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.

Morphological and functional characterization of the oyster Crassostrea gasar circulating hemocytes: Cell types and phagocytosis activity.

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.

Reproduction and population dynamics of the invasive bivalves Mytilopsis sallei and Isognomon bicolor on the Northeast coast of Brazil.

Knowing the reproductive biology and population dynamics of invasive species are essential for environmental conservation and protection of native species. The success of these invasive species is directly linked to their reproductive strategy. Therefore, this study aimed to describe the reproductive cycles and evaluate population parameters of the invasive bivalves Mytilopsis sallei and Isognomon bicolor, and to estimate if those characteristics would favor their population growths in the northeast coast of Brazil. The bivalves were sampled monthly from June 2016 to May 2017, respectively from the Sanhauá River estuary and Jacarapé beach, State of Paraíba, Northeast Brazil. Through histological analyses, reproductive parameters were determined in order to identify sex, gonadal development, minimum size at maturity, and mean gonadal index. The asymptotic growth (L∞) and growth rate (K) parameters were estimated using the von Bertalanffy growth curve, and recruitment patterns and cohorts were projected based on shell length frequency distributions. Mytilopsis sallei presented more than 50% spawning individuals in most months, while animals showing gametogenic gonads were predominant during the season of greatest precipitation. Isognomon bicolor had ripe gonads (about 30%) and spawning individuals (more than 40%) in all months of the year, but unlike M. sallei, it had the highest concentration of ripe individuals in the months of greatest precipitation. Both species showed equal and high growth rates (K = 1.1 yr-¹) and analysis of the cohorts indicated that these populations are established and expanding. The results confirmed the great invasive potential of the two species in their local environments (estuary and marine) in Northeast Brazil and, therefore, their harmful potential for the conservation of native species and the environment in the invaded areas.

Apoptotic and antioxidant effects in HCT-116 colorectal carcinoma cells by a spiro-acridine compound, AMTAC-06.

BACKGROUND: Acridine compounds have been described as promising anticancer agents. Previous studies showed that (E)-1'-((4-chlorobenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-06), a spiro-acridine compound, has antitumor activity on Ehrlich tumor and low toxicity. Herein, we investigated its antitumor effect against human cells in vitro. METHODS: MTT assay was used to assess cytotoxicity of AMTAC-06 (3.125-200 µM) against tumor and non-tumor cells, and the half-maximal inhibitory concentration (IC50) and the selectivity index (SI) were calculated. The effects on the cell cycle (propidium iodide-PI-staining), apoptosis (Annexin V-FITC/PI double staining by flow cytometry), and production of reactive oxygen species, ROS (DCFH assay) were also evaluated. Statistical analysis was achieved using ANOVA followed by Tukey's post-test. RESULTS: AMTAC-06 showed higher cytotoxicity against colorectal carcinoma HCT-116 cells (IC50: 12.62 µM). The SI showed that AMTAC-06 was more selective for HCT-116 cells (HaCaT SI: 1.41; PBMC SI: 0.62) than doxorubicin (HaCaT SI: 0.10; PBMC SI: 0.01). AMTAC-06 (15 and 30 µM) induced an increase in the sub-G1 peak (p < 0.000001) and cell cycle arrest in S phase (p = 0.003547). Moreover, treatment with this compound (15 and 30 µM) resulted in increased early (p < 0.000001) and late apoptotic cells (p < 0.000001). In addition, there was a reduction on ROS production (p < 0.000001). CONCLUSIONS: AMTAC-06 presents anticancer activity against HCT-116 cells by regulating the cell cycle, inducing apoptosis and an antioxidant action.

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.

Anticancer Effect of a Spiro-acridine Compound Involves Immunomodulatory and Anti-angiogenic Actions.

BACKGROUND/AIM: Studies with acridine compounds have reported anticancer effects. Herein, we evaluated the toxicity and antitumor effect of the (E)-1'-((4-chlorobenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-06), a promising anticancer spiro-acridine compound. MATERIALS AND METHODS: The toxicity of AMTAC-06 was evaluated on zebrafish and mice. Antitumor activity was assessed in Ehrlich ascites carcinoma model. Effects on angiogenesis, cytokine levels and cell cycle were also investigated. RESULTS: AMTAC-06 did not induce toxicity on zebrafish and mice (LD50 approximately 5000 mg/kg, intraperitoneally). No genotoxicity was observed on micronucleus assay. AMTAC-06 significantly reduced the total viable Ehrlich tumor cells and increased sub-G1 peak, suggesting apoptosis was triggered. Moreover, the compound significantly decreased the density of peritumoral microvessels, indicating an anti-angiogenic action, possibly dependent on the cytokine modulation (TNF-α, IL-1ß and IFN-γ). No significant toxicological effects were recorded for AMTAC-06 on tumor transplanted animals. CONCLUSION: AMTAC-06 has low toxicity and a significant antitumor activity.

