Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is pre-dominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Efecto de la temperatura y concentración de microplásticos en la tasa de filtración del mejillón Semimytilus algosus (Mytiloida: Mytilidae) / Effect of temperature and microplastic concentration on the filtration rate of the mussel Semimytilus algosus (Myiloida: Mytilidae)

Resumen Introducción: La presencia de microplásticos (MPs, partículas menores a 5 mm) y el incremento de la temperatura en los océanos, vienen generando perturbaciones en la vida marina, que se pueden relacionar con alteraciones en el metabolismo de organismos filtradores, como los mitílidos. Objetivo: Se evalúa el efecto de diferentes temperaturas y concentraciones de MPs sobre la tasa de filtración (TF) de Semimytilus algosus. Métodos: Una muestra de organismos (N = 72) fue expuesta a cuatro temperaturas (17, 20, 23 y 26 °C), y un testigo sin microplásticos (MPs0) y dos concentraciones de MPs (< 125 µm) de 0.125 mg/l (MPs1) y 0.250 mg/l (MPs2), todos en combinación con la microalga Isochrysis galbana (1x106 cel/ml/día) por 21 días. Resultados: A medida que aumentó la concentración de MPs, se redujo la TF de S. algosus. Respecto a la temperatura, durante el día 7 se observó una mayor TF a 23 °C en todos los tratamientos, y para los días 14 y 21 se obtuvieron los menores valores de TF a 23 y 26 °C. La acción conjunta del incremento de temperatura y MPs, afectó negativamente la TF de S. algosus, donde ambos factores ocasionaron el descenso de la TF para todos los tiempos de evaluación. No se registró mortalidad a 17 °C para ningún tratamiento, y en el caso de mitílidos expuestos a MPs1 y temperaturas de 20 y 26 °C se presentó la mayor mortalidad (67 %). Conclusiones: El estudio demuestra el efecto adverso del incremento de temperatura y MPs sobre la TF de S. algosus.

Abstract Introduction: The presence of microplastics (MPs, particles smaller than 5 mm) and the increase in temperature in the oceans, have been generating disturbances in marine life, which can be related to alterations in the metabolism of filter-feeders, such as Mythilids. Objective: The effect of different temperatures and concentrations of MPs on the filtration rate (TF) of Semimytilus algosus is evaluated. Methods: A sample of organisms (N = 72) was exposed to four temperatures (17, 20, 23 and 26 °C), and a control without microplastics (MPs0) and two concentrations of MPs (< 125 µm) of 0.125 mg/l (MPs1) and 0.250 mg/l (MPs2), all in combination with Isochrysis galbana microalgae (1x106 cells/ml/day) for 21 days. Results: As the concentration of MPs increased, the TF of S. algosus decreased. Regarding temperature, during day 7 a higher TF was observed at 23 °C in all treatments, and during days 14 and 21 the lowest TF values were obtained at 23 and 26 °C. The joint action of the increase in temperature and MPs, negatively affected the TF of S. algosus, where both factors caused the decrease in TF for all evaluation times. No mortality was recorded at 17 °C for any treatment, and in the case of mytylids exposed to MPs1 at 20 °C and 26 °C, the highest mortality (67 %) occurred. Conclusions: The study demonstrates the adverse effect of the increase in temperature and MPs on the TF of S. algosus.

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.

I See Golden Mussel! They are Everywhere! Environmental DNA Supports Widespread Dissemination of Limnoperna fortunei in Hydrographic Basins in the Paraná State, Brazil

Abstract The golden mussel is an aquatic invasive species that was introduced in South America in the '90s, including Brazil, and was firstly registered in 2001 in the Paraná state. This bivalve causes macrofouling issues because adhere in substrates and form dense aggregations, affecting especially hydroelectric power plants, and water treatment facilities, apart from the native biodiversity. The present research aimed to diagnose the distribution of Limnoperna fortunei in the Paraná state using environmental DNA (eDNA) from 174 sites from 12 basins, and scientific and technical previous records. L. fortunei eDNA was found in 90 sites from 11 hydrographic basins sampled. Most of the positive samples were detected in Iguaçu (42), Tibagi (15), and Ivaí (10) rivers basins. We also registered the first occurrence for five basins: Cinzas, Itararé, Ivaí, Pirapó e Ribeira rivers. Together, our and previous data recorded L. fortunei in 118 sites, between adult, larvae, and eDNA detection. Moreover, the results evidenced that eDNA is a low coast and reliable tool, and it may be very recommended for L. fortunei early detection and diagnosis. The present research was the greatest and most widespread survey for golden mussel prospection in a state in Brazil, supported a worrying scenario for Parana, with the urgent need for intense and continuous monitoring and prevention actions for controlling and mitigation of the L. fortunei impacts.

