Qualidade microbiológica da água e dos mexilhões Perna perna (Linnaeus, 1758) cultivados em Piúma, Espírito Santo, Brasil / Microbiological quality of water and Perna perna (Linnaeus, 1758) mussels cultivated in Piúma, Espírito Santo, Brazil

RESUMO Este estudo objetivou verificar a qualidade microbiológica da água e dos mexilhões cultivados pela Associação dos Maricultores de Piúma (AMPI), Espírito Santo, Brasil. Foram realizadas sete coletas de água e mexilhões, mensalmente, entre outubro de 2016 e maio de 2017. Os mexilhões foram coletados nos long lines da AMPI, e em cada mês foram coletados 40 mexilhões Perna perna e 100 mL de água do local. O material coletado foi destinado ao laboratório para a realização das análises microbiológicas em duplicata, número mais provável de coliformes totais (CT) e termotolerantes (Ctt), presença ou ausência de Salmonella sp e número de unidades formadoras de colônias de Staphylococcus aureus. Os resultados mostraram que o número de Ctt nas amostras de água no mês de janeiro estavam acima do permitido pela Resolução n° 357 do Conselho Nacional do Meio Ambiente (CONAMA). Já os níveis de Ctt e Staphylococcus aureus na carne dos mexilhões mostraram-se dentro do limite aceitável pela RDC n° 12 da Agência Nacional de Vigilância Sanitária (ANVISA). Entretanto, foram encontradas bactérias com características do gênero Salmonella sp nos meses de dezembro e março nos mexilhões, impossibilitando sua comercialização e seu consumo. Durante esses meses, a cidade tem alto fluxo de turistas. Por fim, recomenda-se a realização das análises microbiológicas continuamente, principalmente no período do verão, época que tem grande fluxo de turistas no município de Piúma e que registrou presença de Salmonella na carne dos mexilhões e níveis de Ctt na água acima do permitido pelas legislações vigentes.

ABSTRACT This study aimed to verify the microbiological quality of water and mussels cultivated by the Piúma Farmers Association (Associação dos Maricultores de Piúma — AMPI), Espírito Santo, Brazil. Seven samples of water and mussels were collected monthly from October 2016 to May 2017. The mussels were collected from the long lines of the AMPI, and in each month 40 mussels Perna perna and 100 mL of water were sampled from the site. The collected material was sent to the laboratory for duplicate microbiological analysis, Most Probable Number of Total (CT) and Thermotolerant (Ctt) coliforms, presence or absence of Salmonella sp and number of Staphylococcus aureus colony forming units. The results showed that the number of Ctt in the water samples in January was higher than that allowed by Resolution 357 of the National Environment Council (Conselho Nacional do Meio Ambiente — CONAMA). The levels of Ctt and Staphylococcus aureus in mussel meat were within the acceptable range by the Brazilian National Environment Council (Agência Nacional de Vigilância Sanitária — ANVISA) Resolution RDC No. 12. However, colonies with characteristics of Salmonella sp were found in December and March in the mussels, making it impossible to sell and consume. This period coincides with a high flow of tourists in the municipality. Finally, it is recommended to perform microbiological analyzes continuously, especially in the summer, where there are a lot of tourists in the city of Piúma, period that showed the presence of Salmonella in the meat of mussels and Ctt levels in water above the allowed current legislation.

Limnoperna fortunei - Updating the geographic distribution in the Brazilian watersheds and mapping the regional occurrence in the Upper Uruguay River basin / Limnoperna fortunei - Atualização da distribuição geográfica nas bacias hidrográficas brasileiras e mapeamento da ocorrência regional na bacia do Alto Rio Uruguai

Abstract: Limnoperna fortunei is an invasive alien species (IAS) that cause serious ecological and economic problems in Brazilian freshwater environments. Due to its high dispersion capacity and the lack of new records in peer-reviewed journals we carried out an extensive survey to update the distribution of L. fortunei in the Brazilian hydrographic basins. We also performed a detailed investigation of its distribution in the Upper Uruguay River basin using a molecular method. We presented new records, showing the invasion in new basins and a wide distribution in the basins previously infested. Additionally, we confirmed that the Upper Uruguay River is fully colonized by the golden mussel, being distributed in the lentic, lotic, and transitional lotic/lentic environments presented in this region. This update is an important tool for the implementation of guidelines and the development of safety protocols and sanitary barriers to avoid the dispersion of this IAS to new environments..

Resumo: Limnoperna fortunei é uma espécie exótica invasora que causa sérios problemas ecológicos e econômicos em ambientes de água doce do Brasil. Devido à sua elevada capacidade de dispersão e à falta de novos registros em publicações científicas, o objetivo deste estudo foi realizar uma extensa pesquisa para entender e alertar sobre o atual cenário de distribuição de L. fortunei nas bacias hidrográficas brasileiras. Também realizamos uma investigação mais detalhada sobre a distribuição da espécie na bacia do Alto Rio Uruguai, utilizando um método molecular. Apresentamos novos registros de ocorrência da espécie, mostrando a invasão em novas bacias e uma ampla distribuição nas bacias anteriormente infestadas. Além disso, confirmamos que o Alto Rio Uruguai está totalmente colonizado pelo mexilhão-dourado, estando distribuído pelos ambientes lênticos, lóticos e de transição existentes na região. Esta atualização se mostra como uma importante ferramenta para a implementação de diretrizes e o desenvolvimento de protocolos de segurança e barreiras sanitárias para evitar a dispersão desta espécie invasora em novos ambientes.

