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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

Microbiota, the microorganisms that colonize living organisms. Oysters rely exclusively on an innate immune system. This study characterized the cultivable gastrointestinal tract microbiota (GTM) of the oyster Crassostrea gasar and evaluated their influence on immune responses. Antibiotics were used to reduce or alter the microbiota during in vitro and in vivo assays. Haemocyte viability, concentration, and phagocytic capacity, as well as the production of reactive oxygen species (ROS), were evaluated in antibiotic-treated and control oysters. Microbiological analysis of the gastrointestinal tract was also performed; bacteria were selected using culture media and were identified by 16S ribosomal DNA. The results showed that the antibiotics eliminated bacteria in vitro but increased their concentrations in vivo . The GTM was composed of 26% amylolytic bacteria, 21% lipolytic bacteria, 18% proteolytic bacteria, 18% cellulolytic bacteria and 17% lactic acid bacteria; there were no differences in the amounts of these bacterial types between the control and treated oysters. Three major bacterial phyla, Proteobacteria, Firmicutes and Actinobacteria, and seven genera, Labrenzia , Pseudomonas , Halomonas , Shewanella , Vibrio , Bacillus , and Micrococcus , were detected. The concentration, viability, and phagocytic capacity of hemocytes and the production of ROS were not significantly altered by antibiotic treatment. In conclusion, the antibiotics altered the number of heterotrophic bacteria without changing hemocyte function, suggesting that this assay could be useful for verifying the influence of microbiota in host-parasite interactions.

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.

International audience; 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, Paraiba 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 x 10(6) cells mL(-1)) were higher in the affected oysters than in unaffected oysters (268 A.U. and 1.5 x 10(6) 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.

Few studies have associated the effects of changes in caloric intake and redox disturbances in the gastrointestinal tract. Therefore, the present study aimed at evaluating the hypercaloric diet consumption influence on the contractile reactivity of intestinal smooth muscle, morphology, and oxidative stress of rat ileum. Wistar rats were randomly divided into groups that received a standard diet and fed with a hypercaloric diet for 8 weeks. Animals were euthanized, and the ileum was isolated to isotonic contraction monitoring. Morphology was evaluated by histological staining and oxidative stress by quantification of malondialdehyde levels and total antioxidant activity. Cumulative concentration-response curves to KCl and carbachol were attenuated in rats fed with a hypercaloric diet compared to those that received a standard diet. In addition, an increase in caloric intake promotes a rise in the thickness of the longitudinal smooth muscle layer of rat ileum and tissue malondialdehyde levels, characterizing lipid peroxidation, as well as a decrease in the antioxidant activity. Thus, it was concluded that the consumption of a hypercaloric diet impairs rat intestinal contractility due to mechanisms involving modifications in the intestinal smooth muscle architecture triggered by redox disturbances.

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 (

Asthma is a chronic inflammatory disease of the airways characterized by immune cell infiltrates, bronchial hyperresponsiveness, and declining lung function. Thus, the possible effects of virgin coconut oil on a chronic allergic lung inflammation model were evaluated. Morphology of lung and airway tissue exhibited peribronchial inflammatory infiltrate, epithelial hyperplasia, and smooth muscle thickening in guinea pigs submitted to ovalbumin sensitization, which were prevented by virgin coconut oil supplementation. Additionally, in animals with lung inflammation, trachea contracted in response to ovalbumin administration, showed a greater contractile response to carbachol (CCh) and histamine, and these responses were prevented by the virgin coconut oil supplementation. Apocynin, a NADPH oxidase inhibitor, did not reduce the potency of CCh, whereas tempol, a superoxide dismutase mimetic, reduced potency only in nonsensitized animals. Catalase reduced the CCh potency in nonsensitized animals and animals sensitized and treated with coconut oil, indicating the participation of superoxide anion and hydrogen peroxide in the hypercontractility, which was prevented by virgin coconut oil. In the presence of L-NAME, a nitric oxide synthase (NOS) inhibitor, the CCh curve remained unchanged in nonsensitized animals but had increased efficacy and potency in sensitized animals, indicating an inhibition of endothelial NOS but ineffective in inhibiting inducible NOS. In animals sensitized and treated with coconut oil, the CCh curve was not altered, indicating a reduction in the release of NO by inducible NOS. These data were confirmed by peribronchiolar expression analysis of iNOS. The antioxidant capacity was reduced in the lungs of animals with chronic allergic lung inflammation, which was reversed by the coconut oil, and confirmed by analysis of peribronchiolar 8-iso-PGF2α content. Therefore, the virgin coconut oil supplementation reverses peribronchial inflammatory infiltrate, epithelial hyperplasia, smooth muscle thickening, and hypercontractility through oxidative stress and its interactions with the NO pathway.

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.

SUMMARYField 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.

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-β negative modulation of β 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.

SUMMARYThe 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.

International audience; 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.

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.