Zoochemical analyses and in vitro antimicrobial activity of crude methanolic extract of Perna viridis

@#<p style="text-align: justify;"><strong>Introduction:</strong> The rise of antibiotic resistance and superbugs drives the search for new antibiotics today. Meanwhile, the green mussel Perna viridis is a cultivated and marketed staple bivalve in the Philippines due to its fast repro-duction, high protein content, and tolerance to environmental variables. Although some studies have analyzed the antimicrobial activity of P. viridis, zoochemical analyses and further evaluation of its antimicrobial activity, such as determining the minimum inhibitory concentration (MIC), remains unexplored.</p><p style="text-align: justify;"><strong>Objectives:</strong> The study evaluated the zoochemicals present in crude methanolic extract of P. viridis by qualitative screening and thin-layer chromatography analysis. It further evaluated the crude extract for its antimicrobial activity against common pathogenic bacteria and plant pathogenic fungi.</p><p style="text-align: justify;"><strong>Materials and Methods:</strong> The zoochemicals in crude methanolic extract of P. viridis were screened using qualitative spotting methods and thin-layer chromatography (TLC). The antimicrobial activity of the extract was evaluated against the bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis, and the fungi Colletotrichum capsici, Lasiodiplodia theobromae, and Rhizopus sp. using disk diffusion assay and two-fold microdilution.</p><p style="text-align: justify;"><strong>Results:</strong> Qualitative screening and thin-layer chromatography analysis of the crude extract revealed detectable amounts of alkaloids, saponins, terpenoids, sterols, and polyphenols. All of the tested bacteria were susceptible to the extract with P. aeruginosa (19.00±0.82 mm) and S. aureus (19.33±0.47 mm) as the most inhibited with MICs of 2.60±0.63 and 3.65±1.69 mg/mL, respectively. However, for the three fungi tested, only the growth of the fungus L. theobromae (7.33±0.94 mm) was inhibited with a MIC of 33.33±11.79 mg/mL.</p><p style="text-align: justify;"><strong>Conclusion:</strong> It can be inferred that the zoochemicals detected in the crude extract of P. viridis contributed to its antimicrobial activity.</p><p style="text-align: justify;"><strong>Key Words:</strong> Antibacterial agent, Antifungal agent, Green mussel, Plant pathogenic fungi, Secondary metabolites</p>

Aislamiento de cepas de Enterococcus hirae productoras de enterocinas a partir del contenido intestinal de mejillón patagónico (Mytilus edulis platensis) Aislamiento de Enterococcus hirae de mejillón patagónico / Isolation of enterocin-producing Enterococcus hirae strains from the intestinal content of the Patagonian mussel (Mytilus edulis platensis)

Resumen Se aislaron del contenido intestinal del mejillón patagónico dos cepas de bacterias ácido lácticas y se caracterizaron por pruebas fenotípicas y moleculares. Los aislamientos se identificaron como Enterococcus hirae y fueron denominados E. hirae 463Me y 471Me. Por técnicas de PCR se identificó el gen de la enterocina P en ambas cepas, mientras que solamente en la cepa 471Me se detectó la enterocina hiracin JM79. Ambas cepas resultaron sensibles a los antibióticos clínicamente importantes y entre los rasgos de virulencia investigados mediante amplificación por PCR solo se pudieron detectar los genes cylL l y cylL s , sin embargo, no se observó actividad hemolítica en la prueba de agar sangre. Los sobrenadantes libres de células resultaron activos contra todas las cepas de Listeria y Enterococcus ensayadas, contra Lactobacillus plantarum TwLb 5 y contra Vibrio anguilarum V10. En óptimas condiciones de crecimiento, ambas cepas mostraron actividad inhibitoria contra Listeria innocua ATCC 33090 después de 2h de incubación. E. hirae 471Me alcanzó una actividad inhibitoria máxima de 163.840UA/ml después de 6h de incubación, mientras que el mismo valor se registró para E. hirae 463Me después de 8h. En ambos casos, la actividad antagonista alcanzó su máximo antes de lograr la fase estacionaria y permaneció estable hasta las 24h de incubación. En nuestro conocimiento, este es el primer informe de aislamiento de cepas bacteriocinogénicas de E. hirae de mejillón patagónico. La alta actividad inhibitoria y la ausencia de rasgos de virulencia indican que estos microorganismos podrían aplicarse en áreas biotecnológicas como la biopreservación de alimentos o las formulaciones probióticas.

Abstract Two bacteriocin-producing lactic acid bacterial strains were isolated from the intestinal content of the Patagonian mussel and characterized by phenotypic and molecular tests. The isolates were identified as Enterococcus hirae and named E. hirae 463Me and 471Me. The presence of the enterocin P gene was identified in both strains by PCR techniques, while enterocin hiracin JM79 was detected only in the 471Me strain. Both strains were sensitive to clinically important antibiotics and among the virulence traits investigated by PCR amplification, only cylL l and cylL s could be detected; however, no hemolytic activity was observed in the blood agar test. Cell free supernatants were active against all Listeria and Enterococcus strains tested, Lactobacillus plantarum TwLb 5 and Vibrio anguilarum V10. Under optimal growth conditions, both strains displayed inhibitory activity against Listeria innocua ATCC 33090 after 2h of incubation. E. hirae 471Me achieved a maximum activity of 163840AU/ml after 6h of incubation, while the same value was recorded for E. hirae 463Me after 8h. In both cases, the antagonist activity reached its maximum before the growth achieved the stationary phase and remained stable up to 24h of incubation. To our knowledge, this is first report of the isolation of bacteriocinogenic E. hirae strains from the Patagonian mussel. The high inhibitory activity and the absence of virulence traits indicate that they could be applied in different biotechnological areas such as food biopreservation or probiotic formulations.

