In vitro effects of the harmful benthic dinoflagellates Prorocentrum hoffmannianum and Ostreopsis cf. ovata on immune responses of the farmed oyster Crassostrea gasar.

Oyster culture is a sustainable solution to food production. However, this activity can be severely impacted by the presence and proliferation of harmful microalgae such as the benthic dinoflagellates Prorocentrum hoffmannianum and Ostreopsis cf. ovata. This study aimed to evaluate the in vitro effects of P. hoffmannianum and O. cf. ovata on immune system cells (hemocytes) of the native cultured oyster Crassostrea gasar. The direct toxicity of both dinoflagellates was first evaluated assessing hemocyte viability exposed to eight concentrations of each HAB species. No reduction in hemocyte viability was found with the exposure to cell culture or the crude extract of P. hoffmannianum, but O. cf. ovata culture induced hemocyte death in a concentration-dependent manner. Ostreopsis cf. ovata concentration that promoted half of maximal reduction in hemocyte viability (EC50) was 779 cells mL-1. Posteriorly, hemocytes were exposed to both dinoflagellate cells and crude extracts to investigate their effects on hemocyte functional parameters. Despite no direct toxicity of the dinoflagellate cells, P. hoffmannianum extract caused a threefold increase in ROS production and decreased the phagocytosis rate by less than half. Ostreopsis cf. ovata cells and crude extracts also triggered an increase in ROS production (two-fold), but the phagocytosis rate was reduced (by half) only in response to the two lower cell concentrations. These results indicate a harmful potential of both dinoflagellates through a direct toxicity (only for O. cf. ovata) and functional impairment of hemocytes (both species) which could expose C. gasar oyster to opportunistic infections.

New phosphodiesterase-4 inhibitors present airways relaxant activity in a guinea pig acute asthma model.

Asthma is a disease characterized by chronic inflammation and hyper responsiveness of airways. We aimed to assess the relaxant potential of phosphodiesterase-4 (PDE4) inhibitors N-sulfonilhidrazonic derivatives on non-asthmatic and asthmatic guinea pig trachea. Firstly, guinea pigs were sensitized and challenged with ovalbumin, and then morphological, and contractile changes were evaluated resulting from asthma, followed by evaluation of relaxant effect of derivatives on guinea pig trachea and the cAMP levels measurement by ELISA. It has been evidenced hypertrophy of airway smooth muscle, inflammatory infiltrate, and vascular abnormalities. Moreover, only sensitized tracheal rings were responsive to OVA. Contractile response to histamine, but not to carbachol, was greater in sensitized animals, however the relaxant response to aminophylline and isoprenaline were the same in non-asthmatics and asthmatics. N-sulfonilhidrazonic derivatives presented equipotent relaxant action independent of epithelium, with exception of LASSBio-1850 that presented a low efficacy (< 50%) and LASSBio-1847 with a 4-fold higher potency on asthmatics. LASSBio-1847 relaxant curve was impaired in the presence of propranolol and potentiated by isoprenaline in both groups. Furthermore, relaxation was potentiated 54- and 4-fold by forskolin in non-asthmatics and asthmatics, respectively. Likewise, LASSBio-1847 potentiated relaxant curve of aminophylline 147- and 4-fold in both groups. The PKA inhibitor H-89 impaired the relaxant potency of the derivative. Finally, LASSBio-1847 increased tracheal intracellular cAMP levels similarly to rolipram, selective PDE4 inhibitor, in both animals. LASSBio-1847 showed to be promising to relax guinea pig trachea from non-sensitized and sensitized guinea pigs by activation of ß2-adrenergic receptors/AC/cAMP pathway.

Supplementation with Spirulina platensis Prevents Damage to Rat Erections in a Model of Erectile Dysfunction Promoted by Hypercaloric Diet-Induced Obesity.

