Application of multiplex real-time polymerase chain reaction assay for simultaneous quantification of Escherichia coli virulence genes in oysters.

Strains of diarrheagenic Escherichia coli (DEC) are involved in foodborne disease outbreaks worldwide, especially the enterohemorrhagic E. coli O157:H7. This study describes two multiplex quantitative real time PCR (qPCR) assays for simultaneous identification and quantification of genes related to virulence of DEC; a triplex reaction for detection and quantification of stxA1, stxA2, and eaeA genes, and a duplex reaction for detection and quantification of eaeA and virA genes. The technique was applied in raw oyster samples for direct quantification of DEC, thereby evaluating the applicability of this methodology for microbiological quality assessment of food. Using custom designed primers and specific MGB probes, a triplex qPCR assay was performed to quantify stxA1, stxA2, and eaeA, and a duplex reaction was performed to quantify virA and eaeA genes. The assays showed high sensitivity, with the detection limit varying between 5 and 17 copies of the genes. The coefficient of determination (R2) of the standard curves was 0.99. The coefficient of variation was < 1% indicated high intra- and inter-assay reproducibilities. The application of this methodology in oyster samples from tropical environment provided direct quantitative data that determined the presence of the genes stxA1 (32.1%), eaeA (28.6%), stxA2 (3.6%), and virA (3.6%). This would prove critical for immediate intervention of control strategies, particularly in oysters that are often ingested as raw food.

First record of Perkinsus chesapeaki infecting Crassostrea rhizophorae in South America.

This study investigated Perkinsus spp. infecting Crassostrea rhizophorae from the Jaguaribe River estuary, Ceará, Brazil. Fragments of gills and rectum of the oysters (n=150) were incubated in Ray's fluid thioglycollate medium (RFTM). Genus Perkinsus-specific PerkITS85/750 PCR assays were performed and their amplicons were sequenced by the Sanger method. The RFTM assays confirmed Perkinsus spp. The sequencing of the amplified fragments from the rDNA internal transcribed spacers (ITS) of Perkinsus spp. confirmed Perkinsus chesapeaki. Neighbor-Joining analyzes place P. chesapeaki identified in this study in a well-supported clade with other isolates of the same species. This is the first record of P. chesapeaki infecting C. rhizophorae in South America.

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.

[Reproductive cycle of the mangrove oyster Crassostrea rhizophorae (Bivalvia: Ostreidae) in Camamu Bay, Bahia, Brasil]. / Ciclo reproductivo del ostión de manglar Crassostrea rhizophorae (Bivalvia: Ostreidae) en la Bahía de Camamu, Bahia, Brasil.

The mangrove oyster Crassostrea rhizophorae is important fishery resource along the entire Brasilian coast with excellent potential for marine culture. The purpose of this paper was to examine the reproductive characteristics of the oyster of the Maraú river estuary in Camamu Bay, Bahia, Brasil. The samples were collected monthly, from September 2006 to August 2007, at two points (I and II) in Camamu Bay. At each site 20 oysters were collected for histological analysis, fixed in Davidson's solution, embedded in paraffin, dehydrated in an ethanol series, sectioned (7 microm thick) and stained with Harris hematoxylin and Eosin (HE). Additionally, 30 oysters were sampled, at each point, for a condition index analysis. The water temperature ranged from 23.5 degrees C to 30 degrees C and the salinity from 15 to 25 ups at Point I (Maraú) and from 25 to 35 at Point II (Tanque Island). The oyster's height ranged from 30 to 92 mm at Point I and from 27 to 102 mm at Point II, with an average of 49.0 mm +/- 9.1 (n = 230) and 49.9 mm +/- 9.9 (n = 237), respectively. Among the sampled oysters at Point I, 59.1% were females, 31.3% males, 1.3% hermaphrodites and 8.2% of the oysters of undetermined sex. At Point II, 66.2% were females, 30.4% males, 0.8% hermaphrodites and 2.5% (n = 237) of undetermined sex. The gonadic stage analysis indicated that the reproduction period of the C. rhizophorae in the Maraú Peninsula was continuous all year, without any regressive phase. The condition index (R) ranged from 8.0% to 17.7%. The peak periods of R coincided with the expressive oyster's percentage in the maturation and liberation gametic stages. The results of these findings will contribute information for the oyster spat collection and to the process installation of the oyster culture in Camamu Bay.

Parasites of three commercially exploited bivalve mollusc species of the estuarine region of the Cachoeira river (Ilhéus, Bahia, Brazil).

This paper reports the parasites found in three commercially exploited bivalve molluscs (Mytella guyanensis, Anomalocardia brasiliana and Iphigenia brasiliana) of an estuarine region of Ilhéus, south of Bahia, Brazil (14 degrees 48'23''S; 39 degrees 02'47''W). Samples of 20 individuals of each species were collected fortnightly from August 2005 to August 2006. A total of 1480 individuals was collected and processed by standard histologic techniques; the histologic sections were stained with Harris haematoxylin and eosin and examined with light microscope. The water temperature in the study area varied from 24 to 30.5 degrees C and the salinity from 0 to 23ppt. Remarkable differences were found in the parasitic community between the three mollusc species involved in the study, which occupied different habitats in the estuarine region of the Cachoeira river. The following parasites were found: intracellular rickettsia-like colonies in digestive epithelia; intracellular gregarine Nematopsis sp. in gills, mantle, gonad, digestive gland and foot muscle; sporocysts of a Bucephalidae trematode in gonads, mantle, gills, digestive gland and foot; unidentified digenetic metacercariae in digestive gland and gonad; metacestodes of Tylocephalum sp. in connective tissue in the digestive gland and in gonad; and an unidentified metazoan in mantle and intestinal lumen. No significant temporal variation in the prevalence of any parasite was detected, which could be due to the narrow temperature range of the region and the absence of patterns of salinity and rainfall variation through the year. The infestation by sporocyst was the only pathological threat detected for the studied populations because of its potential for host castration. The low infection intensity and/or prevalence of the other parasites and the lack of obvious lesions suggest that there is no other serious pathological risk for the studied mollusc populations.