Genetic diversity and differentiation of nine populations of the hard clam (Meretrix petechialis) assessed by EST-derived microsatellites

BACKGROUND: Meretrix petechialis is one of the commercially important marine bivalves. In this study, we selected six highly polymorphic EST-derived microsatellite markers to assess the genetic diversity and population differentiation on nine wild populations of Meretrix petechialis. RESULTS: The number of alleles detected per-locus ranged from 4 to 30 (mean NA = 27.5) with a total of 165 alleles. The mean value of observed and expected heterozygosities varied from 0.717 to 0.861 and from 0.797 to 0.856, respectively. Meanwhile, the result of Neighbor-joining and overall FST = 0.214 (P < 0.01) reveled that M. petechialis populations from GX are the farthest populations, a certain degree of genetic variation among individuals in each population and the genetic differentiation is significant. CONCLUSIONS: GX population has high genetic diversity among individual, and there are certain differences in genetic characteristics among different populations. This study will provide a basis for the domestication and cultivation of genetic diversity of M. petechialis population and the protection of clam germplasm resources.

Reporte de presuntas hipnosporas de Perkinsus sp, en Megapitaria squalida del Golfo de California con la técnica de tinción con tioglicolato / Report of presumptive Perkinsus sp hypnospores in Megapitaria squalida of the Gulf of California with the thioglycollate staining technique

RESUMEN Objetivo. Detectar la presencia de presuntas hipnosporas del protozoario Perkinsus sp. en una población silvestre de la almeja chocolata mexicana (Megapitaria squalida) del sureste del Golfo de California, usando el medio fluido de tioglicolato de Ray (RFTM). Materiales y métodos. Cada mes durante un ciclo anual, se capturaron 30 especímenes con una longitud entre 56.17 y 69.04 mm. La prevalencia e intensidad de la infección y los parámetros del agua se registraron mensualmente desde septiembre 2012 a septiembre 2013. Resultados. Se detectó la presencia de presuntas hipnosporas de Perkinsus sp. en muestras de tejido de la almeja chocolata mexicana usando la prueba RFTM, por la presencia de corpúsculos redondos y oscuros que representan hipnosporas del parásito. Los muestreos mensuales revelaron un rango de prevalencia e intensidad de la infección de 0-43.33% y 1-4 (infección negativa = 0 hipnosporas/preparación, a moderada = 34 hipnosporas/ preparación), respectivamente. Conclusiones. Perkinsus sp. es reportado por primera vez en una población silvestre de M. squalida en la parte más al sureste del Golfo de California. Los resultados indican que el parásito está disperso intraespecíficamente y, potencialmente, parasitaría un nuevo huésped en la región.

ABSTRACT Objective. To detect the presence of presumptive hypnospores of the protozoan Perkinsus sp. in a wild population of the Mexican chocolata clam Megapitaria squalida in the southeastern Gulf of California, using Ray's fluid thioglycollate medium (RFTM). Material and methods. Thirty specimens with size between 56.17 and 69.04 mm were captured each month, during an annual cycle. Infection prevalence and intensity and water parameters were recorded monthly from September 2012 to September 2013. Results. Perkinsus sp. was detected in tissue samples from the Mexican chocolate clam using the RFTM test by the presence of dark round corpuscles that represent parasite's hypnospores. Monthly samplings revealed a prevalence of 0-43.33% and an infection intensity ranging from 1 to 4 (no infection = 0 hypnospores/entire preparation, to moderate = 34 hypnospores/entire preparation). Conclusions. Perkinsus sp. is reported for the first time in a wild population of M. squalida in the southesternmost Gulf of California. The results indicate that this protozoan is dispersed intraspecifically and would now, potentially, parasiting a new host in the region.

Genetics characterization of fresh water clam Spathopsis rubens using ISSR-PCR markers

Spathopsis rubens is freshwater bivalves distributed in the Nile River and its main canals all over Egypt.Four populations of S. rubens on Al-Mahmoudia irrigation canal at Damnhour, Egypt were collected andinvestigated for morphometric characters and genetic diversity by using inter simple sequence repeats (ISSR).The results declared that both the length and height measurements of the collected samples from the differentlocations showed slight significant differences; however, no significant differences between the total weightand the width of the samples have found. High degree of correlation between temperature and total weight,height and length was reported in samples collected from the third location. Genomic DNA from the selectedsamples of each population was extracted and amplified using 10 ISSR primers. The primers (M2, M3, M8,M12, M17, F2, F4, and F9) showed 100% polymorphism. Unweighted pair group method with arithmetic meandendrogram characterized the samples of S. rubens into two main definite clusters. The cluster of genotypes2 and 3 recorded the highest similarity and distance indices at a distance of 0.60859, while genotypes 1 and 3recorded the lowest similarity and distance indices at a distance of 0.1716.