Biosseguridade para sistemas de produção de peixes em tanque-rede em função da colmatação agravada por Limnoperna fortunei / Biosecurity for fishfarming cage systems due to the highly clogged screen aggravated by Limnoperna fortunei

Este trabalho avaliou a colmatação por Limnoperna fortunei em diferentes materiais para confecção de telas de arame, a dinâmica da colmatação pelo molusco e a relação custo-benefício dos materiais usados em tanques-rede. Utilizaram-se amostras de telas de simples torção de malha 19mm, de cinco tipos diferentes de revestimentos, colocadas na barragem de Salto Caxias, no Rio Iguaçu, estado do Paraná, entre julho de 2012 e julho de 2014. A cada seis meses, foi verificado o ganho de peso das telas em razão da colmatação, além da contagem do número de mexilhões aderidos. Após as coletas dos dados, foi realizado o teste estatístico de Kruskal-Wallis para se avaliar o ganho de peso das telas devido à colmatação. Realizou-se também uma pesquisa com 21 empresas para se obter o preço comercializado de telas para confecção de tanques-rede e se avaliar a expectativa de durabilidade dos materiais. Verificou-se que os diferentes materiais avaliados para as telas influenciam na colmatação e aderência do mexilhão-dourado; além disso, constatou-se que a tela mais eficiente quanto ao custo/ano é a de arame galvanizado plastificado e, para baixa colmatação, a de arame Bezinal.(AU)

This research evaluated degree of clogging by Limnoperna fortunei in different materials for wire mesh fabrication, the dynamics of mollusk sealing and the cost-benefit ratio of these materials used in tank-nets. Samples of single-twist screens of 19 mm mesh were used, from five different types of coatings were placed in the Salto Caxias dam on the Iguaçu River in the Paraná state, between July 2012 and July 2014. Every six months, it was verified the weight gain of the screens due to clogging and counting of the number of mussels adhered. After the data collection was performed Kruskal-Wallis statistical test to evaluate the weight gain of the screens due to clogging. In addition, a survey was carried out with 21 companies to obtain the commercialized price of screens for the production of net tanks and to evaluate the expected durability of the materials. The different materials evaluated for the screens influenced the sealing and adhesion of the golden mussel. In addition, the most cost-per-year screen is that of plastic-coated galvanized wire and for low clogging it is the Bezinal wire.(AU)

Qualidade microbiológica do sururu (Mytella charruana) comercializado por catadoras da Baía de Sepetiba, Rio de Janeiro, Brasil / Microbiological quality of sururu (Mytella charruana) marketed by scavengers of the Sepetiba Bay, Rio de Janeiro, Brazil

A preocupação com a qualidade do pescado é de grande importância para todos, pois é um alimento de alto valor nutritivo, mas tem grande susceptibilidade à deterioração e formação de substâncias prejudiciais à saúde, quando as condições de conservação e manipulação não são mantidas. O cultivo de moluscos bivalves além de ser uma fonte alternativa de alimentos, é uma opção para a subsistência das populações ribeirinhas de todo o litoral, à medida que a matriz alimentícia ganha espaço no mercado, principalmente nas regiões nordeste, sul e sudeste. Os sururus são organismos filtradores, capazes de absorver micropartículas em suspensão, as quais podem carrear elevadas concentrações de microrganismos patogênicos, outro fator importante relacionado a este molusco é a má condição de manipulação no beneficiamento, que pode provocar toxinfecções alimentares, ambos provocando danos à saúde. As análises microbiológicas foram realizadas em sururu in natura e após cozimento. Nas amostras analisadas houve ausência de Salmonella spp., com redução na contagem de Staphylococcus coagulase-positiva e de Coliformes Termotolerantes a 45ºC nos produtos cozidos. Os resultados são indicativos de que mesmo havendo redução de contaminação bacteriana, a manipulação das catadoras, após cozimento, é inadequada e que medidas corretivas devem ser adotadas para se obter o produto adequado bacteriologicamente ao consumo.

Fish quality has a great importance concerning to its high nutritional value, due to its great susceptibility to deterioration and occurrence of harmful substances to human health when the storage and handling conditions are not kept properly. The bivalves farming is an alternative source of food and also a source of livelihood for riverside communities, as the product is accepted in the market, mainly in the Northeast, South and Southeast regions. As filter feeders organisms, “sururu” mussels can absorb microparticles which can carry high level of pathogenic micro-organisms. Another important factor is the bad handling conditions both can lead to foodstuff infections. Microbiological analyzes were performed in raw “sururu” mussel and after cooking. The samples analyzed there was no Salmonella sp., a reduction in coagulase-positive Staphylococcus count and thermotolerant coliforms at 45 °C in baked products. Results of the analyzes showed that even with the contamination reduction, the handling of the women staff after cooking is inadequate and corrective measures should be taken to obtain a safe product for the final consumer.