Microbiological Quality and Prevalence of ß-Lactam Antibiotic Resistance Genes in Oysters ( Crassostrea rhizophorae ).

The microbiological quality of oysters reflects the microbiological quality of their habitats because they are filter feeders. The objective of this study was to assess the bacterial composition of the edible oyster Crassostrea rhizophorae in urban and preserved estuaries. Particularly, we assessed the presence of pathogenic bacteria, investigated antibiotic susceptibility in bacterial isolates, and quantified ß-lactam antibiotic resistance genes (blaTEM, blaSHV, and blaKPC) via quantitative PCR of oyster DNA. Our results detected total coliforms, Escherichia coli , and enterobacteria in the oysters from urban estuaries, which is indicative of poor water quality. In addition, our detection of the eaeA and stxA2 virulence genes in 16.7% of E. coli isolates from oysters from this region suggests the presence of multiantibiotic-resistant enteropathogenic and enterohemorrhagic E. coli strains. During periods of low precipitation, increased contamination by E. coli (in winter) and Vibrio parahaemolyticus (in autumn) was observed. In contrast, cultivated oysters inhabiting monitored farms in preserved areas had low levels of bacterial contamination, emphasizing that oyster culture monitoring enhances food quality and makes oysters fit for human consumption. Distinct antibiotic resistance profiles were observed in bacteria isolated from oysters collected from different areas, including resistance to ß-lactam antibiotics. The presence of the blaTEM gene in 91.3% of oyster samples indicated that microorganisms in estuarine water conferred the capability to produce ß-lactamase. To our knowledge, this is the first study to directly quantify and detect ß-lactam antibiotic resistance genes in oysters. We believe our study provides baseline data for bacterial dynamics in estuarine oysters; such knowledge contributes to developing risk assessments to determine the associated hazards and consequences of consuming oysters from aquatic environments containing pathogenic bacteria that may possess antibiotic resistance genes.

Parasites in the mangrove oyster Crassostrea rhizophorae cultivated in the estuary of the Graciosa River in Taperoá, Bahia.

In this work, parasites associated with the mangrove oyster Crassostrea rhizophorae were studied at a cultivation unit in the estuary of the Graciosa River in Taperoá, Bahia. On a monthly basis, between April 2011 and March 2012, 20 oysters were collected, measured and fixed in 10% formaldehyde. In order to identify the parasites, routine histological techniques were used. The specimens were embedded in paraffin and sections, 7 µm thick, were cut. These were then stained with Harris hematoxylin and eosin and were examined using an optical microscope. The parameters analyzed to investigate water quality showed that the temperature ranged from 23.9 °C to 29.3 °C, water salinity from 0.4 to 24.2 Practical Salinity Units PSU and recorded rainfall from 80 mm to 406.4 mm/month. During the parasitological analyses, infestation of the polychaete Neanthes succinea was observed in 41% of the oysters. Through histological techniques, the following parasites were identified: colonies of Rickettsia-like organisms (RLOs); the protozoa Nematopsis sp. and Ancistrocoma sp.; the turbellarian Urastoma sp.; the metacestode Tylocephalum sp. and an unidentified tapeworm. The data collected showed that there was low-intensity infection with and prevalence of parasites, except for Nematopsis sp., thus attesting that these cultivated oysters were in a healthy condition.

Molecular identification of cryptic species of oysters (Genus Crassostrea Sacco, 1897) in the northeast Atlantic coast of Brazil / Identificação molecular de espécies crípticas de ostras (Gênero Crassostrea Sacco, 1897) na Costa Atlântica Nordeste do Brasil

Oysters of the genus Crassostrea Sacco, 1897 are widely distributed worldwide, being important extractive and cultivation resources in Brazil. Because they have high phenotypical plasticity and congeneric similarity, identifications based on shell morphology are not always safe. The goal of this study was to identify the oysters of the Bahia State, northeast Brazil, using the molecular tools Polymerase Chain Reaction, Restriction Fragment Length Polymorphism, DNA sequencing and phylogenetic analysis. Oysters were collected at 12 sampling stations, from October 2014 to March 2015 and included samples of rhizomes (aerial roots)/stems of the red mangrove Rhizophorae mangle L. and in the sediment near to the underground roots of this one, on berths, natural rock outcrops near the mangrove swamp and in three oyster crops. It was confirmed the presence of two species of oysters: Crassostrea rhizophorae (Guilding, 1828) and C. gasar (Deshayes, 1830) and that the latter was genetically identical to C. brasiliana reported in previous studies on the Brazilian coast. There was no co-occurrence of the two species on the same substrate, but these were found in nearby environments at two sampling points. Crassostrea rhizophorae was observed on the rhizomes/stems of R. mangle, as well as on artificial concrete walls (berths). The semi-buried oysters near R. mangles subterranean roots and adhered to small rocks of a rocky outcrop were C.gasar, which was also the exclusive oyster of the crops.(AU)

