desenvolvimento , Alveolados/efeitos dos fármacos , Alveolados/imunologia , Animais , Brasil , Contagem de Células , Sobrevivência Celular , Crassostrea/efeitos dos fármacos , Crassostrea/imunologia , Citometria de Fluxo , Hemócitos/efeitos dos fármacos , Hemócitos/imunologia , Interações Hospedeiro-Parasita/efeitos dos fármacos , Interações Hospedeiro-Parasita/imunologia , Toxinas Marinhas/toxicidade , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Synechocystis/química , Poluentes Químicos da Água/toxicidadeRESUMO
Saxitoxin (STX) is a neurotoxin produced by dinoflagellates in diverse species, such as Alexandrium spp., and it causes paralytic shellfish poisoning (PSP) in humans after the ingestion of contaminated shellfish. Recent studies have suggested that the immune functions of bivalves could be affected by harmful algae and/or by their toxins. Herein, hemocytes are the main effector cells of the immune cellular response. In this study, we evaluated the response of hemocytes from the mussel Mytilus chilensis to STX exposure in a primary culture. Cell cultures were characterized according to size and complexity, while reactive oxygen species (ROS) production was evaluated using a dichlorofluorescein diacetate (DCFH-DA) assay. Finally, phagocytic activity was measured using both flow cytometry and fluorescence microscopy assays. Additionally, gene transcription of candidate genes was evaluated by qPCR assays. The results evidenced that exposures to different concentrations of STX (1-100 nM) for 24 h did not affect cell viability, as determined by an MTT assay. However, when hemocytes were exposed for 4 or 16 h to STX (1-100 nM), there was a modulation of phagocytic activity and ROS production. Moreover, hemocytes exposed to 100 nM of STX for 4 or 16 h showed a significant increase in transcript levels of genes encoding for antioxidant enzymes (SOD, CAT), mitochondrial enzymes (COI, COIII, CYTB, ATP6, ND1) and ion channels (K+, Ca2+). Meanwhile, C-type lectin and toll-like receptor genes revealed a bi-phase transcriptional response after 16 and 24-48 h of exposure to STX. These results suggest that STX can negatively affect the immunocompetence of M. chilensis hemocytes, which were capable of responding to STX exposure in vitro by increasing the mRNA levels of antioxidant enzymes.
Assuntos
Hemócitos/efeitos dos fármacos , Mytilus/efeitos dos fármacos , Fagocitose , Venenos/farmacologia , Saxitoxina/farmacologia , Transcriptoma , Animais , Hemócitos/imunologia , Hemócitos/metabolismo , Mytilus/imunologia , Mytilus/metabolismo , Estresse Oxidativo , Venenos/toxicidade , Saxitoxina/toxicidade , Transcrição GênicaRESUMO
Perkinsus genus includes protozoan parasites of marine mollusks, especially bivalves. In the last four years, this parasite has been detected in mangrove oysters Crassostrea rhizophorae and Crassostrea gasar from the Northeastern region of Brazil. Hemocytes are the key cells of the oyster immune system, being responsible for a variety of cellular and humoral reactions, such as phagocytosis, encapsulation and the release of several effector molecules that control the invasion and proliferation of microorganisms. In Brazil, there is little information on perkinsosis and none on the immune responses of native oysters' species against Perkinsus spp. The objective of this study was to determine the effects of natural infection by Perkinsus sp. on the immunological parameters of mangrove oysters C. gasar cultured in the Mamanguape River Estuary (Paraíba, Brazil). Adults oysters (N = 40/month) were sampled in December 2011, March, May, August and October 2012. Gills were removed and used to determine the presence and intensity of the Perkinsus sp. infection, according to a scale of four levels (1-4), using the Ray's fluid thioglycollate medium assay. Immunological parameters were measured in hemolymph samples by flow cytometry, including: total hemocyte count (THC), differential hemocyte count (DHC), cell mortality, phagocytic capacity, and production of Reactive Oxygen Species (ROS). The plasma was used to determine the hemagglutination activity. The results showed the occurrence of Perkinsus sp. with the highest mean prevalence (93.3%) seen so far in oyster populations in Brazil. Despite that, no oyster mortality was associated. In contrast, we observed an increase in hemocyte mortality and a suppression of two of the main defense mechanisms, phagocytosis and ROS production in infected oysters. The increase in the percentage of blast-like cells on the hemolymph, and the increase in THC in oysters heavily infected (at the maximum intensity, 4) suggest an induction of hemocytes proliferation. The immunological parameters varied over the studied months, which may be attributed to the dynamics of infection by Perkinsus sp. The results of the present study demonstrate that Perkinsus sp. has a deleterious effect on C. gasar immune system, mainly in high intensities, which likely renders oysters more susceptible to other pathogens and diseases.