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)

Perkinsus beihaiensis (Perkinsozoa) in oysters of Bahia State, Brazil / Perkinsus beihaiensis (Perkinsozoa) em ostras do Estado da Bahia

Abstract This study reports the pathogen Perkinsus beihaiensis in oysters of the genus Crassostrea on the coast of the State of Bahia (Brazil), its prevalence, infection intensity and correlation with salinity. Oysters (n = 240) were collected between October and December 2014 at eight sampling stations between latitudes 13°55'S and 15°42'S. The laboratory procedures included macroscopic analysis, histology, culture in Ray's fluid thioglycollate medium (RFTM), Polymerase Chain Reaction (PCR) and DNA sequencing. PCR and sequencing have been used for the genetic identification of oysters as well. Two species of oysters have been identified: Crassostrea rhizophorae and C. brasiliana. In both oyster species P. beihaiensis was the only Perkinsus species detected. In C. rhizophorae, the average prevalence was 82.8% by histology and 65.2% by RFTM. In C. brasiliana, the prevalences were 70.5% and 35.7%, respectively. The higher prevalence of P. beihaiensis in C. rhizophorae was probably influenced by salinity, with which was positively correlated (r> 0.8). In both oysters, P. beihaiensis was located mainly in the gastric epithelium. The infection was generally mild or moderate, without apparent harm to the hosts, but in cases of severe infection, there was hemocytical reaction and tissue disorganization. The generally high prevalence in the region suggests that oysters should be monitored with respect to this pathogen, especially in growing areas.

Resumo Este estudo relata o patógeno Perkinsus beihaiensis em ostras do gênero Crassostrea no litoral do Estado da Bahia (Brasil), sua prevalência, intensidade de infecção e correlação com a salinidade. As ostras (n = 240) foram coletadas entre outubro e dezembro de 2014 em oito estações amostrais entre as latitudes 13°55'S e 15°42'S. Os procedimentos laboratoriais incluíram análise macroscópica, histologia, cultivo em meio de tioglicolato de Ray (RFTM), reação em cadeia da polimerase (PCR) e sequenciamento de DNA. PCR e sequenciamento foram também utilizados para a identificação genética das ostras. Foram identificadas duas espécies de ostras: Crassostrea rhizophorae e C. brasiliana. Em ambas as espécies, P. beihaiensis foi a única espécie de Perkinsus detectada. Em C. rhizophorae, a prevalência média foi de 82,8% por histologia e de 65,2% por RFTM. Em C. brasiliana, as prevalências foram de 70,5% e 35,7%, respectivamente. A maior prevalência de P. beihaiensis em C. rhizophorae foi provavelmente influenciada pela salinidade, com a qual este apresentou correlação positiva (r>0,8). Em ambas as espécies, P. beihaiensis esteve localizada principalmente no epitélio gástrico. A infecção foi geralmente leve ou moderada, sem danos aparentes aos hospedeiros, mas em casos de infecção severa, houve reação hemocitária e desorganização de tecidos. As prevalências geralmente altas na região sugerem que as ostras devam ser monitoradas com relação a este patógeno, principalmente em áreas de cultivo.

Parasitism by a Digenea in Lucina pectinata (Mollusca: Lucinidae) / Parasitismo por um Digenea em Lucina pectinata (Mollusca: Lucinidae)

Abstract Lucina pectinata is an important economic resource in the Brazilian coast. This study reports parasitism caused by a Digenea in this species. The specimens (n = 470) were collected in December 2012 in a mangrove swamp of the Cachoeira River estuary, Bahia, Brazil. They were measured along the anterior-posterior axis (length), and after macroscopic analysis for parasites and diseases cuts of 5 mm were fixated in Carnoy's solution and processed by routine histology technique wherein sessions of 7 μm were stained with Harris hematoxylin and eosin (H&E). The tissues were examined using an optical microscope. The mean length of L. pectinata was 4.0 ± 0.53 cm. Microscopic analysis showed sporocysts containing both germ balls as cercariae of an unidentified Digenea (Platyhelminthes), these in various stages of development. The prevalence was 1.48% (7/470). In a parasitized specimen was macroscopic evidence of tissue densification of gills. The sporocysts were observed in mantle, gills, digestive gland and gonads, with evident alteration/destruction of tissues, including parasitic castration. There were no other parasites found, which is probably related to inaccessibility and chemical conditions in which lives L. pectinata, i.e., between 10 and 20 cm in mangrove sediment.