Erectile dysfunction (ED) is the inability to achieve and/or maintain a penile erection sufficient for sexual satisfaction. Currently, many patients do not respond to the pharmacotherapy. The effects of a supplementation with Spirulina platensis, were evaluated in a model of ED induced by hypercaloric diet consumption. Wistar rats were divided into groups fed with standard diet (SD) or hypercaloric diet (HD) and supplemented with this alga at doses of 25, 50 or 100 mg/kg. Experimental adiposity parameters and erectile function were analyzed. In SD groups, Spirulina platensis reduced food intake, final body mass and adiposity index, and increased the total antioxidant capacity (TAC) of adipose tissue. However, no change was observed in erectile function. In the HD group, without Spirulina supplementation, a decrease in food intake was observed, in addition to an increase of final body mass, weight gain, adipose reserves, and adiposity index. Additionally, reduction in the number and increase in the latency of penile erection and adipose malondialdehyde levels, as well as a reduction in TCA was noted. Furthermore, cavernous contractility was increased, and the relaxing response was decreased. Interestingly, these deleterious effects were prevented by the algae at doses of 25, 50 and/or 100 mg/kg. Therefore, the supplementation with S. platensis prevents damages associated to a hypercaloric diet consumption and emerges as an adjuvant the prevention of ED.

Exposure to 2,4-D herbicide induces hepatotoxicity in zebrafish larvae.

2,4-Dichlorophenoxyacetic acid (2,4-D) herbicide is the main ingredient in over 1500 commercially available products such as Weedestroy® AM40 and DMA® 4 IVM. Although the liver has been identified as one of the organs that are affected by this herbicide, reports on its hepatotoxic effects available in the literature are restricted to rats. Thus, there is a gap in information on other organisms that may be vulnerable to 2,4-D exposure, such as fish. Therefore, the present work aimed to assess the hepatotoxic potential of 2,4-D in fish using zebrafish (Danio rerio) larvae as a model system. For this purpose, its acute toxicity to zebrafish embryos was assessed, as well as its sublethal effects (< LC50) on the activity of enzymes related to oxidative (GST, CAT and GPX) and metabolic (LDH) stress and liver parameters (AST, ALT and ALP) after 48 h of exposure. Morphological analyses of the liver were also assessed in zebrafish larvae. As a result, 2,4-D reduced larvae survival (LC50 15.010 mg/L in 96 h of exposure), induced malformations, altered the activity of LDH, GST and CAT enzymes and significantly increased the activity of all biomarkers for liver damage. Although no changes in the color or size of larval liver were observed, histopathological analysis revealed that treatment with 2,4-D caused severe changes in liver tissue, such as vacuolization of the cytosol, eccentric cell nucleus, loss of tissue architecture and cellular boundaries. Thus, the results showed that 2,4-D altered the enzymatic profile related to oxidative stress, and induces liver damage.

Effects of the toxic dinoflagellates Prorocentrum lima and Ostreopsis cf. ovata on immune responses of cultured oysters Crassostrea gasar.

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.

Disseminated neoplasia in cultured Crassostrea gasar oysters from northeast Brazil.

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 (3.9 × 106 cells mL-1) were higher in the affected oysters than in unaffected oysters (268 A.U. and 1.5 × 106 cells 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.

A Guinea Pig Model of Airway Smooth Muscle Hyperreactivity Induced by Chronic Allergic Lung Inflammation: Contribution of Epithelium and Oxidative Stress.

Asthma is a heterogeneous disease of the airways characterized by chronic inflammation associated with bronchial and smooth muscle hyperresponsiveness. Currently, different murine models for the study of asthma show poor bronchial hyperresponsiveness due to a scarcity of smooth muscle and large airways, resulting in a failure to reproduce smooth muscle hyperreactivity. Thus, we aimed to standardize a guinea pig model of chronic allergic lung inflammation mimicking airway smooth muscle hyperreactivity observed in asthmatics (Asth). Animals were randomly divided into a control group (Ctrl), which received saline (0.9% NaCl), and the Asth group, subjected to in vivo sensitization with ovalbumin (OVA) nebulization. Morphological analysis was performed by hematoxylin-eosin staining. Bronchial hyperresponsiveness was evaluated by nebulization time in the fifth, sixth, and seventh inhalations (NT5-7) and tracheal isometric contractions were assessed by force transducer. Total antioxidant capacity was measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and protein expression by Western blot. Histologically, the Asth group developed peribronchial cellular infiltrate, epithelial hyperplasia and smooth muscle thickening. After the fourth nebulization, the Asth group developed bronchial hyperreactivity. The trachea from the Asth group contracted after in vitro stimulation with OVA, differing from the Ctrl group, which showed no response. Additionally, airway smooth muscle hyperreactivity to carbachol and histamine was observed in the Asth group only in intact epithelium preparations, but not to KCl, and this effect was associated with an augmented production of reactive oxygen species. Moreover, lung inflammation impaired the relaxant potency of isoproterenol only in intact epithelium preparations, without interfering with nifedipine, and it was found to be produced by transforming growth factor-ß negative modulation of ß adrenergic receptors and, furthermore, big-conductance Ca2+-sensitive K+ channels. These effects were also associated with increased levels of phosphatidylinositol 3-kinases but not extracellular signal-regulated kinases 1/2 or phosphorylation, and augmented α-actin content as well, explaining the increased smooth muscle mass. Furthermore, pulmonary antioxidant capacity was impaired in the Asth group. Therefore, we developed a standardized and easy-to-use, reproducible guinea pig model of lung inflammation that mimics airway smooth muscle hypercontractility, facilitating the investigation of the mechanisms of bronchial hyperresponsiveness in asthma and new therapeutic alternatives.