More than two decades after the introduction of Limnoperna fortunei (Dunker 1857) in La Plata Basin / Duas décadas após a chegada de Limnoperna fortunei (Dunker 1857) na Bacia do Prata

Abstract The golden mussel, Limnoperna fortunei, is an Asian freshwater bivalve introduced in South America in the beginning of the 1990's, probably through ballast water releases in La Plata River estuary. It dispersed north through Paraná, Uruguay and Paraguay Rivers. The study evaluated the macroscale (18° to 34° S; 45° to 60° W) distribution of L. fortunei (larval stages) in the main rivers and reservoirs of La Plata Basin. Samplings were performed through 68 µm vertical plankton net hauls. Limnological variables were simultaneously determined. Larvae abundance correlated significantly with oxygen (positively) in summer, with temperature (positively) in winter and with total phosphorus and total nitrogen (both negatively) in winter. We expected densities to decrease towards north (latitudinal gradient) and increase in lentic conditions (reservoirs). Despite maximum density was found near the introduction point (La Plata River) similar value was also observed 2,000 km north (Paraguay River). The first hypothesis was refuted. The second hypothesis was partially accepted. Abundances were consistently higher in summer. Higher trophic conditions and fast flow seem to inhibit population growth. Food resources (chlorophyll a) locally influenced temporal variation. Limnoperna fortunei is widely distributed in La Plata basin, reaching high larval densities superior to 10,000 ind. m-3, in all major sub-basins. The species exhibits a high intrinsic dispersal ability (free planktonic larvae), wide tolerance to environmental factors and dispersion is potentialized by natural dispersion processes (e.g. fauna displacement) and human facilitation (e.g. commercial navigation).

Resumo O mexilhão dourado, Limnoperna fortunei, é um bivalve de água doce originário da Ásia e introduzido na América do Sul no início de 1990, provavelmente através da água de lastro liberada no estuário do rio La Plata. Dispersando-se para o norte através dos rios Paraná, Uruguai e Paraguai. Esse estudo avaliou a distribuição em macroescala (18° a 34° S; 45° a 60° W) de L. fortunei (estágios larvais) nos principais rios e reservatórios da bacia La Plata. Amostragens foram realizadas através de arrastos verticais com rede planctônica de 68 µm. Variáveis limnológicas foram simultaneamente determinadas. A abundância larval correlacionou-se significativamente com oxigênio (positivamente) no verão, com a temperatura (positivamente) no inverno e com fósforo total e nitrogênio total (ambos negativamente) no inverno. Esperávamos um decréscimo da densidade em direção ao norte (gradiente latitudinal) e aumento em condições lênticas (reservatórios). Apesar de a densidade máxima ter sido encontrada próximo do ponto de introdução (La Plata River) valores similares também foram observados 2.000 km ao norte (rio Paraguai). A primeira hipótese foi refutada. A segunda hipótese foi parcialmente aceita. Abundâncias foram consistentemente maiores no verão. Condições eutróficas e fluxo rápido parecem inibir o crescimento populacional. Recurso alimentar (clorofila a) local influenciou a variação temporal. Limnoperna fortunei está amplamente distribuído na bacia de La Plata, alcançando densidades larvais superiores a 10.000 ind. m-3 em todas as principais sub bacias. A espécie possui alta capacidade de dispersão intrínseca (larva planctônica livre), ampla tolerância aos fatores ambientais e os processos de dispersão são potencializados por processos naturais (e.g. deslocamento de fauna) e facilitação humana (e.g. navegação comercial).

Development of double-component rapid curing bioadhesive / 生物医学工程学杂志

Mussel foot proteins (MFp) could cure rapidly under water and adhere to different substrates. It has broad application prospects as an biocompatible bioglue. The soluble recombinant SUMO-MFp fusion protein (SFp3) was efficiently expressed in , and about 5% of tyrosine of SFp3 were converted into DOPA by using mushroom tyrosinase. The adhesion strength of the mixture of DOPA-containing SFp3 (DSFp3) and hyaluronic acid (MW = 1 500 kD) was more than twice that of the cyanoacrylate-based tissue adhesives, Dermabond , and it reached 52% of its maximal strength within 5 minutes on cowhide. A layer-by-layer assembly of hyaluronic acid with DSFp3 was observed to form compact sheet structures through biofilm interferometry assay and scanning electron microscopy. This work provides a solution and theoretical basis for the low adhesion strength and slow curing of protein-based bioglue.