Antitumor Effect of a Novel Spiro-Acridine Compound is Associated with Up-Regulation of Th1-Type Responses and Antiangiogenic Action.

Tumor cells have specific features, including angiogenesis induction, cell cycle dysregulation, and immune destruction evasion. By inducing a T helper type 2 (Th2) immune response, tumor cells may favor immune tolerance within the tumor, which allows progression of cancer growth. Drugs with potential antitumor activity are the spiro-acridines, which is a promising new class of acridine compounds. Herein, the novel spiro-acridine (E)-5'-oxo-1'-((3,4,5-trimethoxybenzylidene)amino)-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-17) was synthesized and tested for antitumor effects. Toxicity evaluation was performed in mice after acute treatment (2000 mg/kg, intraperitoneally, i.p.). The Ehrlich ascites carcinoma model was used to investigate the antitumor activity of AMTAC-17 (12.5, 25, or 50 mg/kg, i.p.) after seven days of treatment. Effects on the cell cycle, angiogenesis, and inflammatory responses were investigated. LD50 (lethal dose 50%) was estimated to be higher than 5000 mg/kg. AMTAC-17 reduced the Ehrlich tumor's total viable cancer cells count and peritumoral micro-vessels density, and induced an increase in the sub-G1 peak. Additionally, there was an increase of Th1 cytokine profile levels (IL-1ß, TNF-α, and IL-12). In conclusion, the spiro-acridine compound AMTAC-17 presents low toxicity, and its in vivo antitumor effect involves modulation of the immune system to a cytotoxic Th1 profile and a reduction of tumor angiogenesis.

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.

Effect of antiprotozoal molecules on hypnospores of Perkinsus spp. parasite.

Perkinsus protozoan parasites have been associated with high mortality of bivalves worldwide, including Brazil. The use of antiproliferative drugs to treat the Perkinsosis is an unusual prophylactic strategy. However, because of their environment impact it could be used to control parasite proliferation in closed system, such as hatchery. This study evaluated the anti-Perkinsus activity potential of synthesized and commercial compounds. Viability of hypnospores of Perkinsus spp. was assessed in vitro. Cells were incubated with three 2-amino-thiophene (6AMD, 6CN, 5CN) and one acylhydrazone derivatives (AMZ-DCL), at the concentrations of 31.25; 62.5; 125; 250 and 500 µM and one commercial chlorinated phenoxy phenol derivative, triclosan (2, 5, 10 and 20 µM), for 24-48 h. Two synthetic molecules (6CN and AMZ-DCL) caused a significant decline (38 and 39%, respectively) in hypnospores viability, at the highest concentration (500 µM), after 48 h. Triclosan was the most cytotoxic compound, causing 100% of mortality at 20 µM after 24 h and at 10 µM after 48 h. Cytotoxic effects of the compounds 6CN, AMZ-DCL, and triclosan were investigated by measuring parasite's zoosporulation, morphological changes and metabolic activities (esterase activity, production of reactive oxygen species and lipid content). Results showed that zoosporulation occurred in few cell. Triclosan caused changes in the morphology of hypnospores. The 6CN and AMZ-DCL did not alter the metabolic activities studied whilst Triclosan significantly increased the production of reactive oxygen species and changed the amount and distribution of lipids in the hypnospores. These results suggest that three compounds had potential to be used as antiprotozoal drugs, although further investigation of their mechanism of action must be enlightened.

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.

Morphological and functional characterization of the hemocytes from the pearl oyster Pteria hirundo and their immune responses against Vibrio infections.