As ostras do gênero Crassostrea Sacco, 1897 são amplamente distribuídas mundialmente, sendo importantes recursos extrativistas e de cultivo no Brasil. Por possuírem alta plasticidade fenotípica e semelhança congenérica, as identificações baseadas na morfologia da concha nem sempre são seguras. O objetivo deste estudo foi identificar as ostras do estado da Bahia, nordeste do Brasil, utilizando as ferramentas moleculares Reação em Cadeia da Polimerase, Polimorfismo do Comprimento de Fragmentos de Restrição, sequenciamento de DNA e análise filogenética. As ostras foram coletadas em 12 estações amostrais, de outubro de 2014 a março de 2015 e incluíram coletas sobre rizomas (raízes aéreas)/caules do mangue vermelho Rhizophorae mangle L. e no sedimento próximo às raízes subterrâneas deste, em atracadouros, afloramentos rochosos naturais próximos ao manguezal e em três cultivos de ostras. Confirmou-se a presença de duas espécies de ostras: Crassostrea rhizophorae (Guilding, 1828) e C. gasar (Deshayes, 1830) e que esta última foi geneticamente idêntica à C. brasiliana, relatada em estudos anteriores na costa brasileira. Não houve co-ocorrência das duas espécies no mesmo substrato, mas em dois pontos amostrais estas foram encontradas em ambientes próximos. Crassostrea rhizophorae foi observada nos rizomas/caules de R. mangle, bem como em paredes artificiais de concreto (atracadouros). As ostras semi-enterradas perto das raízes subterrâneas de R. mangle e aderidas a pequenas rochas de um afloramento rochoso foram C. gasar, que também foi a ostra exclusiva nos cultivos.(AU)

Molecular identification of cryptic species of oysters (Genus Crassostrea Sacco, 1897) in the northeast Atlantic coast of Brazil / Identificação molecular de espécies crípticas de ostras (Gênero Crassostrea Sacco, 1897) na Costa Atlântica Nordeste do Brasil

Oysters of the genus Crassostrea Sacco, 1897 are widely distributed worldwide, being important extractive and cultivation resources in Brazil. Because they have high phenotypical plasticity and congeneric similarity, identifications based on shell morphology are not always safe. The goal of this study was to identify the oysters of the Bahia State, northeast Brazil, using the molecular tools Polymerase Chain Reaction, Restriction Fragment Length Polymorphism, DNA sequencing and phylogenetic analysis. Oysters were collected at 12 sampling stations, from October 2014 to March 2015 and included samples of rhizomes (aerial roots)/stems of the red mangrove Rhizophorae mangle L. and in the sediment near to the underground roots of this one, on berths, natural rock outcrops near the mangrove swamp and in three oyster crops. It was confirmed the presence of two species of oysters: Crassostrea rhizophorae (Guilding, 1828) and C. gasar (Deshayes, 1830) and that the latter was genetically identical to C. brasiliana reported in previous studies on the Brazilian coast. There was no co-occurrence of the two species on the same substrate, but these were found in nearby environments at two sampling points. Crassostrea rhizophorae was observed on the rhizomes/stems of R. mangle, as well as on artificial concrete walls (berths). The semi-buried oysters near R. mangle’s subterranean roots and adhered to small rocks of a rocky outcrop were C.gasar, which was also the exclusive oyster of the crops.

As ostras do gênero Crassostrea Sacco, 1897 são amplamente distribuídas mundialmente, sendo importantes recursos extrativistas e de cultivo no Brasil. Por possuírem alta plasticidade fenotípica e semelhança congenérica, as identificações baseadas na morfologia da concha nem sempre são seguras. O objetivo deste estudo foi identificar as ostras do estado da Bahia, nordeste do Brasil, utilizando as ferramentas moleculares Reação em Cadeia da Polimerase, Polimorfismo do Comprimento de Fragmentos de Restrição, sequenciamento de DNA e análise filogenética. As ostras foram coletadas em 12 estações amostrais, de outubro de 2014 a março de 2015 e incluíram coletas sobre rizomas (raízes aéreas)/caules do mangue vermelho Rhizophorae mangle L. e no sedimento próximo às raízes subterrâneas deste, em atracadouros, afloramentos rochosos naturais próximos ao manguezal e em três cultivos de ostras. Confirmou-se a presença de duas espécies de ostras: Crassostrea rhizophorae (Guilding, 1828) e C. gasar (Deshayes, 1830) e que esta última foi geneticamente idêntica à C. brasiliana, relatada em estudos anteriores na costa brasileira. Não houve co-ocorrência das duas espécies no mesmo substrato, mas em dois pontos amostrais estas foram encontradas em ambientes próximos. Crassostrea rhizophorae foi observada nos rizomas/caules de R. mangle, bem como em paredes artificiais de concreto (atracadouros). As ostras semi-enterradas perto das raízes subterrâneas de R. mangle e aderidas a pequenas rochas de um afloramento rochoso foram C. gasar, que também foi a ostra exclusiva nos cultivos.

Efficiency of anesthetics on female gastropod Littoraria angulifera / Eficiência de anestésicos em fêmeas do gastrópode Littoraria angulifera

The present study evaluated the effect of three anesthetics at three concentrations, menthol (C10H20O) (1%, 1.5%, 2%), eugenol (C10H12O2) (0.5%, 1%, 1.5%) and magnesium chloride (MgCl2) (4%, 7%, 10%) diluted in distilled water, on the gastropod Littoraria angulifera. Twenty females were tested for each concentration with shell height varying between 18.3 and 26.1 mm (n = 180), all collected from the same site in the estuary of Barra do Sargi-Uruçuca, Bahia (Brazil). The time of induction and recovery of the sedative effect, as well as the survival rate, were assessed. The animals were kept at a temperature of 25 ± 1 °C and observations were made every 30 minutes since the beginning of the anesthesia for up to 2 hours. After this period, the animals were observed during the following 15 days for survival evaluation. Only MgCl2 caused a sedative effect on L. angulifera. At 4% and 7% MgCl2 solutions, 20% and 95% of animals were anesthetized after 2 hours, respectively, and at 10% all animals were anesthetized after 1 hour. In conclusion, L. angulifera can be efficiently anesthetized in a 10% MgCl2 solution for 1 h and the recovery occurs after 1 h with no apparent side effects on behavior and survival.