Resumo Lucina pectinata é um importante recurso econômico na costa brasileira. Este estudo relata parasitismo causado por um Digenea nesta espécie. Os exemplares (n = 470) foram coletados em dezembro de 2012 em um manguezal do estuário do Rio Cachoeira, Ilhéus, Bahia. Estes foram medidos quanto ao eixo ântero-posterior (comprimento) e após análise macroscópica quanto a parasitos e enfermidades, cortes transversais de 5 mm foram fixados em solução de Carnoy e processados por técnica rotineira de histologia, sendo que sessões de 7 μm foram coradas com hematoxilina de Harris e eosina (HE). Os tecidos foram examinados em microscopia de luz. A média de comprimento de L. pectinata foi de 4,0 ± 0,53 cm. A análise microscópica evidenciou esporocistos de um Digenea (Platyhelminthes) não identificado, contendo massas germinativas e cercárias, estas em vários estágios de desenvolvimento. A prevalência foi de 1,48% (7/470). Um dos exemplares parasitado apresentou adensamento dos tecidos nas brânquias. Os esporocistos foram observados no manto, brânquias, glândula digestiva e gônadas, causando evidente alteração/destruição de tecidos, inclusive castração parasitária. Não foram observados outros parasitos, o que está provavelmente associado à inacessibilidade e condições químicas em que vive este hospedeiro, isto é, entre 10 e 20 cm no sedimento do manguezal.

Separation and Identification of Microplastics in Digestive System of Bivalves / 分析化学

As a new type of pollutants,microplastics (small pieces of plastic with the longest dimension less than 5 mm) are bringing wide concerns by policy makers and researchers. The impact of microplastics on marine life is closely related to human health. Bivalves, as filter feeding creatures, can easily ingest microplastics in their feeding process. The microplastics in these bivalves can easily enter the human body through the food chain. In this study,a bivalve sample pretreatment method was established for separating the microplastics in their digestive system. Qualitative and quantitative analysis of microplastics was carried out by micro-Fourier transformed infrared spectroscope (μ-FT-IR) and Stereo microscope. By comparing the digestion systems of using 10% KOH and 30% H2O2, respectively, it was found that the digestion system using 10% KOH had better digestion efficiency. The recoveries of polypropylene (PP),polyethylene (PE), polystyrene (PS) and polyvinyl chloride (PVC) ranged from 96.7% to 98.6%, with RSD of less than 3.2%. Therefore, 10% KOH was used to digest organic tissue for separating the microplastics from the bivalves'digestive system. With this digestion system,the microplastics in Chlamys farreri from local markets and Mytilus galloprovincialis from both local markets and wild environments in Qingdao were separated and analyzed. The results showed that microplastics were found in over 80% of the individuals purchased from the market and 40% in the collected wild individuals. The average abundance of microplastics in Chlamys farreri purchased from different markets varied from 5.2 to 19.4 items/individual and 3.2-7.1 items/g, while in Mytilus galloprovincialis, the numbers varied from 1.9 to 9.6 items/individual and 2.0-12.8 items/g. Farmed mussels contained more microplastics (average 1. 9 items/individual, 3. 17 items/g) than wild mussels (average 0.53 items/individual, 2.0 items/g). There were various colors of microplastics. The detected microplastics came from three shapes: fibers, fragments and granules. Fibrous microplastics, being the most dominant ones,accounted for 84.11%. The average size of fibrous microplastics(0.66±0.70 mm) was larger than that of the other two shapes of microplastics. The number of microplastics decreased with increasing microplastic sizes. Microplastics of less than 500 μm coming from different markets were in the range of 26% to 84%. There was great spatial difference in the size of microplastics. The polymer composition of the microplastic was identified by μ-FT-IR. And it was found that the most common polymer component sin the samples were cellophane(CP),followed by polypropylene(PP). In the farmed mussels,PP was the most abundant component. The results showed that the abundance and types of microplastics in cultured or wild bivalves were closely related to their growing environment.

Parasitism by a Digenea in Lucina pectinata (Mollusca: Lucinidae)

Abstract Lucina pectinata is an important economic resource in the Brazilian coast. This study reports parasitism caused by a Digenea in this species. The specimens (n = 470) were collected in December 2012 in a mangrove swamp of the Cachoeira River estuary, Bahia, Brazil. They were measured along the anterior-posterior axis (length), and after macroscopic analysis for parasites and diseases cuts of 5 mm were fixated in Carnoys solution and processed by routine histology technique wherein sessions of 7 m were stained with Harris hematoxylin and eosin (H&E). The tissues were examined using an optical microscope. The mean length of L. pectinata was 4.0 ± 0.53 cm. Microscopic analysis showed sporocysts containing both germ balls as cercariae of an unidentified Digenea (Platyhelminthes), these in various stages of development. The prevalence was 1.48% (7/470). In a parasitized specimen was macroscopic evidence of tissue densification of gills. The sporocysts were observed in mantle, gills, digestive gland and gonads, with evident alteration/destruction of tissues, including parasitic castration. There were no other parasites found, which is probably related to inaccessibility and chemical conditions in which lives L. pectinata, i.e., between 10 and 20 cm in mangrove sediment.