Hemocyte populations of the pearl oyster Pteria hirundo were characterized at morphological, ultrastructural and functional levels. Three main hemocyte populations were identified: hyalinocytes, granulocytes and blast-like cells. Hyalinocytes were the most abundant population (88.2%) characterized by the presence of few or no granules in the cytoplasm and composed by two subpopulations, large and small hyalinocytes. Comparatively, granulocytes represented 2.2% of the hemocyte population and were characterized by the presence of numerous large electron-lucid granules in the cytoplasm. Finally, the blast-like cells (9.5%) were the smallest hemocytes, showing spherical shape and a high nucleus/cytoplasm ratio. Hemocytes exhibited a significant phagocytic capacity for inert particles (38.5%) and showed to be able to produce microbicidal molecules, such as reactive oxygen species (ROS) (ex vivo assays). The immune role of hemocytes was further investigated in the P. hirundo defense against the Gram-negative Vibrio alginolyticus. A significant decrease in the total number of hemocytes was observed at 24 h following injection of V. alginolyticus or sterile seawater (injury control) when compared to naïve (unchallenged) animals, indicating the migration of circulating hemocytes to the sites of infection and tissue damage. Bacterial agglutination was only observed against Gram-negative bacteria (Vibrio) but not against to marine Gram-positive-bacteria. Besides, an increase in the agglutination titer was observed against V. alginolyticus only in animals previously infected with this same bacterial strain. These results suggest that agglutinins or lectin-like molecules may have been produced in response to this particular microorganism promoting a specific recognition. The ultrastructural and functional characterization of P. hirundo hemocytes constitutes a new important piece of the molluscan immunity puzzle that can also contribute for the improvement of bivalve production sustainability.

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.

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.

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.

First report of Perkinsus beihaiensis in wild clams Anomalocardia brasiliana (Bivalvia: Veneridae) in Brazil.

This is the first report of Perkinsus sp. (Bivalvia: Veneridae) infecting wild clams of the species Anomalocardia brasiliana in Brazil. The gill lamellae and rectum of 150 specimens of A. brasiliana collected in the Timonha river estuary (Ceará, Northeastern Brazil) in March 2012 were incubated in Ray's fluid thioglycollate medium (RFTM) for detection of Perkinsus sp. In RFTM, the prevalence of Perkinsus sp. was 14.7% (22/150) and the intensity of infection ranged from very light (1-10 cells across the slide) to light (12-100 cells). The presence of Perkinsus sp. was confirmed by PCR in seven (31.8%) out of 22 RFTM-positive specimens. DNA sequencing confirmed the presence of the genus Perkinsus and the phylogenetic analysis strongly indicated Perkinsus beihaiensis as the species responsible for the infection.

Two Perkinsus spp. infect Crassostrea gasar oysters from cultured and wild populations of the Rio São Francisco estuary, Sergipe, northeastern Brazil.

Brazilian production of bivalve molluscs is small but expanding, especially in the northeastern region where the native oysters Crassostrea rhizophorae and C. gasar are abundant, and tropical weather promotes their rapid growth. Studies on bivalve pathology are scarce in Brazil, with only a few employing techniques for detecting protozoan pathogens listed by the World Organisation for Animal Health (OIE). In 2008, a Perkinsus sp. was reported for the first time in Brazil, infecting C. rhizophorae oysters from a wild population in Ceará state, NE Brazil. Recently P. marinus was detected in the same oyster species in nearby Paraíba state. These findings highlighted the need to expand knowledge on the presence and impacts of Perkinsus spp. on Brazilian oyster populations. The current investigation evaluated Perkinsus sp. infections among wild and cultured C. gasar mangrove oysters from the estuary of the Rio São Francisco, Sergipe state, NE Brazil. Our results show that Perkinsus sp. infections occurred commonly in oysters of both groups, at prevalences that were frequently higher among cultured oysters. Prevalences varied seasonally, with maximum values during summer (January) of 57% and 80% for wild and cultured oysters respectively, and minimum values during winter (July). Results of DNA sequencing, in situ hybridization assays, and phylogenetic analyses showed dual- and single-pathogen infections by P. marinus and/or P. olseni in the tested oysters.

First report of the protozoan parasite Perkinsus marinus in South America, infecting mangrove oysters Crassostrea rhizophorae from the Paraíba River (NE, Brazil).

The present work aimed to study the infection by Perkinsus sp. in the mangrove oysters Crassostrea rhizophorae from the estuary of the Paraíba River (Paraíba State, Brazil). Perkinsosis was detected by incubation of oyster gill pieces in Ray's fluid thioglycollate medium. The monthly prevalence values were all above 70%, thus infection was not likely to be a transient event. Perkinsus sp. parasites isolated from eight oysters were propagated in vitro. PCR-RFLP analysis of in vitro cultured cells as well as the sequences of the rDNA ITS region allowed the identification of the in vitro propagated parasites as Perkinsus marinus. Phylogenetic analyses using rDNA ITS region sequences strongly supported the Perkinsus sp. from Paraíba in a monophyletic group with P. marinus. Thus, the results confirmed the species affiliation of Paraíba Perkinsus sp. as P. marinus. This is the first report of P. marinus in Brazil and South America and the first report of P. marinus naturally infecting C. rhizophorae.