ABCB1 and ABCC1-like transporters in immune system cells from sea urchins Echinometra lucunter and Echinus esculentus and oysters Crassostrea gasar and Crassostrea gigas.

ABC transporters activity and expression have been associated with the multixenobiotic resistance phenotype (MXR). The activity of these proteins leads to a reduction in the intracellular concentration of several xenobiotics, thus reducing their toxicity. However, little attention has been given to the expression of ABC transporters in marine invertebrates and few studies have investigated their role in immune system cells of sea urchins and shellfish bivalves. The aim of the present study was to investigate the activity of the ABC transporters ABCB1 and ABCC1 in immune system cells of sea urchins (coelomocytes) and oysters (hemocytes) from different climatic regions (Brazil and France). Sea urchins and oysters were collected at Paraíba coast; Brazil (Echinometra lucunter and Crassostrea gasar) and Rade of Brest; France (Echinus esculentus and Crassostrea gigas). Coelomocytes and hemocytes were stained with the ABC transporter substrate calcein-AM and dye accumulation analyzed under flow cytometry. Reversin 205 (ABCB1 transporter blocker) and MK571 (ABCC1 transporter blocker) were used as pharmacological tools to investigate ABC transporter activity. A different pattern of calcein accumulation was observed in coelomocytes: phagocytes > colorless spherulocytes > vibrate cells > red spherulocytes. The treatment with MK571 increased calcein fluorescence levels in coelomocytes from both species. However, reversin 205 treatment was not able to increase calcein fluorescence in E. esculentus coelomocytes. These data suggest that ABCC1-like transporter activity is present in both sea urchin species, but ABCB1-like transporter activity might only be present in E. lucunter coelomocytes. The activity of ABCC1-like transporter was observed in all cell types from both bivalve species. However, reversin 205 only increased calcein accumulation in hyalinocytes of the oyster C. gasar, suggesting the absence of ABCB1-like transporter activity in all other cell types, including hyalinocytes from the oyster C. gigas. Additionally, our results showed that C. gigas exhibited higher activity of ABCC1-like transporter in all hemocyte types than C. gasar. The present work is the first to characterize ABCB1 and ABCC1-like transporter activity in the immune system cells of sea urchins E. lucunter and E. esculentus and oysters. Our findings encourage the performing studies regarding ABC transporters activity/expression in immune system cells form marine invertebrates under stress conditions and the possible use of ABC transporters as biomarkers.

Effects of cyanobacteria Synechocystis spp. in the host-parasite model Crassostrea gasar-Perkinsus marinus.

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.

Epizootiology of Perkinsus sp. inCrassostrea gasar oysters in polyculture with shrimps in northeastern Brazil.

Bivalve culture is of considerable economic and social interest in northeastern (NE) Brazil. The polyculture is an alternative approach to traditional monoculture for reducing the environmental impact of shrimp farming and improving oyster culture. Perkinsus marinus and Perkinsus olseni were found infecting oysters in NE Brazil and can threaten oyster production. This study evaluated Perkinsus spp. occurrence in Crassostrea gasar during all production stages. Oyster spats were produced in a hatchery and grown in shrimp ponds in Rio Grande do Norte state. Perkinsus spp. were surveyed by Ray's fluid thioglycollate medium and confirmed by polymerase chain reaction. Prevalence and intensity of infection were determined in oysters until they reached 7 cm. Results showed that the broodstock was already infected by Perkinsus (60%), but the derived spats were Perkinsus-free. Oyster spats acquired Perkinsus infection when transferred to ponds. The prevalence gradually increased in the seven months following placement in ponds (73%), and then decreased to 17% by the tenth month. The infections were initially mild, but intensity increased at the final growth stage. In conclusion, it is possible to produce Perkinsus-free C. gasar oyster spats from infected broodstock, and their culture in shrimp ponds is feasible.