O presente estudo avaliou o efeito de três anestésicos em três concentrações, o mentol (C10H20O) (1%; 1,5%; 2%), o eugenol (C10H12O2) (0,5%; 1%; 1,5%) e o cloreto de magnésio (MgCl2) (4%; 7%; 10%), diluídos em água destilada, sobre fêmeas do gastrópode Littoraria angulifera. Para cada concentração foram utilizadas 20 fêmeas (total = 180), com altura da concha entre 18,3 e 26,1 mm, coletadas no mesmo local, no estuário da Barra do Sargi-Uruçuca, Bahia. Foram observados os tempos de indução e de recuperação do efeito sedativo, assim como a sobrevivência. Os animais foram mantidos a uma temperatura laboratorial de 25 ± 1 °C e as observações foram feitas a cada 30 minutos após a anestesia, durante 2 horas. Após o término do experimento, os animais foram observados por 15 dias quanto à sobrevivência. Apenas o MgCl2 causou efeito sedativo sobre L. angulifera, sendo que a 4% e a 7% observou-se sedação de 20% e de 95% dos animais, respectivamente, após 2 horas de anestesia, enquanto que a 10%, todos os animais foram anestesiados em 1 hora. Em conclusão, L. angulifera pode ser anestesiada de forma eficiente em uma solução de 10% de MgCl2 durante 1 h e a sua recuperação ocorre ao fim de 1 h sem nenhum efeito colateral evidente no comportamento e na sobrevivência.

Efficiency of anesthetics on female gastropod Littoraria angulifera / Eficiência de anestésicos em fêmeas do gastrópode Littoraria angulifera

The present study evaluated the effect of three anesthetics at three concentrations, menthol (C10H20O) (1%, 1.5%, 2%), eugenol (C10H12O2) (0.5%, 1%, 1.5%) and magnesium chloride (MgCl2) (4%, 7%, 10%) diluted in distilled water, on the gastropod Littoraria angulifera. Twenty females were tested for each concentration with shell height varying between 18.3 and 26.1 mm (n = 180), all collected from the same site in the estuary of Barra do Sargi-Uruçuca, Bahia (Brazil). The time of induction and recovery of the sedative effect, as well as the survival rate, were assessed. The animals were kept at a temperature of 25 ± 1 °C and observations were made every 30 minutes since the beginning of the anesthesia for up to 2 hours. After this period, the animals were observed during the following 15 days for survival evaluation. Only MgCl2 caused a sedative effect on L. angulifera. At 4% and 7% MgCl2 solutions, 20% and 95% of animals were anesthetized after 2 hours, respectively, and at 10% all animals were anesthetized after 1 hour. In conclusion, L. angulifera can be efficiently anesthetized in a 10% MgCl2 solution for 1 h and the recovery occurs after 1 h with no apparent side effects on behavior and survival.(AU)

O presente estudo avaliou o efeito de três anestésicos em três concentrações, o mentol (C10H20O) (1%; 1,5%; 2%), o eugenol (C10H12O2) (0,5%; 1%; 1,5%) e o cloreto de magnésio (MgCl2) (4%; 7%; 10%), diluídos em água destilada, sobre fêmeas do gastrópode Littoraria angulifera. Para cada concentração foram utilizadas 20 fêmeas (total = 180), com altura da concha entre 18,3 e 26,1 mm, coletadas no mesmo local, no estuário da Barra do Sargi-Uruçuca, Bahia. Foram observados os tempos de indução e de recuperação do efeito sedativo, assim como a sobrevivência. Os animais foram mantidos a uma temperatura laboratorial de 25 ± 1 °C e as observações foram feitas a cada 30 minutos após a anestesia, durante 2 horas. Após o término do experimento, os animais foram observados por 15 dias quanto à sobrevivência. Apenas o MgCl2 causou efeito sedativo sobre L. angulifera, sendo que a 4% e a 7% observou-se sedação de 20% e de 95% dos animais, respectivamente, após 2 horas de anestesia, enquanto que a 10%, todos os animais foram anestesiados em 1 hora. Em conclusão, L. angulifera pode ser anestesiada de forma eficiente em uma solução de 10% de MgCl2 durante 1 h e a sua recuperação ocorre ao fim de 1 h sem nenhum efeito colateral evidente no comportamento e na sobrevivência.(AU)

Health assessment of the oyster Crassostrea rhizophorae on the southern coast of Bahia, northeastern Brazil.

This study investigated the health of natural stocks of the oyster Crassostrea rhizophorae on the southern coast of Bahia in northeastern Brazil, during summer and winter 2010, at three localities (sampling points) in the estuaries of the Maraú (Camamu Bay) and Graciosa rivers. A total of 180 oysters (30/sampling point/season) were examined macroscopically for the presence of pathogens and anatomical changes. The specimens were subsequently fixed in Davidson solution, processed for paraffin embedding, sectioned and stained with Harris' hematoxylin and eosin. Histological analysis revealed the presence of Rickettsia-like organisms (RLOs), Ancistrocoma, Trichodina, Sphenophrya, Nematopsis, Urastoma, Bucephalus in the sporocyst phase, a nonspecific metacercaria, and a metacestode of genus Tylocephalum. The prevalence of infection was low except for parasitism by Nematopsis sp. which also caused histopathological changes. The presence of Bucephalus sp. caused parasitic castration. These two pathogens significantly affect the health of C. rhizophorae.