Resumo Lucina pectinata é um importante recurso econômico na costa brasileira. Este estudo relata parasitismo causado por um Digenea nesta espécie. Os exemplares (n = 470) foram coletados em dezembro de 2012 em um manguezal do estuário do Rio Cachoeira, Ilhéus, Bahia. Estes foram medidos quanto ao eixo ântero-posterior (comprimento) e após análise macroscópica quanto a parasitos e enfermidades, cortes transversais de 5 mm foram fixados em solução de Carnoy e processados por técnica rotineira de histologia, sendo que sessões de 7 m foram coradas com hematoxilina de Harris e eosina (HE). Os tecidos foram examinados em microscopia de luz. A média de comprimento de L. pectinata foi de 4,0 ± 0,53 cm. A análise microscópica evidenciou esporocistos de um Digenea (Platyhelminthes) não identificado, contendo massas germinativas e cercárias, estas em vários estágios de desenvolvimento. A prevalência foi de 1,48% (7/470). Um dos exemplares parasitado apresentou adensamento dos tecidos nas brânquias. Os esporocistos foram observados no manto, brânquias, glândula digestiva e gônadas, causando evidente alteração/destruição de tecidos, inclusive castração parasitária. Não foram observados outros parasitos, o que está provavelmente associado à inacessibilidade e condições químicas em que vive este hospedeiro, isto é, entre 10 e 20 cm no sedimento do manguezal.

Perkinsus beihaiensis (Perkinsozoa) in oysters of Bahia State, Brazil

Abstract This study reports the pathogen Perkinsus beihaiensis in oysters of the genus Crassostrea on the coast of the State of Bahia (Brazil), its prevalence, infection intensity and correlation with salinity. Oysters (n = 240) were collected between October and December 2014 at eight sampling stations between latitudes 13°55'S and 15°42'S. The laboratory procedures included macroscopic analysis, histology, culture in Ray's fluid thioglycollate medium (RFTM), Polymerase Chain Reaction (PCR) and DNA sequencing. PCR and sequencing have been used for the genetic identification of oysters as well. Two species of oysters have been identified: Crassostrea rhizophorae and C. brasiliana. In both oyster species P. beihaiensis was the only Perkinsus species detected. In C. rhizophorae, the average prevalence was 82.8% by histology and 65.2% by RFTM. In C. brasiliana, the prevalences were 70.5% and 35.7%, respectively. The higher prevalence of P. beihaiensis in C. rhizophorae was probably influenced by salinity, with which was positively correlated (r> 0.8). In both oysters, P. beihaiensis was located mainly in the gastric epithelium. The infection was generally mild or moderate, without apparent harm to the hosts, but in cases of severe infection, there was hemocytical reaction and tissue disorganization. The generally high prevalence in the region suggests that oysters should be monitored with respect to this pathogen, especially in growing areas.

Resumo Este estudo relata o patógeno Perkinsus beihaiensis em ostras do gênero Crassostrea no litoral do Estado da Bahia (Brasil), sua prevalência, intensidade de infecção e correlação com a salinidade. As ostras (n = 240) foram coletadas entre outubro e dezembro de 2014 em oito estações amostrais entre as latitudes 13°55'S e 15°42'S. Os procedimentos laboratoriais incluíram análise macroscópica, histologia, cultivo em meio de tioglicolato de Ray (RFTM), reação em cadeia da polimerase (PCR) e sequenciamento de DNA. PCR e sequenciamento foram também utilizados para a identificação genética das ostras. Foram identificadas duas espécies de ostras: Crassostrea rhizophorae e C. brasiliana. Em ambas as espécies, P. beihaiensis foi a única espécie de Perkinsus detectada. Em C. rhizophorae, a prevalência média foi de 82,8% por histologia e de 65,2% por RFTM. Em C. brasiliana, as prevalências foram de 70,5% e 35,7%, respectivamente. A maior prevalência de P. beihaiensis em C. rhizophorae foi provavelmente influenciada pela salinidade, com a qual este apresentou correlação positiva (r>0,8). Em ambas as espécies, P. beihaiensis esteve localizada principalmente no epitélio gástrico. A infecção foi geralmente leve ou moderada, sem danos aparentes aos hospedeiros, mas em casos de infecção severa, houve reação hemocitária e desorganização de tecidos. As prevalências geralmente altas na região sugerem que as ostras devam ser monitoradas com relação a este patógeno, principalmente em áreas de cultivo.

Genotoxicity and oxidative stress as biomarkers in fresh water mussel, Lamellidens marginalis (Lam.) exposed to monocrotophos.

Monocrotophos (MCP) is an organophosphate pesticide widely used in India for controlling various pests. In this study, we evaluated the oxidative stress and genotoxic potential of MCP on the freshwater mussel Lamellidens marginalis (Lamarck) after 7 days exposure and repair of the damaged DNA after 4 days recovery. The bivalves were exposed to 5.25 mg/L of MCP for 7 days and then allowed to recover for 4 days in pesticide-free water. Increase in the levels of thiobarbituric acid reactive substances was recorded in the gill, muscle, foot and mantle tissues. Cellular antioxidant defences i.e. antioxidant enzyme activities like catalase, superoxide dismutase, glutathione reductase and glutathione-S-transferase were used as biomarkers of oxidative stress. Altered activities of antioxidant enzymes were observed after exposure. There was a significant recovery in the antioxidative enzymes in the tissues after the recovery period. To monitor genotoxicity of MCP, we used micronucleus and comet assay. Increase in Olive tail moment in the gill cells of exposed mussels as compared to that of control ones indicated significant DNA damage. Our findings suggest that the MCP-induced oxidative stress may be contributing partly to genotoxic damage of gill cells. Thus, these biomarkers are found to be useful in evaluating the toxicity of MCP in mussels.