Epizootiology of Perkinsus sp. inCrassostrea gasar oysters in polyculture with shrimps in northeastern Brazil / Epizootiologia de Perkinsus sp. em ostrasCrassostrea gasar em policultivo com camarões no Nordeste do Brasil

Abstract Bivalve culture is of considerable economic and social interest in northeastern (NE) Brazil. The polyculture is an alternative approach to traditional monoculture for reducing the environmental impact of shrimp farming and improving oyster culture. Perkinsus marinus andPerkinsus olseni were found infecting oysters in NE Brazil and can threaten oyster production. This study evaluatedPerkinsus spp. occurrence in Crassostrea gasar during all production stages. Oyster spats were produced in a hatchery and grown in shrimp ponds in Rio Grande do Norte state.Perkinsus spp. were surveyed by Ray’s fluid thioglycollate medium and confirmed by polymerase chain reaction. Prevalence and intensity of infection were determined in oysters until they reached 7 cm. Results showed that the broodstock was already infected by Perkinsus (60%), but the derived spats were Perkinsus-free. Oyster spats acquired Perkinsus infection when transferred to ponds. The prevalence gradually increased in the seven months following placement in ponds (73%), and then decreased to 17% by the tenth month. The infections were initially mild, but intensity increased at the final growth stage. In conclusion, it is possible to produce Perkinsus-free C. gasar oyster spats from infected broodstock, and their culture in shrimp ponds is feasible.

Resumo O cultivo de bivalves é de grande interesse econômico e social no Nordeste (NE) do Brasil. O policultivo é uma alternativa ao monocultivo tradicional de camarões para reduzir o impacto ambiental e melhorar a produção de ostras.Perkinsus marinus e Perkinsus olseni foram identificados infectando ostras no Nordeste do Brasil e representam uma ameaça a produção de ostras. Este estudo avaliou a ocorrência dePerkinsus spp. em Crassostrea gasardurante todas as fases de produção. Sementes de ostras foram produzidas em laboratório e cultivadas em viveiros de camarão no Rio Grande do Norte.Perkinsus spp. foram diagnosticados ​​com o uso do meio de tioglicolato fluido de Ray e confirmado por reação em cadeia da polimerase. A prevalência e intensidade de infecção foram determinadas em ostras até atingirem 7 cm. Os resultados mostraram que os reprodutores encontravam-se infectados porPerkinsus (60%), mas as sementes produzidas estavam livres de Perkinsus. As sementes adquiriram a infecção porPerkinsus quando transferidas para os viveiros. A prevalência aumentou gradualmente nos sete meses após a colocação nos viveiros (73%) e, em seguida, diminuiu para 17% até o décimo mês. As infecções foram inicialmente leves, mas aumentaram até a fase final do crescimento. Em conclusão, é possível produzir sementes de ostras C. gasarlivres de Perkinsus a partir de reprodutores infectados e seu cultivo em viveiros de camarão é viável.

Effects of salinity and temperature on in vitro cell cycle and proliferation of Perkinsus marinus from Brazil.

Field and in vitro studies have shown that high salinities and temperatures promote the proliferation and dissemination of Perkinsus marinus in several environments. In Brazil, the parasite infects native oysters Crassostrea gasar and Crassostrea rhizophorae in the Northeast (NE), where the temperature is high throughout the year. Despite the high prevalence of Perkinsus spp. infection in oysters from the NE of Brazil, no mortality events were reported by oyster farmers to date. The present study evaluated the effects of salinity (5, 20 and 35 psu) and temperature (15, 25 and 35 °C) on in vitro proliferation of P. marinus isolated from a host (C. rhizophorae) in Brazil, for a period of up to 15 days and after the return to the control conditions (22 days; recovery). Different cellular parameters (changes of cell phase's composition, cell density, viability and production of reactive oxygen species) were analysed using flow cytometry. The results indicate that the P. marinus isolate was sensitive to the extreme salinities and temperatures analysed. Only the highest temperature caused lasting cell damage under prolonged exposure, impairing P. marinus recovery, which is likely to be associated with oxidative stress. These findings will contribute to the understanding of the dynamics of perkinsiosis in tropical regions.