Study on the 3,4-Dihydroxyphenylalanine Redox State Characterization Method of Mussel Adhesive Protein / 中国医疗器械杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of using liquid chromatography (HPLC) to characterize the 3, 4-Dihydroxyphenylalanine (DOPA) redox state of mussel adhesive protein (MAP).</p><p><b>METHODS</b>The DOPA and protein contents of MAP were determined by HPLC, Arnow and Bradford methods respectively.</p><p><b>RESULTS</b>With extended oxidation time, the protein contents of MAP samples remained unchanged whereas the DOPA contents declined. The retention times of main peaks in HPLC for both the accelerated oxidation and retained samples shifted as the storage time extended, which could be related to the changes of sample redox state.</p><p><b>CONCLUSIONS</b>The redox state of MAP can be characterized by the change of HPLC peak retention time. HPLC can be used in the research on the MAP redox state, which is beneficial to the product development and quality control.</p>

Physiological response of invasive mussel Limnoperna fortunei (Dunker, 1857) (Bivalvia: Mytilidae) submitted to transport and experimental conditions / Resposta fisiológica do mexilhão invasor Limnoperna fortunei (Dunker, 1857) (Bilvalvia: Mytilidae) submetido ao transporte e condições experimentais

Abstract Successful animal rearing under laboratory conditions for commercial processes or laboratory experiments is a complex chain that includes several stressors (e.g., sampling and transport) and incurs, as a consequence, the reduction of natural animal conditions, economic losses and inconsistent and unreliable biological results. Since the invasion of the bivalve Limnoperna fortunei (Dunker, 1857) in South America, several studies have been performed to help control and manage this fouling pest in industrial plants that use raw water. Relatively little attention has been given to the laboratory rearing procedure of L. fortunei, its condition when exposed to a stressor or its acclimation into laboratory conditions. Considering this issue, the aims of this study are to (i) investigate L. fortunei physiological responses when submitted to the depuration process and subsequent air transport (without water/dry condition) at two temperatures, based on glycogen concentrations, and (ii) monitor the glycogen concentrations in different groups when maintained for 28 days under laboratory conditions. Based on the obtained results, depuration did not affect either of the groups when they were submitted to approximately eight hours of transport. The variation in glycogen concentration among the specimens that were obtained from the field under depurated and non-depurated conditions was significant only in the first week of laboratory growth for the non-depurated group and in the second week for the depurated group. In addition, the tested temperature did not affect either of the groups that were submitted to transport. The glycogen concentrations were similar to those of the specimens that were obtained from the field in third week, which suggests that the specimens acclimated to laboratory conditions during this period of time. Thus, the results indicate that the air transport and acclimation time can be successfully incorporated into experimental studies of L. fortunei. Finally, the tolerance of L. fortunei specimens to the stressor tested herein can help us understand the invasive capacity of this mussel during the establishment process.

Resumo A criação bem sucedida de animais em condições de laboratório para processos comerciais ou experimentais é uma cadeia complexa que inclui vários fatores de estresse (ex. coleta e transporte) que tem como consequência a redução das condições naturais do animal, prejuízos econômicos e resultados biológicos inconsistentes. Desde a invasão do bivalve Limnoperna fortunei (Dunker, 1857) na América do Sul, vários estudos têm sido realizados para ajudar no controle e gestão dessa praga em plantas industriais que utilizam água. Relativamente pouca atenção tem sido dada ao processo de criação de L. fortunei em laboratório, sua condição quando exposta ao estresse e sua aclimatação a condições de laboratório. Considerando estes aspectos, os objetivos deste estudo foram: (i) investigar as respostas fisiológicas de L. fortunei submetidos ao processo de depuração e subsequente transporte (sem água/condição seca) em duas temperaturas, analisando as diferentes concentrações de glicogênio e (ii) monitorar as concentrações de glicogênio nos diferentes grupos, quando mantidos por 28 dias em condições de laboratório. Com base nos resultados obtidos, a depuração não afetou nenhum grupo quando eles foram submetidos a oito horas de transporte. A variação da concentração de glicogênio entre os espécimes do campo quando depurados e não depurados, foi significativa apenas em relação à primeira semana em laboratório para o grupo não depurado e à segunda semana para o grupo depurado. Além disto, a temperatura testada não afetou os grupos submetidos ao transporte. As concentrações de glicogénio foram semelhantes as dos espécimes do campo a partir da terceira semana, o que sugere que os espécimes estão aclimatados às condições de laboratoriais neste período de tempo. Assim, os resultados indicam que o transporte ao ar e o tempo de aclimatação podem ser incorporados com sucesso aos estudos experimentais com L. fortunei. Finalmente, o conhecimento sobre a tolerância de L. fortunei ao estresse pode ajudar a entender a capacidade invasiva deste durante o processo de estabelecimento.