A novel DNase like compound that inhibits virus propagation from Asian Green Mussel, Perna viridis (Linn.).

Viral diseases are not only responsible for health related issues but also exert pressure on the State economy. Tropical and subtropical countries have more prevalence of virus associated pathological conditions such as chickenpox, adenovirus related infections, dengue, chickengunya, infectious mononucleosis, etc. Treatment options with effective antiviral drugs are limited and are unfortunately not free from undesirable effects. The Asian Green Mussel, Perna viridis (Linn.) (Mytilidae) are not only important for their evolutionary significance, high caloric index, ecological role in the sequestration of environmental pollutants especially heavy metals, but also are potential source for extraction of therapeutic and bioactive compounds. On the other hand, generally in bivalves, virus mediated mortality is not uncommon. In this study, we made a maiden attempt of exploring DNAse like bioactivity for natural non-protenacious compound(s) extracted from P. viridis. Crude Methanol Extract (CME) of soft tissue of P. viridis and subsequently its partially purified component (PPC) possess exceptional ability to degrade indiscriminately both low and high molecular weight DNAs. In vitro digestions for1, 2 and 3 h with CME and PPC were found to be comparable to commercial (Sigma-Aldrich) enzyme, DNase I. Bioactive assays conducted to evaluate antimicrobial property, have shown that CME and PPC exclusively inhibit viral propagation. Nonetheless, CME & PPC have no effect on the propagation of bacteria (0 mm ZOI). These results indicate the possibility of a source of potential antiviral drug against DNA Group I viruses. Although our study does not provide any data to correlate to any physiological functions of these substances but provides a clue towards an important role in the biology of mussels. Any conclusion at this stage is premature. However, taking into consideration the significantly high virus mediated mortality in bivalves and the antiviral bioactivity of these substances, it appears that mussels have evolved some mechanisms to counteract some viruses.

Lista anotada de los bivalvos marinos del Perú / Checklist of the marine bivalves from Peru

Se presenta una lista taxonómica actualizada de 401 especies de bivalvos marinos del Perú, distribuidas en 62 familias y 195 géneros. Se considera la nomenclatura y clasificación en el contexto de los cambios recientes, incluyendo los trabajos de Bernard (1983) y Coan & Valentich-Scott (2012). Cada especie de la lista es comentada, teniendo en cuenta la distribución geográfica y el hábitat.

A checklist of the marine bivalve species from Peru is presented, this list has 401 species which are distributed in 65 families and 195 genera. The taxonomical classification and nomenclature is based on classical and current bibliography including Bernard (1983) and Coan & Valentich-Scott (2012). We give distribution and hábitat information about every species.

Limnoperna fortunei Dunker, 1857 larvae in different environments of a Neotropical floodplain: relationships of abiotic variables and phytoplankton with different stages of development / Larvas de Limnoperna fortunei Dunker, 1857 em diferentes ambientes de uma planície de inundação Neotropical: relação com variáveis abióticas e algas fitoplanctônicas em diferentes estágios de desenvolvimento

Abstract Limnoperna fortunei Dunker, 1857 is an Asian invasive freshwater bivalve. Although there need to contain their spread, studies about the biology of the larvae are scarce. We correlated the larval stages of L. fortunei with biotic factors such as phytoplankton and main abiotic variables in lotic environments of the Upper Paraná River floodplain. The four samples were taken quarterly during the year 2012. The Principal component analysis (PCA) showed only spatial differences, as did a Canonical Correspondence Analysis (CCA). High densities of larvae were recorded in all samples the Paraná River and Baía River only in December, especially those in their initial stage. In the biovolume of Class of algae, Bacillarophyceae showed the highest value, but Chlorophycea who was strongly correlated with the density of D-stage larvae. The large variety of phytoplankton, especially microplankton Chlorophyceae, high values of PO4, NH4 and temperature were positively correlated with high densities of D-stage larvae. We conclude that high temperature, and food availability, indicated by phytoplankton community, favored the reproduction of L. fortunei and enhance the ability of specie dispersion due to the increase in the emission of propagules. Therefore, studies that address the biology of golden mussel larvae should be performed in order to prevent its spread.