Chronic aerobic swimming exercise promotes functional and morphological changes in rat ileum.

Several studies have reported the gastrointestinal (GI) effects promoted by the physical exercise. Thus, we aimed to evaluate the influence of swimming exercise on the contractile reactivity, lipid peroxidation and morphology of rat ileum. Wistar rats were divided into sedentary (SED) and groups exercised for two (EX2), four (EX4), six (EX6) or eight (EX8) weeks, 5 days/week. Animals were killed; the ileum was removed and suspended in organ baths where the isotonic contractions were recorded. Lipid peroxidation was evaluated by MDA (malondialdehyde) measurement with TBARS (thiobarbituric acid reactive substances) assay and morphology by histological staining. Cumulative concentration-response curves to KCl were attenuated, as the Emax values were changed from 100% (SED) to 63.1±3.9 (EX2), 48.8±3.8 (EX4), 19.4±1.8 (EX6) and 59.4±2.8% (EX8). Similarly, cumulative concentration-response curves to carbamylcholine hydrochloride (CCh) were attenuated, as the Emax values were changed from 100% (SED) to 74.1±5.4 (EX2), 75.9±5.2 (EX4) and 62.9±4.6 (EX6), but not in the EX8 (89.7±3.4%). However, CCh potency was increased in this latter, as the EC50 was altered from 1.0±0.1×10(-6) (SED) to 2.1±0.4×10(-7) (EX8). MDA concentration was altered only in EX4 (44.3±4.4) compared with SED (20.6±3.6 µmol/l). Circular layer was reduced in SED when compared with the exercised groups. Conversely, longitudinal layer was increased. In conclusion, chronic swimming exercise reduces the ileum contraction, equilibrates the oxidative damage and promotes changes in tissue size to establish an adaptation to the exercise.

Parasites infecting the cultured oyster Crassostrea gasar (Adanson, 1757) in Northeast Brazil.

The oyster Crassostrea gasar is a species widely used as food and a source of income for the local population of the estuaries of Northeast Brazil. Perkinsus marinus and Perkinsus olseni are deleterious parasites for oyster farming and were recently detected in Brazil. In this study, a histopathologic survey of the oyster C. gasar cultured in the estuary of the River Mamanguape (Paraíba State) was performed. Adult oysters were collected in December 2011 and March, May, August and October 2012 and processed for histology and Perkinsus sp. identification by molecular analyses. Histopathological analysis revealed the presence of parasitic organisms including viral gametocytic hypertrophy, prokaryote-like colonies, protozoans (Perkinsus sp. and Nematopsis sp.) and metazoans (Tylocephalum sp. and cestodes). Other commensal organisms were also detected (the protozoan Ancistrocoma sp. and the turbellarian Urastoma sp.). The protozoan parasite Perkinsus sp. had the highest overall prevalence among the symbiotic organisms studied (48.9%), followed by Nematopsis sp. (36.3%). The other organisms were only sporadically observed. Only the protozoan Perkinsus sp. caused alterations in the oysters' infected organs. Molecular analyses confirmed the presence of P. marinus, P. olseni and Perkinsus beihaiensis infecting the oyster C. gasar. This is the first report of P. beihaiensis in this oyster species.

Immunological responses of the mangrove oysters Crassostrea gasar naturally infected by Perkinsus sp. in the Mamanguape Estuary, Paraíba state (Northeastern, Brazil).

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.

Perkinsus sp. infecting oyster Crassostrea rhizophorae (Guilding, 1828) on the coast of Bahia, Brazil.

This study investigated the occurrence of the protozoan Perkinsus in the oyster Crassostrea rhizophorae on the coast of Bahia State, Brazil. The oysters (n = 900) were collected in February-March and July-August 2010. The Ray's fluid thioglycollate medium (RFTM) analysis of gills and rectum revealed hypnospores of Perkinsus sp. with a high mean prevalence (63%). The infection intensity varied from very light to advanced. The polymerase chain reaction confirmed Perkinsus in 87.2% of the RFTM-positive oysters. Histological analysis showed trophozoites and schizonts phagocytized by hemocytes, mainly in the intestine and the stomach epithelium.