More than two decades after the introduction of Limnoperna fortunei (Dunker 1857) in La Plata Basin

Abstract The golden mussel, Limnoperna fortunei, is an Asian freshwater bivalve introduced in South America in the beginning of the 1990s, probably through ballast water releases in La Plata River estuary. It dispersed north through Paraná, Uruguay and Paraguay Rivers. The study evaluated the macroscale (18° to 34° S; 45° to 60° W) distribution of L. fortunei (larval stages) in the main rivers and reservoirs of La Plata Basin. Samplings were performed through 68 µm vertical plankton net hauls. Limnological variables were simultaneously determined. Larvae abundance correlated significantly with oxygen (positively) in summer, with temperature (positively) in winter and with total phosphorus and total nitrogen (both negatively) in winter. We expected densities to decrease towards north (latitudinal gradient) and increase in lentic conditions (reservoirs). Despite maximum density was found near the introduction point (La Plata River) similar value was also observed 2,000 km north (Paraguay River). The first hypothesis was refuted. The second hypothesis was partially accepted. Abundances were consistently higher in summer. Higher trophic conditions and fast flow seem to inhibit population growth. Food resources (chlorophyll a) locally influenced temporal variation. Limnoperna fortunei is widely distributed in La Plata basin, reaching high larval densities superior to 10,000 ind. m-3, in all major sub-basins. The species exhibits a high intrinsic dispersal ability (free planktonic larvae), wide tolerance to environmental factors and dispersion is potentialized by natural dispersion processes (e.g. fauna displacement) and human facilitation (e.g. commercial navigation).

Resumo O mexilhão dourado, Limnoperna fortunei, é um bivalve de água doce originário da Ásia e introduzido na América do Sul no início de 1990, provavelmente através da água de lastro liberada no estuário do rio La Plata. Dispersando-se para o norte através dos rios Paraná, Uruguai e Paraguai. Esse estudo avaliou a distribuição em macroescala (18° a 34° S; 45° a 60° W) de L. fortunei (estágios larvais) nos principais rios e reservatórios da bacia La Plata. Amostragens foram realizadas através de arrastos verticais com rede planctônica de 68 µm. Variáveis limnológicas foram simultaneamente determinadas. A abundância larval correlacionou-se significativamente com oxigênio (positivamente) no verão, com a temperatura (positivamente) no inverno e com fósforo total e nitrogênio total (ambos negativamente) no inverno. Esperávamos um decréscimo da densidade em direção ao norte (gradiente latitudinal) e aumento em condições lênticas (reservatórios). Apesar de a densidade máxima ter sido encontrada próximo do ponto de introdução (La Plata River) valores similares também foram observados 2.000 km ao norte (rio Paraguai). A primeira hipótese foi refutada. A segunda hipótese foi parcialmente aceita. Abundâncias foram consistentemente maiores no verão. Condições eutróficas e fluxo rápido parecem inibir o crescimento populacional. Recurso alimentar (clorofila a) local influenciou a variação temporal. Limnoperna fortunei está amplamente distribuído na bacia de La Plata, alcançando densidades larvais superiores a 10.000 ind. m-3 em todas as principais sub bacias. A espécie possui alta capacidade de dispersão intrínseca (larva planctônica livre), ampla tolerância aos fatores ambientais e os processos de dispersão são potencializados por processos naturais (e.g. deslocamento de fauna) e facilitação humana (e.g. navegação comercial).

O mexilhão Perna perna no Brasil: nativo ou exótico? / The brown mussel Perna perna in Brazil: native or exotic?

A mitilicultura no Brasil se baseia no mexilhão Perna perna (Linnaeus, 1758), que é encontrado em todo o litoral brasileiro, sendo especialmente abundante do Espírito Santo a Santa Catarina. Nos últimos anos, foi lançada a hipótese de que P. perna seja uma espécie exótica no litoral brasileiro. A hipótese se baseia na análise da malacofauna presente em sítios arqueológicos. Todas as contestações levantadas não remetem à uma conclusão, pois precisam de estudos específicos, com metodologias claras, aliando arqueologia, ecologia e biologia molecular. O objetivo deste trabalho foi estudar a condição de nativo ou exótico do mexilhão Perna perna no Brasil, a partir de levantamento dos resultados em sítios arqueológicos, de técnicas moleculares e de datação de conchas com C14. A datação indicou que as amostras de Perna perna do sítio arqueológico Rio do Meio/Jurerê, Florianópolis/SC, têm idade de 720±30 e 780±30 anos. O cálculo do tempo de divergência indicou que a separação das populações brasileiras e africanas ocorreu por volta de 200 mil anos. Os resultados apontam a presença da espécie no território brasileiro muito antes do descobrimento do Brasil pelos portugueses no ano de 1500, indicando que P. perna é de fato uma espécie nativa.

Mussel farming in Brazil is based on brown mussel Perna perna (Linnaeus, 1758), which is found throughout the Brazilian coast, especially abundant from Espírito Santo to Santa Catarina coast. In recent years it was suggested that Perna perna is an exotic species for the Brazilian coast. The hypothesis is based on the analysis of the zooarchaeology studies in archaeological sites in Brazil. All objections raised do not offer a conclusion, because they need specific studies with clear methodology, combining archeology, ecology and molecular biology. The aim of this work was to study the condition of the brown mussel Perna perna in Brazil if native or exotic, from survey results in archaeological sites, molecular techniques and dating of shells with C14. The dating indicated that the shells were 720±30 and 780±30 years, respectively. The calculation of divergence time indicated that the separation of the African and Brazilian mussel populations occurred around 200 thousand years ago. The results indicate the presence of the P. perna species in Brazilian territory long before the discovery of Brazil by the Portuguese in 1500, indicating that P. perna is actually a native species of Brazil.