Resumo Limnoperna fortunei Dunker, 1857 é um bivalve asiático dulcícola invasor. Embora haja necessidade de conter sua dispersão, estudos que abordam a biologia de suas larvas ainda são escassos. Analisou-se as fases larvais de L. fortunei relacionando-as a fatores bióticos como a comunidade fitoplânctônica e às principais variáveis abióticas, em ambientes lóticos da planície de inundação do alto do rio Paraná. As quatro coletas foram trimestrais durante o ano de 2012. A análise de componentes principais (PCA) demonstrou apenas diferenças espaciais, assim como a Análise de Correspondência Canônica (CCA). Altas densidades de larvas foram registradas em todas as coletas no rio Paraná e no rio Baía apenas no mês de dezembro, principalmente para as larvas em estádio inicial. No biovolume das classes de algas, Bacillarophyceae obteve o maior valor, porém Chlorophycea foi a que fortemente correlacionou-se com a densidade de larvas D. A grande variedade de fitoplâncton, em especial de Chlorophyceae microplanctônica, altos valores de PO4, NH4 e temperatura estiveram positivamente correlacionadas com altas densidades de larvas D. Conclui-se que, altas temperaturas, e disponibilidade de alimento, como a comunidade fitoplanctônica, favorecem a reprodução de L. fortunei e aumentam a capacidade de dispersão da espécie devido ao incremento na emissão de propágulos. Portanto, estudos que abordem a biologia das larvas de mexilhão-dourado devem ser realizados a fim de evitar sua propagação.

Determination of Free Inorganic Cadmium Ions in Marine Bivalves by High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry / 分析化学

Abstract A new method was established to determine the free inorganic cadmium ion ( Cd2+) in marine bivalves using high performance liquid chromatography and inductively coupled plasma mass spectrometry ( HPLC-ICP-MS) . The free Cd2+ in shellfish was ultrasonic extracted for 40 min by 10 mmol/L Tris buffer solution ( with 0. 1 mol/L NaCl, pH=7. 5), and the separation of Cd2+ was achieved using an IonPac CS5A analytical column with an IonPac CG5A guard column. The mobile phase consisted of 50 mmol/L C2 H2 O4 and 95 mmol/L LiOH. The new method had a good linear relationship with the correlation coefficient of 0. 999 and the standard recoveries of Cd2+ were all above 84. 6%. The free Cd2+ content in several kinds of marine bivalves was determined using the method and the results showed that the percentage of free inorganic Cd2+ to the total Cd content in samples with high Cd content was higher than those with low Cd content.

Perfil socioeconômico dos marisqueiros e condições higiênicas adotadas na cadeia produtiva de ostra (mollusca, bivalvia) / Socio economic profile of shellfish collector and hygienic conditions in the production chain of oysters (mollusca, bivalvia) / Perfil socioeconómico de marisqueros y condiciones higiénicas adoptadas en la cadena productiva de ostra (mollusca, bivalvia)

O consumo de ostras na forma in natura exige muito cuidado e práticas higiênicas e sanitárias corretas pelos marisqueiros e demais manipuladores desse alimento durante o processo de beneficiamento à comercialização, para que não haja contaminação por microrganismos patogênicos e deteriorantes. O presente estudo teve como objetivo avaliar o perfil socioeconômico de marisqueiros e as condições higiênicas e sanitárias praticadas na cadeia produtiva de ostras do gênero Crassostrea desde a extração até a comercialização. No período de janeiro a abril de 2014, por meio da técnica de amostragem "Bola de Neve" foram selecionados 40 marisqueiros na Ilha de São Luís - MA, os quais foram entrevistados utilizando-se questionário semiestruturado. De acordo com os resultados, a maioria dos marisqueiros, possui ensino fundamental incompleto, faixa etária entre 21 e 30 anos e encontram-se em união estável ou casados. Parte das ostras são extraídas em bancos naturais na área de estudo, enquanto outra parte é proveniente de atravessadores oriundos de outros municípios maranhenses.Após a aquisição, pode demorar até três dias para serem comercializadas. Concluiu-se que as condições higiênicos sanitárias adotadas na atividade de extração de ostras até a comercialização são insatisfatórias, indicando falta de boas práticas em todas as etapas ao longo do processo de produção, o que pode representar riscos à saúde da população apreciadora do consumo in natura desse bivalve. Além disso, indica a necessidade de fiscalização dessa atividade, já que os organismos são transportados de forma inadequada e passam muito tempo até chegar ao consumidor final.

Consumption of fresh oysters requires very careful and correct hygienic and sanitary practices by shellfish collectors and other food handlers during the handling process to marketing, in order to avoid contamination by pathogenic microorganisms or deterioration. This study aimed to evaluate the socio-economic profile of shellfish collectors and the hygienic conditions applied in the production chain of Crassostrea oysters from extraction to marketing. In the period from January to April 2014, through a snowball sampling technique, a total of 40 shellfish collector were selected in Ilha de São Luís ­ MA. These collectors were interviewed using a semi-structured questionnaire. According to the results, most shellfish collector had not completed elementary school, aged between 21 and 30 years and married or co-habiting. Part of the oysters is extracted from natural stocks in the study area, while other oysters come from middlemen who bring them from other cities in Maranhão. After acquisition, they can take up to three days to be marketed. Therefore, it was concluded that although it is an important source of income, hygienic and sanitary conditions adopted in the oyster extraction activity are unsatisfactory, indicating a lack of good practices throughout the production process, which can pose risks to the population who consume this bivalve fresh. It also shows the need for supervision of the activity, since the organisms are improperly transported, spending much time until reaching the end consumer.