Parasitism by Nematopsis sp. (Apicomplexa: Eugregarinida) in Mytella guyanensis at the Marine Extractive Reserve Baía do Iguape, Bahia, Brazil / Parasitismo por Nematopsis sp. (Apicomplexa: Eugregarinida) em Mytella guyanensis na Reserva Extrativista Marinha Baía do Iguape, Bahia, Brasil

Several studies show the presence of protozoa of the genus Nematopsis Schneider, 1892, in many species of bivalves. The pathogenicity of this Apicomplexa is still much debated, which is possibly related to the parasitism degree and host habitat. In this context, this study investigated parasitism by Nematopsis sp. in Mytella guyanensis (Bivalvia: Mytilidae) in a mangrove of the Marine Extractive Reserve Baía do Iguape, Bahia, Brazil. The collections were made monthly, from March 2014 to March 2015, consisting of 30 adults with shell height above 40 mm per month, totaling 360 specimens. The specimens were measured, weighed, opened, and macroscopically examined for identifying parasites and/or signs of morphological changes. After that, they were fixed in Davidsons solution and processed according to classical histological techniques, with inclusion in paraffin, obtaining 5 m-thick sections by microtomy, and stained by Harris Hematoxylin and Eosin (HE). The water temperature ranged from 25.5 to 33.6 ºC and the salinity from 21.1 to 34.3 Practical Salinity Units (PSU). The specimens measured between 41.1 and 68.6 mm and had an average weight of 7.24 g. Macroscopically, there was no presence of the parasite or any morphological changes related its presence. The frequency percentage of Nematopsis sp. was 99.45%. The mantle was the organ with the highest frequency of infection (46

Diversos estudos evidenciam a presença de protozoários do gênero Nematopsis Schneider, 1892 em várias espécies de bivalves. A patogenicidade desse apicomplexo ainda é bastante debatida, a qual possivelmente relaciona-se ao grau de parasitismo e habitat do hospedeiro. Nesse contexto, este estudo investigou o parasitismo por Nematopsis sp. em Mytella guyanensis (Bivalvia: Mytilidae) em um manguezal da Reserva Extrativista Marinha Baía do Iguape, Bahia, Brasil. As coletas foram efetuadas mensalmente, de março de 2014 a março de 2015, constando 30 adultos com altura da concha acima de 40 mm, totalizando 360 espécimens. Os espécimens foram medidos, pesados, abertos e examinados macroscopicamente para a identificação de parasitos e/ou sinais de alterações morfológicas, em seguida fixados em solução de Davidson e processados segundo técnica histológica clássica, com inclusão em parafina, obtenção de cortes de 5 m de espessura por microtomia e coloração por hematoxilina de Harris e Eosina (HE). A temperatura da água variou de 25,5 a 33,6 ºC e a salinidade de 21,1 a 34,3 Unidades Práticas de Salinidade (UPS). Os espécimens mediram entre 41,1 e 68,6 mm e tiveram um peso médio de 7,24 g. Macroscopicamente, não se observou presença do parasito ou qualquer alteração morfológica relacionada à presença do mesmo. A frequência percentual de Nematopsis sp. foi de 99,45%. O manto foi o órgão de

Parasitism by Nematopsis sp. (Apicomplexa: Eugregarinida) in Mytella guyanensis at the Marine Extractive Reserve Baía do Iguape, Bahia, Brazil / Parasitismo por Nematopsis sp. (Apicomplexa: Eugregarinida) em Mytella guyanensis na Reserva Extrativista Marinha Baía do Iguape, Bahia, Brasil

Several studies show the presence of protozoa of the genus Nematopsis Schneider, 1892, in many species of bivalves. The pathogenicity of this Apicomplexa is still much debated, which is possibly related to the parasitism degree and host habitat. In this context, this study investigated parasitism by Nematopsis sp. in Mytella guyanensis (Bivalvia: Mytilidae) in a mangrove of the Marine Extractive Reserve Baía do Iguape, Bahia, Brazil. The collections were made monthly, from March 2014 to March 2015, consisting of 30 adults with shell height above 40 mm per month, totaling 360 specimens. The specimens were measured, weighed, opened, and macroscopically examined for identifying parasites and/or signs of morphological changes. After that, they were fixed in Davidson's solution and processed according to classical histological techniques, with inclusion in paraffin, obtaining 5 µm-thick sections by microtomy, and stained by Harris' Hematoxylin and Eosin (HE). The water temperature ranged from 25.5 to 33.6 ºC and the salinity from 21.1 to 34.3 Practical Salinity Units (PSU). The specimens measured between 41.1 and 68.6 mm and had an average weight of 7.24 g. Macroscopically, there was no presence of the parasite or any morphological changes related its presence. The frequency percentage of Nematopsis sp. was 99.45%. The mantle was the organ with the highest frequency of infection (46.26%), followed by the gonads (18.36%). Among the analyzed organs, all presented the parasite and, in a few severely infected cases, microscopical changes were observed in the mantle conformation. The infection intensity for most of the sessions analyzed was 1 to 3 oocysts/phagocyte; however, in one of the sessions, 9 oocysts/phagocytes were recorded. The parasite had a negative correlation with salinity. The high frequency of Nematopsis sp. should be considered for the sustainability of natural stocks, especially if such parasitism occurs simultaneously with other pathogens and stressful environmental conditions.(AU)