Determination of the content and the molecular weight of Mussel Polysaccharide by GPC / 中国生化药物杂志

Objective To develop a gel permeation chromatography method for determination of content and molecular weight ( Mw ) of Mussel Polysaccharide.Methods Using GPC method, the sample was separated with TSK-gel GMPWXL(7.8 mm ×300 mm) chromatography column which was set at 35℃.The mobile phase was 0.05 mol/mL NaNO3(including 0.05%Na2N3) and the flow rate was 0.6 mL/min.The detector was RID-20AT. Results The average molecular weight of the polysaccharide of Mytilus coruscus was 1 261 411 and the average content was 88.6%by using of the calibration curves of dextrans.The average molecular weight of the polysaccharide of Mytilus edulis was 1 244 062 and the average content was 87.4%. Conclusion The method established in this paper is simple and rapid, accurate and reproducible, which can be used for the quality control of Mussel Polysaccharide.

Investigation on Aspidogastrea infection in freshwater mussels in Anhui Province / 中国血吸虫病防治杂志

Objective To investigate the prevalence of trematode Aspidogastrea in freshwater mussels in Anhui Province, China. Methods The freshwater mussels living in different water areas within Anhui territory were harvested and dissected from April to May,2015. Then the flukes were collected from the pericardial cavities of the clams into a petri dish containing small amount of saline. After rinsing,the flukes were transferred into a bottle containing 70%ethanol for following identification by staining of the specimens prepared as previous protocol. Results A total of 3 007 calms in 12 species,including Unio doug?lasiae(603),Acuticosta chinensis(90),Lamprotula caveat(150),Lamprotula leai(250),Lanceolaria grayana(47),An?odonta pacifica(60),Anodonta woodiana(350),Cuneopsis heudei(100),Solenaia oleivora(150),Cristaria plicata (567),Hyriopsis cumingii(550)and Arconaia lanceolata(90)were dissected,in which 1 467 mussels were infected with the trematode Aspidogastrea,with an infection rate of 48.79%(1 467/3 007). Totally,7 306 flukes belonging to 3 genera under the subclass of Aspidogastrea were isolated. These trematodes were genera of Aspidogaster,Lophotaspis and Cotylaspis. The in?fection density was from 1 to 128 capita for individual positive clam,with a mean infectiosity of 4.98(7306/1467)for each mus?sel. Conclusion The freshwater bivalves living in different water areas in Anhui Province are infected with the trematode Aspi?dogastrea. These flukes were identified as Aspidogaster sp. Lophotaspis sp. and Cotylaspis sp. belonging to subclass Aspidogas?tridae of family Aspidogastrea.

Molecular markers for genetic diversity, gene flow and genetic population structure of freshwater mussel species / Marcadores moleculares para estudo da diversidade genética, fluxo génico e estrutura genética de populações de espécies de mexilhões de água doce

Freshwater mussel species are in global decline. Anthropogenic changes of river channels and the decrease of autochthonous fish population, the natural hosts of mussels larval stages (glochidia), are the main causes. Therefore, the conservation of mussel species depends not only on habitat conservation, but also on the availability of the fish host. In Portugal, information concerning most of the mussel species is remarkably scarce. One of the most known species, Unio pictorum is also in decline however, in the basins of the rivers Tua and Sabor (Northeast of Portugal), there is some indication of relatively large populations. The aforementioned rivers can be extremely important for this species conservation not only in Portugal, but also in the remaining Iberian Peninsula. Thus, it is important to obtain data concerning Unio pictorum bioecology (distribution, habitat requirements, population structure, genetic variability, reproductive cycle and recruitment rates), as well as the genetic variability and structure of the population. Concomitantly, information concerning fish population structure, the importance of the different fish species as “glochidia” hosts and their appropriate density to allow effective mussel recruitment, will also be assessed. The achieved data is crucial to obtain information to develop effective management measures in order to promote the conservation of this bivalve species, the conservation of autochthonous fish populations, and consequently the integrity of the river habitats.