El consumo de ostras en la forma fresca, in natura, requiere gran cuidado y prácticas higiénicas sanitarias correctas por los marisqueros y otros manipuladores de ese alimento durante el proceso de beneficiación a la comercialización, para que no haya contaminación por microorganismos patógenos y de deterioración. Este estudio tuvo como objetivo evaluar el perfil socioeconómico de marisqueros y las condiciones higiénicas sanitarias practicadas en la cadena productiva de ostras del género Crassostrea, desde la extracción hasta la comercialización. En el período de enero a abril de 2014, a través de la técnica de muestreo "Bola de Nieve" se seleccionaron 40 marisqueros en la Isla de São Luís - MA, que fueron entrevistados mediante cuestionario semiestructurado. De acuerdo con los resultados, la mayoría de los marisqueros tiene enseñanza primaria incompleta, con edades comprendidas entre 21 y 30 años y se encuentran en unión estable o casados. Parte de las ostras son extraídas en bancos naturales en el área del estudio, mientras otra parte proviene de los intermediarios de otros municipios de Maranhão. Después de la adquisición, puede tardar hasta tres días para que sean comercializadas. Se concluye que las condiciones higiénicas sanitarias adoptadas en la actividad de extracción de ostras hasta la comercialización son insatisfactorias, indicando falta de buenas prácticas en todo el proceso de producción, lo que puede plantear riesgos a la salud de la población aficionada al consumo in natura de ese bivalvo. Además, indica la necesidad de supervisar esta actividad, ya que los organismos son transportados inadecuadamente y pasan mucho tiempo para llegar al consumidor final.

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...

Determinação de coliformes na água e no tecido mole das ostras extraídas em Cananéia, São Paulo, Brasil / Determination of coliforms in the water and soft tissue of oysters extracted in Cananéia, São Paulo, Brazil

Este estudo objetivou avaliar a qualidade microbiológica da água de várias localidades em que são produzidas ostras Crassostrea sp. no estuário de Cananéia, tanto como nos bivalves, por meio dos valores de coliformes baseados na Técnica dos Tubos Múltiplos. Foram realizadas coletas de água em dez localidades durante os anos de 2005 a 2011. A análise da água apresentou média geométrica de coliformes termotolerantes de 24,67 NMP 100 mL-1 e, nos tecidos das ostras, de 15,53 NMP g-1. Verificou-se uma diferença significativa entre as localidades analisadas quanto à densidade de coliformes totais e termotolerantes na água. Analisaram-se as variações das contagens bacterianas em relação à sazonalidade, verificando-se que o outono apresentou a maior média de coliformes nas ostras, sendo os maiores dados coletados em abril. Houve correlação positiva entre coliformes na água e na ostra.

This study sought to evaluate the microbiological quality of water from the various locations where specimens of the oysters Crassostrea sp. were harvested in Cananéia estuary, as well as of the bivalve itself, through values of coliforms based on the Multiple Tube Technique. During the period from 2005 to 2011, samples of water were collected at ten points. The water analysis presented a geometric mean of thermotolerant coliforms of 24.67 MPN 100 mL-1 and, in the soft tissues of the oysters, of 15.53 MPN g-1. There was a significant difference between the locations analyzed as to the density of total and thermotolerant coliforms in water. After analysis of the variations of bacterial counting with regard to seasonal mean, autumn presented the highest mean numbers of coliforms in oysters, and most data were collected in April. There was a positive correlation between the coliforms found in water and those in the oyster.

Parasites in the mangrove oyster Crassostrea rhizophorae cultivated in the estuary of the Graciosa River in Taperoá, Bahia / Parasitos na ostra-do-mangue Crassostrea rhizophorae cultivada no estuário do rio Graciosa em 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.

No presente trabalho, parasitos associados à ostra-do-mangue Crassostrea rhizophorae foram estudados em uma unidade de cultivo no estuário do rio Graciosa em Taperoá, Bahia. Mensalmente, entre abril de 2011 e março de 2012, 20 ostras foram coletadas, mensuradas e fixadas em formol a 10%. Para a identificação dos parasitos, foram utilizadas técnicas histológicas de rotina, com inclusão em parafina e obtenção de cortes com 7µm, corados com hematoxilina de Harris e eosina e examinados em microscopia de luz. Os parâmetros analisados para a qualidade da água mostraram que a temperatura variou de 23,9 °C a 29,3 °C, a salinidade de 0,4 a 24,2 PSU e a pluviometria de 80 mm a 406,4 mm/mês. Nas análises parasitológicas foram observadas infestações do poliqueta Neanthes succinea em 41% das ostras. A partir das técnicas histológicas foram identificados os seguintes parasitos: colônias de organismos assemelhados a Rickettsiae (RLOs); os protozoários Nematopsis sp. e Ancistrocoma sp.; o turbelário Urastoma sp.; o metacestóide Tylocephalum sp. e um platelminto não identificado. Os dados obtidos mostraram baixa intensidade de infecção e prevalência de parasitos, exceto para Nematopsis sp., atestando a boa condição de saúde das ostras no cultivo.