Diversos estudos evidenciam a presença de protozoários do gênero Nematopsis Schneider, 1892 em várias espécies de bivalves. A patogenicidade desse apicomplexo ainda é bastante debatida, a qual possivelmente relaciona-se ao grau de parasitismo e habitat do hospedeiro. Nesse contexto, este estudo investigou o parasitismo por Nematopsis sp. em Mytellaguyanensis(Bivalvia: Mytilidae) em um manguezal da Reserva Extrativista Marinha Baía do Iguape, Bahia, Brasil. As coletas foram efetuadas mensalmente, de março de 2014 a março de 2015, constando 30 adultos com altura da concha acima de 40 mm, totalizando 360 espécimens. Os espécimens foram medidos, pesados, abertos e examinados macroscopicamente para a identificação de parasitos e/ou sinais de alterações morfológicas, em seguida fixados em solução de Davidson e processados segundo técnica histológica clássica, com inclusão em parafina, obtenção de cortes de 5 µm de espessura por microtomia e coloração por hematoxilina de Harris e Eosina (HE). A temperatura da água variou de 25,5 a 33,6 ºC e a salinidade de 21,1 a 34,3 Unidades Práticas de Salinidade (UPS). Os espécimens mediram entre 41,1 e 68,6 mm e tiveram um peso médio de 7,24g. Macroscopicamente, não se observou presença do parasito ou qualquer alteração morfológica relacionada à presença do mesmo. A frequência percentual de Nematopsis sp. foi de 99,45%. O manto foi o órgão de maior frequência de infecção (46,26%), seguido das gônadas (18,36%). Entre os órgãos analisados, todos apresentaram o parasito, sendo que, para poucos casos severamente infectados, observaram-se microscopicamente modificações na conformação do manto. A intensidade de infecção para a maioria das sessões analisadas foi de 1 a 3 oocistos/fagócitos, contudo em uma das sessões foram registrados 9 oocistos/fagócitos. O parasito apresentou correlação negativa com a salinidade. A elevada frequência do Nematopsis sp. deve ser levada em consideração para a sustentabilidade dos estoques naturais, principalmente se tal parasitismo ocorrer simultaneamente a outros patógenos e condições ambientais estressantes.(AU)

Parasitism by Nematopsis sp. (Apicomplexa: Eugregarinida) in Mytella guyanensis at the Marine Extractive Reserve Baía do Iguape, Bahia, Brazil / Parasitismo por Nematopsis sp. (Apicomplexa: Eugregarinida) em Mytella guyanensis na Reserva Extrativista Marinha Baía do Iguape, Bahia, Brasil

Several studies show the presence of protozoa of the genus Nematopsis Schneider, 1892, in many species of bivalves. The pathogenicity of this Apicomplexa is still much debated, which is possibly related to the parasitism degree and host habitat. In this context, this study investigated parasitism by Nematopsis sp. in Mytella guyanensis (Bivalvia: Mytilidae) in a mangrove of the Marine Extractive Reserve Baía do Iguape, Bahia, Brazil. The collections were made monthly, from March 2014 to March 2015, consisting of 30 adults with shell height above 40 mm per month, totaling 360 specimens. The specimens were measured, weighed, opened, and macroscopically examined for identifying parasites and/or signs of morphological changes. After that, they were fixed in Davidsons solution and processed according to classical histological techniques, with inclusion in paraffin, obtaining 5 m-thick sections by microtomy, and stained by Harris Hematoxylin and Eosin (HE). The water temperature ranged from 25.5 to 33.6 ºC and the salinity from 21.1 to 34.3 Practical Salinity Units (PSU). The specimens measured between 41.1 and 68.6 mm and had an average weight of 7.24 g. Macroscopically, there was no presence of the parasite or any morphological changes related its presence. The frequency percentage of Nematopsis sp. was 99.45%. The mantle was the organ with the highest frequency of infection(AU)

Diversos estudos evidenciam a presença de protozoários do gênero Nematopsis Schneider, 1892 em várias espécies de bivalves. A patogenicidade desse apicomplexo ainda é bastante debatida, a qual possivelmente relaciona-se ao grau de parasitismo e habitat do hospedeiro. Nesse contexto, este estudo investigou o parasitismo por Nematopsis sp. em Mytella guyanensis (Bivalvia: Mytilidae) em um manguezal da Reserva Extrativista Marinha Baía do Iguape, Bahia, Brasil. As coletas foram efetuadas mensalmente, de março de 2014 a março de 2015, constando 30 adultos com altura da concha acima de 40 mm, totalizando 360 espécimens. Os espécimens foram medidos, pesados, abertos e examinados macroscopicamente para a identificação de parasitos e/ou sinais de alterações morfológicas, em seguida fixados em solução de Davidson e processados segundo técnica histológica clássica, com inclusão em parafina, obtenção de cortes de 5 m de espessura por microtomia e coloração por hematoxilina de Harris e Eosina (HE). A temperatura da água variou de 25,5 a 33,6 ºC e a salinidade de 21,1 a 34,3 Unidades Práticas de Salinidade (UPS). Os espécimens mediram entre 41,1 e 68,6 mm e tiveram um peso médio de 7,24 g. Macroscopicamente, não se observou presença do parasito ou qualquer alteração morfológica relacionada à presença do mesmo. A frequência percentual de Nematopsis sp. foi de 99,45%. O manto foi o órgão(AU)

Parasites of economically important bivalves from the southern coast of Bahia State, Brazil.