As espécies de mexilhões de água doce estão em declínio global. Mudanças antropogénicas do canal dos rios e diminuição das populações de peixes autóctones, os hospedeiros naturais do mexilhão nos estágios larvais (glochidia), são as principais causas. Portanto, a conservação de espécies de mexilhão depende não só da conservação dos habitats, mas também da disponibilidade do hospedeiro peixe. Em Portugal, a informação relativa à maioria das espécies de mexilhão é extremamente escassa. Uma das espécies mais conhecidas, Unio pictorum também está em declínio, no entanto, nas bacias dos rios Tua e Sabor (Nordeste de Portugal), existe indicação de populações relativamente grandes. Os rios acima mencionados podem ser extremamente importantes para a conservação das espécies, não só em Portugal, mas também na restante Península Ibérica. Assim, é importante obter dados relativos à bioecologia do Unio pictorum (distribuição, requisitos de habitat, estrutura populacional, variabilidade genética, ciclo reprodutivo e as taxas de recrutamento), bem como a variabilidade e estrutura genética da população. Concomitantemente, informações sobre a estrutura da população de peixes, a importância das diferentes espécies de peixes como hospedeiros e sua densidade adequada para permitir o recrutamento eficaz do mexilhão, também será avaliada. Os resultados obtidos serão cruciais para obter informações que permitam o desenvolvimento de medidas de gestão eficazes, a fim de promover a conservação destas espécies de bivalves, a conservação das populações de peixes autóctones e, consequentemente, a integridade dos habitats fluviais.

Molecular markers for genetic diversity, gene flow and genetic population structure of freshwater mussel species

Freshwater mussel species are in global decline. Anthropogenic changes of river channels and the decrease of autochthonous fish population, the natural hosts of mussels larval stages (glochidia), are the main causes. Therefore, the conservation of mussel species depends not only on habitat conservation, but also on the availability of the fish host. In Portugal, information concerning most of the mussel species is remarkably scarce. One of the most known species, Unio pictorum is also in decline however, in the basins of the rivers Tua and Sabor (Northeast of Portugal), there is some indication of relatively large populations. The aforementioned rivers can be extremely important for this species conservation not only in Portugal, but also in the remaining Iberian Peninsula. Thus, it is important to obtain data concerning Unio pictorum bioecology (distribution, habitat requirements, population structure, genetic variability, reproductive cycle and recruitment rates), as well as the genetic variability and structure of the population. Concomitantly, information concerning fish population structure, the importance of the different fish species as glochidia hosts and their appropriate density to allow effective mussel recruitment, will also be assessed. The achieved data is crucial to obtain information to develop effective management measures in order to promote the conservation of this bivalve species, the conservation of autochthonous fish populations, and consequently the integrity of the river habitats.

As espécies de mexilhões de água doce estão em declínio global. Mudanças antropogénicas do canal dos rios e diminuição das populações de peixes autóctones, os hospedeiros naturais do mexilhão nos estágios larvais (glochidia), são as principais causas. Portanto, a conservação de espécies de mexilhão depende não só da conservação dos habitats, mas também da disponibilidade do hospedeiro peixe. Em Portugal, a informação relativa à maioria das espécies de mexilhão é extremamente escassa. Uma das espécies mais conhecidas, Unio pictorumtambém está em declínio, no entanto, nas bacias dos rios Tua e Sabor (Nordeste de Portugal), existe indicação de populações relativamente grandes. Os rios acima mencionados podem ser extremamente importantes para a conservação das espécies, não só em Portugal, mas também na restante Península Ibérica. Assim, é importante obter dados relativos à bioecologia do Unio pictorum (distribuição, requisitos de habitat, estrutura populacional, variabilidade genética, ciclo reprodutivo e as taxas de recrutamento), bem como a variabilidade e estrutura genética da população. Concomitantemente, informações sobre a estrutura da população de peixes, a importância das diferentes espécies de peixes como hospedeiros e sua densidade adequada para permitir o recrutamento eficaz do mexilhão, também será avaliada. Os resultados obtidos serão cruciais para obter informações que permitam o desenvolvimento de medidas de gestão eficazes, a fim de promover a conservação destas espécies de bivalves, a conservação das populações de peixes autóctones e, consequentemente, a integridade dos habitats fluviais.

Composition and seasonal variation of brachyura and anomura (Crustacea, Decapoda) associated with brown mussel farms at Praia da Cocanha, Brazil

The composition and seasonal variation of brachyuran and anomuran species associated with mussel farms were evaluated at Praia da Cocanha, São Paulo between May 2007 and February 2008. Nine mussel ropes were sampled at random in each quarter, and 1,208 organisms were identified, comprising five families and 28 species. The most numerous species was the porcellanid Pachycheles laevidactylus (18.5%), followed by the xanthids Acantholobulus schmitti (16.6%), Hexapanopeus paulensis (11.3%), Panopeus americanus (10.2%), and Menippe nodifrons (8.4%). The exotic crab Charybdis hellerii was recorded throughout the study period. The ecological descriptors, except Pielou evenness index, varied significantly over the time. The highest abundance and diversity of the species were recorded during November and February. This pattern was reversed for Berger-Parker dominance, with the lowest values recorded in February. The development of epifauna was correlated with the different stages of the mussel farms, since the mean size of mussels and consequently the abundance of epibiotic organisms and the structural complexity on the mussel ropes increased from May (seeding) until February (harvest). Despite this, the temporal population variations in recruitment patterns of the different epibionts should not be overlooked. The results indicated that the mussel farms provided favorable conditions for the development of these crustacean groups, which could be used in environmental monitoring programs and / or be exploited for the aquarium trade.