This study investigated the parasites of three commercially important bivalve species (Crassostrea rhizophorae, Mytella guyanensis and Lucina pectinata) from the southern coast of Bahia, Brazil. A total of 540 specimens were collected in August 2009 and February 2010, at three localities. The bivalve specimens were measured on their longest axis, opened, and macroscopically examined for the presence of parasites or signs of disease. They were then fixed in Davidson' solution and subjected to routine histological processing, with paraffin embedding and H&E staining; next, the specimens were examined under a light microscope. No parasites were observed associated with L. pectinata. Rickettsia-like organisms (RLOs), Sphenophrya sp. (Ciliophora), Nematopsis sp. (Apicomplexa), Urastoma sp. (Turbellaria) and Bucephalus sp. (Digenea) were observed in both C. rhizophorae and M. guyanensis, as well as Ancistrocoma sp. (Ciliophora) and Tylocephalum sp. (Cestoda) in the former. A high prevalence of Nematopsis sp. was seen, but caused no apparent damage to the host. Bucephalus sp. caused the destruction of tissues, with castration, but showed low prevalence. The other parasites occurred in low prevalence and intensity, without causing significant damage.

Perkinsus beihaiensis infecting the oyster Crassostrea rhizophorae under cultivation and in natural stock in Camamu Bay, Bahia, Brazil / Perkinsus beihaiensis infectando a ostra Crassostrea rhizophorae em cultivo e em estoque natural na Baía de Camamu, Bahia, Brasil

This study investigated the infection of Perkinsus beihaiensis (Perkinsozoa) in the oyster Crassostrea rhizophorae, both from a long-line cultivation system and from a nearby intertidal zone of mangrove, both in the state of Bahia, northeastern Brazil. The collections were performed in October and November 2012, and in January 2013. The oysters (n = 300) were measured, examined macroscopically for signs of infection and then submitted to the following laboratory techniques: histology, Ray"s fluid thioglycollate medium assay (RFTM), polymerase chain reaction (PCR) and sequencing, which confirmed the identification of the pathogen. Histological and RFTM analyses showed, respectively, a mean prevalence of 93.3% and of 69%. The infection was usually mild or very mild. There was no significant difference (p > 0.05) between the environments in terms of infection prevalence or severity. This is the first record of P. beihaiensis in the state of Bahia and the second in oysters from Brazil and South America(AU)

Foi investigada a infecção de Perkinsus beihaiensis (Perkinsozoa) na ostra Crassostrea rhizophorae em um sistema de cultivo do tipo espinhel e em um estoque natural de ostras no manguezal adjacente ambos localizados no estado da Bahia, Nordeste do Brasil. As colheitas foram realizadas em outubro e novembro de 2012 e em janeiro de 2013. As ostras (n = 300) foram medidas, examinadas macroscopicamente quanto a sinais da infecção e submetidas às técnicas laboratoriais: histologia, ensaio em meio de cultivo de tioglicolato de Ray (RFTM), reação em cadeia da polimerase (PCR) e sequenciamento, que confirmou a identificação do patógeno. As análises histológicas e o RFTM mostraram, respectivamente, prevalência média de 93,3% e de 69%. A infecção foi geralmente leve ou muito leve. Não houve diferença significativa (p > 0,05) entre os ambientes em termos de prevalência ou severidade da infecção. Este é o primeiro registro de P. beihaiensis no estado da Bahia e o segundo em ostras do Brasil e América do Sul(AU)

Perkinsus beihaiensis infecting the oyster Crassostrea rhizophorae under cultivation and in natural stock in Camamu Bay, Bahia, Brazil / Perkinsus beihaiensis infectando a ostra Crassostrea rhizophorae em cultivo e em estoque natural na Baía de Camamu, Bahia, Brasil

This study investigated the infection of Perkinsus beihaiensis (Perkinsozoa) in the oyster Crassostrea rhizophorae, both from a long-line cultivation system and from a nearby intertidal zone of mangrove, both in the state of Bahia, northeastern Brazil. The collections were performed in October and November 2012, and in January 2013. The oysters (n = 300) were measured, examined macroscopically for signs of infection and then submitted to the following laboratory techniques: histology, Ray"s fluid thioglycollate medium assay (RFTM), polymerase chain reaction (PCR) and sequencing, which confirmed the identification of the pathogen. Histological and RFTM analyses showed, respectively, a mean prevalence of 93.3% and of 69%. The infection was usually mild or very mild. There was no significant difference (p > 0.05) between the environments in terms of infection prevalence or severity. This is the first record of P. beihaiensis in the state of Bahia and the second in oysters from Brazil and South America...

Foi investigada a infecção de Perkinsus beihaiensis (Perkinsozoa) na ostra Crassostrea rhizophorae em um sistema de cultivo do tipo espinhel e em um estoque natural de ostras no manguezal adjacente ambos localizados no estado da Bahia, Nordeste do Brasil. As colheitas foram realizadas em outubro e novembro de 2012 e em janeiro de 2013. As ostras (n = 300) foram medidas, examinadas macroscopicamente quanto a sinais da infecção e submetidas às técnicas laboratoriais: histologia, ensaio em meio de cultivo de tioglicolato de Ray (RFTM), reação em cadeia da polimerase (PCR) e sequenciamento, que confirmou a identificação do patógeno. As análises histológicas e o RFTM mostraram, respectivamente, prevalência média de 93,3% e de 69%. A infecção foi geralmente leve ou muito leve. Não houve diferença significativa (p > 0,05) entre os ambientes em termos de prevalência ou severidade da infecção. Este é o primeiro registro de P. beihaiensis no estado da Bahia e o segundo em ostras do Brasil e América do Sul...

Detection of Perkinsus marinus in the oyster Crassostrea rhizophorae in southern Bahia by proteomic analysis / Detecção de Perkinsus marinus na ostra Crassostrea rhizophorae do sul da Bahia por análise proteômica

This study reports the presence of the pathogen Perkinsus marinus, notifiable to the World Organization for Animal Health (Office International des Èpizooties = OIE) in the oyster Crassostrea rhizophorae in southern Bahia via proteomic analysis. We analyzed Crassostrea brasiliana from a long-line cultivation system and C. rhizophorae from an adjacent mangrove in Porto do Campo, Camamu Bay, Bahia, Brazil. The collections (n = 100) were performed in October 2012. In the laboratory, the oysters were measured and opened to remove the meat, which was steeped in dry ice. For extraction of proteins, adaptation of a protocol used for mussels was used, after which separation in the first dimension was taken by isoelectric focusing (IEF). The peptides were transferred to a Mass Spectrometer. The obtained spectra were analyzed with the ProteinLynx Global Server 4.2 software tool and also by MASCOT (Matrix Science) and compared to the databases of the SWISSPROT and NCBI, respectively. The identification was evidenced by beta-tubulin, Perkinsus marinus ATCC 50983 and protein homology code in the database NCBI = gi / 294889481. This is the first record of P. marinus in Bahia and the fourth in Brazil.

Este estudo relata a presença do patógeno Perkinsus marinus, de notificação obrigatória à Organização Internacional de Epizootias (OIE) na ostra Crassostrea rhizophorae no sul da Bahia, via análise proteômica. Foram analisadas as ostras Crassostrea brasiliana de um cultivo em espinhel e C. rhizophorae de um manguezal adjacente, na localidade de Porto do Campo, Baía de Camamu, Bahia. As coletas (n = 100) foram efetuadas em outubro de 2012. Em laboratório, as ostras foram medidas e abertas para a retirada da carne, que foi macerada em gelo seco. Para a extração das proteínas, foi adotada a adaptação de um protocolo utilizado para mexilhões, após o que foi realizada a separação na primeira dimensão, por focalização isoelétrica (IEF). Os peptídeos foram transferidos para um Espectrômetro de Massas. Os espectros obtidos foram analisados no software ProteinLynx Global Server 4.2 e também pela ferramenta MASCOT (Matrix Science) e comparados com os bancos de dados do SWISSPROT e do NCBI, respectivamente. A identificação foi evidenciada por meio da beta-tubulina, homologia Perkinsus marinus ATCC 50983 e código da proteína no banco de dados NCBI = gi/294889481. Este é o primeiro registro de P. marinusna Bahia e o quarto no Brasil.

Detection of Perkinsus marinus in the oyster Crassostrea rhizophorae in southern Bahia by proteomic analysis / Detecção de Perkinsus marinus na ostra Crassostrea rhizophorae do sul da Bahia por análise proteômica

This study reports the presence of the pathogen Perkinsus marinus, notifiable to the World Organization for Animal Health (Office International des Èpizooties = OIE) in the oyster Crassostrea rhizophorae in southern Bahia via proteomic analysis. We analyzed Crassostrea brasiliana from a long-line cultivation system and C. rhizophorae from an adjacent mangrove in Porto do Campo, Camamu Bay, Bahia, Brazil. The collections (n = 100) were performed in October 2012. In the laboratory, the oysters were measured and opened to remove the meat, which was steeped in dry ice. For extraction of proteins, adaptation of a protocol used for mussels was used, after which separation in the first dimension was taken by isoelectric focusing (IEF). The peptides were transferred to a Mass Spectrometer. The obtained spectra were analyzed with the ProteinLynx Global Server 4.2 software tool and also by MASCOT (Matrix Science) and compared to the databases of the SWISSPROT and NCBI, respectively. The identification was evidenced by beta-tubulin, Perkinsus marinus ATCC 50983 and protein homology code in the database NCBI = gi | 294889481. This is the first record of P. marinus in Bahia and the fourth in Brazil.(AU)

Este estudo relata a presença do patógeno Perkinsus marinus, de notificação obrigatória à Organização Internacional de Epizootias (OIE) na ostra Crassostrea rhizophorae no sul da Bahia, via análise proteômica. Foram analisadas as ostras Crassostrea brasiliana de um cultivo em espinhel e C. rhizophorae de um manguezal adjacente, na localidade de Porto do Campo, Baía de Camamu, Bahia. As coletas (n = 100) foram efetuadas em outubro de 2012. Em laboratório, as ostras foram medidas e abertas para a retirada da carne, que foi macerada em gelo seco. Para a extração das proteínas, foi adotada a adaptação de um protocolo utilizado para mexilhões, após o que foi realizada a separação na primeira dimensão, por focalização isoelétrica (IEF). Os peptídeos foram transferidos para um Espectrômetro de Massas. Os espectros obtidos foram analisados no software ProteinLynx Global Server 4.2 e também pela ferramenta MASCOT (Matrix Science) e comparados com os bancos de dados do SWISSPROT e do NCBI, respectivamente. A identificação foi evidenciada por meio da beta-tubulina, homologia Perkinsus marinus ATCC 50983 e código da proteína no banco de dados NCBI = gi|294889481. Este é o primeiro registro de P. marinus na Bahia e o quarto no Brasil.(AU)